ITALY ITALY Abstract Book

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1 ITALY ITALY Abstract Book

2 Table of Content Oral Presentations.. Page 3 Poster Presentations.. Page 96 Abstract Author Index. Page 336 2

3 NSV 208, Verona Abstract Book ORAL PRESENTATIONS BREAKING AND ENTERING - viral entry Abstract final identifier: HUMAN LEUCOCYTE ANTIGEN DR (HLA-DR) HOMOLOGS ARE CROSS-SPECIES ENTRY RECEPTORS FOR BAT INFLUENZA VIRUSES Silke Stertz *, Umut Karakus, Thiprampai Thamamongood 2, Kevin Ciminski 2, Wei Ran 2, Sira C. Günther, Davide Eletto, Benjamin G. Hale, Adolfo Garcia-Sastre 3, Martin Beer 4, Martin Schwemmle 2 University of Zurich, Zurich, Switzerland, 2 Medical Center University of Freiburg, Freiburg, Germany, 3 Icahn School of Medicine at Mount Sinai, New York, United States, 4 Friedrich-Loeffler-Institute, Greifswald-Insel Riems, Germany Abstract: In 202 and 203 two novel influenza A viruses were discovered in South American bat species. Remarkably, the hemagglutinin of these bat influenza viruses was found not to bind the canonical influenza virus receptor, sialic acid, or any other glycan, despite high sequence and structural homology with conventional influenza A virus hemagglutinins. Using transcriptomic profiling of susceptible versus non-susceptible cells, in combination with genome-wide CRISPR/Cas9-based screening, we identified the MHC-II complex HLA-DR as proteinaceous receptor for bat influenza viruses. CRISPR/Cas9- mediated knockout of HLA-DR rendered susceptible cells completely resistant to bat influenza but not conventional influenza virus infection, whereas ectopic expression of HLA-DR in non-susceptible cells conferred susceptibility to bat influenza virus infection. Expression of HLA-DR homologs from three different bat species, pigs or chickens also conferred susceptibility. Notably, infection of mice with bat influenza virus resulted in robust virus replication in the upper respiratory tract, whereas mice deficient for MHC-II were resistant to infection. Collectively, our data identify HLA-DR homologs from multiple species as receptors for bat influenza virus, suggesting the potential for broad vertebrate tropism. 3

4 BREAKING AND ENTERING - viral entry Abstract final identifier: 2 NECTINS TRANSFER CYTOPLASM BETWEEN CELLS AND CAN SPREAD MEASLES VIRUS TO NEURONS Alex Generous, Oliver Harrison 2, Regina Troyanovsky 3, Mathieu Mateo, Chanakha Navaratnarajah, Ryan Donohue, Christian Pfaller, Alina Sergeeva 2, Indrajyoti Indra 3, Theresa Thornburg 4, Irina Kochektova 4, Matthew Taylor 4, Sergey Troyanovsky 3, Barry Honig 2, Lawrence Shapiro 2, Roberto Cattaneo * Molecular Medicine, Mayo Clinic, Rochester MN, 2 Biochemistry and Molecular Biophysics, Columbia University, New York, 3 Dermatology, Northwestern University, Chicago, 4 Microbiology and Immunology, Montana State University, Bozeman, United States Abstract: We discovered a process mediating intercellular transfer of cytoplasmic materials. Cells expressing the adherens junction protein nectin- (N) take in plasma membrane patches and cytoplasmic materials from cells expressing other nectins. This process is most active during cell adhesion, 2-8 hours post-co-culture. It is most efficient from cells expressing nectin-4 (N4) towards cells expressing N. And, it depends on the N cytoplasmic tail: its deletion prevents transfer, while its exchange with the N4 cytoplasmic tail reverses transfer direction. We term the process nectin-elicited cytoplasm transfer (NECT). Nectin family proteins serve as receptors for positive strand RNA viruses like poliovirus, large DNA viruses like herpes simplex virus, and negative strand RNA viruses such as measles virus (MeV). MeV infections can cause subacute sclerosing panencephalitis (SSPE), a rare but lethal disease. Because of SSPE and of other neurological diseases caused by MeV and related animal viruses, a neuronal receptor has been postulated but no consensus candidate has emerged. We show that N4-expressing, MeV-infected epithelial cells transmit infection to axons of N-expressing primary neurons that cannot be infected by MeV particles. Thus NECT can spread MeV infections to neurons, circumventing the need for a neuronal receptor. We have evidence that NECT functions through an endocytic pathway, and seek to understand how and where MeV ribonucleocapsids escape it. 4

5 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: 3 STRUCTURE OF FULL-LENGTH INFLUENZA HAEMAGGLUTININ Donald J. Benton *, Andrea Nans 2, Lesley J. Calder 2, Davide Corti 3, Steven J. Gamblin, Peter B. Rosenthal 2, John J. Skehel Structural Biology of Disease Processes Laboratory, 2 Structural Biology of Cells and Viruses Laboratory, The Francis Crick Institute, London, United Kingdom, 3 -, Humabs BioMed SA, Bellinzona, Switzerland Abstract: Influenza A virus Haemagglutinin (HA) is the surface glycoprotein responsible for receptor binding and membrane fusion. Previous structural characterisation of HA has been limited to a soluble ectodomain which does not contain membrane proximal or transmembrane regions. We present structures, determined by cryo-em, of full-length detergentsolubilised HA in isolation and complexed with a Fab fragment from an infectivity neutralising, H subtype-specific antibody, that recognises the ecto-domain-transmembrane domain junction. The structures of the ectodomain compare favourably in resolution with those previously determined by x-ray crystallography. Our description of the previously undetermined parts of the molecule may be important in relation to the immune recognition of the membrane proximal region of HA in antibody binding and antibody induction. 5

6 BREAKING AND ENTERING - viral entry Abstract final identifier: 4 STRUCTURAL BASIS OF LOW-DENSITY LIPOPROTEIN RECEPTOR RECOGNITION BY VSV GLYCOPROTEIN Laura Belot, Nikolic Jovan, Pierre Legrand 2, Hélène Raux, Yves Gaudin, Aurelie Albertini * Institut de Biologie Intégrative de la Cellule, CNRS, 2 Synchrotron SOLEIL, GIF SUR YVETTE, France Abstract: Vesicular stomatitis virus (VSV) is an oncolytic rhabdovirus and its glycoprotein G is widely used to pseudotype other viruses for gene therapy. VSV G mediates both virus attachment to its receptor and fusion of the viral envelope with the endosomal membrane. Low-density lipoprotein receptor (LDL-R) serves as a major entry receptor for VSV. We demonstrate that VSV G is able to independently bind two distinct cystein-rich (CR) domains (CR2 and CR3) of LDL-R with similar affinities (Kd ~ 5µM). The biological relevance of this interaction was demonstrated by the ability of both CR2 and CR3 to inhibit VSV infection. We obtained two crystal structures of G in its pre-fusion conformation in complex with CR2 and CR3 revealing that the binding sites of CR2 and CR3 on G are identical. CR domains recognition by VSV G involves basic residues pointing toward the calcium-coordinating acidic residues present in each CR. Mutations of two of these residues abolish G ability to bind to LDL-R without impairing G fusion activity. This demonstrates that it is possible to decouple G fusion activity and receptor recognition. We also show that although VSV can use alternative receptors of the LDL-R family, G mutants affected in their CR domain binding site cannot rescue a recombinant VSV lacking the G gene. Those data indicate that the only receptors of VSV are members of the LDL-R family and that G has specifically evolved to interact with their CR domains. This work provides structural insights on the interaction between G and host cell receptors and paves the way for the design of recombinant G with an altered tropism. 6

7 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: 5 DELINEATING INTERACTION OF FILOVIRAL GP WITH ITS ENDOSOMAL RECEPTOR NPC BY IN SITU PROXIMITY LIGATION Eva Mittler *, Rohit K. Jangra, Tanwee Alkutkar, Kartik Chandran Microbiology and Immunology, ALBERT EINSTEIN COLLEGE OF MEDICINE, New York, United States Abstract: Filoviruses are emerging zoonotic pathogens that cause outbreaks of lethal hemorrhagic fever in sub-saharan Africa. The development of effective countermeasures against these agents is hindered by our limited understanding of filovirus-host molecular interactions required for viral entry and infection. For host cell entry, virions traffic to late endosomes/lysosomes (LE/LY), where the viral surface glycoprotein GP gains access to multiple essential host factors: GP is proteolytically processed by cathepsin B/L, and a cleaved form of GP (GPCL) binds to the critical intracellular receptor Niemann-Pick C (NPC). Mechanistic studies of this indispensable binding step have been limited to a truncated, soluble form of a single domain in NPC, domain C, as robust cell-based assays assessing interaction of GPCL with full-length NPC in its native context have been lacking. Here, we developed an in situ assay to monitor GPCL:NPC binding in infected cells. Subcellular visualization of this interaction at single molecule resolution was based on the principle of DNA-guided, antibodymediated in situ proximity ligation. GPCL:NPC interaction was restricted to the lumina of NPC + LE/LY, and was blocked by disruption of GP s proteolytic cleavage or GPCL:NPC interface formation. Testing the effect of FDA-approved small molecule inhibitors on proximity ligation revealed that drug treatments significantly disrupted virus entry and GPCL:NPC binding by distinct mechanisms. In summary, our in situ proximity ligation assay allows us to monitor GPCL:NPC engagement in intact cells. We are employing it to define the cellular and viral requirements for this interaction and to delineate the mechanisms of action of small molecules with anti-filovirus activity. 7

8 BREAKING AND ENTERING - viral entry Abstract final identifier: 6 HOST FACTOR REQUIRED FOR IN VIVO PATHOGENICITY OF NEW WORLD HANTAVIRUSES Rohit K. Jangra *, Andrew S. Herbert 2, Lucas T. Jae 3, Rong Li 4, Lara M. Kleinfelter, Megan M. Slough, Eva Mittler, Ana I. Kuehne 2, James Pan 5, Sarah L. Barker 5, Ariel S. Wirchnianski, Sushma Bharrhan, Anna Z. Wec, J. Maximilian Fels, Melinda Ng, Nicolás A. Muena 6, Nicole D. Tischler 6, Sachdev S. Sidhu 7, Jason Moffat 5, 7, Zhongde Wang 4, Thijn R. Brummelkamp 3, John M. Dye 2, Kartik Chandran Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, 2 United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, MD, United States, 3 Netherlands Cancer Institute, Amsterdam, Netherlands, 4 Department of Animal, Dairy and Veterinary Sciences, Utah State University,, Logan, UT, United States, 5 Donnelly Centre and Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada, 6 Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile, 7 Canadian Institute for Advanced Research, Toronto, ON, Canada Abstract: Hantaviruses are rodent-borne RNA viruses that cause a severe cardiopulmonary syndrome (HCPS) in humans in the New World. Currently, no vaccines or therapeutics are available against these viruses. Host factors that mediate hantavirus infection and disease in vivo remain unknown. Using a genetic screen in human haploid cells, we identified a cadherin-superfamily protein, previously associated with hereditary respiratory disease, as an entry receptor for HCPScausing Andes and Sin Nombre viruses. This cadherin is highly expressed in pulmonary endothelial cells, which are major hantavirus targets in vivo, and its genetic depletion substantially reduced infection. Cadherin directly engaged the viral glycoprotein via its first extracellular cadherin (EC) domain to mediate virus-cell attachment and internalization, and viral infection was sensitive to soluble EC and EC-targeting monoclonal antibodies. Finally, Cadherin knockout Syrian hamsters, generated by CRISPR/Cas9 genome engineering, were strongly protected from lethal Andes virus challenge, highlighting its pathogenicity relevance and therapeutic potential. [RKJ, ASH, LTJ & RL contributed equally] 8

9 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: 7 ANCHORING THE SPRING: THE ROLE OF TRANSMEMBRANE DOMAIN INTERACTIONS IN VIRAL FUSION PROTEIN FUNCTION Rebecca E. Dutch *, Chelsea Barrett, Stacy Webb Molecular and Cellular Biochemistry, UNIVERSITY OF KENTUCKY, Lexington, United States Abstract: Enveloped viruses utilize surface glycoproteins to bind and fuse with a target cell membrane. The zoonotic Hendra virus (HeV), a member of the Paramyxoviridae family, utilizes the HeV attachment protein (G) and fusion protein (F) to perform these critical functions. Upon triggering, the trimeric F protein undergoes a set of large, irreversible conformation changes to drive membrane fusion. We have shown that the transmembrane domain (TM) of the F protein, separate from the rest of the protein, is present in a monomer-trimer equilibrium, and that specific sequences drive this association. This TM-TM association contributes to the stability of the pre-fusion form of the protein, supporting a role for the TM-TM interactions in control of F protein conformational changes. To determine the impact of disrupting TM-TM interactions, constructs expressing the HeV F TM with limited flanking sequences were synthesized. Co-expression of these constructs with HeV F resulted in dramatically reductions in the stability of F protein expression and ablation of fusion activity. In contrast, no effects were observed when the HeV F TM constructs were co-expressed with the non-homologous parainfluenza virus 5 (PIV5) fusion protein, indicating a requirement for specific interactions. To further examine this, a TM peptide homologous to the PIV5 F TM domain was synthesized. Addition of the peptide prior to infection inhibited viral infection with PIV5, but did not significantly affect infection of human metapneumovirus, a related virus. These findings indicate that TM-TM interactions are a critical stabilizer for the pre-fusion form of viral fusion proteins, and suggest that disruption of these interactions inactivates F protein function, likely by prematurely triggering F protein conformational changes. 9

10 BREAKING AND ENTERING - viral entry Abstract final identifier: 8 UNCOVERING A NEW PARAMYXOVIRAL ATTACHMENT GLYCOPROTEIN ROLE BEYOND FUSION TRIGGERING Hector C. Aguilar *, I Abrrey Monreal, Victoria Ortega, J. Lizbeth R. Zamora Microbiology and Immunology, Cornell University, Ithaca, NY, United States Abstract: Paramyxoviruses have two glycoproteins that in concert promote viral-cell membrane fusion during viral entry and cell-cell membrane fusion during the pathognomonic syncytia formation. The attachment glycoprotein (HN, H, or G) has two known functions:. binding the target cell surface receptor, and 2: subsequently triggering the fusion protein (F) to initiate its own conformational cascade. The F cascade proceeds from pre-fusion to pre-hairpin intermediate (PHI) to postfusion conformations, executing membrane fusion. Recently, we identified conformational changes in Nipah virus (NiV) G that link receptor binding to F triggering, and identified a role of the NiV G stalk domain in F triggering. Further, we found G and F mutants whose fusogenicity negatively correlates with the avidity of G/F interactions, supporting a model in which dissociation of a G/F bidentate interaction allows membrane fusion. In this study, we found several hypofusogenic mutants in the NiV G stalk that expressed well at the cell surface, bound receptors at wild-type levels, but relatively increased the G oligomeric strength, and bound F with higher avidities. Surprisingly, in a heterologous cell-cell fusion assay, some of these G mutants trapped the membrane fusion cascade at the hemifusion stage, whereby only lipids, but not cytoplasmic contents were mixed. Importantly, these G mutants were capable of F triggering, defined as changing F from pre-fusion to PHI conformations. Combined, these data suggest that G has a role in a later F conformational step beyond the PHI, and that detachment of G from F helps F undergo such conformational step(s) to allow full membrane fusion. This role is novel for the paramyxoviral attachment glycoproteins. 0

11 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: 9 HANTAVIRUS GC HOMOMERIC DIMER AND TRIMER CONTACTS DIRECT SPIKE DISSOCIATION AND MEMBRANE FUSION Eduardo A. Bignon, Pablo Guardado-Calvo 2, Félix Rey 2, Nicole Tischler * Molecular Virology Laboratory, FUNDACIÓN CIENCIA & VIDA, Santiago, Chile, 2 Unité de Virologie Structurale, Département de Virologie, Institut Pasteur, Paris, France Abstract: Rodent-transmitted hantaviruses are human pathogens that expose (Gn/Gc)4 tetrameric spikes on viral particles. During hantavirus cell entry, these surface glycoproteins mediate receptor binding and membrane fusion. The recent Gc crystal structures from the Orthohantavirus genus confirmed that it is a class II viral fusion protein and the crystals further revealed a homo-dimeric association of Gc at neutral ph and a homo-trimeric assembly at low ph. The Gc dimer interface connects adjacent spikes and consists of four strictly conserved Gc residues, which form a polar network of interactions with inter-chain ionic and hydrogen bonds. These residues are further involved in inter-chain contacts of the low ph Gc homotrimer. Here we aimed to functionally characterize the observed Gc dimer and trimer interface, by assessing the spike stability and fusion phenotype of interface mutants. The substitution of the conserved charged residues lowered or increased the temperature stability of the spikes and changed the ph required for the spike dissociation and fusion activation. Further, all Gc dimer interface contacts were crucial for the membrane fusion activity, and their substitution showed a decreased Gc homotrimer stability, arresting fusion after the trimerization step. Together, our results functionally confirm that the residues forming the Gc homodimer induce spike assembly during the exit of the virus from the cell, while during viral cell entry, these residues direct acid-induced spike dissociation and fusion by stabilizing the post-fusion homotrimer.

12 BREAKING AND ENTERING - viral entry Abstract final identifier: 0 ANTIGENIC ARRANGEMENT OF THE DYNAMIC HANTAVIRAL SURFACE Ilona Rissanen *, 2, Robert Stass, Sai Li, Antra Zeltina, Jussi Hepojoki 3, Karl Harlos, Jayna Raghwani 4, Oliver Pybus 4, Robert Gilbert, Juha Huiskonen, Thomas Bowden Division of Structural Biology, University of Oxford, Oxford, United Kingdom, 2 Helsinki Institute of Life Science, 3 Department of Virology, University of Helsinki, Helsinki, Finland, 4 Department of Zoology, University of Oxford, Oxford, United Kingdom Abstract: Despite the risks presented by zoonotic hantaviruses, there exists a paucity of knowledge regarding the pathobiology of viral infection, and both the treatment and prevention options remain extremely limited. Early stages of hantavirus infection are regulated by the glycoprotein lattice presented on the hantaviral surface. The two glycoproteins forming the lattice, Gn and Gc, are responsible for host recognition and entry, and are targeted by the neutralizing humoral immune response. Through structural investigation of the Gn glycoprotein from three genetically distinct hantavirus species, we reveal that the fold of the Gn is highly conserved and can form discrete oligomeric configurations. We describe the molecular-level arrangement of the Gn and Gc glycoproteins, as displayed on the hantavirus surface, and address the function of the hantaviral envelope during host cell entry. Our analysis reveals the antigenic determinants of the humoral immune response and provides insights into the structural transitions that the envelope undergoes during endocytosis of the virus into the host cell. 2

13 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: RECEPTOR TYROSINE KINASES ARE PROMISING TARGETS IN LASSA VIRUS ANTIVIRAL THERAPY Chiara Fedeli *, Giulia Torriani, Hector Moreno, Gert Zimmer 2, Gisa Gerold 3, Stefan Kunz Institute od Microbiology, CHUV, Lausanne, 2 Institute of Virology and Immunology, Mittelhäusern, Switzerland, 3 TWINCORE, Institute for Experimental Virology, Hannover, Germany Abstract: Lassa Virus (LASV) causes a severe viral hemorrhagic fever with high mortality in humans. The main LASV receptor is dystroglycan (DG) in its functional O-glycosilated form. However, glycosylation of DG does not always correlate with LASV tropism in vivo, suggesting alternative receptors. In absence of functional DG, LASV can hijack phosphatidylserine receptors of the Tyro3/Axl/Mer (TAM) family and enter cells via apoptotic mimicry. We showed that productive LASV entry via the receptor tyrosine kinase (RTK) Axl involves virus-induced activation of Axl signalling and macropinocytosis. Here we investigated the role of Axl RTK activity for LASV entry as a function of DG modification, using recombinant lymphocytic choriomeningitis virus expressing LASV glycoprotein (rlcmv-lasvgp) as a BSL2 surrogate.rather unexpected, we found that the specific Axl RTK inhibitor R428 potently inhibits LASV entry in different cell types with EC50 in the mid-nanomolar range, independently of the functional glycosylation of DG. Notably, LASV entry via functionally glycosylated DG into human epithelial cells did not induce Axl activation but was still highly sensitive to R428, suggesting a role of Axl RTK activity as an essential permissive signal. Combination of R428 with the hepatocyte growth factor (HGFR) inhibitor EMD24063 resulted in a pronounced synergistic anti-viral effect, indicating non-redundant roles of these RTKs in LASV entry. Used in combination with ribavirin, a nucleoside analogue used to treat human Lassa fever in the clinic, the RTK inhibitors showed additive anti-viral effects. Our studies provide a rationale to target RTKs in combinatorial therapy against human Lassa fever. 3

14 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 2 STRUCTURE AND FUNCTION OF PHENUIVIRIDAE CAP-SNATCHING ENDONUCLEASES Juan Reguera *, 2, Sana Lessoued 2, Gabriel Bragagnolo 2, Maria Mate 2 INSERM, 2 AFMB, Aix Marseille University/CNRS, Marseille, France Abstract: Segmented negative stranded viruses (snsvs) perform transcription initiation by cap snatching. By using a viral endonuclease they snatch short capped nucleotides from cellular mrnas to subsequently use them for priming transcription. In the last two years the crystal structures of the cap snatching endonucleases of several highly pathogenic viruses have been reported, broadening our understanding of the diversity of the cap snatching mechanism and some basic common features that allow the transcription of this large group of animal, plant and human pathogens. Here we present the structural and functional characterization of the Toscana cap-snatching endonuclease, the first reported for a Phenuivirdae, a family of Bunyavirales order including highly pathogenic arboviruses such as Rift Valley Fever Virus. The structure reveals new unexpected features differing from cap snatching endonucleases of other related virus such Influenza, Lassa, Hantaan or La Crosse, while maintaining some basic core folding features and enzymatic activity. Our results confirm that the in vitro endonuclease activity of cap snatching endonucleases is dependent of the presence of a catalytic histidine in the active site, validating the proposed classification of these enzymes as His+ and His-. This classification is critical for the design of strategies for antiviral discovery targeting cap snatching. Thus, the results of our study pave the way for the discovery of specific antivirals targeting the viral transcription of Orthophlebovirus. Our study evidences the large folding diversity among snsvs cap snatching endonucleases and shows the potential for discovery of antivirals with broad spectra for snsvs. 4

15 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 3 MECHANISM OF POLYMERASE REGULATION BY A 5 HOOK-LIKE STRUCTURE IN THE GENOMIC RNA OF MACHUPO VIRUS Jesse D. Pyle *, Sean P. J. Whelan * Harvard Medical School, Boston MA USA Abstract: The atomic structures of the influenza (Orthomyxoviridae) and La Crosse virus (Peribunyaviridae) RNAdependent RNA polymerases (RdRPs) revealed that the 5 terminal 0-nucleotides of the viral genome RNA (vrna) are bound as a hook like structure proximal to the RdRP active site. This 5 hook RNA appears to stabilize the catalytic residues of the RdRP. For influenza virus, mutagenesis of the element results in changes in gene expression but the underlying mechanism remains to be determined. Using an in vitro biochemical assay for RdRP function, we probe the requirement for a similar 5 hook structure in arenaviruses, which like the orthomyxoviruses and peribunyaviruses have segmented negativesense RNA genomes. Working with the purified 250 kda large polymerase (L) of Machupo virus (MACV), we demonstrate that the 5 terminal 2-nucleotides of the vrna stimulate RdRP activity >6-fold. The stimulation is specific to the MACV RNA sequence, and requires quasi-complementary interactions between the 5 and 3 vrnas. The presence of the 5 hook also renders the polymerase dependent on magnesium as the divalent cation in common with other polymerases further validating the importance of this RNA ligand in polymerase function. A 5 hook-like structure is thus a shared requirement for function in polymerases of segmented negative-strand RNA viruses. 5

16 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 4 STRUCTURAL INSIGHTS INTO THE MECHANISMS OF INFLUENZA A VIRUS RNA REPLICATION Haitian Fan *, Itziar Serna Martin, Jeremy Keown 2, Narin Hengrung 3, Robert P. Rambo 4, Jonathan M. Grimes 2, Ervin Fodor Sir William Dunn school of pathology, 2 Division of Structural Biology, University of Oxford, Oxford, 3 The Francis Crick Institute, London, 4 Diamond Light Source Ltd, Didcot, United Kingdom Abstract: Transcription and replication of the influenza A virus single-stranded viral RNA (vrna) genome is carried out by the viral RNA-dependent RNA polymerase, composed of the PB, PB2, and PA subunits. Transcription of vrna into mrna is primed by short capped RNA fragments derived from host capped RNAs by the cap-snatching activity of the viral polymerase and occurs in association with host RNA polymerase II. On the other hand, replication of vrna is a primer independent process and proceeds through a complementary RNA (crna) replicative intermediate. However, the molecular mechanisms of vrna replication, which has been reported to require polymerase dimerisation, remain largely unknown. Here we report the high-resolution crystal structures of polymerases from human A/NT/60/968 (H3N2) and avian A/duck/Fujian/0/2002 (H5N) influenza viruses in their apo forms. In solution, both polymerases form dimers of heterotrimers and analysis of the crystal structure revealed a dimerisation interface which has been confirmed by mutagenic studies as well as small-angle X-ray scattering (SAXS). Dimerisation is mediated mostly by the PA C-terminal domain but involves also regions of PB and PB2. Furthermore, a nanobody, a single-domain antibody, raised against the viral polymerase, was found to inhibit polymerase dimerisation. Cryo-EM and crystal structures of the nanobody-polymerase complex confirmed that that nanobody binds near the dimerisation interface. Inhibition of polymerase dimerisation by nanobody resulted in reduced polymerase activity in ribonucleoprotein reconstitution assays. We propose that the polymerase dimer we have identified could play a role in RNA genome replication. 6

17 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 5 TRANSLATIONAL REGULATION OF BORNA DISEASE VIRUS Akiko Makino *, Yutaro Yamamoto, Yuya Hirai 2, Keizo Tomonaga KYOTO UNIVERSITY, Kyoto, 2 Osaka Dental University, Osaka, Japan Abstract: Borna disease virus (BoDV), which belongs to the order Mononegavirales, establishes a persistent infection in the nucleus. Because BoDV replicates in the nucleus, the viral translation is separated from its transcription in space and time. This feature indicates that BoDV may control the quality and quantity of translating mrnas to suppress virus production and maintain the persistent infection in the nucleus. However, translational regulation of BoDV has not been understood in detail. To elucidate translational regulation of BoDV, we performed screening of shrna library using BoDV-infected cells and identified insulin-like growth factor 2 (IGF2) as a host factor associated with viral particle production. Knockdown of IGF2 enhanced translation activity and particle production of BoDV, and overexpression of IGF2 in IGF2-knockdown cells reduced BoDV translation. On the other hand, treatment of recombinant IGF2 protein to BoDV-infected cells had no effect on the viral production. These results suggest that not IGF2 protein, but its mrna plays a role in the regulation of BoDV translation. We therefore assessed the involvement of IGF2 mrna binding proteins (IGF2BPs), which regulate IGF2 mrna translation. Overexpression of IGF2BP2 enhanced the BoDV translation and particle production, whereas it decreased expression level of viral mrnas in infected cells, like IGF2-knockdown. The RNA recognition motif and RhoGAP domains of IGF2BP2 were dispensable for its enhancing effect on viral translation. We also showed that IGF2BPs were co-immunoprecipitated with mrnas and N protein of BoDV in infected cells. These results suggest that BoDV mrnas undergo translational regulation of IGF2BP2 in competition with IGF2BPs-binding mrnas, such as IGF2, for regulation of viral particle production in infected cells. 7

18 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 6 VIRAL N6-METHYLADENOSINE PROMOTES REPLICATION AND GENE EXPRESSION OF PNEUMOVIRUSES Miaoge Xue, Boxuan Zhao 2, Mijia Lu, Scott Zhang 2, Xueya Liang, Zhike Lu 2, Yuanmei Ma, Anzhong Li, Mark Peeples 3, Chuan He 2, Jianrong Li * Department of Veterinary Biosciences, THE OHIO STATE UNIVERSITY, Columbus, 2 Department of Chemistry, The University of Chicago, Chicago, 3 Center for Vaccines and Immunity, The Research Institute at Nationwide Children s Hospital, Columbus, United States Abstract: N 6 -methyladenosine (m 6 A) is the most prevalent internal modification of mrnas in most eukaryotes. Likewise, viral RNAs may acquire m 6 A methylation during replication within these cells. The m 6 A modification is installed by host m 6 A methyltransferases and is reversible by RNA demethylases. The biological functions of m 6 A are mediated through m 6 A binding proteins that specifically recognize and bind the methylated adenosine on RNA. Here, we show that genome, antigenome, and mrnas of human respiratory syncytial virus (RSV) and human metapneumovirus (hmpv), two medically important pneumoviruses, are modified by m 6 A within discreet regions and that these modifications enhance viral replication and gene expression. Notably, overexpression of the m 6 A binding proteins, YTHDF-3, significantly enhanced viral RNA synthesis, translation, replication, and viral release. Knockdown of m 6 A methyltransferases decreased viral replication and gene expression whereas knockdown of m 6 A demethylases had the opposite effect. Subsequently, the m 6 A sites in the viral G protein mrna, the most abundant m 6 A modified gene transcript of both RSV and hmpv, were inactivated by mutagenesis. The resultant recombinant RSVs and hmpvs were defective in replication, gene expression, and spread in A549 cells and primary well differentiated human airway epithelial (HAE) cultures. Moreover, a small molecule that inhibited S-adenosyl-Lhomocysteine (SAH) hydrolase, thereby reducing the cellular SAH pool and viral RNA m 6 A, also inhibits viral replication in HAE cells. Collectively, our results identify a novel mechanism for regulating pneumovirus replication and gene expression and identify m 6 A methylation as a target for controlling infection by pneumoviruses and perhaps other non-segmented negative-sense (NNS) RNA viruses. 8

19 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 7 PROTEOMIC ANALYSIS IDENTIFIES HUMAN E3 UBIQUITIN LIGASE RBBP6 AS A NEGATIVE REGULATOR OF EBOLA VIRUS INFECTION Christopher Basler *, Jyoti Batra, Judd F. Hultquist 2, John VonDollen 2, Laura Satkamp 2, Gwendolyn M. Jang 2, Olena Shtanko 3, Manu Anantpadma 3, Robert Davey 3, Nevan J. Krogan 2 Center for Microbial Pathogenesis, Georgia State University, Atlanta, 2 Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, 3 Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, United States Abstract: Ebola virus (EBOV) is a member of Filovirdae family of negative-sense RNA viruses. EBOV infection causes severe hemorrhagic fever in humans, but little is understood as to how this virus exploits the host machinery to replicate successfully. To better define this process, we used affinity tag-purification mass spectrometry (AP-MS) to generate an EBOV-host protein-protein interaction (PPI) map. We uncovered 93 high-confidence EBOV-human PPIs, including one between the EBOV VP30 protein and the host ubiquitin ligase human retinoblastoma binding protein 6 (RBBP6). EBOV VP30 acts as a regulator of viral RNA synthesis including through interactions with the viral nucleoprotein (NP). RBBP6 is a large multidomain protein that interacts with a variety of host molecules. We mapped the interaction to a 23 amino acid stretch within RBBP6 that was both necessary and sufficient for VP30 binding. Furthermore, a co-crystal structure of RBBP6 peptide bound to VP30 C-terminus solved to.5 A resolution revealed that RBBP6 usurps the NP binding cleft on VP30. Knockdown of endogenous RBBP6 enhances viral RNA synthesis and increases EBOV infectivity, while over-expression of either RBBP6 or RBBP6 peptide results in strong inhibition. Common between RBBP6 and the region of NP that binds VP30 is a PPxPxY motif. Strikingly, three other VP30 interactors identified by the PPI screen, hnrnpl, hnrnpul and PEG0, possess the same motif as RBBP6 and NP, and can also modulate EBOV RNA synthesis. Cumulatively, these data unexpectedly identified host negative regulators of EBOV replication and point to the VP30-NP interaction as a therapeutic target. 9

20 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 8 ADVANCED ANALYSES ON THE ROLE OF VP30 AND RNA STRUCTURES IN EBOLA VIRUS TRANSCRIPTION S. Bach *, A. Grünweller, S. Becker 2, N. Biedenkopf 2, R. K. Hartmann IPC, 2 IfV, PHILIPPS-UNIVERSITÄT, Marburg, Germany Abstract: Ebola virus (EBOV) genome replication requires the polymerase L, NP and VP35. Transcription of viral mrnas additionally requires transcription factor VP30. The transcription start regions (TSR) of all EBOV genes are embedded in potential 5'-UTR secondary structures of varying stability on the genomic RNA or mrna level. We used mono- and bicistronic minigenomes to investigate the role of these secondary structures in viral transcription and their dependency on VP30. The TSR of the first NP gene and its complementary spacer sequence separate the replication promoter elements PE and 2. Through replacing the NP TSR:spacer hairpin structure with those of the internal EBOV genes, we found that the spacing between PE and 2 has to be a multiple of 6 nt (termed rule of six) not only for efficient replication [Weik et al., 2005, J Virol] but also efficient transcription initiation. This suggests that the replication promoter directs replication and transcription. In line with this, exchange of 5'-UTRs at the TSR of the 2 nd EBOV gene was permissive toward deviations from the rule of six. We further showed (i) that, for efficient transcription initiation, insertions between PE and 2 are limited to 48 nt; (ii) stabilization of 5 -UTR hairpin structures by GC-rich helical extensions eliminated reporter gene activity; (iii) a 2-nt deletion of the NP spacer, which eliminates any potential to form RNA structures at the TSR in (-) and (+) RNAs, still allowed transcription and, remarkably, did not abolish VP30 dependency of transcription. We propose that a major function of VP30, which primarily binds ssrna [Schlereth et al., 206, RNA Biol], is to induce and support a conformational switch of the RNA:L/35 complex required for transcription initiation. 20

21 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 9 REPURPOSING CANONICAL ANTIVIRAL RESPONSES TO PROMOTE TRANSLATION OF INFLUENZA VIRUS MESSENGER RNAS Vy G. Tran, Mitch P. Ledwith, Thiprampai THAMAMONGOOD 2, Adoflo Garcia-Sastre 3, Martin Schwemmle 2, Adrianus C. Boon 4, Michael S. Diamond 4, Andrew Mehle * Medical Microbiology and Immunology, University of Wisconsin Madison, Madison, United States, 2 Institute of Virology, Medical Center, University of Freiburg, Freiburg, Germany, 3 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, 4 Departments of Medicine, Molecular Microbiology, and Pathology & Immunology, Washington University School of Medicine, St Louis, United States Abstract: Cells infected by influenza virus mount a large-scale antiviral response and most ultimately initiate cell death pathways in an attempt to suppress viral replication. During a CRISPR knockout screen designed to query post-entry steps during infection, we identified a large class of presumptive antiviral factors, including IFIT2, as important enhancers of influenza virus replication. IFIT2 is an interferon stimulated gene with well-established antiviral function for some viruses. We show here that IFIT2 is instead repurposed by influenza virus to support viral replication. IFIT2 interacts with viral RNAs and the viral nucleoprotein to stimulate viral gene expression. IFIT2 recruits viral mrnas to translating ribosomes, enhancing production of viral proteins and ultimately replication of the virus. IFIT2 also stimulates virally-induced apoptosis. Cells lacking IFIT2 fail to undergo apoptosis and survive infection. Remarkably, genetic and chemical ablation of apoptosis further reduced viral yield. Our results suggest that influenza virus has evolved to exploit the antiviral and apoptotic cellular environment, redirecting these classically antiviral events into pro-viral effectors. Moreover, it is highly unlikely that the repurposing of antiviral genes is specific to influenza A virus, but rather is generalizable to additional viruses and other antiviral viral proteins defining a new interface between the virus and host. 2

22 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 20 BINDING TO ANP32 IS REQUIRED BUT NOT SUFFICIENT FOR INFLUENZA A VIRUS POLYMERASE ACTIVITY Jason Long *, Wendy Barclay, Bhakti Mistry, Ecco Staller, Jocelyn Schreyer Virology, Imperial College London, London, United Kingdom Abstract: Influenza pandemics are sparked by zoonotic infections from animal influenza A viruses in humans. Thankfully, these events are rare due to species specific barriers that the virus encounters when it switches host species. Host protein ANP32 underlies the important host barrier that prevents avian influenza polymerases functioning in human cells. Avian influenza polymerases have learnt to utilise avian ANP32A which encodes a 33aa insertion between the LRR and LCAR domains of the protein that is absent in mammals, flightless birds and ANP32B from birds and mammals. Mutations in PB2, most famously E627K, enable the polymerase to utilise the shorter ANP32 forms in human cells. We have shown that the influenza polymerase heterotrimer binds to ANP32 proteins in the absence of RNA, and that this binding is required but not sufficient for activity. We have mapped the minimal requirements of ANP32 proteins to support influenza polymerase and conducted experiments to understand at what stage of virus replication the interaction occurs. Understanding how the polymerase interacts with essential host proteins may enable the identification of novel small molecule inhibitors and help map mutations required by the polymerase to overcome the species barrier, informing surveillance and disease control. 22

23 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 2 INCOMPLETE GENOMES OF INFLUENZA A VIRUS DRIVE ABUNDANT REASSORTMENT Anice C. Lowen *, Nathan T. Jacobs, Kara L. Phipps, John Steel Department of Microbiology and Immunology, Emory University, Atlanta, United States Abstract: Evolution of influenza A virus (IAV) poses a continued threat to public health by allowing evasion of immunity and emergence of pandemic strains. The segmentation of the IAV genome contributes to this evolution by facilitating diversification through reassortment. Using genetically tagged parental viruses to ensure unbiased detection of all progeny genotypes, we found that reassortment is a routine feature of IAV infection, even in vivo. In cell culture, where MOI can be more tightly controlled, reassortment levels greatly exceeded predictions based on the random distribution of two eightsegmented parental viruses into cells. Modeling suggested that high reassortment might be due a dependence on coinfection for productive infection. In agreement with this prediction, single cell analysis of cells infected at low MOI revealed that each of the segments of A/Panama/2007/99 (H3N2) virus was present with a frequency of ~0.56. Thus, Pan/99 viral genomes in singly infected cells were complete with a frequency of only =0.0. Strain variation in reassortment phenotype allowed identification of the polymerase and nucleoprotein genes as viral determinants of genome completeness. Importantly, comparison of a given strain in multiple cell lines revealed that incomplete genome levels are cell type dependent. This host-dependent phenotype suggests that segments are lost after delivery of the viral genome to the cell, through interaction with host factors. Rather than dead-end products, however, incomplete viral genomes appear to be complemented frequently through co-infection. In summary, our data suggest that inefficiencies early in the life cycle often render IAV genomes incomplete; that IAV infection is typically mediated by a multi-particle infectious unit; and that abundant reassortment results. 23

24 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 22 DIFFERENCES BETWEEN TRANSCRIPTION AND REPLICATION INITIATION MECHANISMS OF THE PNEUMO- AND PARAMYXOVIRIDAE Afzaal Shareef, Paul Jordan 2, Michael Mawhorter, Tessa Cressey, Sarah Noton, Kartikeya Nagendra, Jerome Deval 2, Rachel Fearns * Microbiology, Boston University School of Medicine, Boston, 2 Alios Biopharma, Johnson and Johnson, South San Francisco, United States Abstract: The polymerase of the non-segmented negative strand RNA viruses (nsnsvs) initiates both transcription and genome replication from the same promoter region. We have previously shown that the polymerase of respiratory syncytial virus (RSV), a member of the Pneumoviridae, accomplishes this by initiating RNA synthesis from two sites on its promoter: position U to begin replication and position 3C to begin transcription. Here we used purified RSV polymerase in an in vitrorna synthesis assay to study the mechanism by which this occurs. We show that initiation at U and 3C occurred independently of each other and that the polymerase preferred to initiate at 3C, but initiation site selection could be modulated by the relative concentrations of ATP versus GTP. We then used a similar assay system to investigate if other nsnsv promoters and polymerases have similar properties. The polymerase of human metapnuemovirus, another pneumovirus, also initiated at positions U and 3C of its promoter. In contrast, the polymerase of human parainfluenza virus 3 (HPIV3), a paramyxovirus, only initiated at position U of its own promoter. However, evidence indicates that the HPIV3 polymerase could initiate internally on an RSV promoter sequence. These findings suggest that the pneumo- and paramyxoviruses have different initiation mechanisms, but that these differences are largely driven by differences in the promoter sequences, rather than by differences in polymerase structure. 24

25 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 23 NEXT GENERATION SEQUENCING REVEALS NOVEL ASPECTS OF PARAMYXOVIRUS TRANSCRIPTION AND REPLICATION AND THE SWITCH BETWEEN ACUTE AND PERSISTENT INFECTIONS. Richard Randall *, Elizabeth Wignall-Fleming 2, Dan Young, Elizabeth M. Randall, Andrew Davison 2, Steve Goodbourn 3 UNIVERSITY OF ST ANDREWS, St Andrews, 2 University of Glasgow, Glasgow, 3 St George's, London, United Kingdom Abstract: Following a high moi, maximum amounts of viral mrna are observed between 2 8h p.i., when the viral mrna contributes 4-8% of the total mrna of the cell. Maximum levels of virus genomes/ antigenomes are observed at 24h p.i. Whilst the ratio of genomes to antigenomes in infecting virus is ~ 9:, by 6 h p.i. the number of antigenomes exceeds the number of genomes, but this ratio reverts in favour viral genomes by 2h p.i. As virus replication begins before 6h p.i., virus replication must occur before there are high overall levels of NP within infected cells. Immunofluoresence suggests a model in which virus transcription and replication initially occurs at a limited number of local foci within an infected cell, before the virus spreads to initiate new foci of replication throughout the cell. At later times p.i., whilst >90% of the cells survive the infection, virus transcription and replication become represses such that the level of virus mrnas contribute approximately 0.% of the total mrna. Repression of virus replication at late times p.i. is linked to the phosphorylation status of the P protein. Thus, virus replication is not switched off, and infected cells die, following infection with a recombinant virus with a single amino acid substitution of serine to phenylalanine at position 57 on P. We speculate that in vivo, during acute phases of virus infection variants of PIV5 will be selected for in which virus replication cannot switched off. However, as the adaptive immune response develops, variants in which virus replication can be repressed will be selected, leading the establishment of prolonged/ persistent infections. 25

26 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 24 STRUCTURE GUIDED TUNING OF VESICULAR STOMATITIS VIRUS RNA DEPENDENT RNA POLYMERASE FIDELITY Louis-Marie Bloyet *, Sean P. Whelan Microbiology and Immunobiology, Harvard Medical School, Boston, United States Abstract: Reflecting the error prone nature of RNA replication, RNA viruses exist as a quasispecies a swarm of sequences around a core consensus sequence. Experimental evidence supports the hypothesis that quasispecies diversity influences viral pathogenesis by for example providing variants necessary to overcome intrinsic host barriers to infection. This has led to efforts to rationally attenuate RNA viruses through manipulation of their intrinsic polymerase error rates. Reducing error rates diminishes quasispecies diversity, restricting the ability of the virus to overcome barriers. Increasing polymerase error rates pushes the virus swarm past a point of recovery through a process termed error catastrophe. Mutations in other viral polymerases that influence error rate map to the active-site region, the nucleoside triphosphate (NTP) entrance channel, and other regions that likely operate through an allosteric mechanism. Guided by the structure of the vesicular stomatitis virus (VSV) large polymerase protein, we generated a panel of 25 mutants and screened them in a minigenome assay which identified 6 functional mutants. We introduced each of those into an infectious cdna clone of VSV and recovered 4 of 6 variants and used frequency of escape to a neutralizing antibody, and incorporation of incorrect nucleotides in an in vitro transcription assay to compare polymerase error rates. Substitution F54Y in the template entrance channel reduced polymerase fidelity 2-fold, whereas K535R in the NTP entrance channel increased fidelity. Catalytic site region substitutions N763K and E766D reduced fidelity.5 and 25-fold respectively. Substitutions that influence polymerase error rate by directly impacting the active site and NTP entrance channel may aid in rationally attenuating other mononegavirales. 26

27 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 25 TO V, OR NOT TO V? CYCLIC SELECTION OF GENOMIC QUASISPECIES WITH ALTERNATIVE CODING CAPACITIES IN DUAL-TROPIC MEASLES VIRUS Ryan C. Donohue *, Christian K. Pfaller, Roberto Cattaneo Molecular Medicine, MAYO CLINIC, Rochester, United States Abstract: RNA virus genomes are quasispecies differing by one or more nucleotides from the consensus sequence. Genomic diversity is instrumental for virus escape from immune selection, and for adaptation to new hosts. It is less clear whether it also contributes to virus adaptation to different tissues within a host. Towards answering this question, we operated with measles virus (MeV), which replicates first in immune cells and then in respiratory epithelia. We mimicked its infection by adapting the virus cyclically to human lymphocytic (Granta-59) and epithelial cells (H358) or exclusively in either cell line. MeV adapted to Granta-59 cells replicated poorly on H358 cells. Thus, we performed deep-sequencing genome analyses. Strikingly, adaptation to Granta-59 cells reproducibly resulted in genomic variants with different combinations of four mutations within an -nucleotide region of the phosphoprotein gene. This sequence mediates polymerase slippage and insertion of a guanosine in the corresponding mrna, resulting in expression of an interferon antagonist (V protein) in place of a polymerase co-factor (P protein). Granta-59-adapted MeV produces minimal amounts of edited transcripts, while parental and H358-adapted viruses produce edited and non-edited transcripts at a : ratio. Granta-59-adapted MeV expresses much more P than V protein, while the other viruses express P and V at similar levels. We have rescued recombinant viruses carrying individual mutations and confirmed a shift the P to V expression ratio towards enhanced P production. Thus, we discovered a mechanism that may accelerate the speed of MeV replication in lymphocytic cells. It is important to assess whether tissue-specific quasispecies adaptation occurs within infected hosts. 27

28 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 26 INFLUENZA A VIRUS GENOMIC HETEROGENEITY REGULATES SUPERINFECTION POTENTIAL AND THE CELLULAR RESPONSE TO INFECTION Chris Brooke * University of Illinois, Urbana, United States Abstract: The vast majority of influenza A virus (IAV) virions express highly variable, incomplete subsets of viral genes, and cannot undergo productive replication without complementation. Despite being the primary product of IAV infection, the role that these semi-infectious particles (SIPs) play in shaping viral dynamics and the host response to infection remains poorly understood. Here, we used a novel single virion infection approach to reveal that SIPs play a direct role in determining the frequency of superinfection. We show that superinfection susceptibility is determined by the total number of viral genes expressed within a cell, independent of their identity. IAV particles that express a complete set of viral genes potently inhibit superinfection, while SIPs that express incomplete subsets of viral genes do not. As a result, viral populations that contain more SIPs undergo more frequent superinfection. Finally, we used single cell RNAseq to dissect how viral genomic heterogeneity within IAV populations shapes the host transcriptional response to infection. Altogether, these studies reveal viral genomic heterogeneity as a major factor that governs both superinfection susceptibility and the overall cellular response to infection. 28

29 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 27 NUCLEAR IMPORT OF THE RABIES VIRUS P PROTEIN IS REQUIRED FOR INHIBITION OF RIG-I-LIKE RECEPTOR- MEDIATED TYPE I INTERFERON INDUCTION Marco Wachowius, Verena Pfaffinger, Maximilian F. Eizinger, Karl-Klaus Conzelmann * Max von Pettenkofer Institute Virology and Gene Center, LMU Munich, München, Germany Abstract: The rabies virus phosphoprotein P plays essential roles in various aspects of the cytoplasmic viral life cycle, including viral RNA synthesis and RNA encapsidation, as well as in counteracting the antiviral immune response. Although P is accumulating almost exclusively in the cytoplasm, it is long known to undergo nucleocytoplasmic shuttling, as directed by various conserved nuclear import and export signals. However, the biological relevance of nuclear shuttling has remained unclear up to now. We here provide experimental evidence that nuclear import of P is absolutely required for inhibition of RIG-I-dependent IFN induction both upon plasmid transfection and in the context of virus infection. Deletion of nuclear import signals of P proteins from a vaccine strain and a virulent clinical isolate rendered the proteins defective in preventing IRF3 phosphorylation and transcription of IFN-β. Recombinant RABV carrying shuttling-defective P effectively induced IFN. The defect was cured by addition of an ectopic NLS from SV40, and an extra NLS even improved the IFN inhibitory capacity of wt P protein. Mechanisms involved in priming of RABV P for IFN inhibition are being analyzed. 29

30 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 28 NOVIRHABDOVIRUS NV PROTEINS ACT AS ANTAGONISTS OF THE HOST ANTIVIRAL DEFENSE Stéphane Biacchesi *, Emilie Mérour, Laury Baillon, Didier Chevret, Michel Brémont INRA, Jouy en Josas, France Abstract: The non virion (NV) protein expression is critical for pathogenesis of viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV) in trout. We further described that a single amino acid change in VHSV NV found in field isolates highly attenuated in trout had a tremendous impact on the virulence, underlying the major function of this protein. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomics that allowed us identifying several NV-associated cell partners. We showed that both NV proteins specifically interact with a serine/threonine protein phosphatase, PPMB, which has been shown in mammals to negatively regulate the host antiviral response via dephosphorylating TBK. We demonstrated that NV and PPMB efficiently counteract RIG-I- and TBK-dependent IFN and ISG promoter induction in fish cells and, hence, the establishment of an antiviral state. We further showed that PPMB dephosphorylates TBK, as previously observed for its mammalian orthologs, and that the expression of VHSV NV re-located PPMB in close vicinity of mitochondria, a subcellular compartment important for the recruitment and the activation of TBK, and led to TBK dephosphorylation. In contrast, IHNV NV did not lead to TBK inactivation meaning that IHNV NV has another mechanism of action. These data bring new insights on the function of the NV expressed by two major pathogens for aquaculture and ecosystems worldwide. Although both NV share a same function, their mechanism of action seems to be distinct. All together, these findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPMB to subvert innate immune response. 30

31 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 29 MECHANISMS OF IMMUNE EVASION BY RSV NON-STRUCTURAL PROTEINS Daisy W. Leung * Pathology & Immunology, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, St. Louis, United States Abstract: Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections, morbidity, and mortality in the pediatric, the elderly, and immunocompromised populations worldwide. Despite decades of intensive research, prophylactic and therapeutic treatment options are limited and in need of improvement. RSV encodes for two nonstructural (NS) proteins that are unique among non-segmented negative strand viruses and are important, multifunctional immune antagonists. NS and NS2 are involved in host immune suppression, including inhibition of Type I interferon (IFN) induction and signaling, as well as inhibition of the NF-kB pathway and apoptosis. However, limited information on the mechanistic aspect of how these proteins function or their therapeutic potential for antiviral and vaccine development has not been fully explored. Here we describe our recent novel structural findings on NS and NS2, which now provides a framework to mechanistically dissect the function of these well described, but poorly defined viral proteins. We also provide insights to target NS and NS2 for therapeutic development to limit RSV-associated morbidity and mortality. 3

32 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 30 A BARCODED LIBRARY UNVEILS NS-DRIVEN INFLUENZA VIRUS TROPISM Raquel Muñoz-Moreno *, Carles Martínez-Romero, Asiel Arturo Benitez, Christian Forst 2, Daniel Blanco-Melo, Raffael Nachbagauer, Vinod Balasubramaniam, Ilseob Lee, Sadaf Aslam, Maryline Panis, Ignacio Mena, Juan Ayllón, David Sachs 2, Florian Krammer, Benjamin R. Tenoever, Adolfo García-Sastre, 3, 4 Microbiology, Icahn School of Medicine at Mount Sinai, 2 Genetic and Genomic Sciences, 3 Mount Sinai School of Medicine, 4 Institute of Global Health and Emerging Pathogens, Icahn School of Medicine at Mount Sinai, New York, United States Abstract: Influenza NS protein is a key virulence factor that inhibits type I interferon (IFN), thus allowing the virus to replicate efficiently in host cells. In this work, a PR8 backbone has been used to generate a library of recombinant viruses, each expressing a different NS. This approach allowed us to obtain a representative library of recombinant PR8 viruses containing NS sequences encompassing the known NS evolutionary landscape from different viral strains, countries, years and hosts. Different sequences were collected from the Influenza Research Database (IRD) based on different structural and functional Sequence Feature Variant Types (SFVT). The NS library was designed by using a modified split NS segment where we inserted a 22-nucleotide barcode (BC) that allows for the monitoring and quantitation of the different recombinant NS viruses through the Illumina Miseq deep sequencing platform after infecting different systems. Equivalent levels of each unique virus were combined to obtain the NS-barcoded library. Egg allantoic fluid, MDCK supernatants and lungs collected from mice previously infected with the NS library were analyzed. The study revealed the emerging groups of NSs that are best adapted, the ones that tend to disappear as the infection progresses as well as the impact of the host antiviral pathways in determining the fitness of influenza A viruses according to their specific NS sequences. 32

33 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 3 MARBURG VIRUS PROTEIN VP30 REGULATES THE IRE/XBP-DEPENDENT UNFOLDED PROTEIN RESPONSE TO ENSURE EFFICIENT VIRAL REPLICATION Cornelius Rohde *, Stephan Becker, Verena Krähling Institute of Virology, Philipps University, Marburg, Germany Abstract: Too many unfolded proteins impose stress on the endoplasmic reticulum (ER) leading to the activation of three distinct signaling cascades, referred to as unfolded protein response (UPR). The most conserved among them is triggered by IRE. IRE is activated upon ER stress resulting in XBP mrna splicing and the translation of the transcription factor XBPs (XBP spliced). XBPs is transported into the nucleus to bind cis-acting UPR elements (UPRE) and thereby enhances the expression of many genes to restore ER homeostasis. Under non-upr conditions XBP mrna is not spliced, resulting in the translation of XBPu (XBP unspliced). XBPu recruits its own mrna to the ER membrane to facilitate IRE-mediated splicing. The aim of our study was to examine if and how Marburg virus (MARV) interacts with the IRE-dependent UPR. IRE activation can be detrimental or beneficial for viral replication. Here we show that the ectopic expression of MARV GP induced UPR via the IRE-XBP axis while MARV infected cells did not show hallmarks of IRE-dependent UPR activation. These seemingly contradictory results could be reconciled by the finding that MARV VP30 counteracted the GP-activated UPR. In addition, VP30 also regulated UPRE activation upon stimulation by other potent UPR inducers. Co-immunoprecipitation studies revealed that VP30 interacts with XBPu; this interaction was RNA-dependent. Using IRE knock-out cells and artificial stimulation of UPR we could show that the growth of MARV is influenced by activation of UPR. These results support the idea that tight regulation of the IRE-dependent UPR by MARV VP30 via interaction with XBPu is needed for an efficient viral propagation. 33

34 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 32 SUBVERSION OF LYMPHOCYTE RESPONSE AND MODULATION OF GENE EXPRESSION AND SIGNALING BY EBOLA VIRUS Patrick Younan, Mathieu Iampietro, Ndongala Lubaki, Rodrigo Santos, Palaniappan Ramanathan, Andrew Nishida 2, Mukta Dutta 2, Michael Katze 2, Fabian Gusovsky 3, Richard Koup 4, Alexander Bukreyev * University of Texas Medical Branch, Galveston National Laboratory, Galveston, 2 University of Washington, Seattle, 3 Eisai Inc., Andover, 4 National Institutes of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, United States Abstract: Fatal outcomes of Ebola virus (EBOV) infections are preceded by a sepsis-like syndrome and lymphopenia despite T cells being resistant to EBOV infection. To determine the effects of EBOV interferon-inhibiting domains (IIDs) on cell-mediated responses, we used a panel of recombinant strains of EBOVs with point mutations disabling the VP24 and/or VP35 IIDs. The viruses were used for infection of human dendritic cells (DCs) co-cultured with T cells. We found that IIDs block activation and proliferation of T cells as a result of their role in suppressing maturation of DCs and limiting the formation of immunological synapses. We demonstrated that IIDs block phosphorylation of TCR adapters and downstream signal molecules. Similarly, we showed that IIDs suppress activation of B and NK cells. Furthermore, we demonstrated that EBOV binds to CD4 + T cells through interaction of GP with TLR4 leading to T cell death. Transcriptome analysis revealed that the addition of EBOV to CD4 + T cells results in the upregulation of interferon signaling, pattern recognition receptors and NFκB pathways. Transcriptome analysis and specific inhibitors identified apoptosis and necrosis as mechanisms of the T cell death. Lastly, TLR4 antagonist Eritoran protected mice from lethal EBOV challenge by alleviating the cytokine storm. Thus EBOV IIDs cause a global suppression of cell-mediated responses, including T, B and NK cells, as a consequence of the deficient DC maturation. In addition, EBOV GP subverts the immune response by triggering lymphopenia through direct and indirect mechanisms and also causes the cytokine storm. 34

35 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 33 EPIGENETIC CONTROL OF INFLUENZA VIRUS MEDIATED BY H3K79 HISTONE METHYLATION: ROLE IN INTERFERON-INDUCED ANTIVIRAL RESPONSE Laura Marcos-Villar, 2, Juan Diaz-Colunga, Noelia Zamarreño, Ana Falcon, 2, Amelia Nieto *, 2 Biologia Molecular y Celular, CENTRO NACIONAL DE BIOTECNOLOGIA-CSIC, 2 CIBERES, Madrid, Spain Abstract: Studying possible epigenetic changes elicited by influenza virus infection, we identified a marked increase on methylation of lysine 79 of histone 3 (H3K79). There is a histone methylase named DotL, which exclusively methylates H3K79 residue and an inhibitor specific for this histone methylase. The use of this specific inhibitor or the silencing of DotL methylase, increase influenza virus replication. In addition, in conditions of DotL downregulation there are decreased nuclear translocation of NF-kB complex, and IFN-b, Mx and ISG56 expression. In agreement with a role of H3K79 methylation controlling the antiviral signaling, influenza virus replication was unaffected in IFN pathway-compromised DotL-inhibited cells. DotL down-regulation did not cause significant changes on viral replication of an influenza virus lacking NS protein (delns), which is unable to counteract the antiviral response. In addition, delns infection caused a lower increase on H3K79 methylation compared with the wild type virus. These results indicate that epigenetic methylation of H3K79 might have an important role in controlling interferon-induced signaling against viral pathogens. Accordingly, the analysis of cellular RNAs expression modified by influenza virus replication in DotL down-regulated cells, showed a reduced expression of genes that have a pivotal role controlling the antiviral response. 35

36 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 34 THE INFLUENZA A VIRUS HOST SHUTOFF RNASE PA-X USURPS HOST MRNA PROCESSING MECHANISMS Denys Khaperskyy, Brittany Porter, Lea Gaucherand 2, Craig McCormick, Marta Gaglia * 2 Microbiology and Immunology, Dalhousie University, Halifax, Canada, 2 Molecular Biology and Microbiology, TUFTS UNIVERSITY SCHOOL OF MEDICINE, Boston, United States Abstract: The influenza A-encoded RNase PA-X modulates innate immune responses. PA-X deficiency exacerbates lung inflammation during infection, but this stronger response fails to clear the virus, instead causing increased morbidity and mortality. A mechanistic understanding of PA-X effects on the cell transcriptome is required to determine how PA-X modulates inflammatory responses. We previously showed that PA-X selectively degrades transcripts that are made by host RNA Pol II and undergo canonical 3 end processing, and that Pol II transcripts have varying sensitivity to PA-X. We hypothesize that PA-X binds mrna processing factors to access substrates, and that the differential sensitivity of RNAs reflects differential engagement of this machinery. We are using two approaches to test this hypothesis: transcriptome-wide profiling of PA-X targets and proteomic analysis of PA-X-interacting proteins. By comparing host RNA levels in cells infected with wild-type and PA-X-deficient viruses or overexpressing PA-X, we discovered that RNA splicing is a determinant of susceptibility to PA-X. We found a negative correlation between exon number and RNA levels. Also, intronless RNAs were largely resistant to PA-X. Using proximity-dependent biotinylation, we determined that the PA-X C-terminal domain interacts with several cellular proteins involved in RNA metabolism, particularly splicing and alternative polyadenylation. Together, our transcriptomic and proteomic data support a model in which PA-X interacts with cellular mrna processing pathways to target subsets of host RNAs. This mechanism of action sets PA-X apart from other viral host shutoff RNases that selectively target mrnas based on interactions with actively translating mrnas in the cytoplasm. 36

37 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 35 TRIM28/KAP IS A PKR-CONTROLLED NEGATIVE REGULATOR OF THE INNATE IMMUNE RESPONSE TO HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES Tim Krischuns, Vanessa Gerlt, Carolin Nordhoff, Joschka Willemsen 2, Marco Binder 2, Sebastian Schloer 3, Stephan Ludwig, Linda Brunotte * Institute of Virology Muenster (IVM), Westfaelische Wilhelms-University Muenster, Muenster, 2 German Cancer Research Centre (DKFZ), Division virus-associated carcinogenesis, Heidelberg, 3 Institute of Medical Biochemistry (IMB), Westfaelische Wilhelms-University Muenster, Muenster, Germany Abstract: TRIM proteins are well known for their multifaceted immune regulatory functions mainly brought by the highly conserved N-terminal ubiquitin-ligase domain. In contrast to most other TRIM family members TRIM28/KAP is a transcriptional co-repressor involved in many cellular processes such as DNA-damage response, cell cycle regulation, cancer development, reactivation of retroviruses and also immune regulation. TRIM28 co-repressor activity is alleviated by stress-induced phosphorylation at serines 473 and 827 leading to the expression of repressed genes. Our experimental data provide the first evidence that TRIM28 is a negative regulator of the innate immune response and selectively phosphorylated during infection with the highly pathogenic avian influenza A virus strains A/FPV/Bratislava/79 (H7N7) and a A/Thailand/(KAN-)/2004 (H5N) but not by the human prototype HN strain A/Puerto Rico/8/934. Interestingly, phosphorylation is induced by viral RNA, however, in a RIG-I-independent manner. In contrast, we could demonstrate that TRIM28 phosphorylation is largely dependent on protein kinase R (PKR) and induction of the stress kinase p38 and its downstream effector MSK during viral infection, as evidenced by using CRISPR/Cas9-technology and specific kinase inhibitors. Elevated levels of Type-I-IFNs as well as prototypic pro-inflammatory cytokines in HPAIV-infected TRIM28- KO-cells corroborate the role of TRIM28 as a negative regulator of the innate immune response. We hypothesize that PKR/p38/MSK-mediated phosphorylation of TRIM28 provides a novel RIG-I-independent signaling pathway contributing to elevated cytokine expression during HPAIV infection. In conclusion, our data indicate an extended role of PKR and TRIM28 as modulators of the innate immune response towards influenza A viruses beyond todays understanding. 37

38 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 36 H5N INFLUENZA A VIRUS PB-F2 RELIEVES HAX--MEDIATED RESTRICTION OF AVIAN VIRUS POLYMERASE PA IN HUMAN LUNG CELLS Béryl Mazel-Sanchez *, Ines Boal Carvalho, Filo Silva, Ronald Dijkman 2, Mirco Schmolke Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, 2 Institute of Virology and Immunology, University of Bern, Bern, Switzerland Abstract: Highly pathogenic IAV from avian hosts were first reported to directly infect humans 20 years ago. However, these are rare events and our understanding of factors promoting or restricting zoonotic transmission is still limited. One accessory protein of IAV, PB-F2, was associated with pathogenicity of pandemic and zoonotic IAV. This 90-amino-acidshort peptide does not harbour an enzymatic function. We thus identified host factors interacting with H5N PB-F2, which could explain its importance for virulence. PB-F2 binds to HCLS associated protein X (HAX-), a recently identified host restriction factor of the PA subunit of IAV polymerase complexes. We demonstrate that the PA of a mammalian adapted HN IAV is resistant to HAX- imposed restriction while the PA of an avian origin H5N IAV remains sensitive. We also showed HAX- sensitivity for PAs of A/Brevig Mission//98 (HN) and A/Shanghai//203 (H7N9), two avian origin zoonotic IAV. Inhibition of H5N PA by HAX- can be alleviated by PB-F2 through direct competition. Accordingly, replication of PB-F2 deficient H5N IAV is attenuated in presence of high amounts of HAX-. Mammalian adapted HN and H3N2 viruses do not display this dependence on PB- F2 for efficient replication in presence of HAX-. We propose that PB-F2 plays a key role in zoonotic transmission of avian H5N IAV into humans. 38

39 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 37 IDENTIFICATION OF A NEW INTERFERON-INDUCIBLE PROTEIN INHIBITING ENDOSOME MEDIATED VIRAL ENTRY THROUGH REGULATION OF THE VACUOLAR H+-ATPASE Tomas Doyle, Olivier Moncorgé 2, Darja Pollpeter, Boris Bonaventure 2, Marine Tauziet 2, Michael H. Malim, Caroline Goujon * 2 King's College, London, United Kingdom, 2 IRIM, CNRS, Montpellier, France Abstract: Type interferon (IFN) induces an antiviral state through the regulation of numerous IFN-stimulated genes (ISGs), with some acting broadly while others display distinctive substrate specificity. Following IFN treatment, influenza A virus (IAV) infection is largely inhibited, and both the MX GTPase and the integral membrane protein IFITM3 contribute substantially to suppression. However, these ISGs do not fully account for the IFN-induced restriction of IAV, and we have identified a new ISG playing an important role. CRISPR/Cas9 knock-out of this gene partially rescues IAV infection in the presence of IFN. Conversely, the ectopic expression of this gene potently inhibits IAV infection. Importantly, VSV-G- and rabies-pseudotyped lentiviruses are also inhibited by expression of this gene, in contrast to lentiviruses bearing their natural Env glycoproteins, suggesting that this factor could prevent endosome-mediated entry of viruses. Step-wise dissection of IAV entry showed that this gene does not prevent acid-induced conformational changes in the viral haemaglutinin but inhibits fusion of the viral and endosomal membranes. Critically, combinations of knock-out and overexpression experiments have shown that IFITM3, which also inhibits viral entry, and our gene of interest are functionally independent. Ectopic expression of this gene globally increases the ph of intracellular vesicles and the activity of lysosomal proteases. Finally, interactions with several cytoplasmic subunits of the vatpase and the product of our gene provide support for a model in which IFNmediated regulation of the vacuolar H+ ATPase (vatpase) restricts IAV infection. 39

40 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 38 DEFINING HOST RESTRICTION FACTORS THAT MODULATE RESPIRATORY VIRUS ENTRY AND EXIT FROM INFECTED CELLS. Patrick Reading *, 2, Sarah Londrigan, Fernando Villalon-Letelier Department of Microbiology and Immunology, The University of Melbourne, 2 WHO Collaborating Centre for Reference and Research on Influenza, Peter Doherty Institute for Infection and Immunity, Melbourne, Australia Abstract: Acute respiratory tract infections are important causes of morbidity and mortality worldwide, particularly in infants and the elderly. Influenza A virus (IAV), human metapneumovirus (HMPV) and respiratory syncytial virus (RSV) are major causes of viral respiratory disease. Respiratory viruses infect airway epithelial cells, resulting in virus amplification and spread. Viruses such as IAV and RSV also infect cells of the immune system, such as airway macrophages (AMΦ), however virus replication is generally blocked in these cells. Recent studies in our laboratory used RNA-seq to investigate differences in expression of host factors between AMΦ and airway epithelial cells, in the presence or absence of IAV infection, in an attempt to identify putative restriction factors that may block virus infection. Based on these results we have focused on particular gene families where certain members were expressed at high levels in AMΦ, but not in epithelial cells. We have used overexpression and/or knockdown approaches to screen families of membrane-associated RING-CH (MARCH) ubiquitin ligases, interferon-inducible transmembrane (IFITM)-family proteins and T-cell immunoglobulin and mucin (TIM)- domain family proteins for antiviral activity against IAV, RSV and HMPV. Preliminary data indicates that overexpression of MARCH8 (but not other MARCH-family proteins) does not alter IAV entry, but does inhibit virus release from infected cells. Moreover, MARCH8 expression also inhibited replication of other respiratory viruses such as RSV. Current studies in the laboratory aim to define the mechanisms underlying the antiviral activity of MARCH8, as well as defining the role of endogenous MARCH8 in limiting respiratory virus infection. 40

41 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 39 TRIM25 TARGETS EBOLA VIRUS RIBONUCLEOPROTEIN TO SENSITIZE IT TO ZAP-MEDIATED RESTRICTION Rui P. Galao *, Harry Wilson, Kristina Schierhorn, Franka Debeljak, Chad Swanson, Stuart Neil Infectious Diseases, King's College London, London, United Kingdom Abstract: As part of the early host antiviral defence, RNA viruses are sensed by pattern recognition receptors whose activation triggers a signalling cascade that ultimately results in the expression of type-i interferons (IFN-I), and consequent upregulation of hundreds of IFN-stimulated genes (ISGs). Although Ebola virus (EBOV) has evolved strategies to counteract antiviral responses, we observed that IFN-I treatment of target cells impacted the propagation of EBOV transcription and replication competent virus-like particles (trvlp). Using a well-characterized library of human ISGs we identified TRIM25 as potently restricting EBOV trvlp replication, and its deletion by CRISPR/Cas9 markedly alleviated viral sensitivity to IFN-I. TRIM25 is an E3-ubiquitin ligase known to ubiquitinate the RNA sensor RIG-I to facilitate its interaction with MAVS, thus modulating downstream signalling of the IFNresponse. Here, we uncovered an antiviral mechanism for TRIM25 that requires MDA5 and MAVS, but is independent of RIG-I and downstream pro-inflammatory signalling through canonical sensing of cytoplasmic viral RNA. Instead, TRIM25 is recruited to incoming viral particles after cell entry and interacts with the EBOV ribonucleoprotein complex, leading to ubiquitination of the viral nucleoprotein (NP), its own auto-ubiquitination and to a considerable reduction of NP-associated viral genome levels. This is turn sensitizes the virus to the Zinc Antiviral Protein (ZAP), the potency of which can be modulated by the CG dinucleotide content of the trvlp genome. These findings suggest that TRIM25 couples cytoplasmic RNA sensing to direct restriction of EBOV via ZAP. The uncovered antiviral mechanism indicates that TRIM25 may act as a direct antiviral mediator, suggesting that the full scope of TRIM25 antiviral roles is still to be fully defined. 4

42 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 40 INHIBITION OF THE CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS-ENCODED DEUBIQUITINASE BLOCKS VIRAL REPLICATION Florine E. Scholte *, Brian L. Hua, Stephen R. Welch, Laura K. McMullan, Stuart T. Nichol, Scott D. Pegan 2, Christina F. Spiropoulou, Éric Bergeron Virus Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, 2 Pharmaceutical and Biomedical Sciences, University of Georgia, Athens, United States Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne bunyavirus (Nairoviridae) that can cause a fatal hemorrhagic syndrome. CCHFV has a broad geographic distribution, and no licensed vaccines or effective antiviral treatments are available. The CCHFV L segment encodes a multifunctional protein, containing the viral RNA-dependent RNA polymerase (RdRp), and an ovarian tumor-like (OTU) cysteine protease. The OTU domain can remove ubiquitin (Ub) and ubiquitin-like interferon-stimulated gene-5 (ISG5) conjugates. Ub and ISG5 are involved in post-translational regulation of many signaling pathways, including innate immune responses. Therefore, CCHFV can manipulate the immune responses by reversing Ub/ISG5 conjugation, and we demonstrated that the OTU domain suppresses RIG-I-mediated type I interferon responses during infection. Disrupting OTU activity should block OTU-mediated immune suppression, and consequently enhance immune responses to CCHFV. To address the therapeutic potential of OTU inhibition, we used a synthetic Ub variant (UbV-CC4) previously shown to form high affinity complexes with CCHFV-OTU. We demonstrate that UbV-CC4 completely reversed the immunosuppressive activity of overexpressed CCHFV-OTU, and blockage of OTU during viral infection resulted in a >4-log reduction in wild-type CCHFV infectious titers. In contrast, CCHFV mutants unable to bind Ub were insensitive to UbV-CC4, suggesting that inhibition requires direct binding to OTU. Interestingly, the strong inhibition of wild-type CCHFV replication did not solely rely on enhanced immune responses, as blocking OTU resulted in reduced RdRp activity, likely by affecting L protein levels. Taken together, we demonstrate that targeting CCHFV-OTU robustly inhibits CCHFV replication and is a promising target for antiviral therapies targeting viral RdRps. 42

43 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 4 HANTAAN VIRUS DOWNREGULATES CELL SURFACE EXPRESSION OF DEATH RECEPTOR 5 VIA THE 26S PROTEASOME PATHWAY AND INHIBITS TRAIL-MEDIATED INDUCTION OF APOPTOSIS Carles Solà Riera *, Shawon Gupta 2, Kimia Maleki, Clas Ahlm 3, Hans-Gustaf Ljunggren, Niklas Björkström, Jonas Klingstrom Department of Medicine, KAROLINSKA INSTITUTET, Stockholm, Sweden, 2 Department of Infectious Diseases, University Hospital Heidelberg, Heidelberg, Germany, 3 Department of Clinical Microbiology, Umeå University, Umeå, Sweden Abstract: Tumor necrosis factor related apoptosis-inducing ligand (TRAIL) is an important factor of our immune system to combat cancer and virus-infected cells. Cytotoxic lymphocytes induce TRAIL-mediated apoptosis to specifically kill targeted cancer and virus-infected death receptor (DR) 4 and DR5 expressing cells. Hantaviruses, order Bunyavirales, cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome (HCPS) with up to 40% case fatality rates. Increased levels of soluble TRAIL (strail) have previously been observed in Hantaan virus (HTNV) infected HFRS-patients, and in HCPS-patients. Here we show that HTNV induces TRAIL in endothelial cells, as well as production and secretion of TRAIL by NK cells co-incubated with infected cells. As TRAIL is a potent apoptosis-inducing ligand, we next analyzed the effect of soluble TRAIL on infected cells. HTNV-infected cells strongly resisted TRAIL-mediated apoptosis. When investigating possible mechanisms behind this phenomenon we observed that HTNV causes downregulation of DR5 from the cell surface. Early after infection, an almost complete depletion of cellular DR5 was observed. Production of DR5 was then resumed, including the appearance of the short DR5 isoform. Interestingly, the newly produced DR5 accumulated in lysosomes, not reaching the cell surface. Inhibition of E3 ubiquitin ligase by the chemical SMER-3 blocked the internalization and degradation of DR5 via the 26S proteasome pathway, restoring DR5 to the cell surface. The finding that hantaviruses inhibit TRAIL-mediated apoptosis, together with our previous finding that hantaviruses inhibit cytotoxic granulemediated apoptosis, shows that these viruses have a strong capacity to inhibit cytotoxic lymphocyte-mediated killing of infected cells. 43

44 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: 42 REPLICATION HETEROGENEITY DRIVES DISTINCT CELLULAR RESPONSES TO INFLUENZA A INFECTION IN VIVO Elizabeth Fay *, 2, Louisa Sjaastad 3, Jessica Fiege 2, 3, Ian Stone 3, Matthew Markman 3, Marissa Macchietto 4, Steven Shen 4, Ryan Langlois, 2, 3 Biochemistry, Molecular Biology, and Biophysics, 2 Center for Immunology, 3 Microbiology & Immunology, 4 Institute for Health Informatics, University of Minnesota, Minneapolis, United States Abstract: Influenza A virus (IAV) infects a broad range of cell types within the respiratory tract. The cells initially targeted by the virus in a naïve host are the site of primary replication and virus spread. These cells are difficult to detect using replication competent IAV as the virus rapidly spreads to secondary cells. To overcome this obstacle we utilize a fluorescence expressing single cycle IAV (sciav) to identify the cells initially infected in the mouse lung. Using this tool, we observed two distinct populations of epithelial cells during the early stages of infection: cells with high virus replication and cells with low virus replication. This suggests that some cells within the lung are innately permissive to virus replication while others are able to blunt replication. We have determined that this phenotype is not due to coinfection of a cell with multiple virions. The level of replication is also not dependent on epithelial cell type. There are distinct cellular pathways that are significantly impacted in each population of infected cells compared to uninfected cells. Additionally, there are subsets of interferon-stimulated genes that are specifically upregulated in each population. These data indicate that different levels of virus replication may activate distinct cellular responses to infection. Fluorescent reporter sciavs could be used to further elucidate the mechanism of cellular permissibility and the early innate immune response to IAV infection. 44

45 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 43 MOLECULAR ANATOMY OF THE INFLUENZA VIRION Naina Nair, Terry Smith 2, Swetha Vijayakrishnan 3, Daniel Goldfarb 3, Elizabeth Sloan 3, David Bhella 3, Edward Hutchinson * 3 Glasgow School of Art, Glasgow, 2 University of St Andrews, St Andrews, 3 MRC-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom Abstract: As influenza virions are complex and irregularly shaped, no single method can describe their structure completely. We applied a multidisciplinary approach, combining proteomics, lipidomics, cryo-electron tomography and molecular modelling, to reconcile quantitative data about the virion s components with their atomic structures and the virion s overall shape and produce a highly detailed, pseudo-atomic model of the virions of influenza A/WSN/33 virus (WSN). We first considered the viral envelope. This was relatively crowded, with a tenth of its area occupied by protein transmembrane domains, including low levels of the M2 splice variant M42. Its lipid contained similar levels of phosphatidylcholine and sphingomyelin to cell lysates, with more phosphatidylserine and less phosphatidylglycerol. The envelope s inner surface area matched the total membrane-binding surface of the viral matrix protein lying beneath it. This area of the bacilliform virions observed budding from cells and the spherical virions often observed in solution was comparable, suggesting that the former can relax into the latter. We next considered the virion interior. This contains a substantial amount of host protein along with the viral genome and the immunosuppressive protein NS. In budding and bacilliform virions the genome forms a well-ordered array of ribonucleoproteins (RNPs), but we showed that when virions collapse into spheres this must force the RNP array to become disordered. Integrating qualitative and quantitative data in this way clarified and corrected our understanding of the molecular anatomy of influenza virions. This multidisciplinary approach is applicable to many other complex structures, including other enveloped virions. 45

46 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 44 ASSEMBLY AND STRUCTURE OF MEASLES VIRUS NUCLEOCAPSID PARTICLES Sigrid Milles, Malene R. Jensen, Ambroises Desfosses 2, Guy Schoehn, Irina Gutsche, Rob W. Ruigrok *, Martin Blackledge IBS, CEA-CNRS-Univ Grenoble Alpes, 2 IBS, CEA-CNRS-Univ Grenoble Alpes,, Grenoble, France Abstract: Paramyxoviridae such as Measles, are important human pathogens. Their RNA genomes are packaged into long helical nucleocapsids, comprising thousands of copies of the nucleoprotein (N) binding the entire genome. The N-RNA complex provides the template for replication and transcription by the viral polymerase complex and as such presents a promising target for viral inhibition. Elucidation of mechanisms regulating transcription and replication have been hampered by the inability to controllably assemble nucleocapsids (NCs). We recently demonstrated self-organization of N into NC-like particles upon addition of RNA to N, chaperoned by a peptide from the viral phosphoprotein (P). This offers a tool for investigating NC assembly, as well as establishing a platform for biotechnological applications. NMR and fluorescence spectroscopy reveal biphasic assembly kinetics. Assembly is seen to strongly depend on RNA-sequence, and we use cryoelectron microscopy to determine atomic resolution structures of NCs assembled on different RNA sequences to understand this phenomenon. Full length, tetrameric P comprises a 300 amino acid intrinsically disordered N-terminal domain whose role is poorly understood. An integrated structural dynamics study is used to delineate its importance in NC assembly Milles, Jensen, Communie, Maurin, Schoehn, Ruigrok, Blackledge. Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence. Angew Chemie Intl Edition, 28, (206) 46

47 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 45 ULTRASTRUCTURE OF THE INFLUENZA VIRUS RIBONUCLEOPROTEIN COMPLEXES PRODUCING VIRAL RNAS Masahiro Nakano *, Keiko Shindo, Yukihiko Sugita 2, Yukiko Muramoto, Yoshihiro Kawaoka 3, Matthias Wolf 4, Takeshi Noda Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 2 Institute for Protein Research, Osaka University, Osaka, 3 Institute of Medical Science, University of Tokyo, Tokyo, 4 Okinawa Institute of Science and Technology Graduate University, Okinawa, Japan Abstract: The influenza A virus genome is composed of eight segmented single-stranded negative-sense RNAs (vrnas). Each vrna is encapsidated by multiple nucleoproteins (NPs) and an RNA-dependent RNA polymerase to form a ribonucleoprotein complex (vrnp). The vrnp shows a twisted rod-shaped configuration, where an NP-vRNA strand is folded back and coiled on itself to form a double-stranded helix. The vrna is either transcribed into mrna or replicated into complementary RNA (crna) while complexed with RNPs. However, the configuration of vrnps while performing these functions remains unknown. Here, we first isolated vrnps from influenza A virions and confirmed by RT-qPCR that the vrnps were able to produce both mrna and crna in vitro. Then, we analyzed the configuration of vrnps during an in vitro RNA synthesis reaction by using high-speed atomic force microscopy (HS-AFM) and cryo-electron microscopy (cryo-em). Two different vrnp configurations associated with newly synthesized RNAs were observed. The vrnps associated with structured RNA had a twisted rod-shaped configuration similar to vrnps not undergoing RNA synthesis. In contrast, vrnps associated with a looped RNA were deformed and did not retain their helical configuration. In addition, it was demonstrated that the looped RNA was double-stranded, and composed of a template vrna and a progeny crna. Our findings provide important insights into viral RNA synthesis by vrnps and suggest mechanisms for transcription and replication of the influenza virus genome. 47

48 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 46 NEGRI BODIES ARE VIRAL FACTORIES WITH PROPERTIES OF LIQUID ORGANELLES Jovan Nikolic, Cécile Lagaudrière-Gesbert, Romain Le Bars, Nathalie Scrima, Danielle Blondel, Yves Gaudin * INSTITUT DE BIOLOGIE INTÉGRATIVE DE LA CELLULE, CNRS, Gif sur Yvette, France Abstract: Replication of Mononegavirales occurs in viral factories which form inclusion in the host-cell cytoplasm. For rabies virus (RABV), those inclusions are called Negri Bodies (NBs). NBs concentrate both viral nucleoproteins (N) and phosphoproteins (P). They also constitute the site of synthesis of viral RNAs (messengers, antigenomes and genomes). We have demonstrated that NBs have characteristics similar to those of liquid organelles: they are spherical, they fuse to form larger structures, and they disappear upon hypotonic shock. Their liquid phase was confirmed by FRAP experiments. Live-cell imaging indicates that viral nucleocapsids are ejected from NBs and transported along microtubules to form either new virions or secondary viral factories. Co-expression of RABV N and P proteins results in cytoplasmic inclusions recapitulating NBs properties. This minimal system reveals that an intrinsically disordered domain and the dimerization domain of P are essential for NB-like structures formation. We suggest that formation of liquid viral factories by phase separation is common among Mononegavirales and allows specific recruitment and concentration of viral proteins but also the escape to cellular antiviral response. 48

49 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 47 NEW TECHNIQUES TO VISUALIZE THE MAMMARENAVIRUS (LCMV) LIFE CYCLE REVEAL A RAB5C-POSITIVE COMPARTMENT AS A POTENTIAL SITE FOR GENOME REPLICATION AND VIRAL PARTICLE PRE-ASSEMBLY Emily A. Bruce *, Benjamin R. King, 2, Christopher M. Ziegler, Philip L. Eisenhauer, Daniel Zenklusen 3, Jason Botten Medicine, University of Vermont, Burlington, 2 Institute for Systems Genetics, Department of Cell Biology, New York University School of Medicine, New York City, United States, 3 Departement de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Canada Abstract: While arenaviruses, including lymphocytic choriomeningitis mammarenavirus (LCMV), are known to bud at the plasma membrane, relatively little is known about the late stages of the viral life cycle, including the process of viral particle assembly. We have developed a fluorescence in situ hybridization (FISH) assay and a recombinant LCMV that encodes an HA-tagged matrix protein (Z) to permit the robust detection of genomic RNAs and Z protein respectively, in individual cells. At the peak of viral particle production, we show that the viral S segment genomic RNA, along with viral nucleoprotein, coalesces into a perinuclear foci that is located near the cellular microtubule organizing center and colocalizes with the small GTPase Rab5c and the viral glycoprotein (GPC). In addition, we report that during the peak of infection, the LCMV matrix protein Z is also found concentrated in a perinuclear compartment. In cells stably expressing GFP-Rab5C, the matrix protein colocalizes with GFP-Rab5c in both perinuclear foci and cytoplasmic puncta. We propose that the virus is using the surface of a cellular membrane-bound organelle as a site for the pre-assembly of viral components, including genomic RNA, glycoprotein, and matrix prior to their transport to the plasma membrane, where new particles will bud. As Rab5c is required for efficient LCMV production and is redistributed during the late stages of LCMV infection, we propose that Rab5c plays a role in coordinating the assembly and/or trafficking of viral components to the site of budding. 49

50 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 48 KEY LCMV-HOST INTERACTIONS REQUIRED FOR DEFECTIVE INTERFERING PARTICLE PRODUCTION AND THE HIGHLY DYNAMIC STATE OF VIRAL REPLICATION AND TRANSCRIPTION DURING PERSISTENCE. Christopher M. Ziegler, Benjamin R. King 2, Emily A. Bruce, Philip L. Eisenhauer, Marion E. Weir 3, David J. Shirley 4, Joseph P. Klaus 5, Dimitry N. Krementsov 6, Aubin Samacoits 7, Christophe Zimmer 7, Daniel Zenklusen 8, Florian Mueller 7, Bryan A. Ballif 9, Jason Botten * Medicine, University of Vermont, Burlington, 2 Cell Biology, New York University School of Medicine, New York, 3 Biology, University of Vermont, 4 Data Science, Ixis, Burlington, 5 Immunology and Microbial Science, The Scripps Research Institute, La Jolla, 6 Medical Laboratory and Radiation Sciences, University of Vermont, Burlington, United States, 7 Unité Imagerie et Modélisation, Institut Pasteur, Paris, France, 8 Departement de Biochimie et Médecine Moléculaire, Université de Montréal, Montréal, Canada, 9 Biology, University of Vermont, Burlington, United States Abstract: Lymphocytic choriomeningitis arenavirus (LCMV) causes disease in humans but establishes an asymptomatic, lifelong infection in reservoir rodents. To better understand how rodent-borne viruses like LCMV maintain a persistent infection without compromising the fitness of the host rodent, we investigated (i) the host and viral machinery needed for the production of defective interfering (DI) particles and (ii) the dynamics of virus genome replication and transcription at the single cell level during persistence. Our studies show that LCMV uses divergent pathways to create infectious virus particles versus DI particles. In particular, LCMV s only late domain, PPXY, and a functional ESCRT pathway are critical for the production of DI particles, but not for infectious virus. Further, phosphorylation of the PPXY motif appears essential for DI particle production, suggesting that reversible phosphorylation may regulate the rate of DI particle production independent of standard virus. Finally, our smfish studies suggest that most LCMV-infected cells, despite supporting high levels of virus replication and transcription in the first days following infection, ultimately clear infection as evidenced by a progressive loss of viral RNA and antigen. Further, during persistence, the majority of cells appear to exhibit repeating cyclical waves of viral transcription and replication followed by clearance of viral RNA. Thus, our data suggest that infected cells spontaneously clear infection and become reinfected by viral reservoir cells that remain in the population. Collectively, our studies support a model whereby LCMV spread can be restricted through multiple mechanisms, including (i) host kinasedriven DI particle production and (ii) the establishment of short-lived, transient infections in permissive host cells. 50

51 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 49 RESTRICTED INTRANEURONAL TRANSPORT OF FIELD RABIES VIRUS GLYCOPROTEIN Verena te Kamp *, Tobias Nolden 2, Sabine Nemitz, Michael Christen, Luca Zaeck, Stefan Finke FRIEDRICH-LOEFFLER-INSTITUT, Greifswald - Insel Riems, Germany, 2 Vira Therapeutics, Innsbruck, Austria Abstract: Rabies virus (RABV) is a neurotropic virus that enters the central nervous system (CNS) through infection of peripheral neurons and subsequent trans-synaptic spread to next-order neurons. Beside the common knowledge of exclusively retrograde RABV transport we have shown glycoprotein G dependent anterograde transport of newly replicated virus in axons of peripheral dorsal root ganglion (DRG). However, it is so far unknown whether anterograde transport is limited to peripheral sensory neurons due to neuron specific sorting mechanisms or whether virus strain/isolate specific sequences contribute to the directionality of virus transport in infected neurons. We compared the distribution of different RABV G proteins in peripheral DRG and CNS neurons. G protein of cell culture adapted RABV exhibited efficient surface transport with accumulation at the plasma membranes of cell soma and dendritic/axonal sites. By contrast, infection with RABV field isolate led to accumulation of G in the cell body with only few G particles at plasma membranes of DRG and hippocampus neurons. This indicates that retention and limited surface transport of G protein is characteristic for field virus. By use of chimeric RABV it could be shown, that G protein retention was mediated by the ectodomain. However, adaptation of field virus replication to cell culture by sequential passages led to the identification of three amino acid replacements required for efficient surface transport, counteracting G protein retention. Based on these data we propose a model, in which limited and highly regulated transport of RABV G protein to synaptic budding sites is characteristic for pathogenic field viruses and therefore an important factor in RABV pathogenesis. 5

52 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 50 NUCLEOTIDE RESOLUTION MAPPING OF INFLUENZA A VIRUS NUCLEOPROTEIN-RNA INTERACTIONS REVEALS THE LANDSCAPE OF VIRAL RNA FEATURES REQUIRED FOR REPLICATION Graham Williams, Dana Townsend, Kristine Wylie, Preston Kim, Gaya Amarasinghe, Sebla Kutluay, Adrianus Boon * WASHINGTON UNIVERSITY, Saint Louis, United States Abstract: Influenza A virus nucleoprotein (NP) associates with all eight negative-sense genomic RNA segments during virus replication. Although the positioning of protein components within viral ribonucleoprotein complexes (RNPs) is defined, the native interaction of NP with the viral RNA (vrna) and the mechanisms by which a complete multi-segment genome assembles remain elusive. Here, we applied photoactivatable ribonucleoside enhanced crosslinking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP bound short fragments of RNA (~2 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding was reduced at specific locations within the viral genome including regions previously identified as required for viral genome segment packaging. Synonymous, structural mutations in these low-np binding regions impacted genome packaging and resulted in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions had no effect. Finally, we demonstrate that the sequence conservation of low-np binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells. 52

53 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 5 FINE MAPPING OF INFLUENZA A VIRUS INTRA AND INTERSEGMENT RNA INTERACTIONS Naoki Takizawa * Laboratory of Virology, INSTITUTE OF MICROBIAL CHEMISTRY (BIKAKEN), Tokyo, Japan Abstract: Influenza A virus genome consists of eight single-stranded negative-sense viral RNA (vrna) segments and one set of eight segments is packaged together into a single virus particle. The vrna is complexed with viral RNA polymerase complexes and nucleoproteins (NP) to form a viral ribonucleoprotein complex (vrnp) and 3 and 5 sequences of vrna are annealed to form a hairpin structure. Classically, the interaction between NP and vrna is considered to be a uniform and to melt secondary structure of vrna. However, it has recently been shown that NP dose not bind vrna uniformly and direct RNA-RNA interactions between segments are required for packaging of the eight vrna segments. Although electron microscopy studies revealed the architecture of vrnp and configuration of vrnps in virion, the intra and intersegment vrna interaction sites are not fully identified. To elucidate intra and intersegment vrna structures more detail, we employed the mapping RNA interactome in vivo (MARIO) like method for mapping the intra and intersegment proximal RNA sites and the psoralen analysis of RNA interactions and structures (PARIS) like method for mapping the intra and intersegment vrna interactions. The genome-wide contact maps were generated by both method and intra and inter vrnp architectures were reconstituted form the contact map. While intrasegment architecture of vrnp can be reconstituted from both MARIO and PARIS experiments, intersegment architecture and 7+ configuration of vrnps in the virion can be reconstituted only from PAIRS experiment. Our data reveal the segment contact networks by direct RNA-RNA interactions and the role of intersegment interactions for genome packaging will be discussed. 53

54 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: 52 VISUALIZING THE INTRACELLULAR ASSEMBLY OF INFLUENZA VIRAL RNA USING CONFOCAL AND LIGHT SHEET LIVE CELL MICROSCOPE Seema Lakdawala * University of Pittsburgh, Pittsburgh, United States Abstract: Influenza A viruses (IAV) contain eight negative-sense RNA gene segments that replicate in the nucleus, export to the cytoplasm as subcomplexes and travel along RabA recycling endosomes to the plasma membrane for budding. However, the role of host cytoskeletal proteins in IAV assembly are still poorly understood. To study viral RNA (vrna) transport and assembly dynamics within a single cell, we utilized two imaging techniques: ) multi-color fluorescent in situ hybridization and 2) a custom light-sheet microscope with isotropic resolution along the x, y and z directions. Treatment of cells with nocodazole, a microtubule (MT) depolymerizing drug, did not alter the release of infectious virus but decreased the co-localization of RabA and vrna, suggesting the presense of a RabA and MT independent transport mechanism. To determine whether the transport dynamics of vrna were altered in the absence of MT we used a virus encoding a GFPtagged polymerase (PA-GFP) protein, as a surrogate for vrna. Nocodazole treatment only slightly lowered the speed and straightness of PA-GFP movement, yet the speeds were still fast enough to indicate directed movement. This observation demonstrates that MT are not critical for vrna transport. Additionally, we used cell lines stably expressing a GFP-tagged RabA protein to characterize the movement of RabA during IAV infection. We observed that nocodazole treatment drastically inhibited the movement of RabA. In contrast, IAV infection significantly altered the straightness, speed and arrest coefficient of RabA movement in infected cells. In this study, we have used multiple microscopy tools to provide novel insights into the transport and assembly processes of IAV vrna and have described the presence of a RabA independent transport mechanism. 54

55 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 53 HA STABILITY GOVERNS INFLUENZA A VIRUS REPLICATION, VIRULENCE, HOST RANGE, AND PANDEMIC POTENTIAL Charles J. Russell *, Guohua Yang, Marion Russier 2 Infectious Diseases, St. Jude Children's Research Hospital, Memphis, United States, 2 Max Planck Institüt für Biochemie, Martinsreid, Germany Abstract: Various hosts differ in extracellular and intracellular ph. The influenza A virus HA protein couples changes in ph to activation, but HA acid stability varies by subtype and host species. Seasonal influenza viruses have HA proteins that are generally more stable (ph ) while exotic viruses like H5N and H7N9 are less (ph ). To dissect the biological impact of HA stability, we generated A/TN/560-/09 (HN) variants including wild-type (ph 5.5), Y7H (6.0), and R06K (5.3). We experimentally infected ferrets, swine, and mice. In ferrets, Y7H lost airborne transmissibility until acquiring mutations that stabilized the HA protein to ph 5.3. This parallels the human 2009 pandemic, during which early isolates had moderate stability (ph ) and later isolates were stabilized (ph ). Swine can serve as a bridging host between avians and humans. We found naturally occurring swine isolates range in activation ph from Experimental infections showed swine tolerate a wide range of HA activation ph for growth and transmission (at least ph ), overlapping the ranges of avian and human viruses. This suggests swine are an ideal host for humanizing HA receptor-binding specificity and HA stability and emphasizes the need for swine surveillance. In mice, the destabilized Y7H variant was attenuated due to susceptibility to extracellular inactivation and diminished antagonization of type I interferon responses in dendritic cells. In addition to being a ph sensor that triggers membrane fusion at the right time and place, our findings show the HA protein needs to shift its stability so that influenza viruses can adapt to unique extracellular and intracellular environments in different host species. 55

56 DAMAGING AND SPREADING pathogenesis Abstract final identifier: 54 IMPACT OF OBESITY ON INFLUENZA VIRUS; PATHOGENESIS, VIRAL POPULATIONS AND TRANSMISSION Stacey Schultz-Cherry *, Victoria Meliopoulos, Brandi Livingston, Rebekah Honce, Erik Karlsson 2, Elodie Ghedin 3 Infectious Diseases, St Jude Children's Research Hospital, Memphis, United States, 2 Pasteur Institute, Phnom Penh, Cambodia, 3 New York University, New York City, United States Abstract: Obesity is a known risk factor for increased disease severity during influenza virus infection. Using diet-induced and genetically obese mouse models our group and others have shown that this increased disease severity is due to enhanced acute lung injury and respiratory distress syndrome leading to pneumonia. We found that the virus spreads more rapidly into the deep lung of obese hosts and persists longer in infected animals; often up to 8 days post-infection. Preliminary data from a human cohort studies suggests that similar trends may be seen in infected people. To better understand how prolonged viral persistence or shed impacts the viral populations within obese hosts, we developed a diet-induced obese (DIO) ferret model. Young male ferrets placed on a defined high fat diets gained more weight, had increased visceral fat accumulation, and higher circumference or BMI as compared to ferrets fed a regular diet after 6 to 8 weeks. As seen with the obese mouse models, DIO ferrets were more susceptible to influenza infection and had enhanced clinical disease, increased viral titers and spread throughout the lungs as well as prolonged shed. Surprisingly, we found that a virus that was unable to transmit in lean ferrets could transmit even by respiratory routes in one hundred percent of DIO ferrets. Next generation sequencing highlighted a significant change in the viral population of obese animals that may lead to the generation of minor viral variants that are not only capable of enhanced spread but also increased transmission. Studies are underway to define these populations and determine if obese hosts are super-spreaders of influenza virus. 56

57 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 55 MULTIROUTE MORBILLIVIRUS ENTRY: DISEASE INFORMS VACCINE DELIVERY Linda J. Rennick, Sham Nambulli, Natasha L. Tilston-Lunel, Rory D. de Vries 2, Rik L. de Swart 2, W. Paul Duprex * Microbiology, Boston University School of Medicine, Boston, United States, 2 Viroscience, Erasmus Medical Center, Rotterdam, Netherlands Abstract: Morbilliviruses represent some of the most transmissible pathogens on the planet. Attaining an understanding of primary pathogenesis and tropism is critical for the development of novel countermeasures. To extend standard reverse genetics approaches we developed a pipeline for the de novo synthesis of morbillivirus genomes based on sequences obtained directly from clinical material. To dissect multiroute morbillivirus entry we generated virologically identical but phenotypically distinct recombinant (r) canine distemper viruses (CDV) and measles viruses (MV) expressing different fluorescent reporter proteins for in vivo competition and airborne transmission studies in ferrets and macaques. Animals simultaneously received three viruses expressing green, red or blue fluorescent proteins via conjunctival (ocular, Oc), intranasal (IN) or intra-tracheal (IT) inoculation. Single lymphocytes expressing multiple fluorescent proteins were abundant in peripheral blood and lymphoid tissues, demonstrating the occurrence of double and triple virus infections for MV and CDV. Multicolor fluorescence in situ hybridization was used to determine if RNA persisted in vivo following virus clearance, seroconversion and disappearance of clinical signs of disease. We show both morbilliviruses can use multiple entry routes in parallel, that co-infection of cells during viral dissemination in the host is common, that cell-to-cell spread in vivo is the norm and that airborne transmission resulted in replication of a single-colored virus, which was the dominant virus in donor animals. Intranasal infection was inefficient, paralleling what is seen when rmv vaccines are delivered using this delivery route in macaques. 57

58 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 56 INTERFERON LAMBDA PREVENTS THE SPREAD OF RESPIRATORY VIRUSES FROM THE UPPER RESPIRATORY TRACT TO THE LUNGS AND RESTRICTS VIRUS TRANSMISSION IN MICE Daniel Schnepf *, Jonas Klinkhammer, Liang Ye, Marilena Schwaderlapp, Hans H. Gad 2, Rune Hartmann 2, Dominique Garcin 3, Tanel Mahlakõiv, Peter Stäheli Institute of Virology, Medical Center University of Freiburg, Freiburg im Breisgau, Germany, 2 Department of Molecular Biology and Genetics, Aarhus University, Aarhus, Denmark, 3 Department of Microbiology and Molecular Medicine, University of Geneva, Geneva, Switzerland Abstract: Host factors restricting the spread of viruses from the upper respiratory tract to the lungs and limiting virus transmission to the next host are poorly characterized. Previous infection studies in which influenza A viruses (IAVs) were administered directly to the lungs of mice suggested a surprisingly minor role of type III interferon in the defense against respiratory viruses. We analyzed the contribution of type I (IFN-α/β) and type III interferon (IFN-λ) using a mouse model in which the virus is selectively administered to the upper airways, mimicking a natural respiratory virus infection. Mice lacking functional IFN-λ receptors (Ifnlr -/- ) no longer restricted influenza or Sendai virus dissemination from the upper airways to the lungs. Ifnlr -/- mice shed significantly more infectious virus particles via the nostrils and transmitted respiratory viruses much more efficiently to naïve contacts compared with wild-type mice or mice lacking functional type I IFN receptors. Prophylactic treatment with IFN-α or IFN-λ inhibited initial virus replication in all parts of the respiratory tract, but only IFN-λ conferred long-lasting antiviral protection in the upper airways and blocked virus transmission. Thus, IFN-λ has a decisive and non-redundant function in the upper airways that greatly limits transmission of respiratory viruses to naïve contacts. 58

59 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 57 BREACHING THE OVINE PLACENTAL BARRIER BY RIFT VALLEY FEVER VIRUS Judith Oymans *, 2, Paul Wichgers Schreur, Lucien van Keulen, Jet Kant, Jeroen Kortekaas, 2 Department of Virology, Wageningen Bioveterinary Research, Lelystad, 2 Laboratory of Virology, Wageningen University, Wageningen, Netherlands Abstract: Rift Valley fever virus (RVFV), an arbovirus of the order Bunyavirales, causes severe disease in ruminants and occasionally humans. Outbreaks, which are thus far mainly confined to the African continent, are characterised by newborn fatalities and abortion storms in ruminants. The capacity of RVFV to transmit from mother to foetus is well recognized, however the exact route, susceptible cells and mechanisms triggering abortion are unknown. We have infected pregnant ewes with RVFV and studied the dissemination of virus at 4, 6 and 7 days post infection (dpi) in both ewe and foetal tissues. We found that the pregnant ewes became viremic at dpi and that viremia peaked at 3 dpi. The foetuses of the ewes which were necropsied at 4 dpi were still alive, while nearly all foetuses of ewes necropsied at 6 and 7 dpi had succumbed to the infection. At 7 dpi, two out of three remaining ewes aborted. Using real-time PCR and immunohistochemistry, we showed that maternal epithelial cells of the placenta are primary target cells of RVFV. Moreover, we observed that RVFV infects foetal trophoblasts when the placental barrier is breached. Interestingly, in half of the dead foetuses viral RNA and viral antigen were detected in liver and brain samples, while in all placentas both the maternal epithelium and the foetal trophoblast layer were infected. This implies that abortion can be caused directly by infection of the foetus, or indirectly by damage to the placenta. Furthermore, these results suggest that infection of the maternal epithelium is the first and most important step in crossing the placental barrier. How this barrier is breached is subject of ongoing research. Graphical Abstract: 59

60 DAMAGING AND SPREADING pathogenesis Abstract final identifier: 58 MECHANISMS AND CONSEQUENCES OF CELLULAR SURVIVAL FROM INFLUENZA VIRUS INFECTION Jessica K. Fiege *, Ryan A. Langlois Microbiology and Immunology, University of Minnesota, Minneapolis, United States Abstract: Influenza A virus (IAV) is a seasonal pathogen with the potential to cause devastating pandemics. IAV infects a variety of cells in the respiratory tract causing damage to the lungs. CD8 + T cells are the primary immune cell population responsible for IAV clearance. CD8 + T cells must balance controlling the spread of infection without causing excessive pathology. We have developed an IAV expressing Cre recombinase which permanantly labels infected cells in a Creinducible reporter mouse. Using this system, we have identified reporter + epithelial cells that survive both lytic IAV replication and CD8 + T cell-mediated clearance, and are henceforth termed survivor cells. How survior cells are able control IAV infection and elude the immune response has yet to be elucidated. We are investigating mechanisms by which survivor cells evade killing by CD8 + T cells. Additionally, the role of survivor cells in lung injury and is still unknown. We have observed proliferation of survivor cells, and at later time points after IAV clearance, a larger percentage of survivor cells have divided when compared to their uninfected counterparts. Fluorescent microscopy imaging revealed clusters of survivor cells in the lung in the weeks after IAV clearance, suggesting survivor cells are proliferating to assist in lung recovery. Together these data indicate that CD8 + T cells spare previously infected cells to minimize lung injury and aid in long-term pulmonary recovery. 60

61 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 59 CHARACTERIZATION OF EBOLA VIRUS DEFECTIVE GENOMES IN THE TESTES OF PERSISTENTLY INFECTED NON-HUMAN PRIMATES Beata Boczkowska, Taylor Weary *, Kendra Alfson, Jennifer Delgado, Gloria Rodriguez, Anthony Griffiths Virology & Immunology, TEXAS BIOMEDICAL RESEARCH INSTITUTE, San Antonio, United States Abstract: Persistent infection of individuals that survive Ebola virus disease has been reported but occurs at an unknown frequency. Interestingly, there is evidence of sexual transmission via semen from survivors suggesting the involvement of testes, which are an immune privileged site. It is important to understand the molecular basis of persistence to identify improved diagnostics and therapies. Deep sequencing of nucleic acids harvested from testes during the acute phase of EBOV infection showed an enrichment of reads in the trailer region of the genome, which for other viruses has been shown to be characteristic of defective genomes. Given the association of defective viral genomes and persistence, we deep sequenced nucleic acids harvested from the testes of drug treated animals that survived EBOV infection. This is a model used by other groups to study persistent EBOV disease. Sequencing showed some evidence of enrichment of the trailer region but coverage was poor. We amplified this region of the genome by PCR and deep sequenced the product. This permitted identification of defective viral genomes in the testes of >90% of surviving animals. Further characterization identified the break and reinitiation points of copy-back defective genomes. These data suggest that defective viral genomes may play a role in EBOV persistence. 6

62 DAMAGING AND SPREADING pathogenesis Abstract final identifier: 60 STUDIES INTO THE MECHANISM OF MEASLES-ASSOCIATED IMMUNE SUPPRESSION DURING AN OUTBREAK OF MEASLES IN THE NETHERLANDS Brigitta M. Laksono, Rory D. de Vries, Pieter L. Fraaij 2, Menno C. van Zelm 3, Wilhelmina L. Ruijs 4, Marion P. Koopmans, Albert D. Osterhaus, Rik L. De Swart * Viroscience, 2 Pediatrics, 3 Immunology, ERASMUS MC, Rotterdam, 4 Centre for Infectious Disease Control, National Institute of Public Health and the Environment, Bilthoven, Netherlands Abstract: Measles is associated with immune suppression, leading to increased susceptibility to opportunistic infections. Based on observations in experimentally infected non-human primates, we previously hypothesised that immune suppression is caused by infection and depletion of CD50 + lymphocytes, referred to as immune amnesia. In 203, a large measles outbreak among unvaccinated individuals in the Dutch Orthodox Protestant community provided a unique opportunity to test this hypothesis in measles patients. We performed an observational cohort study in unvaccinated children aged 4-7 years. Nose- and throat swabs and a blood sample were collected from acute measles patients (cohort A), or paired blood samples were collected from healthy children before and after measles (cohort B). 26 children were included in cohort A, of whom 23 had laboratory-confirmed measles and 4 were sampled before onset of rash. Phenotyping of MVinfected cells in PBMC, by combining lineage markers with intracellular staining of MV nucleoprotein, identified MV-infected B-cells and memory T-cells between five days before and two days after onset of rash. 90 children were included in cohort B, resulting in collection of 77 paired blood samples from children with lab-confirmed measles and 35 paired PBMC samples suitable for multi-colour flow cytometry. We detected a significant reduction in peripheral memory B-cells and increased frequencies of regulatory T-cells and transitional B-cells after measles. These findings support the hypothesis that measles causes immune amnesia. 62

63 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 6 VIRAL ENTRY PROPERTIES REQUIRED FOR FITNESS IN HUMANS REVEALED THROUGH RAPID GENOMIC CHANGE DURING VIRAL ISOLATION Cyrille Mathieu *, Sho Iketani, Ryan C. Shean 2, Marion Ferren, Negar Makhsous 2, Dolly B. Aquino 2, Bert Rima 3, Matteo Porotto, Alexander Greninger 2, Anne Moscona Center for Host-Pathogen Interaction, Microbiology & Immunology, Pediatrics, Physiology & Biophysics, Columbia University Medical Center, New York, 2 Laboratory Medicine, University of Washington, Seattle, United States, 3 Centre for Experimental Medicine, The Queen's University of Belfast, Belfast, Northern Ireland, United Kingdom Abstract: Human parainfluenza viruses cause a large burden of respiratory illness. While much research relies upon viruses grown in cultured immortalized cells, parainfluenza 3 (HPIV3) evolves in culture and properties required for fitness in vivo differ when compared to cultured strains. In our genome-wide survey of HPIV3 adaptations to culture using metagenomic next-generation sequencing of matched pairs of clinical samples and primary culture isolates, non-synonymous changes arose during primary viral isolation, almost entirely in the genes encoding the two surface glycoproteins the receptor binding hemagglutinin-neuraminidase (HN) or the fusion protein (F). We recovered genomes from 95 HPIV3 primary culture isolates and 23 HPIV3 strains directly from clinical samples. HN mutations arising during primary viral isolation resulted in substitutions at HN s dimerization site, a site critical for activation of viral fusion during entry. Alterations in HN dimer interface residues known to differ between cultured and clinical strains occurred rapidly, with H552 and N556 being critical residues for adaptation. A novel cluster of residues at a different face of the HN dimer interface also emerged and imply a role in HPIV3-mediated fusion in vivo. Functional characterization of these culture-associated HN mutations in a clinical isolate background revealed acquisition of the fusogenic phenotype associated with cultured HPIV3; the HN-F complex showed enhanced fusion and decreased receptor-cleaving activity, and engineered viruses bearing alterations at these sites reveal the critical nature of these residues for growth in airway. These results highlight the notion that even brief exposure to immortalized cells may affect key viral properties, and underscore the balance of features of the HN-F complex and the sites at the HN dimer interface required for fitness in humans. 63

64 DAMAGING AND SPREADING pathogenesis Abstract final identifier: 62 GENETIC DETERMINANTS OF SEVERE RESPIRATORY SYNCYTIAL VIRUS INFECTIONS IN INFANTS Martin Wetzke, Sibylle Haid 2, Chris Lauber 3, Daniel Todt 2, 4, Lars Kaderali 5, Robert Geffers 6, Ehsan Vafadarnejad 7, Bettina Wiegmann 8, Eike Steinmann 2, 4, Emmanuel Saliba 7, Thomas F. Schulz 9, Gesine Hansen, Thomas Pietschmann * 2 Pediatric Pneumology, Allergology and Neonatology, Hannover Medical School, 2 Experimental Virology, TWINCORE, Hannover, 3 Medical Informatics and Biometry, Carl Gustav Carus Faculty of Medicine, TU Dresden, Dresden, 4 Molecular and Medical Virology, Ruhr-University Bochum, Bochum, 5 Bioinformatics, University of Greifswald, Greifswald, 6 Genome Analytics, Helmholtz Centre for Infection Research, Braunschweig, 7 Single Cell Analysis, HIRI, Würzburg, 8 Cardiothoracic, Transplantation and Vascular Surgery, 9 Virology, Hannover Medical School, Hannover, Germany Abstract: Respiratory syncytial virus (RSV) is the most common cause of lower respiratory tract infections in infants. Disease severity varies widely among children and ranges from mild upper respiratory symptoms to severe bronchiolitis. Genetic factors governing disease severity are incompletely defined. 0 children aged between 0-2 years and suffering from severe acute RSV infections were subjected to whole exome sequencing (WES). Since interferon-regulated immune responses are critical for the defense of RSV infections and the course of the disease, we focused on 542 genes that either trigger expression of interferons, or contribute to interferon signaling or are controlled by interferons. In total 30,039 variants mapped to these genes. Heterozygosity and homozygosity counts in our cohort as well as in an ethnically matched sub-cohort of the Exome Aggregation Consortium (ExAC) were used to calculate the significance of association of variants with severe RSV infection. Collectively, 28 coding polymorphisms mapping to 84 genes were significantly associated with severe RSV infection. Associated genes expressed in primary human airway epithelial cells were silenced and the impact on RSV infection was quantified. Moreover, their expression upon RSV infection of air-liquid interface cultures of human airway epithelial cells was quantified with single cell resolution. More than six novel viral restriction or dependency factors were identified including proteins involved in cellular ER-stress response and regulation of ER-associated protein degradation (ERAD), in inflammatory cytokine signaling and a protein kinase activated by double-stranded RNA which mediates the effects of interferon in response to viral infection. This integrated approach provides a new paradigm for discovery of genetic traits and protein functions affecting the course and outcome of infectious diseases. 64

65 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 63 THE EGYPTIAN ROUSETTE GENOME, PROVEN HOST FOR MARBURG VIRUS, REVEALS UNEXPECTED FEATURES OF BAT ANTIVIRAL IMMUNITY Gustavo Palacios *, Jonathan Towner 2, Stephanie Pavlovich 3, Mariano Sanchez-Lockhart, Tom Kepler 3 Center for Genome Sciences, USAMRIID, Frederick, 2 Viral Special Pathogens Branch, CDC, Atlanta, 3 Boston University, Boston, United States Abstract: Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection rather than enhanced potency of antiviral defences may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans. Keywords: genome 65

66 DAMAGING AND SPREADING pathogenesis Abstract final identifier: 64 BIPHASIC VASCULAR BREAKDOWN AND INFLUX OF NEUTROPHILS INTO THE BRAIN DURING RIFT VALLEY FEVER VIRUS ENCEPHALITIS Amy Hartman *, Michael Kujawa, Joseph Albe Center for Vaccine Research, UNIVERSITY OF PITTSBURGH, Pittsburgh, United States Abstract: Rift Valley Fever Virus (RVFV) is a vector-borne infection endemic to Africa. However recent outbreaks in the Arabian Peninsula have expanded its potential range. RVFV causes a number of clinical outcomes in people, including hemorrhagic fever and encephalitis with associated morbidity and mortality. Encephalitis is an understudied clinical outcome in people. We use a rat model to better understand the neuropathogenesis of this viral infection. After aerosol infection with the pathogenic ZH50 strain of RVFV, Lewis rats develop a uniformly lethal encephalitic disease with neurological symptoms. The pathogenic events between infection and lethal disease are not well defined. An important question is the timing of breakdown of the brain vasculature in relation to other pathogenic events such as virus invasion and replication, infiltration of immune cells, and inflammatory cytokine expression. We found that early, transient opening of the blood brain barrier in the olfactory bulb occurs concomitantly with the detection of virus in the brain. Using in vivo imaging (IVIS), widespread vascular breakdown in the brain, however, was a late event that corresponded temporally to high levels of virus, expression of matrix metalloproteinase 9 (MMP-9), and an influx of immune cells, which were primarily neutrophils with some macrophages and lymphocytes. There was also evidence of microglia activation. Intracellular staining for viral glycoprotein showed infected cells were neurons, macrophages, and neutrophils. In addition to limiting virus replication, prevention of vascular leakage and infiltration of leukocytes late in infection will likely be an important component for prevention of lethal neurological disease. 66

67 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 65 CARDIAC DISORDERS AND SUDDEN DEATH CAUSED UPON HEART INFECTION BY HUMAN PATHOGENIC INFLUENZA A VIRUS Jasmina Vasilijevic, David Filgueiras-Rama 2, 3, 4, Jose Jalife 2, 3, 5, Sami N. Noujaim 6, Celia Gutiérrez, Noelia Zamarreño, Jose M. Alfonso 2, Alejandro Bernabé 2, Christian P. Cop 2, Daniel G. León 2, Daniel Calle 2, 7, Manuel Desco 8, 9, 0, Jesús Ruiz- Cabello, 2, Amelia Nieto, 2, Ana Falcon *, 2 Molecular and Cellular Biology, National Center for Biotechnology (CNB-CSIC), 2 Myocardial Pathophysiology, National Center for Cardiovascular Research (CNIC), 3 Cardiovascular Diseases, Center fo Biomedical Research (CIBER), 4 Cardiac Electrophysiology Unit, Hospital Clínico San Carlos, Madrid, Spain, 5 Center for Arrhythmia Research, University of Michigan, Ann Arbor, 6 Morsani College, University of South Florida, Tampa, United States, 7 Instituto de Investigación Sanitaria, Hospital Gregorio Marañón, 8 Imaging, National Center for Cardiovascular Research (CNIC), 9 Bioengineering and Aerospace Engineering, University Carlos III of Madrid, 0 Mental Health, Center fo Biomedical Research (CIBER), Madrid, CIC biomagune, IKERBASQUE, Basque Foundation for Science, San Sebastian, 2 Respiratory Diseases, Center fo Biomedical Research (CIBER), Madrid, Spain Abstract: Influenza A virus (IAV) infection has been associated with important cardiovascular complications such as myocarditis, heart failure and acute myocardial infarction, besides respiratory alterations. Sudden death has also been described in infected patients, even without apparent respiratory damage, which could be attributed to heart failure. However, direct infection of cardiac tissue by IAV has been rarely detected. This has led to the assumption that cardiac pathology is derived from lung damage and inflammation caused by the infection. We have explored the relationship between structural and functional cardiac damage and the infective capacity in heart tissue of mice by viruses of different pathogenicity. Heart damage was evaluated by sequential electrocardiograms, cardiac magnetic resonance imaging, connexin43 protein expression and ATP levels in infected versus control animals. We demonstrate that the viruses with higher pathogenicity replicate best in cardiac cell cultures. Moreover, differences in pathogenicity of human viruses did correlate directly with the capacity to replicate in the heart, cause electrical conduction disorders and induce sudden death in infected animals. The data offer a new paradigm toward better treatment(s) of IAV-related heart disease and indicates that direct heart infection should be considered clinically in addition to the indirect effects derived from respiratory pathology. Graphical Abstract: 67

68 DAMAGING AND SPREADING pathogenesis Abstract final identifier: 66 ABERRANT VIRAL RNAS LINK AVIAN AND PANDEMIC INFLUENZA VIRUS VIRULENCE TO ERRONEOUS RNA POLYMERASE ACTIVITY Aartjan Te Velthuis *, 2, Joshua Long, David Bauer, Rebecca Fan 3, Hui-Ling Yen 3, Jane Sharps 4, Marian Killip, Jurre Siegers 5, Maria Jose Oliva-Martin 4, Rick Randall 6, Emmie de Wit 7, Debby Van Riel 5, Leo Poon 3, Ervin Fodor Pathology, UNIVERSITY OF OXFORD, Oxford, 2 Pathology, UNIVERSITY OF CAMBRIDGE, Cambridge, United Kingdom, 3 University of Hong Kong, Hong Kong, China, 4 UNIVERSITY OF OXFORD, Oxford, United Kingdom, 5 Erasmus Medical Centre, Rotterdam, Netherlands, 6 University of St Andrews, St Andrews, United Kingdom, 7 NIH, Hamilton, United States Abstract: Infections with highly pathogenic influenza viruses result in a dysregulation of the innate immune response and severe disease. To investigate the molecular mechanism underlying this process, we analysed human lung cells and lung tissues of mice and ferrets infected with the 98 HN pandemic virus or H5N strains. In all experiments, we found a new type of influenza RNA that is <25 nt in length and generated from viral genome segments through a copy-choice mechanism that maintains the 5' and 3' terminal sequences of the genome segments, but deletes internal sequences. We call these RNAs mini viral RNAs (mvrnas). mvrnas were preferentially bound by RIG-I over other viral RNAs and extremely potent inducers of the immune response. Moreover, they were only formed by influenza viruses that were not adapted to humans, such as zoonotic avian or pandemic viruses. We believe that these results provide an important advance in our understanding of the molecular basis of influenza virus lethality and that this new RNA is a biomarker for highly pathogenic influenza virus infections in humans. 68

69 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: 67 INFLUENZA A VIRUS M2 PROTEIN INTERACTS WITH CELLULAR NA,K-ATPASE: DOES IT HAVE A PATHOPHYSIOLOGICAL ROLE IN INFLUENZA PNEUMONIA? Jessica Schulze *, Christin Peteranderl 2, Irina Kuznetsova 3, Stephan Pleschka 3, Susanne Herold 2, Thorsten Wolff Unit 7 Influenza and Other Respiratory Viruses, Robert Koch Institute, Berlin, 2 Department of Internal Medicine II, Universities of Giessen and Marburg Lung Center, 3 Institute of Medical Virology, Justus Liebig University, Giessen, Germany Abstract: Influenza A Virus (IAV) infections of the lower respiratory tract can induce viral pneumonia resulting in acute lung injury (ALI/ARDS) with fatal outcome. Characteristics of an IV-induced pneumonia are an alveolar epithelial cell (AEC) damage and accumulation of edema fluid in the alveolar compartment impairing gas exchange. Depending on a sodium gradient established by the basolateral Na,K-ATPase (NKA) and the apical epithelial sodium channel (ENaC) edema fluid is removed from the alveolar space under normal conditions. However after IV-infection a decreased alveolar fluid clearance was observed. In primary AEC it was shown that an IAV-infection leads to a mistargeting of the NKAα-subunit to the apical cell membrane, but to a reduced NKA expression in the non-infected neighbouring cells. Co-immunoprecipitation studies identified the viral M2 protein as a binding partner of NKAα. To study the pathophysiological implications of this virus-host interaction we characterized the NKA binding site in the viral M2 protein. In a mutational approach we were able to identify three amino acids in the cytoplasmic tail abutting the transmembrane domain as critical for NKAα binding. A recombinant seasonal IAV with impaired NKAα binding was slightly attenuated for replication in vitro and ex vivo. Currently the impact of this virus on NKAα relocalization and edema clearance is investigated in a polarized Calu 3 cell model. In addition to established roles in intracellular genome release and virus budding our data suggest a further function of the IV M2 protein in relocalizing the cellular NKAα, which is likely to contribute to pathophysiological effects in IV infection. Investigating the impact of the M2/NKA interaction on the impaired edema clearance could help to better understand this outcome of an IV infection in future. 69

70 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 68 IN VIVO IMAGING CAPTURES THE PATHOPHYSIOLOGICAL CHANGES AND DYNAMICS OF IMMUNE CELLS IN INFLUENZA VIRUS-INFECTED MOUSE LUNG Hiroshi Ueki *, I-Hsuan Wang, Satoshi Fukuyama, Hiroaki Katsura, Tiago J. D. S. Lopes, 2, Matthias Gunzer 3, Gabriele Neumann 2, Yoshihiro Kawaoka, 2 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo, Japan, 2 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, United States, 3 Institute for Experimental Immunology and Imaging, University Hospital, University Duisburg Essen, Essen, Germany Abstract: Influenza virus is a respiratory pathogen that causes pandemics and seasonal epidemics. The pathophysiological changes and dynamics of immune cells in influenza virus-infected lungs are poorly understood. In this study, we established an in vivo imaging system that combines two-photon excitation microscopy and fluorescent influenza viruses of different pathogenicity (backbones of H5N A/Vietnam/203/2004 or A/Puerto Rico/8/34 [HN; PR8]). This approach allowed us to monitor and correlate several parameters and pathophysiological changes including the spread of infection, pulmonary permeability, perfusion speed, the number of recruited neutrophils, and neutrophil motion in the lungs of live mice. Several pathophysiological changes were larger and occurred earlier in mice infected with a highly pathogenic H5N influenza virus compared to those in mice infected with a PR8 strain. Time-lapse imaging analysis also revealed that neutrophil movement in the pulmonary capillaries was interlaced with slow (=<50 µm/s) and rapid (>50 µm/s) motions under naïve conditions. In contrast, influenza virus-infected lung showed a temporal increase in pulmonary neutrophil numbers, and neutrophil movement changed to longer durations of the slow motion after the climax of neutrophil recruitment. We also made realtime observations of cell-cell interactions with morphological changes between infected cells and neutrophils or alveolar macrophages infiltrating the alveoli of infected lungs. These findings demonstrate the potential of our in vivo imaging system to provide novel information about the pathophysiological consequences of virus infection. 70

71 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 69 STOPPING MEASLES IN ITS TRACKS - EFFICACY OF AN ORALLY BIOAVAILABLE ANTIVIRAL AGAINST MEASLES VIRUS IN SQUIRREL MONKEYS Kevin Wittwer, Kristin Pfeffermann, Danielle Anderson 2, Sabine Santibanez 3, Annette Mankertz 3, Richard K. Plemper 4, Veronika von Messling *, 2 Veterinary Medicine, Paul-Ehrlich-Institut, Langen, Germany, 2 Emerging Infectious Diseases, Duke-NUS Medical School, Singapore, Singapore, 3 Infectious Diseases, Robert-Koch-Institut, Berlin, Germany, 4 Biomedical Sciences, Georgia State University, Atlanta, United States Abstract: Despite ongoing elimination efforts, measles virus (MeV) continues to circulate, and reduced compliance with vaccine recommendations has led to a resurgence of cases and even outbreaks in industrialized countries. Therapeutic options are thus urgently needed. Towards this we assessed the potential of an orally bioavailable small molecule inhibitor of the viral polymerase in squirrel monkeys. These New World monkeys are highly susceptible to MeV infection and reproduce disease severity and clinical signs seen in patients. Animals were treated twice daily in a prophylactic regimen starting the day before or therapeutically at different times after infection with a recent German MeV field isolate. The drug was generally well-tolerated and reached high plasma concentrations. Prophylactic treatment effectively blocked infection, and treatment start in the incubation period greatly reduced or even prevented clinical disease. Treatment after onset of clinical signs had no effect on the course of disease, but stopped virus shedding from the respiratory tract and may thereby reduce transmission. While robust antibody titers were detected in the therapeutic groups, antibody levels in prophylactically treated animals were variable. These results demonstrate promising efficacy and support further clinical development of this candidate drug. 7

72 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 70 REMDESIVIR (GS-5734) PROTECTS NONHUMAN PRIMATES AGAINST PATHOGENIC FILOVIRUSES Travis Warren *, Alison Hogg 2, Robert Jordan 2, Laura Gomba, Jessica Weidner, Veronica Soloveva, Pamela Wong 2, Iva Trantcheva 2, Scott Sellers 2, Darius Babusis 2, Danielle Porter 2, Roy Bannister 2, Richard Mackman 2, Tomas Cihlar 2, Sina Bavari US Army Medical Research Institute of Infectious Diseases, Frederick MD, 2 Gilead Sciences Inc, Foster City CA, United States Abstract: The W. Africa Ebola virus (EBOV) outbreak highlighted the lack of approved therapeutics and vaccines for filovirus infection. Remdesivir (RDV) is a monophosphoramidate prodrug of a modified adenosine nucleoside analog with potent in vitro antiviral activity against multiple pathogenic RNA virus families including filoviruses, coronaviruses, and paramyxoviruses. Here, we present key findings from nonhuman primate efficacy studies, in which animals were infected with pathogenic filoviruses and treated with various intravenously administered regimens of RDV. Key Filovirus Antiviral Efficacy Findings in Nonhuman Primates Virus Challenge Route RDV Regimen(s)* Treatment Initiation Survival(# survivors/# per group) EBOV Makona/204 IM 0 mg/kg for 2 days 3 days PI Vehicle: 0 (0/6) RDV: 00% (6/6) EBOV Kikwit/995 IM (A) 0 mg/kg loading dose + 5 mg/kg for days (B) 5 mg/kg for 2 days 4 days PI Vehicle: 6.7% (/6) (A) RDV: 00% (6/6) (B) RDV: 83.3% (5/6) EBOV Kikwit/995 Mucosal (aerosol) 0 mg/kg loading dose+ 5 mg/kg for days 4 days PI Vehicle: 6.7% (/6) RDV: 66.7% (4/6) EBOV Kikwit/995 IM (A) 0 mg/kg loading dose+ 5 mg/kg for days (B) 7.5 mg/kg loading dose+ 5 mg/kg for days MARV Angola/2005 IM 0 mg/kg loading dose+ 5 mg/kg for days 5 days PI Vehicle: 20% (/5) (A) RDV: 50% (3/6) (B) RDV: 66.7% (4/6) 5 days PI Vehicle: 0% (0/6) RDV: 83.3% (5/6) *, all regimens are qd IV. PI, Post infection; IV, intravenous; IM, Intramuscular. Phase studies with RDV in normal human subjects have been completed. The safety and efficacy is currently being investigated in a Phase 2 study in male survivors. 72

73 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 7 THE MECHANISM OF RESISTANCE TO FAVIPIRAVIR IN INFLUENZA Daniel Goldhill *, Pinky Langat, Robert Fletcher, Angie Lackenby 2, Aartjan te Velthuis 3, Wendy Barclay Imperial College, 2 Public Health England, London, 3 Cambridge University, Cambridge, United Kingdom Abstract: The evolution of drug resistance is a major problem for currently licensed antivirals for influenza. New antivirals with a high barrier to resistance are needed to treat influenza. Favipiravir is a novel antiviral that has undergone phase III clinical trials and is licensed in Japan to treat pandemic influenza. Favipiravir is a nucleoside analogue which causes mutations. Until now, no resistance to favipiravir has been reported for influenza viruses despite several passaging studies. In this study, we experimentally evolved a prototypical phn influenza A virus in the presence of favipiravir and identified the first mutations causing drug resistance for influenza. We showed that a single mutation in the polymerase subunit PB gave resistance to favipiravir but at a cost to polymerase activity and viral fitness. A mutation in PA compensated for the cost in polymerase activity and restored viral fitness. We used structural modelling, next generation sequencing and in vitro assays to demonstrate the mechanism by which resistance was conferred. We showed that resistant influenza polymerase does not incorporate favipiravir and this prevents favipiravir from causing mutations. We showed that this resistance mechanism was conserved across different subtypes of influenza and propose that this mechanism could be generalised to other viruses. 73

74 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 72 IDENTIFICATION OF CALCIUM CHANNEL BLOCKER AS AN ANTI-SEVERE FEVER WITH THROMBOCYTOPENIA SYNDROME VIRUS (SFTSV) COMPOUND Shuzo Urata *, 2, Jiro Yasuda, 2 NAGASAKI UNIVERSITY, INSTITUTE OF TROPICAL MEDICINE, 2 National Research Center for the Control and Prevention of Infectious Diseases (CCPID), Nagasaki, Japan Abstract: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS virus (SFTSV), which has a high mortality rate. SFTSV belongs to Phenueviridae, Bunyavirales. Currently, no licensed vaccines or therapeutic agents have been approved for treating SFTSV infected patients. Our aim is to identify effective compounds which can be used to treat SFTSV infected patients. Out of approximately 650 compounds from an FDA-approved compound library, 6 compounds, which belong to L-type calcium channel blocker, exhibited strong anti-sftsv effects. We further examined to rule out the target of the calcium channel blocker, Manidipine, on SFTSV replication and propagation. With a time-of addition experiment, we found that it targets replication/transcription step of SFTSV. We also found that this anti-viral effect by Manidipine is not restricted only to SFTSV, but also adopted to other negative strand RNA viruses which belong to different virus families. With the same concentration, the inhibitory effect on infectious SFTSV production by Manidipine was higher than that of T-705 (favipiravir). Inhibiting the activation of calcineurin, a downstream effector of calcium channel, by FK506 and Cyclosporin A, also reduced SFTSV propagation. These results showed that calcium signaling is important for SFTSV replication and could be a target to combat SFTSV. Since calcium channel blockers have been used as a medication to decrease blood pressure in patients with hypertension in clinic, it might be possible to apply these calcium channel blockers to treat SFTS patients. These results also enhance our understanding of the SFTSV replication mechanism and may contribute to the development of novel therapies for SFTSV infection. 74

75 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 73 BUNYAVIRUS VACCINE DEVELOPMENT BOOSTED BY NOVEL INSIGHTS INTO GENOME REPLICATION AND PACKAGING Paul Wichgers Schreur *, 2, Lucien van Keulen, Jet Kant, Sandra van de Water, Nadia Oreshkova, 2, Judith Oymans, 3, Jeroen Kortekaas, 2, 3 Department of Virology, Wageningen Bioveterinary Research, 2 BunyaVax, Lelystad, 3 Laboratory of Virology, Wageningen University, Wageningen, Netherlands Abstract: Bunyaviruses have evolved to replicate in both insects, ticks and mammals. An important step in the bunyavirus life-cycle is the packaging of the tri-segmented RNA genome into maturing virions. Using single-molecule RNA fluorescence in situ hybridization (FISH) we showed that the zoonotic Rift Valley fever virus (RVFV), in contrast to multi-segmented viruses like influenza virus, uses a non-selective genome packaging strategy to produce progeny. Remarkably, the overall efficiency of genome packaging was higher in virus grown in insect cells compared to mammalian cells, suggesting that genome-segment incorporation may differ between hosts. We took advantage of the non-selective nature of RVFV genome packaging and designed two-segmented replicons that lack the M-genome segment but are able to infect and replicate for a single round in vivo when used as a vaccine. These RNA particles are currently developed into a vector platform suitable for counteracting emerging viruses and cancer. In addition, the flexibility in RVFV genome packaging enabled the rescue of four-segmented variants (RVFV-4s). RVFV-4s is based on the splitting of the M-genome segment into two M-type segments either encoding the Gn or the Gc glycoprotein. RVFV-4s is currently used as a RVFV vaccine and induces a strong protective immune response in young sheep. The vaccine was also shown to be safe in pregnant ewes and protected the animals from RVFV induced abortion. Altogether, these studies show that by exploring fundamental aspect of bunyavirus replication and genome packaging effective vaccines can be developed. Graphical Abstract: 75

76 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 74 CONVERGENT IMMUNOLOGICAL SOLUTIONS TO ARGENTINE HEMORRHAGIC FEVER VIRUS NEUTRALIZATION Antra Zeltina, Stefanie A. Krumm 2, Mehmet Sahin 3, Daniel D. Pinschewer 3, Katie Doores * 2, Thomas Bowden Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, 2 Department of Infectious Diseases, King's College London, London, United Kingdom, 3 Division of Experimental Virology, Department of Biomedicine, University of Basel, Basel, Switzerland Abstract: New World arenaviruses cause substantial public health concerns and economic dangers when transmitted from their rodent reservoirs to humans. Transmission is facilitated by specific interactions between viral attachment glycoprotein, GP, a key target for neutralizing antibodies (nab), and the cell surface human transferrin receptor (htfr). We present the structural basis for how the mouse-derived nab OD0 disrupts this inter- action by targeting the receptorbinding surface of GP from Junín virus (JUNV), a hemorrhagic fever arenavirus endemic in central Argentina, and describe the elicitation of nabs using a rlcmv system displaying arenaviral GPs and their isolation via B-cell sorting. Comparison of our structure with that of a previously reported nab complex (JUNV GP GD0) reveals overlapping epitopes but highly distinct antibody-binding modes. Both antibodies present a key tyrosine residue, albeit on different chains, that inserts into a central pocket on JUNV GP and mimics the host TfR contacts. These data provide a molecular-level description of how antibodies, derived from different germlines, arrive at equivalent immunological solutions to virus neutralization. 76

77 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: 75 STRUCTURAL BASIS FOR RECOGNITION OF THE CENTRAL CONSERVED REGION OF RSV G BY NEUTRALIZING HUMAN ANTIBODIES Johannes P. Langedijk *, Harrison G. Jones 2, Tina Ritschel, Gabriel Pascual 3, Just P. Brakenhoff 4, Elissa Keogh 3, Polina Furmanova-Hollenstein, Ellen Lanckacker 5, Jay Wadia 3, Anthony Williamson 3, Dirk Roymans 5, Angelique van 't Wout 4, morgan Gilman 2, Jason McLellan 2 viral vaccines, JANSSEN VACCINES AND PREVENTION, Leiden, Netherlands, 2 of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, 3 Janssen Prevention Center, Janssen, La Jolla, United States, 4 Janssen Prevention Center, Janssen, Leiden, Netherlands, 5 Janssen Infectious Diseases, Janssen Pharmaceutica, Beerse, Belgium Abstract: Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infections, and yet there remains no effective treatment or vaccine. The virion is decorated with the fusion glycoprotein and the attachment glycoprotein G, which binds to CX3CR on human airway epithelial cells. RSV G is a major target of the humoral immune response, and antibodies that target the central conserved region have been shown to neutralize RSV and protect against severe RSV disease in animal models. However, the molecular underpinnings for antibody recognition of this region have remained unknown. Therefore, we isolated a large panel of broadly reactive human antibodies directed against the central conserved region of RSV G, demonstrated complement-dependent neutralization for both HRSV type A and B and we mapped the epitopes using peptide libraries in full detail and demonstrated several different conserved epitopes covering the complete surface of the central conserved domain. Strong complement-independent neutralization of RSV infection could only be demonstrated in human bronchoepithelial cell cultures. The antibodies protected cotton rats from severe RSV disease. High-resolution crystal structures of two antibodies in complex with the domain revealed two conformational epitopes that require proper folding of the cystine noose located in the C-terminal part of the central conserved region. Comparison of these structures with a recently determined structure of fractalkine (CX3CL) in complex with a viral homolog of CX3CR (US28) suggests that RSV G would bind to CX3CR in a mode that is distinct from that of fractalkine. These results build on recent studies demonstrating the importance of RSV G in antibody-mediated protection from severe RSV disease, and the structural information presented here should guide development of new vaccines and therapies for RSV 77

78 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 76 HEADS WIN! HEAD-SPECIFIC B CELLS AND ANTIBODY DOMINATE THE IMMUNE RESPONSE IN A MISMATCHED PRIME-BOOST VACCINE STRATEGY S Jegaskanda, 2, S Andrews 3, A K. Wheatley 2, J W. Yewdell 4, A McDermott 3, K Subbarao * Lab of Infectious Diseases, NIAID, NIH, Bethesda, United States, 2 Microbiology and Immunology, University of Melbourne, Melbourne, Australia, 3 Vaccine Research Center, 4 Lab of Viral Diseases, NIAID, NIH, Bethesda, United States Abstract: Prime-boost vaccination with matched pandemic live-attenuated influenza vaccines (plaiv) and inactivated subunit vaccine (pisv) establishes long-term immune memory. Here, we asked whether mismatched plaiv-isv would generate stem-specific memory B cells (MBCs) and broadly neutralizing antibodies (NAbs). We vaccinated monkeys with H5N plaiv, H5N plaiv-pisv or H5N LAIV followed by seasonal trivalent influenza vaccine (TIV); the HN component has a mismatched HA head but conserved stem. We measured serum NAbs and HA-specific B cell responses using HAspecific B cell probes. H5N plaiv elicited both H5 + -specific and dual (H5 + H + )-specific B cells, reflecting HA head and stem specificity, respectively in the peripheral blood and mediastinal lymph nodes (LN). The frequency of head-specific MBCs was higher than stem-specific MBCs. Following matched pisv boost, the frequency of both head- and stem-specific B cells increased in the blood, axillary LN and spleen but only H5-specific NAbs were detected. In contrast, mismatched boost elicited a small early expansion of stem-specific MBCs in the periphery but predominantly H5 + or H + head-specific B cells in the LN. Stem-specific germinal centre B cells were predominantly found in the spleen and peripheral blood. However, broadly NAbs were not detected. Thus, mismatched boost generated a higher frequency of head- than stemspecific MBCs and did not induce broadly cross-neutralizing Abs. These results have significant implications for vaccine strategies that aim to generate stem-specific NAbs but include the HA head. 78

79 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 77 THE POTENTIAL OF HUMAN MONOCLONAL ANTIBODIES THAT RECOGNIZE THE INFLUENZA A(HN)PDM09 VIRUS HEMAGGLUTININ RECEPTOR-BINDING SITE AS ANTI-INFLUENZA AGENTS. Atsuhiro Yasuhara *, Seiya Yamayoshi, Maki Kiso, Shinya Yamada, Yoshihiro Kawaoka, 2, 3 Institute of Medical Science, University of Tokyo, Tokyo, Japan, 2 School of Veterinary Medicine, University of Wisconsin- Madison, Madison, United States, 3 International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo, Japan Abstract: Antiviral therapy can reduce the burden of seasonal influenza and provides the first line of defense against pandemic influenza before vaccines are available. However, because influenza virus rapidly mutates to escape from neutralizing antibodies, one of the main disadvantages of monoclonal antibodies (mabs) as antiviral treatments is the potential emergence of escape mutants. Here we isolated three human mabs from volunteers vaccinated with seasonal influenza vaccine or H5N pre-pandemic vaccine, and characterized them by evaluating their in vivo protective efficacy, generating escape mutant viruses, and examining the fitness of the escape mutants in vitro. We found that all three mabs possessed high in vivo efficacy against lethal infection of mice with A(HN)pdm09 virus. Mutant viruses that escaped from these mabs rarely appeared, and the escape mutations were identified in conserved residues of the receptor-binding site (RBS) of hemagglutinin (HA). Furthermore, the escape mutant viruses showed significantly lower replication in vitro than their parental viruses, indicating that the mutant viruses would be unlikely to dominate. These results suggest that mabs that recognize the functionally conserved residues within the RBS rarely produce escape mutant viruses. Such mabs could fulfill the need for anti-influenza agents with a low propensity for the emergence of escape mutants. 79

80 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 78 COORDINATE DE-IMMUNIZATION OF MEASLES VIRUS ENVELOPE GLYCOPROTEINS DRIVES THE EMERGENCE OF ANTIGENIC VARIANTS Miguel Ángel Muñoz Alía *, Claude P. Muller 2, Stephen J. Russell Molecular Medicine, Mayo Clinic, Rochester, United States, 2 Department of Infection and Immunity, Luxembourg Institute of Health, Esch-Sur-Alzette, Luxembourg Abstract: Measles virus (MeV) is monotypic and in 70 years measles genotypes evading the vaccine-driven polyclonal response against the virus surface glycoproteins H and F have not emerged. The basis of the remarkable antigenic stability in the measles coat remains unknown. We here show that systematic ablation of 30 known epitopes from the 8 known antigenic sites on the MeV H glycoprotein resulted in resistance to neutralization by anti-h reactive polyclonal antibodies in H-immune sera from humans, mice, rabbits and guinea pigs. Viruses incorporating the fully mutated H glycoprotein interacted poorly with MeV receptors SLAM and Nectin-4, leading to virus entry and syncytia formation solely via receptor CD46, due to an increased in binding avidity. MeVs incorporating the fully de-immunized H protein could still be efficiently neutralized by MeV-immune sera not depleted of anti-f antibodies, underscoring the importance of F-reactive antibodies in protective anti-mev immune responses. Substitution of MeV F for the homologous F from Canine Distemper Virus, resulted in the generation of MeV Stealth, which behaved as a new MeV serotype, showing increased resistance to neutralization by serum from vaccinated humans or from previously MeV infected subjects. All viruses had the Moraten vaccine backbone and were considered BSL2 agents. Our work sheds new light on the mechanisms that constrain the plasticity of the MeV coat, and helps to explain why global vaccine coverage has not led to the emergence of new serotypes. Also, the MeV Stealth here described may aid to close the gap of measles susceptibility in infants, allowing effective vaccination in the presence of transplacentally transferred maternal anti-mev antibodies during the first few months of life, and may further broaden the applicability and safety properties of MeV-based cancer therapies in the clinic. Graphical Abstract: 80

81 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 79 LIVE-ATTENUATED RESPIRATORY SYNCYTIAL VIRUS (RSV) VACCINE CANDIDATE WITH NS2 DELETION AND GENETICALLY STABILIZED TEMPERATURE SENSITIVITY MUTATION IS SAFE, IMMUNOGENIC, AND GENETICALLY STABLE IN RSV SERONEGATIVE CHILDREN Ursula Buchholz *, Cindy Luongo, Ruth Karron 2, Peter Collins RNA Viruses Section, Laboratory of Infectious Diseases, NIAID, NIH, Bethesda, MD, 2 Center for Immunization Research, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, United States Abstract: Respiratory syncytial virus (RSV) is the leading viral cause of pediatric respiratory disease, and there is no vaccine. Non-replicating RSV vaccines are contraindicated for RSV naïve recipients due to the risk of priming for enhanced disease upon subsequent infection with community-acquired RSV. Live intranasal vaccines do not prime for enhanced RSV disease, but development is challenging, because immunogenicity is thought to be inversely correlated with attenuation. We generated the RSV vaccine candidate NS2/ 33/I34L by reverse genetics, combining a deletion of the RSV interferon antagonist NS2 with the genetically stabilized temperature-sensitivity mutation 33/I34L in the L ORF, increasing vaccine safety by limiting replication at higher temperatures of the lower respiratory tract. In a Phase study in 6-24 month-old RSV-seronegative children, NS2/ 33/I34L was poorly infectious at a low dose of 0 5 PFU [0 vaccinees (V)/5 placebo recipients (P)]. At a dose of 0 6 PFU (20V/0P), 80% and 90% of recipients shed vaccine detected by culture and RT-qPCR, respectively (median peak titers.7 log0 PFU/mL; 3.6 log0 copies/ml), and 90% and 80% had 4-fold increases in RSV F IgG and neutralizing serum antibody titers, respectively. Antibody titers were durable over the following RSV season, and the vaccine primed for strong anamnestic responses to wild-type RSV. No mutations were detected in vaccine isolates, showing that the vaccine was genetically stable. These results put NS2/ 33/I34L into the window between previous over- and under-attenuated NS2 candidates, and on fast track for larger studies. 8

82 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 80 RESPONSE TO RE-EMERGENCE OF INFLUENZA A(H7N9) VIRUSES David Wentworth *, Guaniri Mateu-Petit, Li Wang, Xudong Lin, Adam Johnson, Terianne Wong, Jaber Hossain, Pavani Bondugula, Yunho Jang, Joyce Jones, Sharmi Thor, Genyan Yang, Han Di, Erin Hodges, Claudia Pappas, Nicole Brock, Xiangjie Sun, Terrence Tumpey, Taronna Maines, James Stevens, Bin Zhou, C. Todd Davis, Vivien Dugan Influenza Division, Centers for Disease Control and Prevention, Atlanta, United States Abstract: A zoonotic lineage of influenza A(H7N9) viruses first emerged in China in February 203 and subsequently caused annual epidemic waves resulting in,567 human infections (as of 26 Feb 208). A dramatic increase in zoonotic infections (n=766), geographic dissemination, and genetic diversification of the hemagglutinin (HA) was observed in the 5th wave (207/208). The HA evolution generated antigenic variants that escaped neutralization from antisera to existing vaccines and gave rise to a subclade of highly pathogenic viruses. Risk assessment indicated the 5th wave H7N9 viruses posed a considerable pandemic threat, therefore new candidate vaccine viruses (CVVs), against both low pathogenic viruses (i.e., A/Hong Kong/25/207-like) and high pathogenic viruses (i.e. A/Guangdong/7SF003/206-like), were recommended at the WHO influenza vaccine consultation meeting in March 207. Good laboratory practice and reverse genetics approaches were used to engineer multiple candidate vaccines designed to increase HA protein yield and/or breadth of host immune response. Extensive characterization, including antigenic analysis, genetic stability, and ferret pathogenicity studies were rapidly conducted. The IDCDC-RG56B CVV developed against the predominant 5th wave viruses (A/Hong Kong/25/207-like), was available to vaccine manufacturers worldwide in May 207. Another CVV (IDCDC-RG56N) that showed increased HA yield and induced cross-reactive antisera to both low- and high pathogenic viruses became available for distribution in November 207. Finally, human clinical trials to analyze the response to an inactivated vaccine generated from IDCDC-RG56B were initiated in February

83 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 8 HN VACCINE EFFECTIVENESS IN LIVE ATTENUATED INFLUENZA VACCINE IS DRIVEN BY VIRAL REPLICATIVE FITNESS IN THE HUMAN RESPIRATORY TRACT: SELECTION OF A NEW AND IMPROVED VACCINE CANDIDATE Oliver Dibben *, Amy Hawksworth, Jonathan Crowe, Robert Lockhart, Shaun Cooper, Lydia Ritter, Kasia Schewe, Laura Hill, Andrew Nyborg 2, Raburn Mallory 2, Helen Bright Flu-BPD, MEDIMMUNE, Liverpool, United Kingdom, 2 Clinical Development, MEDIMMUNE, Gaithersburg, United States Abstract: In the 203/4 and 205/6 influenza seasons, reduced vaccine effectiveness (VE) was observed for the pandemic 2009 HN (HNpdm09) component of the quadrivalent live attenuated influenza vaccine (Q/LAIV). This resulted in the hypothesis that HNpdm09 LAIV strains might possess a replicative defect, relative to clinically efficacious pre-2009, seasonal HN (pre-2009) strains. A broad-based investigation was launched in response to this concern. Initial observations comparing pre-2009 and HNpdm09 LAIV strains in single and multi-cycle infectivity assays indicated that HNpdm09 LAIV suffered from reduced multi-cycle infectivity. A fully differentiated primary human nasal epithelial cell model then confirmed that the replicative fitness of clinically suboptimal HNpdm09 LAIV strains was reduced. To assess the impact of this change in vivo, a novel ferret protection model was developed. Using this model, it was shown that the reduced fitness of suboptimal 205/6 HNpdm09 vaccine strain, A/Bolivia/559/203 (BOL3), led to a marked reduction in protective efficacy, relative to pre-2009 A/New Caledonia/20/999. Importantly, BOL3 also showed a pronounced reduction in efficacy in trivalent (T/LAIV) and Q/LAIV formulations when compared to monovalent vaccination. This suggested that incorporation of a poorly fit HNpdm09 strain into a T/LAIV or Q/LAIV vaccine could result in inter-strain interference, leading to reduced VE. Using these tools, a new HNpdmo09 LAIV strain, A/Slovenia/2903/205 (SLOV5), with improved replicative fitness and protective efficacy in ferrets, was developed. Finally, the benefits of these improvements were clinically validated by demonstrating significantly improved SLOV5 Q/LAIV shedding and immunogenicity relative to suboptimal BOL3 in US children. 83

84 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 82 LASSA VIRUS GLYCO-PROTEIN CONFORMATIONAL CHANGES A POSSIBLE ROLE FOR IMMUNE EVASION Aliza Katz *, Ron Diskin Structural Biology, WEIZMANN INSTITUTE, Rehovot, Israel Abstract: Lassa virus (LASV) of the Arenaviridae family is the causative of fatal hemohrahgic fever (FHF) in West Africa. These membrane coated viruses have a single surface protein complex which is referred to as the glyco-protein complex (GPC). The GPC is arranged as a trimer where each protomer is capped with the Glyco Protein (GP), a protein responsible for host cell receptor recognition and internalization. The GPC serves as an immunogene once encountered by the immune system. Obtaining a vaccine and treatment against this pathogen is a high priority goal due to its fatality and therefore also harboring the danger of it being exploited as a biological weapon. GP of LASV was purified and injected into mice to test immunogenicity along side with GP of Junin Virus (JUNV), another pathogenic Arenavirus. Both GPLASV and GPJUNV were immunogenic in mice yet only GPJUNV immunized sera can neutralize pseudo viruses and recognize GP in the integral GPC. The structure of GPLASV was solved by our lab and more recently the GPCLASV became available showing conformational changes between the GP component in the loose form and as part of a complex. Based on our results of the GP being immunogenic yet not neutralizing or recognizing the GP on the GPC, we suggest that the GPLASV serves as an immunological decoy to raise irrelevant antibodies. Interestingly, soluble GPLASV was detected in LASV patients supporting our hypothesis of loose GP serving as decoy. Elucidating the mechanism of LASV immune evasion will further help eradicate this deadly illness. 84

85 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: 83 MEASLES WILD TYPE AND VACCINE VIRUSES: REPLICATION AND IMMUNE RESPONSES IN RHESUS MACAQUES Diane E. Griffin *, Wen-Hsuan W. Lin, Robert J. Adams 2 Molecular Microbiology and Immunology, JOHNS HOPKINS BLOOMBERG SCHOOL OF PUBLIC HEALTH, 2 comparative medicine, Johns Hopkins University School of Medicine, Baltimore, United States Abstract: Measles wild type and vaccine viruses: Replication and immune responses in rhesus macaques Wen-Hsuan W. Lin, Robert Adams and Diane E. Griffin W. Harry Feinstone Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, MD 2205 Infection with wild type measles virus (wt-mev) is an important cause of childhood mortality and also induces life-long protective immunity. Despite the antigenic similarities between wt-mev and live-attenuated measles virus vaccine (LAMV), the protective immunity after measles vaccination is not as robust or long-lived. To identify mechanisms that may contribute to the longevity of immune responses induced by wt-mev infection, we compared virus replication and immune responses after respiratory infection with wt-mev or LAMV in rhesus macaques. Analysis of viral RNA in various tissues revealed distinct in vivo tropisms between the two strains of virus. Wt-MeV caused efficient hematogenous spread and prolonged presence of viral RNA, while LAMV caused transient localized infection in the respiratory tract. Type I interferon was not induced by either wt-mev or LAMV, but early after infection with wt-mev plasma levels of cytokines IL-2 and MIF and chemokines CCL2, CCL, CCL22 were elevated. Infection with wt-mev and LAMV induced comparable levels of MeVspecific IFN-g-producing T cells, but significantly higher levels of neutralizing and anti-h antibody and more long-lived plasma cells were observed after wt-mev infection. Therefore, distinct patterns of in vivo virus replication, efficiency of hematogenous spread and persistence of viral RNA are associated with the altered virulence and the level and longevity of the humoral response. 85

86 EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 84 MEGATAXONOMY OF NEGATIVE-SENSE RNA VIRUSES: PHYLUM _NEGARNAVIRICOTA_ Jens H. Kuhn * NIH/NIAID/DCR, INTEGRATED RESEARCH FACILITY AT FORT DETRICK, Frederick, United States Abstract: (-)ssrna viruses, informally grouped in Baltimore class V, are currently classified in the orders Bunyavirales and Mononegavirales, the unassigned families Arenaviridae, Ophioviridae, and Orthomyxoviridae, and the unassigned genus Deltavirus. Recent metagenomic studies revealed the existence of hundreds of novel (-)ssrna viruses in highly divergent organisms. These viruses either cluster with the existing taxa (e.g., bunyaviruses in citrus, melon, and kinetoplastids; hantaviruses in fish) or are highly divergent (e.g., chǔviruses, qínviruses, yuèviruses). With exception of hepatitis D virus (genus Deltavirus), all these viruses are homophyletically connected via their RNA-dependent RNA polymerases. This connection led to a proposal to the International Committee on Taxonomy of Viruses (ICTV) to replace the unofficial Baltimore class V with a phylum that unifies all current taxa and contains the necessary ranks for classification of all currently described (-)ssrna viruses excluding hepatitis D virus. I will outline the phylum as recently proposed, present foreseeable and imminent changes to the phylum based on novel discoveries, and discuss current taxonomic challenges. I will explain the taxonomic process from official proposal writing via Study Group deliberations to ICTV Executive Committee decisions. Finally, I will call on the entire (-)ssrna virology community to become engaged in this process to ensure that classification and nomenclature of all (-)ssrna viruses is done swiftly, as accurately as possible on an annual basis, and with maximum input from all experts in a transparent manner. 86

87 NSV 208, Verona Abstract Book EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 85 ISOLATION, CHARACTERIZATION, AND RAPID ADAPTATION OF TICK-BORNE ARENAVIRUS Hector Moreno Borrego *, Alberto Rastrojo Lastras 2, Katherine Sayler 3, Stefan Kunz Microbiologie, Centre Hospitalier Universitaire Vaudoise (CHUV), Lausanne (Switzerland), Switzerland, 2 Department of Virology, Centro de Biología Molecular Severo Ochoa, CBMSO-CSIC, madrid, Spain, 3 Department of Wildlife Ecology and Conservation, UF/IFAS, University of Florida UF/IFAS, Gainesville, United States Abstract: The New World Arenaviruses are diverse family of emerging negative strand RNA viruses comprised of Clades A, B, C, and D (former rec A/B). North American Clade D viruses, including Whitewater Arroyo (WWAV), Tamiami (TAMV), Bear Canyon, Skinner Tank, and Catarina virus, are carried in nature by members of the Sigmodontinae and Neotominae subfamilies of Cricetidae rodents. TAMV was isolated in 970 from the cotton rat Sigmodon hispidus in Florida and clusters phylogenetically closely with WWAV, which has been associated with human disease. Recent field studies resulted in the first isolation of the New World Arenavirus Tacaribe (TCRV), from host-seeking Amblyomma americanum ticks in Florida. Using next generation sequencing (NGS), we detected genetic traces of TAMV (TAMV-FL) in these tick-derived isolates. The ability of TAMV to prevent super-infection of TCRV but not vice versa, allowed enrichment of TAMV with concomitant extinction of TCRV by serial passaging in human cells, evidenced by NGS and immunofluorescence. The new tick-borne TAMV-FL isolate shares only 85% of homology with available TAMV reference sequence, suggesting strong selection and adaptation, and excluding the possibility of a laboratory contamination. During serial passaging, we further observed rapid de novo selection of two mutations (T569A and G582A) located in the putative interaction region between the TAMV envelope glycoprotein and its cellular receptor, transferrin receptor, suggesting rapid human adaptation. In summary, this study provides first evidence for a more dynamic host range of TAMV that may include ticks as transmission hosts and reveals the capacity for rapid human adaptation. Graphical Abstract: 87

88 EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 86 CHARACTERISATION OF A NOVEL PARAMYXOVIRUS ISOLATED FROM PTEROPID BAT URINE Rebecca Johnson *, 2, Mary Tachedjian, Hans Netter 3, Glenn A. Marsh CSIRO Health and Biosecurity, Australian Animal Health Laboratory, Geelong, 2 Department of Microbiology, Faculty of Medicine, Nursing and Health Sciences, Monash University, Clayton, 3 Victorian Infectious Diseases Reference Laboratory, Melbourne Health, Melbourne, Australia Abstract: The role of bats as reservoir hosts for zoonotic pathogens makes the discovery of novel bat-borne viruses necessary to prepare for emerging infectious diseases. Characterisation of these viruses improves our understanding of their zoonotic potential and prevents virus spillover from having a significant public health and economic impact. Previous surveillance of Australian fruit bats has resulted in the identification of multiple paramyxoviruses, therefore pteropid bat urine was analysed for the presence of novel viruses. This led to the isolation of Alston paramyxovirus (AlsPV), which allowed further characterization of the virus to better understand its potential to cause disease. Whole genome sequencing showed that AlsPV belongs to the genus Rubulavirus and is closely related to Parainfluenza virus 5, the causative agent of respiratory disease in dogs. Experimental infection of ferrets and mice resulted in the shedding of infectious virus in respiratory secretions of ferrets between days 3-0 with a rise in neutralizing antibodies detected after day 0. AlsPV could be isolated from upper respiratory tract tissues and viral RNA could be detected in the brains of all infected ferrets between days 3-0 as well as the brains of two mice at day 2 post infection in the absence of clinical symptoms. The isolation of AlsPV provides a greater understanding of the viral diversity present in Australian bats. These results show that AlsPV replicates in ferrets at sites relevant to virus transmission in addition to infecting the central nervous system. AlsPV has the potential to cause zoonotic infection, indicating the need to learn more about the transmission of this virus and highlighting the importance of the surveillance of bats as a source of emerging viruses. 88

89 NSV 208, Verona Abstract Book EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 87 INVESTIGATION OF THE BIOLOGICAL ROLE OF AN ENDOGENOUS BORNAVIRUS-LIKE ELEMENT IN MINIOPTERID BAT GENOMES Mukai Yahiro *, 2, Horie Masayuki, 3, Yuki Koboyashi 4, Kojima Shohei, 2, Maeda Ken 5, Tomonaga Keizo, 2 Institute for Frontier Life and Medical Sciences, Kyoto University, 2 Graduate School of Biostudies, Kyoto University, 3 Hakubi Center for Advanced Research, Kyoto University, Kyoto, 4 Nihon University Veterinary Research Center, Kanagawa, 5 Joint Faculty of Veterinary Medicine, Yamaguchi University, Yamaguchi, Japan Abstract: Endogenous bornavirus-like N elements (EBLNs) are DNA sequences derived from the N genes of ancient bornaviruses present in diverse mammals. Interestingly, some EBLNs have been co-opted to encode functional proteins by their hosts. However, the biological significance of EBLNs, especially in non-model organisms, are largely unknown. In this study, we searched EBLNs in bats that potentially encode proteins, and investigated their biological functions. We found that an EBLN in the natal long-fingered bat (Miniopterus natalensis), named miebln-, retained an intact ORF that negative selection has operated. We further showed that miebln- is expressed as an mrna in many tissues. These strongly indicate that miebln- encode a functional protein in the bats. To understand its function, we first evaluated the anti-bornaviral effect of miebln- because an EBLN in the squirrels inhibits BoDV replication. We revealed that miebln- inhibits neither the activity of BoDV polymerase nor BoDV infection. We next performed proteomic analyses to identify interaction partners of miebln-. We found that miebln- interacts with several RNA-binding proteins, such as MOV0 and IGF2BP3 in an RNA-dependent manner. We are currently investigating the biological significance of these interactions. The bornaviral N protein binds to viral RNA to form the nucleocapsid. Our data suggest that miniopterid bats had acquired a bornaviral N gene and may have utilized its RNA-binding property for their adaptation. Thus, this study would propose a novel concept that mammals can acquire riboviral genes and utilize their original properties for cellular functions. 89

90 DAMAGING AND SPREADING pathogenesis Abstract final identifier: 88 NIPAH VIRUS W PROTEIN MODULATES THE NF-ΚB SIGNALING PATHWAY BY TARGETING A HOST SCAFFOLD PROTEIN François Enchery, Claire Dumont, Aline Linder, Noemie Aurine, Louis-Marie Bloyet, Cyrille Mathieu 2, Denis Gerlier, Chloe Journo, Branka Horvat * Interanational center for Infectiology Research-CIRI, Lyon, France, 2 Center for Host-Pathogen Interaction, Department of Pediatrics, Columbia University Medical Center, New York, United States Abstract: Nipah virus (NiV) is a highly pathogenic zoonotic paramyxovirus responsible for regular outbreaks in South-East Asia and associated with high mortality and human-to-human transmission. NiV produces three nonstructural proteins, C, V and W, known to function as virulence factors. NiV-W was shown to inhibit the chemokine response in vitro and to modulate the inflammatory reaction in vivo, however, the mechanism of its action remains unclear. Here, we report that the NiV-W protein represses IL-β and TNFα-induced activation of the canonical NF-κB signaling pathway, known as the principal regulator of inflammation. This function requires the C-terminal domain of W, and is dependent on the nuclear accumulation of W. In contrast to wild-type NiV, inhibition of the NF-κB pathway was found to be impaired upon infection with a recombinant W-deficient NiV. Moreover, using a mass spectrometry approach, we identified an intracellular partner of NiV-W, which functions as a cellular scaffold protein playing an important role in intracellular signaling, including in the negative feedback regulation of NF-κB. We further identified a specific mutation within NiV-W that leads to the loss of interaction with this intracellular partner, and, as a consequence, of the capacity of W to inhibit NF-κB. This mechanism gives an additional insight into NiV-W-induced inhibition of chemokine production and leukocyte recruitment. Altogether, our results suggest that the W protein regulates the NF-κB pathway and consequently modulates the NiV-induced inflammatory response, paving the way toward a better understanding of the immunopathogenesis of this severe viral infection. 90

91 NSV 208, Verona Abstract Book EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 89 AVIAN INFLUENZA VIRUS M SEGMENT GENE EXPRESSION IS DYSREGULATED IN MAMMALIAN CELLS, LEADING TO A BLOCK IN AUTOPHAGY AND RESTRICTED VIRAL GROWTH John Steel *, Shamika Danzy, Ketaki Ganti, Brenda M. Calderon, Anice C. Lowen Microbiology and Immunology, EMORY UNIVERSITY, Atlanta, United States Abstract: Influenza A virus (IAV) M segment encodes matrix and M2 proton channel proteins from alternatively spliced mrnas, but the contribution of these products to host adaptation remain unclear. To investigate, we generated isogenic viruses that encode avian-, swine, or human-derived M segments. In a guinea pig model, avian M-encoding viruses exhibited poorer infectivity, lower titers, slower growth and less efficient transmission than viruses possessing a mammalian M segment. Avian and mammalian cell culture data revealed growth restriction of viruses with avian M segments specifically in mammalian cells. As decreased replication correlated with increased expression of M2 relative to M, we determined whether altered fitness was due to over-expression of M2, or to amino acid differences that exist between human- and avian-adapted M or M2 proteins, using viruses encoding chimeric M segments. We separated synonymous and nonsynonymous changes to produce viruses encoding human M and/or M2 proteins on avian vrna background, or avian M and/or M2 proteins on human vrna background. Growth of viruses in mammalian cells was inversely correlated with M2 expression, irrespective of amino acid composition. Analysis of LC3B lipidation and visualization of autophagosomes in infected cells further suggested that high levels of M2 lead to autophagic block. Our data suggest that adaptive change(s) in the M segment are needed to maintain low expression of M2 in mammalian cells and that, in the absence of such changes, excess M2 limits viral growth by blocking autophagy. These results point to novel roles for regulation of viral gene expression and interaction with cellular autophagy components in the host species range of IAV. 9

92 EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 90 ASSESSMENT OF THE ZOONOTIC POTENTIAL OF THE BAT INFLUENZA A VIRUS H8N Kevin Ciminski *, Wei Ran, Jinhwa Lee 2, Marco Gorka 3, Ebrahim Hassan, Anne Pohlmann 3, Donata Hoffmann 3, Wenjun Ma 2, Tony Schountz 4, Martin Beer 3, Martin Schwemmle Institute of Virology, University Medical Center Freiburg, Freiburg, Germany, 2 Department of Diagnostic Medicine/Pathobiology, Kansas State University, Manhattan, United States, 3 Institute of Diagnostic Virology, Friedrich- Loeffler-Institut, Greifswald-Insel Riems, Germany, 4 Arthropod-borne and Infectious Diseases Laboratory, Colorado State University, Fort Collins, United States Abstract: Recently two novel influenza A genomic sequences designated as H7N0 and H8N were found in New World fruit bats. Although these bat derived influenza A viruses (IAVs) overall resemble classical IAVs, their hemagglutinin (HA) and neuraminidase (NA) surface glycoprotein analogs are strikingly different. Here, we show that H8N productively replicates in the Neotropical Jamaican fruit bat (Artibeus jamaicensis) and is transmitted to naïve contact bats, thereby causing transient flu-like symptoms. Sequencing of isolated H8N from infected donor and contact bats, confirmed predominant presence of HA and NA wild-type sequences. However, following serial in vitro passaging in non-bat cells we only identified virus variants with enhanced viral growth that harbored mutations in HA and were characterized by a loss of the NA ectodomain. In mice, replication of H8N was exclusively confined to the upper airways and resulted similarly in the selection of mutant variants with a truncation in NA and various mutations in HA. We next generated a H8N variant (rp) that was selected upon passaging in vitro, harboring two amino acid mutations in HA (K70R and N250S) plus a premature stop codon in NA (G07X) and infected ferrets to assess the zoonotic potential of this mutant virus. Only limited replication in the upper respiratory tract and lung as well as no transmission to naïve contact ferrets was observed. These findings suggest that although bat influenza A viruses can increase their replication properties by losing the NA head domain, they are only poorly adapted to non-bat mammalian species. 92

93 NSV 208, Verona Abstract Book EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 9 SELECTIVE PRESSURE ON RESPIRATORY SYNCYTIAL VIRUS BEARING CODON-PAIR DEOPTIMIZED F AND G ORFS GENERATES INTERNAL-DELETION GENOMES THAT PARADOXICALLY RESCUE VIRAL REPLICATION Cyril Le Nouen *, Thomas McCarty, Michael Brown 2, Lijuan Yang, Cindy L. Luongo, Peter L. Collins, Ursula J. Buchholz RNA Viruses Section, NIH/NIAID/LID, Bethesda, 2 Pacific Biosciences inc., Menlo Park, United States Abstract: Recoding viral genomes by introducing numerous synonymous but suboptimal substitutions provide new types of live-attenuated vaccine candidates. These should have a low risk of de-attenuation because of the many changes involved. However, their genetic stability under selective pressure is largely unknown. For viruses intended for vaccine use, it is essential to evaluate in depth the stability of attenuation. Human respiratory syncytial virus (RSV) is the most important viral agent of pediatric respiratory disease. A vaccine is not available yet. Codon-pair deoptimized (CPD) versions of RSV are attenuated and temperature sensitive (ts). We subjected the ts CPD RSV Min B that contains 69 synonymous mutations in the F and G surface glycoproteins ORFs to serial passage in vitro at progressively increasing temperature. During passage, Min B quickly exhibited a restoration of replication fitness and syncytia formation, the latter a hallmark of RSV F expression. Comprehensive sequence analysis of virus populations by whole-genome deep sequencing, coupled with long-range PCR, revealed the presence of large-deletion (LD) viral genomes that accumulated after only a few passages. The shortest LD viral genomes were about 5 kb (one third of the RSV genome) and encoded the CPD F gene in first genome promoter proximal position. Two representative LD viral genomes were chemically synthetized de novo. Phenotypic analysis revealed that both expressed high levels of F proteins and complemented in trans Min B replication. Thus, the LD viral genomes are a new type of internal-deletion defective genome that enhanced rather than interfered with the replication of Min B. This study provides new insights on the adaptability of genome-scale recoded RNA viruses and describes a novel mechanism of adaptation of RNA viruses. 93

94 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: 92 A NEW OPTICALLY CONTROLLABLE MEASLES VIRUS VECTOR Maino Tahara *, Moritoshi Sato 2, Yuichiro Nakatsu, Kenzaburo Tani 3, Makoto Takeda Department of Virology III,, National Institute of Infectious Diseases, Musashimurayama, 2 Graduate School of Arts and Sciences, 3 The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Abstract: Measles virus (MeV) vector can transfer multiple genes into human cells efficiently without affecting the host genome. We recently have developed novel MeV gene transfer vector which is non-transmissible, can transfer multiple genes simultaneously. We could successfully generate induced pluripotent stem cells from human fibroblasts or peripheral blood T cells using this vector. Although MeV as gene transfer vector is clearly beneficial, no method has been developed to control viral growth. We set out to design a method of controlling viral growth. Now, we developed optically controllable MeV. This MeV replicated efficiently when cells were illuminated by blue light. When infected cells were kept in dark, MeV hardly replicated. We believe our new optically controllable MeV vector has the potential to revolutionize gene transfer technologies. 94

95 NSV 208, Verona Abstract Book EMERGING AND EVOLVING viral discovery, emergence and evolution Abstract final identifier: 93 LOSS OF RNAI WAS REQUIRED FOR THE INVENTION OF NEGATIVE-STRANDED RNA VIRUSES Benjamin Tenoever * Microbiology, ICAHN SCHOOL OF MEDICINE, New York, United States Abstract: Viruses are thought to be living relics of the ancient world yet there is an unequal distribution of viral groups amongst domains and kingdoms. DNA viruses dominate the early prokaryotic world whereas (+)RNA viruses rose to prominence early after eukaryogenesis. Diversification of (+)RNA viruses resulted in the invention of (-)RNA viruses sometime following the appearance of multicellular life. As the emergence of these viral groups follow a trajectory of changing cellular antiviral systems, we set out to determine if this was responsible for the late evolutionary appearance of (-)RNA viruses. Here we define the capacity of different RNA viruses to cope with RNAi, the defense of early eukaryotes, and monitored their propensity to evade this system. Serial passage of viral populations revealed that, while (+)RNA viruses could rapidly excise genomic material and evade targeting, (-)RNA viruses were neutralized. These data suggest that (- )RNA viruses required a multicellular eukaryotic host that was deficient in RNAi and explains their delayed evolutionary invention and increased prevalence in vertebrates. Graphical Abstract: 95

96 POSTER PRESENTATIONS BREAKING AND ENTERING - viral entry Abstract final identifier: P00 INSIGHT INTO THE FUSION MECHANISM GLEANED FROM AN EBOLA VIRUS GLYCOPROTEIN MUTANT THAT DOMINATED THE PANDEMIC WE Diehl, D Mu, L Odongo 2, K Szymanska 2, M Cabot 2, L Feneant 2, B Ganser-Pornillos 3, J M. White 2, Jeremy Luban * Mol Med, UMass Med, Worcester, 2 Cell Biology, 3 Mol Phy & Biol Phys, Univ of Virginia, Charlottesville, United States Abstract: GP-A82V, a clade-defining mutation from the pandemic, enhances virion infectivity (Cell 67:088). Though located near the NPC receptor (PDB:5FB), GP-A82V points away from NPC. Attempting to find explanation for increased infectivity we noticed that GP-A82V abuts GP-R85, and that GP-R85 is tightly coordinated within a charged pocket. We hypothesized that EBOV fusion to the target cell membrane requires a conformational switch in which GP-R85 is expelled from the charged pocket, permitting it to contact NPC-D502, a residue required for infectivity. According to this model, GP-A82V would enhance infectivity by destabilizing interactions that retain GP-R85 within the charged pocket. To test our model, 55 mutants were engineered in the GP pocket, 8 of which enhanced infectivity of pseudotyped lentiviral vectors, as least as well as did GP-A82V. Each of the 8 mutants is predicted to destabilize GP-R85 charge interactions, and all increased resistance to inhibitors of EBOV entry, including a cathepsin inhibitor, an NPC inhibitor, and a neutralizing antibody. While none of these mutations altered EBOV GP-NPC binding affinity, they were better than WT GP at entering cells bearing NPC-D502 mutants with lowered affinity for GP. These data are consistent with a model in which the charged pocket regulates a GP-R85 conformational switch required for fusion and with GP-A82V destabilizing the pocket. Ongoing attempts to to solve structures of the GP mutants are hoped to provide further evidence in support of our model. 96

97 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P002 THE HEMAGGLUTININ-NEURAMINIDASE (HN) HEAD DOMAIN AND THE FUSION (F) PROTEIN STALK DOMAIN OF THE PARAINFLUENZA VIRUSES AFFECT THE SPECIFICITY OF THE HN-F INTERACTION Masato Tsurudome *, Morihiro Ito, Machiko Nishio 2 Medical Sciences, Chubu University, Kasugai, 2 Microbiology, Wakayama Medical University, Wakayama, Japan Abstract: Membrane fusion by the parainfluenza viruses is induced by virus-specific functional interaction between the attachment protein (HN) and the fusion (F) protein. This interaction is thought to be mediated by transient contacts between particular amino acids in the HN stalk domain and those in the F head domain. However, we recently found by chimeric analyses of the HN proteins of human parainfluenza virus 2 (HPIV2) and simian virus 4 (SV4) that replacement of specified amino acids at or around the dimer interface of the HN head domain significantly affected the F protein specificity. We then intended to further investigate this issue in the present study, revealing that the HPIV2 HN protein can be converted to an SV4 HN-like protein by substituting at least three amino acids in the HPIV2 HN head domain with the SV4 HN counterparts in addition to the replacement of the stalk domain. Concomitantly, we also found that the PIV5 F protein can be converted to an SV4 F-like protein by substituting 5 amino acids in the PIV5 F stalk domain as well as the 2 amino acids in the head domain with those of the SV4 F protein. These results indicate that mutations of specified amino acids in the HN head domain and the F stalk domain can modify the specificity of the HN-F interaction, suggesting that the conformations of the HN stalk domain and the F head domain are dependent on the structures of the HN head domain and the F stalk domain, respectively. 97

98 BREAKING AND ENTERING - viral entry Abstract final identifier: P003 STRUCTURE-GUIDED IDENTIFICATION OF MORBILLIVIRUSES WITH ZOONOTIC POTENTIAL Nurshariza Abdullah, Stephen C. Graham 2, Jamie Birch, Jamie Kelly, Daniel Gonçalves-Carneiro, Tim J. Mitchell 3, Robin N. Thompson 4, Katrina A. Lythgoe 4, Nicola Logan 5, Margaret J. Hosie 5, Vassiliy N. Bavro 6, Brian J. Willett 5, Michael P. Heaton 7, Dalan Bailey * Virus Programme, The Pirbright Institute, Guildford, 2 Department of Pathology, University of Cambridge, Cambridge, 3 Microbiology and Infection, University of Birmingham, Birmingham, 4 Zoology, University of Oxford, Oxford, 5 Centre for Virus Research, University of Glasgow, Glasgow, 6 Microbiology, University of Essex, Essex, United Kingdom, 7 Genetics, Breeding, and Animal Health Research Unit, US Department of Agriculture, NE, United States Abstract: Morbilliviruses infect a broad range of mammalian hosts including ruminants, carnivores and humans. The recent eradication of rinderpest virus (RPV), as well as active campaigns for measles virus (MeV), have raised significant concerns that other morbilliviruses may emerge in so-called vacated ecological niches. Seeking to assess the zoonotic potential of non-human morbilliviruses we identified that peste des petits ruminants virus (PPRV) - the small ruminant morbillivirus - is restricted at the point of entry into human cells due to deficient interactions with human SLAMF the immune cell receptor. Using a structure-guided approach, as well as quantitative receptor usage assays, we characterised a single gain-of-function amino acid change, mapping to the receptor-binding domain (RBD) in the PPRV Haemagglutinin (H) protein, which overcomes this restriction. The same mutation allowed escape from cross-protective, human-patient, anti-mev antibodies, raising concerns that PPRV is a pathogen with zoonotic potential. Through structural modelling the mechanistic nature of the PPRV restriction was identified as charge incompatibility and steric hindrance at the protein-protein interaction interface. Significantly, this research was performed entirely using surrogate virus entry assays, negating the requirement for in situ derivation of a human-tropic PPRV. 98

99 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P004 NATURAL GLYCAN RECEPTORS FOR INFLUENZA VIRUS IN HUMAN LUNG Lauren Byrd-Leotis *, 2, Nan Jia 2, Chao Gao 2, Jamie Heimburg-Molinaro 2, Richard Cummings 2, David Steinhauer Department of Microbiology and Immunology, Emory School of Medicine, Atlanta, 2 Department of Surgery, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, United States Abstract: Influenza A viruses bind sialylated N-glycans, with species specificity correlating to the sialic acid linkage conformation. Avian viruses recognize α2,3-linked sialic acid and human and swine viruses prefer α2,6. These observations derive from binding studies on erythrocyte agglutination, synthetic receptor analogs, or undefined surface markers on cells or tissues, and the preferences are predicted to reflect the availability of sialic acid linkage at the site of infection as assayed by lectin binding. Here we present the first examination of the N-glycome of the human lung for identifying the natural receptors for avian and mammalian influenza viruses. We found that the viruses exhibit differential binding profiles, binding to a wide variety of receptors or to a select number. In order to more fully characterize the glycan recognition, we focused on A/Pennsylvania/08/2008 and found that fractions containing bi-antennary sialylated glycans +/- core fucose comprised the highest binding structures. We found many α2,3- and α2,6-linked sialylated glycans bound by virus, but remarkably, all viruses also displayed sialic acid independent binding to phosphorylated, non-sialylated glycans. Our results also suggest that the standard lectin for identifying α2,3-linked sialic acid, MAL-I, can bind to terminal galactose, and therefore MAL-I alone may not be a useful indicator of sialic acid linkage at the site of infection. Moreover, we extended our analysis to include human H3N2 seasonal viruses isolated during the past decades that have progressively lost erythrocyte agglutination capabilities and discovered that these strains also lost binding to sialylated receptors during antigenic drift, while maintaining strong binding to phosphorylated glycans. 99

100 BREAKING AND ENTERING - viral entry Abstract final identifier: P005 THE CRITICAL ROLE OF HEMAGGLUTININ (HA) IN TRANSMISSION OF DUCK-ORIGIN H5NX LOW-PATHOGENIC AVIAN INFLUENZA VIRUSES IN CHICKENS Gert Zimmer *, Daniel Brechbühl, Nicolas Ruggli, Samira Locher INSTITUTE OF VIROLOGY AND IMMUNOLOGY (IVI), Mittelhäusern, Switzerland Abstract: In wild waterfowl avian influenza viruses (AIV) primarily replicate in the gastrointestinal tract and are shed into the environment at large quantities facilitating transmission to domestic poultry. Continuous circulation of H5 and H7 AIVs in domestic poultry may favour the evolution of highly pathogenic AIVs (HPAIV). To understand the adaptation process of AIV in poultry, we experimentally infected chickens via the intratracheal route with a number of low-pathogenic AIV (LPAIV) H5 and H7 isolates from wild waterfowl. While most H7 LPAIV were transmitted to contact animals without prior adaptation, none of the H5 LPAIV was passed to the sentinels. Interestingly, H5N and H5N8 HPAIV isolates were not transmitted to sentinel chickens if the HA proteolytic cleavage site was changed from a polybasic to a monobasic motif. Genetic reassortment of a non-transmittable H5N virus with either a transmittable H7N7 virus or a chicken-adapted H5N2 virus revealed that the hemagglutinin (HA) is critical for efficient virus shedding and transmission. Mutations in HA changing the ph threshold of fusion, the proteolytic cleavage site or potential glycosylation sites did not enhance virus transmission. However, recombinant H5N harboring the globular HA head domain from a chicken-adapted H5N2 was efficiently transmitted. Mutational analysis demonstrated that adaptations in the receptor-binding pocket (30-loop, 90-helix, and 220-loop) of HA are critical for H5 virus transmission in chickens. These findings suggest that duck-origin H5 viruses exhibit different receptor-binding activities than chicken-adapted viruses. Whether these adaptive mutations have an impact on receptor specificity or receptor affinity is currently under investigation. 00

101 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P006 TWO POINT MUTATIONS IN THE HANTAAN VIRUS GLYCOPROTEIN COMPLEX AFFORD THE GENERATION OF A HIGHLY INFECTIOUS REPLICATION-COMPETENT RECOMBINANT VSV Megan Slough *, Kartik Chandran, Rohit K. Jangra Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, United States Abstract: Hantaviruses are highly prevalent rodent-borne zoonotic viruses. Currently, no FDA-approved, specific antiviral drugs or vaccines are available, and the requirement for high biocontainment laboratories limits hantavirus research. Recombinant vesicular stomatitis viruses (rvsvs) bearing Gn/Gc glycoproteins from New World hantaviruses have proven valuable as tools to investigate hantavirus entry under BSL-2 containment. However, no Old World rvsvs have been described. In our initial experiments, rvsvs bearing New World hantavirus Gn/Gc were readily rescued from cdnas, but their counterparts bearing Old World, Hantaan virus (HTNV), were refractory to rescue and only grew to low titers. However, serial passage of the rescued rvsv-htnv Gn/Gc in Vero cells markedly increased viral infectivity and spread of infection. This gain in viral fitness correlated with the acquisition of two point mutations; I532K in the cytoplasmic tail of Gn, and S094L in the stem region of Gc. Re-rescue of rvsv-htnv Gn/Gc, as well as VSV pseudotyping by trans-complementation, verified the mutations were determinative, and contribute to viral infectivity in a synergistic manner. Mechanistic studies revealed the mutations only modestly enhanced protein production and did not alter Gn and Gc co-localization. Rather, they re-localized HTNV Gn/Gc to the cell surface and significantly enhanced Gn/Gc incorporation into budding VSV particles. Our results suggest an enhancement of cell surface expression of other viral glycoprotein(s), by cognate mutations, could enable the generation of other hard-to-rescue rvsvs. The robust replication-competent rvsv-htnv Gn/Gc reported herein may have utility as a vaccine candidate. 0

102 BREAKING AND ENTERING - viral entry Abstract final identifier: P007 HANTAVIRUS ENTRY INTO HUMAN RESPIRATORY EPITHELIAL CELLS INVOLVES MACROPINOCYTOSIS Giulia Torriani *, Jennifer Mayor, 2, Gert Zimmer 3, Nicole Tischler 4, Stefan Kunz, Sylvia Rothenberger, 2, Olivier Engler 2 Institute of Microbiology, CHUV/UNIL, Lausanne, 2 Spiez Laboratory, Spiez, 3 Institute of Virology and Immunology, Mittelhäusern, Switzerland, 4 Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile Abstract: Hantaviruses are emerging rodent-borne viruses of the Bunyaviridae family associated with severe human diseases with high mortality. The current lack of a licensed vaccine and the limited therapeutic options make the development of novel efficacious anti-viral agents to combat hantaviruses an urgent need. The identification of cellular factors hijacked by hantaviruses in order to enter host cells is a promising approach for the development of novel strategies to combat pathogenic hantaviruses. Considering the biosafety restrictions linked to work with live pathogenic hantaviruses and that virus cell attachment and entry are mediated exclusively by the viral envelope, we established a pseudotype platform for Hantaan virus (HTNV) and Andes virus (ANDV). In a first approach, we screened a library of 90 small molecule kinase inhibitors in our hantavirus pseudotype platform using a semi-high-throughput assay. Our screen identified specific sets of cellular kinases required for cell entry of HTNV and ANDV that only partially overlapped, indicating important virusspecific differences. We complemented our unbiased screen with a targeted approach including a panel of well-defined diagnostic inhibitors against cellular factors involved in endocytosis. Entry of both, HTNV and ANDV critically depended on sodium-proton exchangers (NHE) and actin, which are major hallmarks of macropinocytosis. HTNV and ANDV further showed differential dependence on known regulatory factors of macropinocytosis.promising candidate anti-viral drugs are currently followed up and validated using live pathogenic hantaviruses in the new high containment facilities at Spiez Laboratory, Switzerland. 02

103 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P008 HANTAVIRUSES ENTER INTO HUMAN AIRWAY EPITHELIUM USING PHOSPHATIDYLSERINE RECEPTORS TIM- AND AXL Jennifer Mayor *, 2, Giulia Torriani, Gert Zimmer 3, Nicole Tischler 4, Stefan Kunz, Sylvia Rothenberger, 2, Olivier Engler 2 Institute of Microbiology, CHUV/UNIL, Lausanne, 2 Spiez laboratory, Spiez, 3 Institute of Virology and Immunology, Mittelhäusern, Switzerland, 4 Molecular Virology Laboratory, Fundación Ciencia & Vida, Santiago, Chile Abstract: Hantaviruses are emerging human pathogens leading to serious diseases and represent a challenge for public health. Currently, there are no effective antivirals or licensed vaccines against hantaviruses. Viral attachment and entry represent the first steps in virus transmission and are promising targets for anti-viral therapeutic intervention. We investigated the largely unknown receptor used in human airway epithelium of the Old World Hantaan virus (HTNV) and New World Andes virus (ANDV). We established and validated a hantavirus pseudotype system based on replication competent and propagation deficient recombinant vesicular stomatitis virus as a suitable BSL2 surrogate model to study hantavirus entry into the host cell. We hypothesized that hantaviruses may use apoptotic mimicry to invade human respiratory epithelial cells and examined the role of the major classes of cellular phosphatidylserine (PS) receptors, the T- cell immunoglobulin and mucin (TIM) molecules and receptor tyrosine kinases of the Tyro3/Axl/Mer (TAM) family. Using antibody perturbation and other techniques, we found that cell entry of ANDV critically depended on Axl and TIM-, HTNV depended mainly on TIM-.In line with previous studies, HTNV, but not ANDV was able to use the abundant glycosaminoglycan heparan sulfate as co-receptor. In sum, our studies demonstrate for the first time that hantaviruses use PS receptors and hence apoptotic mimicry to invade human airway epithelium, which explains why these viruses can easily break the species barrier. 03

104 BREAKING AND ENTERING - viral entry Abstract final identifier: P009 STRUCTURAL BASIS FOR DIFFERENTIAL EPHRIN-MEDIATED HOST-CELL ENTRY PATHWAYS OF HENIPAVIRUSES Rhys Pryce *, Kristopher Azarm 2, Robert P. Rambo 3, Benhur Lee 2, Thomas A. Bowden Division of Structural Biology, University of Oxford, Oxford, United Kingdom, 2 Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States, 3 Diamond Light Source, Harwell Science and Innovation Campus, Didcot, United Kingdom Abstract: The capability of a paramyxovirus to specifically attach to a human cell surface receptor is a key determinant of cross-species spillover. Here, using a multi-faceted macromolecular crystallography and X-ray foot-printing mass spectrometry approach, we dissect the molecular aspects of host cell attachment for the recently identified Cedar henipavirus (CedV). The CedV attachment glycoprotein (CedV-G) is one of only two antigens displayed on the viral envelope; responsible for viral attachment to cellular membranes it is the primary determinant of host species and cell type tropism. We show that CedV-G displays the canonical six-bladed β-propeller fold utilised by the closely related and highly pathogenic henipaviruses, Hendra (HeV) and Nipah (NiV). Furthermore, we identify the putative ephrin receptor binding site and demonstrate that it is both conserved and compatible with ephrin recognition. XF-MS analysis of CedV-G-ephrinB2 complexes supports a model for a largely conserved receptor binding mode across henipaviruses. These data provide a structural template for understanding the interplay between receptor tropism and pathogenicity within this genus of important human pathogens. 04

105 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P00 SINGLE NUCLEOTIDE POLYMORPHISMS IN HUMAN NIEMANN-PICK C INFLUENCE ENTRY OF FILOVIRUSES INTO CELLS Tatsunari Kondoh *, Michael Letko 2, Vincent Munster 2, Rashid Manzoor, Junki Maruyama, Wakako Furuyama, Hiroko Miyamoto, Asako Shigeno, Daisuke Fujikura, Yoshihiro Takadate, Reiko Yoshida, Manabu Igarashi, Heinz Feldmann 2, Andrea Marzi 2, Ayato Takada Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan, 2 Laboratory of Virology, Division of Intramural Research, NIAID, NIH, Rocky Mountain Laboratories, Hamilton, United States Abstract: Niemann-Pick C (NPC), a host cell receptor involved in the envelope glycoprotein (GP)-mediated entry of filoviruses into cells, has been believed to be a major determinant of cell susceptibility to filovirus infection. It is known that proteolytically digested Ebola virus (EBOV) GP interacts with two protruding loops in domain C of NPC. Using previously published structural data and the single nucleotide polymorphisms (SNPs) database, we identified ten naturally occurring, missense SNPs in the loop regions of human NPC. To investigate the potential effects of these SNPs on filovirus infection, we generated NPC knockout Vero E6 cell lines and newly introduced human NPC with SNP substitutions. Their susceptibility was then examined by using vesicular stomatitis viruses (VSVs) pseudotyped with filovirus GPs and infectious EBOV. Although none of the SNPs tested here completely abolished viral infectivity, some of the substitutions resulted in reduced susceptibility to filoviruses as indicated by the lower infectivity and smaller plaque/focus sizes of the viruses. In particular, substitutions at amino acid positions 424, 425, 502, and 508 (P424A, S425L, D502E, and D508N) in NPC reduced the entry of VSV pseudotyped with GPs from multiple filovirus species. Interestingly, P424A/D508N and S425L/D502E substitutions seemed to be important for the reduced entry of ebolavirus and marburgvirus, respectively. Taken together, we conclude that human NPC SNPs may likely affect host cell susceptibility to filoviruses. 05

106 BREAKING AND ENTERING - viral entry Abstract final identifier: P0 DUAL-REGULATORY ROLE OF THE MORBILLIVIRUS ATTACHMENT PROTEIN HEAD-TO-STALK LINKER MODULE IN MEMBRANE FUSION TRIGGERING Michael Herren *, Neeta Shrestha, Marianne Wyss, Andreas Zurbriggen, Philippe Plattet Division of Experimental and Clinical Research, University of Bern, Bern, Switzerland Abstract: Host cell entry by morbilliviruses (e.g. Measles virus (MeV) or Canine Distemper Virus (CDV)) is coordinated by two interacting envelope glycoproteins; a tetrameric attachment (H) protein and a trimeric fusion (F) protein. The ectodomain of H-tetramers consists of stalk, connector and head domains that adopt F-triggering-permissive native structures. The precise functional role of the C-terminal modules of the H stalk domain (termed linker and connector ), although hypothesized to assume flexible structures to sustain putative receptor-induced structural rearrangements, remains largely unexplored. To gain mechanistic insights, we conducted a thorough non-conservative mutagenesis-scan analysis of the MeV and CDV H-linker/connector domains. Our data provide evidence that substituting a hydrophobic residue encompassed within the linker module (H-I46) into amino acids without hydrophobic side chains translated into the assembly of covalent H-tetramers, which were fusion triggering-deficient despite proper intracellular trafficking, receptorbinding and F-binding bioactivities. Remarkably, we next demonstrated that covalent H-tetramerization per se was not the only mechanism preventing F-activation: the neutral glycine mutant (H-I46G), which exhibited strong covalent tetramerization propensity, retained limited fusion promotion activity, and, conversely, charged H-I46 mutants additionally carrying alanine-substitution of natural cysteines, therefore unable to form covalently-linked tetramers, were fusion activation-defective. In summary, our data suggest a dual-regulatory role of isoleucine 46 of the morbillivirus H-stalk linker module: it secures the formation of natural non-covalent dimer-of-dimers and contributes to receptor-induced F-triggering activity. 06

107 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P02 EBOLA VIRUS ENTRY AND EGRESS IN POLARIZED EPITHELIAL CELLS Manasi Ajit Tamhankar *, Dawn M. Gerhardt 2, Richard S. Bennett 2, Nicole Murphy 2, Peter B. Jahrling 2, Lisa E. Hensley 2, Jean L. Patterson Virology & Immunology, TEXAS BIOMEDICAL RESEARCH INSTITUTE, San Antonio, 2 Division of Clinical Research, Integrated Research Facility/ NIAID, Frederick, MD, United States Abstract: Currently, no FDA-approved vaccines or treatments are available for Ebola virus disease, and therapy remains largely supportive. Ebola virus (EBOV) has broad tissue tropism and can infect a variety of cells including epithelial cells. Epithelial cells differ from most other cell types by their polarized phenotype and barrier function. In polarized cells, the apical and basolateral membrane domains are demarcated by tight junctions. Polarized cells also have specialized sorting machinery, which results in a difference in composition of the two membrane domains. These specialized functions of sorting can have important consequences for viral infections. Differential localization of a viral receptor can restrict virus entry to a particular membrane while, polarized sorting can lead to a vectorial virus release. To elucidate the characteristics of EBOV entry and egress in polarized cells, we first characterized the polarized Caco-2 model on semipermeable transwells using measurement of transepithelial resistance and microscopy. Our data in Caco-2 cells indicate that EBOV preferentially infects from the basolateral route, and this preference may be influenced by the extent of polarity. Infection and egress occurs without changes in cellular permeability. Further, our data shows that polarized distribution of heparan sulfate, a known viral attachment factor, may be responsible for causing the basolateral preference shown by the virus during entry in Caco-2 cells. Treatment with iota-carrageenan, which blocks virus interaction with cellular heparan sulfate significantly reduced cell susceptibility to infection. Our results, thus, also show the potential of heparan sulfate as a therapeutic target during EBOV infection 07

108 BREAKING AND ENTERING - viral entry Abstract final identifier: P03 BIOCHEMICAL AND STRUCTURAL CHARACTERIZATION OF VSV (NEW JERSEY STRAIN) GLYCOPROTEIN Abbas Abou Hamdan, Laura Belot, Aurélie Albertini, Yves Gaudin * INSTITUT DE BIOLOGIE INTÉGRATIVE DE LA CELLULE, CNRS, Gif sur Yvette, France Abstract: Vesiculoviruses entry into host cells is mediated by the unique viral glycoprotein G, anchored in the viral membrane. First, G is involved in receptor recognition. Then, after endocytosis of the viral particle, G mediates the fusion between the viral and endosomal membranes. The fusion is triggered by a low ph-induced conformational change from a trimeric pre- to a trimeric post-fusion conformation. For the moment, only crystal structures of VSV Indiana and Chandipura virus glycoproteins have been determined. In this study, we have characterized the ectodomain of glycoprotein of VSV New Jersey strain, which shares 52% aminoacid sequence identity with that of VSV Indiana. The ectodomain was generated by thermolysin-limited proteolysis of viral particles and was shown to interact with CR2 and CR3 of LDL-R receptor. Using Electron Microscopy, we showed that this ectodomain undergoes a similar structural transition from a pre- to a post fusion form when the ph is lowered. At low ph, we observed rosette-like shapes in which the post-fusion trimers associate via their hydrophobic fusion loops. We performed different crystal conditions screen that allows us to obtain several crystal forms at different ph conditions probably corresponding to distinct structural conformations of the ectodomain. The resolution of those crystalline structures is under way. 08

109 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P04 INFLUENZA VIRUS ENTRY VIA INTERPLAY BETWEEN PLATELET-DERIVED GROWTH FACTOR RECEPTOR BETA AND GM3 GANGLIOSIDE: A VALID ROUTE FOR HOST-TARGETED ANTIVIRAL INTERVENTION Pieter Vrijens, Els Vanstreels, Sam Noppen, Seppe Cambier, Roberto Ronca 2, Paola Chiodelli 2, Manon Laporte, Evelien Vanderlinden, Sandra Liekens, Annelies Stevaert *, Lieve Naesens Rega Institute for Medical Research, KU Leuven - University of Leuven, Leuven, Belgium, 2 Experimental Oncology and Immunology, Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy Abstract: The possible resistance of influenza virus against the existing antiviral drugs calls for new therapeutic targets such as host cell factors associated with virus replication. We here evaluated a diverse panel of 276 protein kinase inhibitors in a multicycle antiviral assay in Madin-Darby canine kidney (MDCK) cells. The receptor tyrosine kinase (RTK) inhibitor Ki875 displayed robust anti-influenza A and B activity and was selected for mechanistic investigations. Ki875 efficiently blocked the post-binding internalization of influenza virus in different cell lines including four Chinese hamster ovary (CHO) cell lines with abundant expression of platelet-derived growth factor β (PDGFRβ). For two other RTKs, i.e. fibroblast growth factor receptor-4 and vascular endothelial growth factor receptor-2, no association with influenza entry was seen. The significantly higher virus uptake in CHO-K compared to CHO-wild type cells indicated stimulation by GM3 ganglioside yet suppression by GM ganglioside. This GM3/PDGFRβ-controlled virus internalization involved PDGFRβ phosphorylation combined with receptor desialylation by the viral neuraminidase. It coincided with strong activation of the Raf/MEK/Erk cascade, but not of PI3K/Akt or phospholipase C-γ. The PDGFRβ-Erk loop was fully suppressed by Ki875. We conclude that influenza virus efficiently hijacks the GM3-enhanced PDGFRβ signaling pathway to penetrate the cells, providing an opportunity for host cell-targeting antiviral intervention. 09

110 BREAKING AND ENTERING - viral entry Abstract final identifier: P05 VIRAL ENTRY AND REPLICATION OF BAT-DERIVED MUMPS VIRUS Nadine Krüger *, Sarah Hüttl, Kathleen Voigt 2, Georg Herrler 2, Claes Örvell 3, Markus Hoffmann 4 Division of Virology, Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, 2 Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany, 3 Division of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden, 4 Infection Biology Unit, Deutsches Primatenzentrum GmbH, Leibniz-Institut für Primatenforschung, Göttingen, Germany Abstract: Mumps is a highly contagious childhood disease with usually mild symptoms caused by mumps virus (MuV), a member of the family Paramyxoviridae. In rare events, mumps can result in complications like encephalitis or meningitis. In 202, the genome of a virus with close phylogenetic relatedness to human MuV was detected in African fruit bats (batmuv). However, in the absence of an infectious isolate, all recent efforts to characterize batmuv were based on directed expression of the fusion (F) and hemagglutinin-neuraminidase (HN) glycoproteins, or chimeric MuVs harboring batmuv F and HN. Although these studies provided initial insights in the functionality of the batmuv glycoproteins, important aspects such as the host range, replication competence or virulence of batmuv remained elusive. Here, we generated recombinant authentic batmuv and analyzed this virus regarding its ability to replicate in different mammalian cells and host cell factors that are required for viral entry. Our data show that batmuv can replicate in cells of human, non-human primate and bat origin. The replication of batmuv most likely occurs in cytoplasmic inclusion bodies the formation of which is mediated by the interaction of the nucleo- and phosphoprotein. The proteolytic cleavage of batmuv F by cellular furin, as well as the binding of batmuv HN to sialic acids expressed on the surface of target cells is a necessity for viral entry. Infection can be inhibited by cross-neutralization of antisera directed against human MuV strains. 0

111 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P06 A GENOME-WIDE GENETIC SCREEN UNCOVERS A ROLE FOR HEPARAN SULFATE IN LCMV ENTRY Mark J. Bakkers *, Sarah Hulsey Stubbs, Sean P. Whelan MBIB, Harvard Medical School, Boston, United States Abstract: Lymphocytic Choriomeningitis Virus (LCMV) the prototypic arenavirus is associated with fatal infection in human transplant recipients, pregnancy termination and birth defects. As an infection model, studies of murine LCMV discovered and shaped our understanding of MHC restriction, T cell memory development and exhaustion, and persistent viral infections. Over 20 years ago, it was recognized that alpha-dystroglycan (α-dg) is the receptor for LCMV and the related Lassa fever virus (LASV), yet both viruses infect cells in the absence of α-dg. Using haploid genetic screens, we previously found that LASV, but not LCMV, undergoes a ph-triggered receptor switch during its internalization to engage LAMP to facilitate infection. To further interrogate the host requirements for LCMV entry, we performed a genome-wide CRISPR screen in the human lung cell line A549 using recombinant vesicular stomatitis virus (VSV) in which the endogenous glycoprotein was replaced by that of LCMV. Cells that survived infection were enriched for grna s targeting α-dg and heparan sulfate (HS) biosynthesis pathways, implicating both in LCMV entry. Using gene-edited cells in which we inactivated the host gene DAG to block all α-dg expression, B4GALT7 to block HS biosynthesis, or both, we confirmed the use of HS by LCMV. A closely related virus isolated from a cluster of transplant recipients, Dandenong, does not use HS for entry. Guided by atomic structures we performed a mutagenic analysis of the respective glycoproteins to define the residues in LCMV GP that confer binding to HS. This study demonstrates that LCMV attaches to the cell surface via α-dg and HS. We also hypothesize that a further internal "LAMP-like" molecule is required for cell entry during LCMV and Dandenong infection.

112 BREAKING AND ENTERING - viral entry Abstract final identifier: P07 CHARACTERIZATION OF HANTAVIRUS ENTRY THROUGH LIVE-CELL IMAGING Lara M. Kleinfelter *, Jennifer S. Spence, Rohit K. Jangra, Kartik Chandran Albert Einstein College of Medicine, Bronx, United States Abstract: Hantaviruses are global emerging pathogens that cause hemorrhagic fever with renal syndrome in the Old world and hantavirus cardiopulmonary syndrome in the New world with fatality rates as high as 2% and 60%, respectively. The development of antiviral drugs and vaccines against these agents is hindered by a poor understanding of the hantavirus entry pathway. To better understand the entry process and the roles of cellular host factors, we developed a live cell imaging system to observe hantavirus entry in real time. Fusion kinetics of fluorescently-labeled recombinant VSV particles bearing glycoproteins of Andes or Hantaan virus, virulent New world and Old world hantaviruses, respectively, in primary human endothelial cells matched those of other late-fusing viruses, though Andes virus requires more time to fuse than Hantaan virus. Furthermore, we defined the compartments where Andes and Hantaan virus GP-dependent membrane fusion occurs. Together, these experiments support the model that hantavirus fusion occurs late in the endocytic pathway. In addition, we are also characterizing the roles of known hantavirus receptors during entry. 2

113 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P08 STRUCTURAL CLASSES OF OLD WORLD ARENAVIRUS GP ATTACHMENT GLYCOPROTEIN Weng M. Ng *, Rhys Pryce, Antra Zeltina, Kamel El Omari 2, Armin Wagner 2, Thomas A. Bowden Division of Structural Biology, University of Oxford, Oxford, 2 Diamond Light Source Ltd, Harwell Science and Innovation Campus, Didcot, United Kingdom Abstract: Arenaviruses categorize into two serogroups: Old World and the New World, which differ genetically and by geographical distribution. The arenaviral surface attachment glycoprotein, GP, is responsible for host cell receptor recognition and is a primary determinant of zoonosis that enables the transmission of hemorrhagic fever arenaviruses from rodent hosts to humans. Previous crystallographic analyses of arenaviral GP glycoproteins have revealed two distinct phdependent conformational states. Here, our high-resolution crystal structures of the GP glycoproteins of Old World Loei River virus (LORV) and New World Whitewater Arroyo virus (WWAV), obtained at both neutral and acidic ph, suggest that these rearrangements are limited to Old World arenaviruses. We believe that Old World arenaviral GP glycoproteins presented on the mature virions adopt a structural class discrete to that formed upon detachment from the GP2 fusion protein during host cell entry. This detached conformation may constitute an immunological decoy and contribute to an ineffective humoral immune response during infection. Furthermore, the ability of detached New World arenaviral GP glycoproteins to bind receptors and neutralizing antibodies suggests that they are unlikely to undergo such conformational rearrangements. These data have implications for the development of detection diagnostics and immunogens specific to Old World arenaviruses. 3

114 BREAKING AND ENTERING - viral entry Abstract final identifier: P09 ARCHITECTURE OF THE METASTABLE HANTAVIRUS ENVELOPE Robert Stass *, Ilona Rissanen, Sai Li, Thomas A. Bowden, Juha T. Huiskonen Department of Structural Biology (Strubi), UNIVERSITY OF OXFORD, Oxford, United Kingdom Abstract: Members of the genus Hantaviridae are ssrna viruses present in rodent, bat, shrew, and mole reservoirs worldwide that cause hemorrhagic fever with renal syndrome and hantavirus cardiopulmonary syndrome upon zoonosis into humans. Hantaviruses are pleomorphic, enveloped viruses that exhibit a tetragonal lattice of glycoproteins on their surface comprised of Gn and the class-ii fusion protein, Gc. Using a non-pathogenic hantavirus, Tula virus, we utilize cryoelectron tomography, combined with previously reported crystal structures of Gn and Gc glycoproteins, to provide the best currently available model of the Gn-Gc glycoprotein spike assembly. We also demonstrate the collapse of this lattice upon exposure to low ph, mimicking its entry into the endocytic pathway. Comparison of our hantaviral Gn-Gc spike model with other viruses highlights the diverse modes of assembly available to class-ii fusion protein displaying viruses. 4

115 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P020 CHARACTERIZATION OF DETERMINANTS FOR HUMAN RESPIRATORY SYNCYTIAL VIRUS SPECIES SPECIFIC CELL ENTRY Svenja M. Wiechert *, Dortothea Bankwitz, Christine Krempl 2, Sibylle Haid, Sebastian Blockus, Luisa Stroeh 3, Thomas Krey 3, Thomas Pietschmann Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, 2 Institute of Virology and Immunobiology, Julius-Maximilian-University, Wuerzburg, 3 Institute of Virology, Hannover Medical School, Hannover, Germany Abstract: Human Respiratory syncytial virus (HRSV) and Pneumonia virus of mice (PVM) belong to the genus of Orthopneumoviruses within the family of Pneumoviridae. Both viruses display a pronounced species and tissue tropism as they infect the respiratory tract of humans and mice, respectively. The determinants that govern tropism of these viruses are poorly defined. HRSV associated lower respiratory tract disease is a leading cause of hospitalizations and in-hospital deaths in young children. Existing mouse models of RSV infection do not precisely mirror the pathophysiology of human disease. We hypothesize that species-specific HRSV cell entry limits susceptibility of mice to HRSV infection. To dissect mechanisms and determinants of species-specific HRSV cell entry, we recently developed infectious lentiviral pseudo particles decorated with HRSV G, F and SH proteins. To complement this system, we cloned codon optimized genes of PVM F, G and SH. PVM G protein expression was confirmed by immunofluorescence microscopy using sera from PVM G immunized rabbits. For detection of PVM F, F-specific monoclonal antibodies were created and used to detect F protein expression in transfected cells and incorporation of F protein onto secreted lentiviral particles. Surprisingly, PVM F protein carrying lentiviral pseudo particles were non-infectious in various human and rodent cell lines. Notably, mobility of F protein species in transfected cells differed from the one of F protein detected in PVM-infected BHK cells suggesting that aberrant post-translational modification (e.g. proteolytic processing) may be responsible for production of non-infectious particles. Mechanistic and functional assays to pinpoint the relevance of post-translational modifications for infectivity of PVM pseudo particles and to dissect the species tropism of HRSV are ongoing and will be presented. 5

116 BREAKING AND ENTERING - viral entry Abstract final identifier: P02 THE NOVEL CEDAR HENIPAVIRUS ATTACHMENT GLYCOPROTEIN DISPLAYS IDIOSYNCRATIC USAGE OF THE HUMAN EPHRIN CELL SURFACE RECEPTORS Kristopher Azarm *, Rhys Pryce 2, Thomas A. Bowden 2, Benhur Lee Icahn School of Medicine at Mount Sinai, New York, United States, 2 Division of Structural Biology, University of Oxford, Oxford, United Kingdom Abstract: The prototypical henipaviruses (HNVs), Hendra virus (HeV) and Nipah virus (NiV), are responsible for outbreaks of severe disease in domestic animals and humans. Zoonotic transmission of these pathogens is dependent upon the interaction of an attachment glycoprotein (HNV-G) with highly conserved ephrin receptors (EFNs). All extant HNVs whose receptors have been functionally defined use EFNB2. In 202, a novel HNV, Cedar henipavirus (CedV), was isolated from a pteropid bat in Australia. Although overall sequence conservation of CedV-G with the extant HNV-Gs is very low (24-3%), we see a substantial increase in conservation at the putative receptor binding site (47-54%). Thus, we examined whether CedV-G exhibited a conserved mode of binding to EFNB2. Moreover, given the low overall sequence conservation with extant HNV-Gs, we also sought to determine whether any of the other EFNs might serve as alternative receptors. Through functional analyses, we show that CedV-G uses not only EFNB2 as an entry receptor, but also EFNB. Soluble EFNB2 and EFNB inhibit entry of a vesicular stomatitis virus pseudotyped with the CedV envelope glycoproteins (CedVpp) into susceptible cells, and stable expression of EFNB2 or EFNB in an EFN-negative cell line is sufficient to confer entry. Moreover, soluble EFNB2 and EFNB were able to compete CedVpp entry on both EFNB2- and EFNB-expressing cells, suggesting that CedV-G interacts with EFNB2 and EFNB via an overlapping site. These results indicate CedV-G displays an idiosyncratic usage of EFN host receptors. Given that EFN receptor usage dictates viral tissue tropism and neurovirulence, this alternative receptor usage could partially explain why CedV is non-pathogenic in small animal models. 6

117 NSV 208, Verona Abstract Book BREAKING AND ENTERING - viral entry Abstract final identifier: P022 POTASSIUM ACTIVATES THE FUSION MACHINERY OF HAZARA VIRUS THROUGH SPIKE CONFORMATIONAL CHANGE Emma Punch *, Samantha Hover 2, Jack Fuller, Henry Blest, Roger Hewson 3, Juan Fontana 4, Jamel Mankouri, John Barr SMCB, University of Leeds, 2 SMCB, Uniersity of Leeds, 3 Tropical Medicine and Health, PHE, 4 ABSL, University of Leeds, Leeds, United Kingdom Abstract: All enveloped viruses enter cells via fusion of viral and host membranes. Fusion is mediated by virus-encoded spikes that respond to the changing endosomal environment, which triggers spike conformational changes that drive the fusion process. Several fusion triggers have been identified including ph, membrane composition and endosome-resident proteins and these cues dictate when and where viral fusion occurs. We recently showed infection of enveloped bunyaviruses required elevated potassium ion concentration [K+] encountered during transit through maturing endosomes, resulting from cellular K+ channel influx. Here, we reveal the molecular basis for K+ requirement using cryo-electron tomography to compare the ultrastructure of model bunyavirus Hazara virus, exposed to low and high [K+]. We show that exposure to K+ alone results in spike extension and induces spike-membrane interactions, thus identifying K+ as a newlydefined fusogenic trigger. We further show that preventing K+ influx through channel blockade prevents virus replication, thus identifying cellular K+ channels as new antiviral targets. 7

118 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P023 COMPETING LYSINE MODIFICATIONS IN INFLUENZA VIRUS PROTEINS. Edward Hutchinson *, Ervin Fodor 2 MRC-University of Glasgow Centre for Virus Research, Glasgow, 2 Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom Abstract: Ubiquitination is a common post-translational modification (PTM) of lysine, regulating protein stability and function. Using mass spectrometry, we found that influenza virions contained tens of ubiquitin molecules, linked through both the K48 and K63 positions. We also found that ubiquitin was conjugated to viral proteins. As only a limited number of ubiquitination sites had been mapped in influenza virus proteins, we used our data to map ubiquitinated lysine residues in the proteins of influenza A and B viruses. In doing so, we discovered that viral lysines are subject to a variety of PTMs including formylation, methylation and acetylation. In the context of cellular proteins, the effects of ubiquitin are known to be modulated by other lysine PTMs. These competing PTMs therefore suggest a potential regulatory lysine code for influenza proteins. The modified lysines in viral proteins are typically surface exposed, as would be expected from regulatory PTMs, and highly conserved, underlining their importance to the virus. We found that KR mutations, which prevent PTMs, often have little effect on viral growth in tissue culture. However, KR mutations to any of a cluster of modified lysines in the RNA-binding groove of the viral nucleoprotein (K84, K229 and K273) substantially reduced viral replication. Evidence for multiple competing PTMs at these and other sites suggests that a balance of modifications may fine-tune the activity of viral proteins. 8

119 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P024 ROLE OF THE NUCLEOCAPSID IN REGULATION OF VIRAL RNA SYNTHESIS Ming Luo *, Ryan H. Gumpper, Weike Li GEORGIA STATE UNIVERSITY, Atlanta, United States Abstract: The nucleocapsid of vesicular stomatitis virus (VSV) serves as the template for viral RNA synthesis. Codon usage bias (CUB) of VSV shows that 50% of the codons have the opposite usage frequency compared to the host. Based on the interaction of the genomic RNA with the nucleocapsid N protein, we hypothesize that its CUB plays a role in regulation of viral RNA synthesis. A minigenome assay has been set up in which the high or low frequency codon usage in the VSV N gene was changed to match those of the host. The results showed that change to either high or low frequency usage dramatically reduced the level of vrna and mrna. In addition, the ratios among the three RNA species were also changed, indicating imbalance between transcription and replication. Study of the smallest number of codons that have a visible effect on viral RNA synthesis and the underlining mechanism for reduction of viral RNA synthesis is ongoing. One of the reasons may be the local stability of the nucleocapsid. Using a unique melting curve assay, we showed that the stability of the nucleocapsid is directly related to the release of the sequestered RNA. Nucleocapsid-like particles (NLP) are more stable when poly(ra) is incorporated or less stable when poly(ru) is incorporated than wtnlp. Stabilization of the nucleocapsid by a RNA binding polyamide as shown by a co-crystal structure could inhibit viral RNA synthesis. Mutagenesis studies also showed that mutations in a helix-loop motif, located next to the sequestered genomic RNA, in the N protein resulted in lower viral RNA synthesis. Compensatory mutations in the L protein are required to restore the activity and to rescue the recombinant VSV. All data suggest that the interactions of the N protein with the sequestered genomic RNA in the nucleocapsid play major roles in regulation of viral RNA synthesis. 9

120 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P026 NUCLEAR IMPORT AND ASSEMBLY OF INFLUENZA VIRUS REPLICATION MACHINERY Amélie Donchet, Jean-Marie Bourhis, Rob W. Ruigrok, Bernard Delmas 2, Mariette Ducatez 3, Thibaut Crepin * IBS, Univ. Grenoble Alpes, CEA, CNRS, Grenoble, 2 Unité de Virologie et Immunologie moléculaires, INRA, Jouy-en-Josas, 3 IHAP, Université de Toulouse, INRA, ENVT, Toulouse, France Abstract: The specific nuclear replication of influenza viruses implies an efficient nucleocytoplasmic transport of viral components during the viral cycle. The nuclear import of neosynthesized viral proteins hijacks the cellular importins-a/β pathway, using several strategies. Whereas both PB2 and the nucleoprotein (NP) widely target the importins-a -3, the nuclear transport of the two others polymerase subunits (PA and PB) is performed through a ternary complex with the importin-β RanBP5 4,5. We have covered many aspects of the interaction between influenza virus replication machinery and the cellular importinsa/β pathway partners, in order to detail the assembly of this intricate viral architecture. Following our data on the complex between influenza B NP (B/NP) and the human importin-a7 6, we have detailed the inherent properties associated to the NP of the newly identified influenza D virus using several methods of the structural biology. The comparison of the X-ray structures of B/NP and D/NP reveals specific issues for the interaction with the cellular importins-a, that will be discussed 7. In parallel, our work cover also the aspect of the interaction between RanBP5 and the viral PA-PB subcomplex prior its assembly with PB2. We have recently shown that RanBP5 impairs the vrna recognition by the viral subcomplex 8. We will present our recent results on the specific interaction between the human importin-β and its viral cargo 9.. Tarendeau et al., (2007) NSMB; 2. Gabriel et al., (20) Nat Com; 3. Nakada et al., (205) Sci Rep; 4. Deng et al., (2006) J Virol; Hutchinson et al., (20) J Gen Virol; 6. Labaronne et al., (207) Sci Rep; 7. Donchet et al., in prep; 8. Swale et al., (206) Sci Rep; 9. Swale et al., in prep 20

121 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P027 DEPHOSPHORYLATION OF RESPIRATORY SYNCYTIAL M2- PROTEIN BY THE CELLULAR PHOSPHATASE PP IS REQUIRED FOR ITS MRNA BINDING ABILITY Jean-Francois Eleouet *, Charles-Adrien Richard 2, Vincent Rincheval 3, Christophe Cardone 4, Camille Esneau, Sergei Nekhai 5, Marie Galloux, Marie-Anne Rameix-Welti 3, Christina Sizun 4 Unite de Virologie et Immunologie Moleculaires, INRA Universite Paris-Saclay, 2 Unite de Virologie et Immunologie Moleculaires, INRA, Jouy en Josas, 3 UMR73, INSERM-Universite de Versailles St Quentin, Montigny le retonneux, 4 ICSN, CNRS-Universite Paris-Saclay, Gif-sur-Yvette, France, 5 Center for Sickle Cell Disease, Howard University, Washington D.C., United States Abstract: The M2- protein of respiratory syncytial virus (RSV) is essential for viral transcription. Previous reports suggested that dynamic regulation of M2- phosphorylation is critical for its function. M2- phosphorylation depends on the presence of the RSV phosphoprotein P, which is a multifunctional protein and the main cofactor of the large RNA polymerase L protein; formation of the P-M2- complex is required for viral transcription. However mechanisms involved in M2- phosphorylation and dephosphorylation in vivo have not been clarified. Using site-directed mutagenesis and NMR, we identified an RVxF-like motif located upstream of the M2- binding domain and involved in the capture of the host cell protein phosphatase- (PP) by P and its recruitment to cytoplasmic inclusion bodies (IBs) where viral RNA synthesis occurs. We further show that the P-PP complex regulates M2- dephosphorylation and RSV transcription. In the absence of PP from inclusion bodies, M2- was excluded from IBs associated granules (IBAGs) formed by viral mrna. These results suggest that M2- functions not only as a transcription antiterminator but plays also a critical role at late transcription steps. Graphical Abstract: 2

122 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P028 THE ISOLATION AND CHARACTERIZATION OF A N -P COMPLEX OF THE RESPIRATORY SYNCYTIAL VIRUS OPENS THE WAY TO THE RATIONAL DESIGN OF NEW ANTIVIRALS Jean-Francois Eleouet *, Ronan Le Goffic, Vanessa Gaillard 2, Origène Nyanguile 2, Marie Galloux Unite de Virologie et Immunologie Moleculaires, INRA, Jouy en Josas, France, 2 peptide and Protein Technologies, HSE- SO Valais, Sion, Switzerland Abstract: The RNA genome of respiratory syncytial virus is encapsidated by the viral nucleoprotein N at all steps. This RNA-N complex is a template for the viral RNA-dependent RNA polymerase. Polymerization of N along the neo-synthesized genomic and anti-genomic RNAs requires the supply of neo-synthesized N protein. This N protein is maintained monomeric and RNA-free through the interaction with the N-terminal residues of RSV phosphoprotein P that plays the role of a chaperone, forming a soluble N0-P complex. In order to isolate a stable N0-P complex suitable for structural and functional studies, we used the N-terminal peptide of P (P40) to purify truncated forms of the N protein. We show that deletion of the 30 first N-terminal residues of N (NΔ30) impairs N oligomerization, and that the C-terminal arm of N is involved in the inhibition of N-RNA interaction in solution. Biochemical and functional analysis of RSV N mutants revealed the critical role of C-terminal acidic and hydrophobic residues of N in the folding of the C-arm close to the RNA groove, and the similarity of the binding surface of P40 on RSV N compared to hmpv N0-P complex. Altogether, these data led us to propose new antiviral approaches targeting the RSV N0-P complex. More specifically, we rationally designed peptides that mimic the P peptide and that were shown to be active in cellula and in vivo in a mouse model. Finally, an RNA-free, chimeric construct composed of the full-length N and the N-terminal region of P was purified and used for in vitro encapsidation studies. This protein represents a new tool to validate the specificity and efficacy of our compounds. Graphical Abstract: 22

123 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P029 MECHANISM OF RESPIRATORY SYNCYTIAL VIRUS RNA SYNTHESIS Dongdong Cao, Yunrong Gao, Bo Liang * Department of Biochemistry, Emory University School of Medicine, Atlanta, United States Abstract: Cryo-electron microscopy (cryo-em) is especially well suited for molecular systems traditionally challenging for structural characterization, including membrane proteins and large and heterogeneous assemblies. One such challenging system is the RNA synthesis machinery of a class of pathogenic and sometimes deadly non-segmented negative-sense (NNS) RNA viruses, including rabies, measles, Ebola, Marburg, and respiratory syncytial virus (RSV). Central to the life of these viruses is RNA synthesis, which is carried out by the RNA polymerase (the multifunctional enzyme). However, the structural and functional basis of the RNA synthesis machinery of these viruses remains largely unclear. The Liang laboratory is dedicated to understanding the mechanism of the RNA synthesis machinery of RSV, the top leading cause of severe pediatric respiratory tract diseases worldwide. The Liang laboratory focuses on establishing an RNA synthesis platform for RSV, elucidating how this RNA synthesis machine functions, and identifying potential antiviral therapeutic targets for more effective treatment. Our immediate research goal is to decipher the molecular architecture of the RSV RNA synthesis machine using single particle cryo-em and x-ray crystallography. This could lead to the development of effective antiviral drugs to block the RSV activity. 23

124 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P030 STRUCTURAL BASIS FOR REGULATION OF FILOVIRAL NUCLEOPROTEIN (NP) BY VP35 DURING VIRAL RNA SYNTHESIS Gaya Amarasinghe * Pathology and Immunology, WASHINGTON UNIVERSITY SCHOOL OF MEDICINE, St. Louis, United States Abstract: Ebola virus and Marburg virus are two members of the zoonotic filovirus family likely maintained in nature in bats. These viruses cause highly lethal hemorrhagic fever in humans (filoviral hemorrhagic fever (FHF)), with case fatality rates approaching 90 percent during some outbreaks. Fatal outcomes correlate with increasing viremia over time and are associated with shock, convulsions and disseminated intravascular coagulation. Viral antagonism of host innate immunity is important for infection and a key protein called viral protein 35 (VP35) performs multiple immune evasion functions. Ebola virus nucleoprotein (enp) assembles into higher-ordered structures that form the viral nucleocapsid (NC) and serve as the scaffold for viral RNA synthesis and its activity is controlled, in part by VP35. However, molecular insights into the NC assembly process are lacking. Using a hybrid approach, we characterized the NC-like assembly of enp, identified novel regulatory elements, and described how these elements impact function. We will describe significant structural differences between Ebola viral NP and Marburg viral NP proteins, including differences in how the VP35 binding site in NP is modulated. Importantly, our in vitro studies are consistent with viral life cycle models, such as minigenome and trvlp assays, supporting physiological relevance and biological significance. We will also provide data to support how these basic biochemical insights provide a framework for much needed small molecule therapeutic development. 24

125 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P03 FIRST IN HUMAN STUDIES OF ONCOLYTIC VESICULAR STOMATITIS VIRUSES ENCODING INTERFERON FOR CANCER THERAPY Nanda Packiriswamy, Bethany Brunton, Jamie Bakkum-Gamez 2, Stephen Russell, Martha Lacy 3, Kah-Whye Peng * Molecular Medicine, 2 Gynecologic Surgery, 3 Hematology, Mayo Clinic, Rochester, United States Abstract: The Indiana strain of Vesicular Stomatitis Viruses (VSV) are being developed as anticancer drugs for the treatment of a variety of malignancies. To ensure tumor selective replication and spread, we have designed the VSV to encode interferon beta. Expression of IFNb also serves as a STING angonist to activate host immunity against the cancer. The sodium iodide symporter (NIS) is inserted as a reporter gene into the viral genome to enable noninvasive monitoring of viral spread using PET/CT imaging. Two Phase I clniical trials testing VSV-IFN-NIS are ongoing after intravenous administration of the virus in hematological malignancies and endometrial cancer. It is a classical 3+3 phase I trial, starting at 5x0 9 TCID50 through 5x0 TCID50, given as a single IV dose. The primary objective is safety and tolerability; secondary objectives include monitoring the PK of viral replication through SPECT/CT imaging with NIS gene, viremia, virus shedding, preliminary efficacy, changes in the immune profile of peripheral blood leukocytes, and immunohistochemistry for immune cell infiltrates in tumors. To date, nine patients have received IV VSV-IFNβ-NIS; three with EC and six with hematologic malignancies. The highest dose administered to date is.7x0 0 TCID50 and dose escalation is ongoing. Multiple cytokines increased at 4h post infusion of virus, but most returned to baseline levels by 24h. Viremia was detectable in all patients at the end of infusion, and to varying levels at 30 mins,, 2, 4, 24, 48h or 72 hours post virus infusion. No persistent viremia was observed. No infectious virus was recovered in buccal swabs or urine and neutralizing anti-vsv antibodies were present by day 29. Extensive immune phenotyping for immune cells showed a trend towards increased PD- expression on CD8+ cells. Dose escalation is ongoing and updated results will be reported. 25

126 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P032 START-SNATCHING: A POTENTIAL NOVEL MECHANISM FOR TRANSLATION INITIATION IN SEGMENTED NEGATIVE-SENSE RNA VIRUSES Elizabeth Sloan *, Veronica Valentina Rezelj, Ingeborg Van Knippenberg, Quan Gu, Edward Hutchinson CVR, MRC-University of Glasgow, Glasgow, United Kingdom Abstract: A number of segmented negative sense RNA viruses use cap-snatching to initiate transcription. As start codons have been found close to the cap in host mrnas, we asked whether cap-snatching might append additional start codons to viral mrnas. We analysed published deep sequencing data of influenza cap-snatched sequences and found that >6% contained start codons. To determine if these could initiate translation we used transfection-based minireplicon systems, in which viral polymerases transcribe a luciferase gene flanked by viral UTRs. We assessed three distantly related capsnatching viruses, influenza A virus (IAV), Oropouche virus and Heartland virus (HRTV). Even after mutating the luciferase start codon we detected luciferase expression from all three minireplicon systems, providing evidence of functional start sites within cap-snatched sequences. Focusing on IAV, we next analysed mass spectra from viral proteins and identified peptides that mapped to the UTR of segment 5, upstream of any encoded start codons, consistent with translation initiation in the cap sequence, or start-snatching. Upon further investigation of published IAV sequences, we discovered out-offrame ORFs which lack an encoded start codon at the 5 end of a number of viral genome segments. These are highly conserved, suggesting they may be of biological importance. Overall, this study has found evidence for a novel model for viral translation initiation, which may be used by at least three negative stranded RNA viruses. 26

127 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P033 STRUCTURAL INSIGHTS INTO REPTARENAVIRUS CAP-SNATCHING MACHINERY Maria Rosenthal *, Nadja Gogrefe, Dominik Vogel, Juan Reguera 2, Bianka Rauschenberger, Stephen Cusack 3, Stephan Guenther, Sophia Reindl Virology, BERNHARD NOCHT INSTITUTE FOR TROPICAL MEDICINE, Hamburg, Germany, 2 AFMB UMR 7257 Aix- Marseille Univ/CNRS, Marseille, 3 Grenoble Outstation, European Molecular Biology Laboratory, Grenoble, France Abstract: Arenaviruses occur worldwide and can cause severe, often fatal hemorrhagic fever in humans. Vaccines and effective treatments are not available. Arenaviruses replicate in the cytoplasm of infected cells and since they cannot synthesize cap-structures they use a mechanism called cap-snatching to steal cap structures from host mrnas for viral transcription. This mechanism is an attractive drug target, as it is essential for virus replication and virus specific. However, the arenaviral components of this mechanism are poorly defined compared to influenza virus, the prototypic cap-snatching virus. We present the first crystal structures of two putative components of the California Academy of Sciences arenavirus cap-snatching machinery, namely the isolated N- and C-termini of the viral RNA polymerase (L protein). The N-terminus harbors what looks like a functional cap-snatching endonuclease. The L protein C-terminus, despite complete sequence divergence, shows overall structural similarity to the C-terminal region of influenza virus polymerase PB2 subunit, suggesting a common evolutionary origin. A domain clearly related to the PB2 cap-binding domain is present, although capbinding could not be biochemically demonstrated. The determined structures provide the basis for future research to unravel the details of the arenavirus cap-snatching mechanism and its potential as a target for drug development. 27

128 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P034 A MECHANISM FOR THE ACTIVATION OF THE INFLUENZA VIRUS TRANSCRIPTASE Itziar Serna Martin *, Narin Hengrung 2, Max Renner 3, Jane Sharps, Ervin Fodor, Jonathan Grimes 3 Dunn School of Pathology, UNIVERSITY OF OXFORD, Oxford, 2 Francis Crick Institute, London, 3 Structural Biology Division, University of Oxford, Oxford, United Kingdom Abstract: The influenza virus RNA polymerase (FluPol), a heterotrimer composed of PB, PB2 and PA subunits (P3 in influenza C), performs both transcription and replication of the viral RNA genome. Transcription depends on capped RNA primers, generated from nascent cellular transcripts by the cap-snatching activity of the polymerase. Access to nascent capped transcripts for cap-snatching is ensured by the interaction between FluPol with the C-terminal domain (CTD) of RNA polymerase II. We have determined the co-crystal structure of influenza C virus polymerase (FluPolC) bound to a Ser5- phosphorylated CTD (ps5-ctd) peptide, identifying novel CTD binding sites distinct from those characterised in FluPolA and FluPolB. The position of the CTD-binding site at the interface of PB, P3 and the flexible PB2 C-terminal domains suggests that CTD-binding stabilizes the transcription-competent conformation of FluPolC. In agreement with this, FluPolC in vitro activity assays show that both the cleavage of capped RNA and capped primer-dependent transcription initiation are greatly enhanced when ps5-ctd peptide is present. Mutagenesis of amino acid residues in the regions identified to be involved in CTD binding resulted in the specific inhibition of viral mrna synthesis, confirming their importance. These data, in combination with cryo-em analysis of the vrna promoter bound FluPolC, have allowed us to propose a model where presence of promoter RNA and an interaction with the ps5-ctd of Pol II are key for the activation of the influenza virus transcriptase. 28

129 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P035 N-TERMINAL ACETYLATION BY NATB IS REQUIRED FOR THE SHUTOFF ACTIVITY OF INFLUENZA A VIRUS PA-X Kohei Oishi *, Seiya Yamayoshi, Hiroko Kozuka-Hata 2, Masaaki Oyama 2, Yoshihiro Kawaoka Virology, 2 Medical Proteomics Laboratory, University of Tokyo, Minato-ku, Japan Abstract: N-terminal acetylation is a major posttranslational modification in eukaryotes catalyzed by N-terminal acetyltransferases (NATs), NatA through NatF. Although N-terminal acetylation modulates diverse protein functions, little is known about its roles in virus replication. Influenza virus PA-X possesses endonuclease activity that suppresses (i.e., shuts off) host protein expression; however, the host proteins involved in this shutoff activity remain unknown. We found that NatB, which comprises NAA20 and NAA25, is involved in the shutoff activity of PA-X. The shutoff activity of wild-type PA-X was suppressed in NatB-deficient cells, and a PA-X mutant that was not acetylated by NatB showed reduced shutoff activity compared with wild-type PA-X. These data show that N-terminal acetylation by NatB is required for the shutoff activity of PA-X. To examine whether NatB is involved in the N-terminal acetylation of PA-X, PA-X expressed in wild-type or NatBdeficient cells, was analyzed for N-terminal modification. All detected N-terminal peptides of PA-X expressed in wild-type cells were N-terminally acetylated, whereas N-terminal acetylation was detected in approximately 50% of N-terminal peptides of PA-X derived from NatB-deficient cells. These data suggest that NatB is involved in the N-terminal acetylation of PA-X. We also evaluated the importance of N-terminal acetylation of PA, because PA-X shares its N-terminal amino acid sequence with PA. Viral polymerase activity was reduced in NatB-deficient cells, whereas mutant PA that was not acetylated by NatB lost its functions in the viral polymerase complex. Taken together, our findings demonstrate that N-terminal acetylation plays roles in both virus protein function and replication. 29

130 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P036 IDENTIFICATION OF AN EBOLA VIRUS VP30-SPECIFIC KINASE THAT REGULATES VIRAL TRANSCRIPTION Yuki Takamatsu *, Nadine Biedenkopf, Verena Krähling, Larissa Kolesnikova, Sandro Halwe, Stefan Baumeister 2, Stephan Becker Institute of Virology, Philipps University Marburg, 2 Department of Parasitology, Faculty of Biology, Philipps University Marburg, Marburg, Germany Abstract: Ebola virus (EBOV) protein VP30 is phosphorylated predominantly at six N-proximal serine residues (S29-S3, S42, S44, and S46). While the EBOV polymerase complex composed of the polymerase L and VP35 executes genome replication, viral transcription requires VP30, an EBOV-specific transcriptional activator. Reversible phosphorylation of VP30 regulates EBOV polymerase function. Nonphosphorylated VP30 promotes genome transcription, whereas phosphorylated VP30 favors genome replication. Although phosphatases which dephosphorylate VP30 are identified as PP and PP2A, VP30- specific kinases remained elusive. Here we identified a VP30-specific kinase that is able to phosphorylate VP30 in vitro and in cellulo at the important position S29, to interact with VP30 in immunoprecipitation, colocalized with VP30 in viral inclusion bodies and regulate viral transcription. Our findings imply a novel therapeutic approach which focuses on VP30 phosphorylation. 30

131 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P037 FUNCTIONAL CHARACTERIZATION OF THE HANTAVIRUS S SEGMENT UNTRANSLATED REGION Janne Tynell *, Jonas Klingström MedH, KAROLINSKA INSTITUTET, Stockholm, Sweden Abstract: Functional characterization of the hantavirus S segment untranslated region Janne Tynell & Jonas Klingström Center for Infectious Medicine, Department of Medicine Huddinge, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden Hantaviruses are rodent- and insectivore-borne viruses estimated to cause over human cases annually around the world. The trisegmented hantavirus RNA genome contains 3-4 open reading frames (ORFs) coding for the N, Gn, Gc and RNA-dependent RNA polymerase (RdRp) proteins as well as the non-structural NSs protein found in some hantavirus species. In addition to the ORFs, the hantavirus S segment contains a large untranslated region (UTR) comprising more than 20% of the entire segment. In an effort to assign functional significance to the S segment UTR, we have performed in vitro RNA pulldown assays and mass spectrometry analysis on human umbilical vein endothelial cell (HUVEC) lysates to identify possible interactions between host cell proteins and the S segment UTR. We present data obtained with various different pulldown techniques using S UTRs from the Puumala and Andes hantaviruses and discuss the significance of our results for hantavirus pathogenesis. 3

132 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P038 DETAILED MAPPING OF THE VARIOUS POLYMERASE COFACTOR FUNCTIONS WITHIN THE PHOSPHOPROTEIN OF MEASLES VIRUS Louis-Marie Bloyet, Antoine SCHRAMM 2, Carine Lazert 3, Sonia Longhi 2, Denis Gerlier * CIRI INSERM U, CNRS UMR5307, UNIVERSITY LYON, ENS LYON, UNIV LYON, Lyon, 2 Architecture et Fonction des Macromolécules Biologiques (AFMB) UMR 7257, CNRS, Aix Marseille University Marseille, Marseille, 3 CIRI, International Center for Infectiology Research, Inserm, U, Université Claude Bernard Lyon, CNRS, UMR5308, Ecole Normale Supérieure de Lyon, Univ Lyon, Lyon, France Abstract: The phosphoprotein (P) of Mononegavirales plays a central role in viral expression and multiplication. In Measles virus, P associates with nascent nucleoprotein (N) via an α-more located at its N-terminus, to form the monomeric N 0 P complex. N 0 P is used as substrate for genome encapsidation by the polymerase. Via its C-terminal X domain, P dynamically tethers the L protein endowed with polymerase enzymatic activities on the helicoidal nucleocapsid (NC). NC is made of a continuous homopolymer of N protein wrapping the genomic RNA. The binding strength of XD-NC interaction tightly regulates the efficiency of polymerase scanning and/or transcription re-initiation at each intergenic junction. P associates with L to yield a functional polymerase complex. Its polymerase cofactor activity can be decomposed in at least three components: P provides HSP90-mediated chaperone and proper assistance to the folding of L into a fully functional polymerase; P allows the polymerase to dynamically bind to its NC template; and P enables the polymerase to be processive all along the genome. A protein complementation assay between a large panel (> one hundred) of P variants (e.g. truncated, chimeric and mutated forms) and L protein fragments indicated a major binding site located at the C terminus of P. L folding assays revealed the requirement of the C-ter of P multimerisation domain (PMD). Minigenome assays and recombinant bigbis viruses coupled to biochemical, biophysical and structural studies unveiled a critical contribution of protein dynamics to the polymerase cofactor activity. The conserved modular organisation of P within the Paramyxoviridae family argues for a common functional organization. 32

133 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P039 VIRAL TRANSCRIPTION DRIVES THE FORMATION OF VIRAL RNA GRANULES WITHIN RESPIRATORY SYNCYTIAL VIRUS INCLUSION BODIES V Rincheval, Mickael Lelek 2, Charles-Adrien Richard 3, Jing Jing Cao 4, Camille Bouillier, Delphine Sitterlin, Sabine Blouquit-Laye, Marie Galloux 3, Christophe Zimmer 2, Ralf Altmeyer 4, Jean-Francois Eleouet 3, Marie-Anne Rameix-Welti * UMR 73, INSERM - Université de Versailles St Quentin, Montigny-le-Bretonneux, 2 UMR 369, Institut Pasteur - CNRS, Paris, 3 UR892 VIM, INRA, Jouy-en-Josas, France, 4 Shandong University-Helmholtz Institute of Biotechnology, Qingdao, China Abstract: We investigated functional organization of respiratory syncytial virus (RSV) cytoplasmic inclusion bodies (IBs). We found that viral RNA synthesis mainly occur in IBs and that newly synthetized viral mrna concentrate in IBs sub compartments called IB associated granules (IBAG). Interestingly confocal microscopy and super-resolution microscopy revealed that M2-, a RSV transcription antiterminator, accumulated in IBAGs when the other components of the viral polymerase (N, P, L) are excluded therefrom. Using a dicistronic minigenome system we found that IBAG formation is strictly dependent on viral RNA synthesis, but may occur in the absence of M2-. Indeed, when omitting M2-, FISH experiments revealed some IBAGs when using probes against the first gene (that can be transcribed without M2-), but not probes against polya or the second gene. IBAGs are highly dynamic structures, which seem to release their content periodically into the cytosol. Noteworthy, we found that RSV IBs exhibit properties of liquid organelles: spherical shape, fusion to form larger spherical structures, disappearance upon osmotic shock. Thus, IBAG formation could be regarded as a liquid-liquid phase separation resulting from accumulation of viral mrna in IBs. The strict dependence of IBAG formation on viral RNA synthesis but not on M2- suggests that viral mrna are the driving force of IBAG formation. Surprisingly, however, Cyclopamine, described as an RSV inhibitor targeting M2-, was found capable of quickly disrupting IBAGs in RSV infected cells. 33

134 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P040 INFLUENZA VIRUS RNA-POLYMERASE: TEMPERATURE-SENSITIVE MUTANTS IN PB DISPLAY A DEFECT IN NUCLEAR TARGETING OF THE PA-PB DIMER Bruno Da Costa, Laura Sedano, Nathalie Lejal, Ronan Le Goffic, Rob Ruigrok 2, Thibaut Crépin 2, Bernard Delmas * Unité VIM, INRA, Jouy-en-Josas, 2 IBS, Université de Grenoble, CEA, CNRS, Grenoble, France Abstract: The influenza virus RNA-dependent RNA polymerase catalyses genome replication and transcription within the cell nucleus. Efficient nuclear import and assembly of the polymerase subunits, PB, PB2, and PA are critical steps in replication and secondary transcription. We previously found that the PA linker plays a key role in nuclear targeting of the PA-PB dimer. Temperature-sensitive mutants in the linker are defective in the transport of the PA-PB dimer into the nucleus at restrictive temperature (39.5 C), resulting in a poor replication/transcription activity and suggesting an alteration of folding kinetic parameters. In this study, we generated numerous PB mutants engineered at the PA linker interface, the PB nuclear location signal, at positions known to promote a ts-phenotype to FluMist vaccine strains or at buried positions in the PB subunit or in the PA-PB dimer. Several PB mutants exhibited temperature-sensitivity with a reduced growth at 39.5 C versus 37 C/33 C. The ts-phenotype was also associated with a reduced efficiency of replication/transcription as measured in a minireplicon assay and to a defect in the transport of the PA-PB dimer into the nucleus at restrictive temperature. Complementation assays using PB mutants, wt-pa and importin-beta IPO5 revealed PB residues associated to the formation of a stable complex and involved in the efficient transport of PA-PB dimer into the nucleus. Taking our results as a whole, we propose that ts-sensitivity marks a local misfolding of the PB subunit that results in a defect of the stability of the PA-PB complex and consequently an inability of the PA-PB-IPO5 complex to assemble and reach the nucleus. 34

135 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P04 OPTIMISING INFLUENZA A REPORTER VIRUSES BY IN VIVO PASSAGING Monique Spronken *, Altagracia Russel, Ron Fouchier, Kirsty Short, 2 Viroscience, Erasmus Medical Centre, Rotterdam, Netherlands, 2 School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia Abstract: Influenza A reporter viruses are a valuable tool to study fundamental research questions regarding virus tropism and replication. To date, several influenza A reporter virus systems have been described that differ in attenuation, stability and expression levels. Additionally, it was shown that these systems are either dysfunctional or inferior when incorporated in human and avian circulating virus strains. In order to use reporter viruses to answer key research questions it s essential that they can be detected by live in vivo imaging. Luminescent influenza A reporter viruses have been successfully used in live in vivo imaging experiments. However, this requires the administration of expensive substrates and it can t be used for single cell detection and analysis. Therefore, the ideal reporter virus is fluorescently labelled so it can be used for both in vivo imaging and single cell analysis. With this in mind, we sought to improve the replication and signal strength of our previously described fluorescently labelled influenza viruses. We inserted reporters in the red spectrum (mcardinal and Katushka_2S) into the A/PR/8 PA gene segment containing promotor up mutations and a duplicated packaging region. The resultant A/PR/8 2UP_PA_mCardinal reporter virus was then passaged four times in C57BL/6 mice. This resulted in enhanced virus replication and increased percentages of reporter expression in the lungs of infected mice. Full sequencing of viral genomes from infected lungs showed a double peak at amino acid position 640 of the PB gene segment. This mutation was introduced in PB and the 2UP_PA_mCardinal virus was further studied in several in vitro assays to assess reporter expression levels, minireplicon activity and virus replication. As this mutation also enhanced reporter expression in vitro, it was further assessed in mice and preliminary experiments show enhanced replication and reporter expression. 35

136 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P042 RIFT VALLEY FEVER PHLEBOVIRUS NUCLEOPROTEIN PROTEOMIC STUDIES IDENTIFY IMPORTANT WNT PATHWAY INTERACTIONS Timothy J. Mottram *, Margus Varjak, Benjamin Brennan, Alain Kohl MRC-University of Glasgow Centre For Virus Research, Glasgow, United Kingdom Abstract: Rift Valley fever phlebovirus (RVFV, Phenuviridae, Bunyavirales) is an important pathogen of both humans and livestock. RVFV transmission by mosquitoes across sub-saharan Africa and the Arabian Peninsula has a significant impact on the socio-economics of these areas. Research interests have primarily focused on identifying interacting partners of the non-structural protein (NSs), encoded within the viral S RNA segment. However, the interaction partners of the nucleocapsid protein (N), remain largely unknown. Using a proteomics-based approach, we identified 24 potential mammalian hostderived N protein interaction partners. Following an sirna screen utilising a viral minigenome system, the cellular proteins: β-catenin, Polyadenylate binding protein 4, Scaffold attachment factor B and Annexin A2 appeared to be important for the formation of functional ribonucleoprotein (RNP) complexes. As β-catenin is a known effector molecule of the WNT pathway, analysis following RVFV infection and minigenome transfection on the WNT pathway indicated a cell specific inhibition of the pathway. Additionally, β-catenin knockout resulted in reduced viral replication indicating an important host-viral interaction. This was evidenced by a direct interaction of β-catenin with RVFV N protein and evidence of relocalisation of β-catenin from the plasma membrane. Understanding the fundamental biology, followed by further characterisation of these interactions will aid future development of new intervention strategies 36

137 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P043 IDENTIFICATION OF MUMPS VIRUS NUCLEOPROTEIN RESIDUE ENHANCES PRODUCTION OF DEFECTIVE INTERFERING PARTICLES Jacquline C. Risalvato *, James R. Zengel 2, Ming Luo 3, Biao He Department of Infectious Disease, University of Georgia College of Veterinary Medicine, Athens, 2 Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, 3 Department of Chemistry, Georgia State University Center for Diagnostics and Therapeutics, Atlanta, United States Abstract: Mumps virus (MuV) is a negative-sense single-stranded RNA virus belonging to the family Paramyxoviridae. A human pathogen, MuV is responsible for acute infection of the parotid glands, and can cause severe cases of encephalitis, meningitis, and deafness. The nonsegmented RNA genome of MuV is encapsidated by the nucleocapsid protein (NP), which forms the ribonucleoprotein (RNP) complex which serves as a template for RNA synthesis. To make RNA accessible to the viral polymerase, a conformational change within NP must occur. Crystal structure analysis of the NP of parainfluenza virus 5 (PIV5), a paramyxovirus closely related to MuV, indicates that an α-helix close to the RNA genome becomes flexible when RNA is removed. This region of the NP is likely responsible for the conformational change which allows the polymerase to access RNA for transcription and replication. To examine the functionality of MuV s NP, point mutations were made in MuV NP protein corresponding to PIV5 at sites G85P, A97Q, Q200R, and regions denoted as Top (N63G, P39D, A97Q), Tip (P09E, N2G, A24R), and Bottom (G2S, S29T, P43N, R93Q, R304Q). The Top MuV mutant exhibited normal growth kinetics at low multiplicity of infections (MOIs); however, at high MOI s the virus could not efficiently replicate. Further analysis indicates that production of defective interfering (DI) particles was enhanced in the mutant virus. Understanding the production of DI particles, which can lead to increased interferon production, will invariably lead to a better understanding of MuV pathogenesis as well as its replication/transcription process. 37

138 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P044 ALTERNATIVE SPLICING UNMASKS AN ENDOPLASMIC RETICULUM TARGETING SIGNAL OF BORNA DISEASE VIRUS NUCLEOPROTEIN Shohei Kojima *, 2, Ryo Sato 3, Mako Yanai, 2, Yumiko Komatsu 2, 4, Masayuki Horie 2, 5, Keizo Tomonaga, 2, 6 Graduate School of Biostudies, 2 Lab. of RNA viruses, 3 Faculty of Medicine, KYOTO UNIVERSITY, 4 K-CONNEX, 5 Hakubi Center, 6 Graduate School of Medicine, KYOTO UNIVERSITY, Kyoto, Japan Abstract: Borna disease virus (BoDV) is a non-segmented negative strand RNA virus which establishes intranuclear infection in mammals. BoDV exploits host pre-mrna splicing machinery to express polycistronic transcripts of M, G and L. However, the detailed analysis of splice sites in other transcripts was not performed. Therefore, we set out to comprehensively analyze the splice junctions of BoDV mrna using deep sequencing. We discovered that the transcripts encoding nucleoprotein (N) undergo mrna-splicing. There were two introns within the N ORF, and spliced transcripts retained the protein reading frame, suggesting that the N gene expresses at least two truncation isoforms of N, named N2 and N3, by splicing. While full length N and N2 mainly localized in the nucleus, N3 translocated to the endoplasmic reticulum (ER) and was cleaved into a shorter form by host protease. We also found that the N protein intrinsically harbors a signal peptide and the truncation of full-length N into N3 works as a molecular switch for ER targeting. Lastly, we unveiled both N2 and N3 inhibited the BoDV minigenome activity, indicating that the N isoforms can regulate BoDV replication. Some reports previously showed that BoDV generates a N-terminally truncated isoform of N, which is important for elaborate control of BoDV polymerase activity. Our study adds novel insights into the molecular framework for how BoDV generates N isoforms of different cellular localization. 38

139 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P045 THE OLIGOMERIC STATE OF MARBURG VIRUS VP40 INFLUENCES ITS ROLE IN MODULATING VIRAL TRANSCRIPTION AND REPLICATION Alexander Koehler *, Sebastian Pfeiffer, Larissa Kolesnikova, Stephan Becker Institute of Virology, Philipps University Marburg, Marburg, Germany Abstract: Marburg virus matrix protein, VP40, is crucial for viral assembly, budding and release; it counteracts the innate immune response and inhibits viral replication and transcription. VP40 is a peripheral membrane protein, which is partially membrane-associated forming an ordered VP40 lattice beneath the plasma membrane and the viral envelope, and partially soluble being diffusely distributed throughout the cell and detected also in viral inclusion bodies. Structural studies have suggested that the transition from VP40 monomers to oligomers is accompanied by the switch from a soluble to a membrane-associated form, the latter is essential for viral assembly and budding. It remains unclear whether the oligomeric state or membrane-binding of VP40 or both are important for the regulation of the innate immune response and/or viral replication and transcription. In the present study several mutations have been introduced into VP40 resulting in the formation of monomeric or dimeric forms of VP40; or oligomeric forms of VP40 with a reduced interaction with membranes. Monomeric or dimeric VP40 mutants co-localized partially with the viral inclusions and inhibited viral transcription and replication only weakly. VP40 mutants that formed higher oligomers but lack membrane association induced significant rearrangements of perinuclear inclusion bodies and strongly inhibited viral replication. Intriguingly, these VP40 mutants did not co-localize with the inclusion bodies, but were arranged along ER-bound ribosomes. Our results indicate that the oligomeric state of VP40 and lack of plasma membrane transport of VP40 oligomers, regulates VP40 s capacity to inhibit viral transcription and replication. 39

140 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P046 TOWARDS THE DEVELOPMENT OF REVERSE GENETICS SYSTEMS FOR TOMATO SPOTTED WILT VIRUS Richard Kormelink, Andre Bertran *, Magdalena Mazur Laboratory of Virology, WAGENINGEN UNIVERSITY, Wageningen, Netherlands Abstract: TOWARDS THE DEVELOPMENT OF REVERSE GENETICS SYSTEMS FOR TOMATO SPOTTED WILT VIRUS André Bertran, Magdalena J. Mazur and Richard Kormelink Laboratory of Virology, Wageningen University and Research, the Netherlands The order of Bunyavirales comprises a large group of emerging arthropod-borne viruses that primarily infect animals and of which some even pose a worldwide biological threat. During the last two decades various reverse genetics systems have been established to generate (mutant) viruses from cdna clones for many different non-segmented and segmented (- )ssrna viruses including bunyaviruses. Infectious clones are now available for almost all type species of the bunyaviruses, with the exception of the hantaviridae and tospoviridae. Here, we present our strategies towards the development of minireplicon systems for Tomato spotted wilt (TSWV), the prototype of the plant-infecting bunyaviruses, in mammalian and insect cell lines. To generate bona fide viral RNA templates, two distinct expression systems are being tested, namely T7 RNA polymerase and RNA polymerase I/II (pol I/ pol II). For both systems, we developed templates that allow qualitative (fluorescent reporter proteins) and quantitative (firefly and Renilla luciferase) measurements of minireplicon activity. As the replication /transcription activity is dependent on the formation of viral ribonucleoproteins (RNPs), the viral RNA templates are co-transfected with helper plasmids coding for the viral nucleocapsid (N) and polymerase (L) proteins. Using immunostaining we have successfully detected the N and L proteins in both mammalian and insect cells, and showed that they localize to cytoplasmic bodies. Current activities aim to establish RNP-dependent expression of the reporter genes and will be discussed. 40

141 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P047 ROLE OF HOST PHOSPHATASE PP2A IN FILOVIRUS REPLICATION CYCLE Nadine Biedenkopf *, Cornelius Rohde, Thomas Kruse 2, Emil P. T. Hertz 2, Jakob Nilsson 2, Stephan Becker Institute of Virology, PHILIPPS-UNIVERSITÄT MARBURG, Marburg, Germany, 2 University of Copenhagen, Copenhagen, Denmark Abstract: Ebolavirus and Marburg virus, both belonging to the family of Filoviridae, are the causative agents of a severe fever with high fatality rates among humans. The only nonpathogenic member within the Filoviridae is Ebolavirus subtype Reston Ebolavirus. Little is known regarding the molecular mechanisms these viruses utilize in order to transcribe and replicate their genome. Ebolavirus transcription is strongly dependent on dephosphorylation of the essential viral transcription factor VP30. Recently, we could show that the nucleoprotein NP recruits the host phosphatase PP2A-B56 via a B56 binding motif (LxxIxE) in order to dephosphorylate and thereby activate VP30. A peptide inhibitor of the LxxIxE motif resulted in inhibition of Ebolavirus transcription. Interestingly, the B56 binding motif LxxIxE is also found in other members of the family Filoviridae, including Marburg and Reston Ebolavirus. The role of VP30 dephosphorylation for transcription of these viruses is currently not understood. Here, we present data indicating that dephosphorylation of VP30 by PP2A B56 plays an important role for transcription of Reston Ebolavirus and Marburgvirus, as well. 4

142 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P048 STRUCTURAL CHARACTERISATION OF THE METAPNEUMOVIRUS TRANSCRIPTASE COMPONENTS P AND M2- BY MODELLING, CRYSTALLOGRAPHY, AND SAXS Max Renner *, Jonathan Grimes, Cédric Leyrat 2 Division of Structural Biology, UNIVERSITY OF OXFORD, Oxford, United Kingdom, 2 Institut de Génomique Fonctionnelle, Montpellier, France Abstract: The phosphoprotein (P) is the essential cofactor of the RNA-dependent RNA polymerase (L) of unsegmented, (- )RNA viruses. P serves as a bridge between the viral polymerase and its nucleoprotein-rna template and prevents unproductive encapsidation of host nucleic acids by the nucleoprotein (N). In addition, P recruits the viral transcription antiterminator M2-, which is crucial for the synthesis of full-length mrnas in pneumoviruses. P proteins of negative strand viruses form multifunctional homomultimers with large intrinsically disordered regions (IDRs). In human metapneumovirus (HMPV) P forms homotetramers via a stable central coiled-coil domain (Pcore) flanked by IDRs. Here we combined x-ray crystallography with small angle x-ray scattering (SAXS) and molecular modelling to characterise the P protein and its interaction with the transcription antiterminator M2-. Our structural description of P and its complex with M2- captures the dynamic and flexible character of pneumoviral transcriptase components and highlights the presence of transiently stable elements within the IDRs. 42

143 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P049 CHARACTERIZING DEFECTIVE VIRAL GENOMES OF CANINE DISTEMPER VIRUS Natasha L. Tilston-Lunel *, Linda J. Rennick, Stephen R. Welch 2, Sham Nambulli, Christina F. Spiropoulou 2, Stuart T. Nichol 2, W. Paul Duprex Microbiology, NEIDL, Boston University School of Medicine, Boston, 2 VSPB, Centers for Disease Control and Prevention, Atlanta, United States Abstract: The error-prone nature of RNA-dependent RNA polymerases (RdRp) drives the diversity we observe in RNA viral populations. A sub-species of this diversity are defective viral genomes (DVGs) which arise due to premature termination of genome synthesis by the RdRp and its reinitiation either onto the same template or onto the newly synthesized strand. Replication competent DVGs alter the dynamics of a viral population by their ability to interfere with standard virus replication and/or by stimulating type-i IFN induction. Such DVGs are known as defective interfering (DI) genomes and these have been studied for many RNA viruses. Dissecting molecular mechanisms that contribute to DI generation and inhibition could potentially allow us to harness the interfering traits for use as vaccine adjuvants or broad-spectrum antivirals. An idea that has been proposed and studied for influenza virus. Here, we present work carried out using canine distemper virus (CDV). CDV is a tractable biosafety level-2 (BSL-2) paramyxovirus. Using recombinant (r) CDV RI we cultured various independently generated rcdv RI stocks across 0 passages in Vero-cCD50 cells. Passages were subjected to a combination of Sanger sequencing and next-generation sequencing to identify DVGs arising naturally in vitro. Isolated DVGs were cloned and assessed for their ability to interfere with rcdv RI replication in vitro. Further, using a CDV minigenome expressing a fluorescent reporter we demonstrate that a synthetic DVG can be maintained along with the full-length virus over several passages in an IFN competent canine B-cell line (CLBL-). We have an established pipeline to engineer natural and synthetic DIs which is transferrable to other paramyxoviruses. Understanding the common mechanisms of inhibition by natural DIs could potentially be powerful against the continuously evolving nature of RNA viruses. 43

144 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P050 RABIES VIRUS L-PROTEIN C-TERMINAL DOMAINS ARE STRUCTURALLY COORDINATED BY THE N-TERMINUS OF THE VIRAL PHOSPHOPROTEIN Joshua Horwitz *, Stephen Harrison 2, Sean Whelan MBIB, 2 BCMP, Harvard Medical School, Boston, United States Abstract: The 250 kda rabies virus large protein (L) contains all of the enzymatic activities necessary for mrna transcription and genome replication. The template for RNA synthesis is the viral genomic RNA completely coated by a sheath of the viral nucleocapsid protein (N), and L must bind the viral phosphoprotein (P) to engage the N-RNA template. Using an in vitro assay comprising purified rabies L protein and chemically synthesized short template RNA, we previously defined a minimal region of P that stimulates the RNA dependent RNA polymerase activity of rabies virus L. Using electron cryo-microscopy we previously provided an atomic model of L for the related vesicular stomatitis virus. L comprises five distinct domains, including three enzymatic domains: an N-terminal RNA-dependent RNA polymerase (RdRP) and RNA capping enzyme (Cap), and three globular C-terminal domains, including a connector domain (CD), methyltransferase domain (MT), and C-terminal domain (CTD). Using negative-stain electron microscopy (nsem) of rabies virus L we show that the globular C-terminal domains of L are highly flexible relative to the N-terminal RdRP and Cap domains in the absence of P. We identify a minimal region of P that coordinates these C-terminal domains atop the RdRP and Cap domains. Additionally, by fusing GFP to the N-terminus of P, we can approximate the location of the N-terminus of P with respect to the C-terminal domains of L. 44

145 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P05 EFFECT OF MUTATIONS IN THE GENE-END SEQUENCE ON RSV TRANSCRIPTION Jean-Francois Eleouet, Charles-Adrien Richard * Unité de Virologie et Immunologie Moléculaires, INRA - Université Paris-Saclay, Jouy en Josas, France Abstract: The respiratory syncytial virus (RSV) genome is a single strand, negative sense RNA of about 5 kb that is packaged by the nucleoprotein (N) and maintained as a left-handed helical N-RNA ribonucleoprotein complex (RNP). This RNP is the template for two distinct activities: RNA replication and RNA transcription that generates 0 capped and polyadenylated mrnas. RNA transcription is carried out by the viral RNA-dependent RNA polymerase complex (RdRp), composed of the viral N, P, L and M2- proteins. RSV transcription proceeds through sequential stop-and-restart events, in which the RdRp recognizes gene start (GS) and gene end (GE) sequences that flank each gene and direct initiation and termination of transcription, respectively. By increasing the processivity of the RdRp, RSV transcription antiterminator protein M2- prevents premature transcription termination. M2- is an RNA binding protein that binds preferentially to GE and A-rich sequences present on RSV mrnas. The exact mechanism of how M2- improves transcription and its relation with its RNA binding abilities remain to be clarified. In this work, the effect of GE sequence variation was analyzed by using a bicistronic RSV minigenome coding for Gaussia and Firefly luciferases. Relative expression of the Gaussia and Firefly luciferase reporters were compared between wild type or mutated GE sequences in the absence or in the presence of M2-. The results highlighted the critical role of some nucleotides in the GE sequence for efficient transcription termination of the first gene and reinitiation at the second GS signal. 45

146 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P052 THE C-TERMINAL FRAGMENT OF THE RESPIRATORY SYNCYTIAL VIRUS PHOSPHOPROTEIN INHIBITS THE VIRAL POLYMERASE ACTIVITY Koyu Hara *, Kenichiro Yaita, Takahito Kashiwagi, Hiroshi Watanabe Department of Infection Control and Prevention, KURUME UNIVERSITY SCHOOL OF MEDICINE, Kurume, Japan Abstract: Respiratory syncytial virus (RSV) can lead to serious lower respiratory tract illness, especially for infants and older adults. However, an effective antiviral therapy or vaccine has not been developed. Here, we demonstrate that a fragment of RSV phosphoprotein (P) has an ability to inhibit the viral polymerase activity and suggest a significant potential to treat RSV infection. Viral ribonucleoprotein (RNP) was reconstituted by transfecting plasmids expressing P, RNA polymerase (L), nucleoprotein (N), M2- and vrna-like luciferase RNA into BHK-2 derived BSR-T7/5 cells, stably expressing T7 RNA polymerase (gifted from Dr. Jean-François Eléouët). Concurrently, a plasmid expressing serial P fragments was transfected as a competitor and the luciferase activity was measured at 24h post transfection to evaluate the polymerase activity. We found that C-terminal half fragment of P severely inhibited the polymerase activity. A precise deletion analysis identified the three domains essential for the inhibitory activity: the oligomerization domain (aa 30-50), N-binding domain (aa 60-80) and L binding domain (aa 22-24). When the TAPtagged P fragment was co-expressed with each component of RNP and purified by TAP-tag method, the P fragment was able to pull-down P, L and M2-. Specifically, a strong interaction with the full-length P was observed. The P protein forms homotetramers and acts as an essential cofactor of the viral polymerase L by recruiting L to the nucleocapsid. Our results suggest that the P fragment primarily targets the full-length P, possibly interfering the tetramer formation, thereby inhibiting the polymerase activity. 46

147 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P053 INVESTIGATION OF A PROTEIN KINASE RESPONSIBLE FOR THE PHOSPHORYLATION OF MEASLES VIRUS NUCLEOPROTEIN Akihiro Sugai *, Hiroki Sato, Misako Yoneda, Chieko Kai The Institute of Medical Science, Laboratory Animal Research Center, THE UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: Measles virus (MeV) N and P proteins are known to be phosphorylated to modify viral gene expression and viral genomic stability. It has been reported that Casein kinase II phosphorylates the P protein, while a responsible protein kinase for the phosphorylation of the N protein has not been identified. The N protein is typically phosphorylated in its tail-domain at S479 and S50. The mechanism phosphorylating these sites of the N protein, however, has not been clarified. In this study, we conducted in vitro kinase assay in the presence or absence of various protein kinase inhibitors to identify a responsible protein kinase for the phosphorylation of the N protein. We first purified non-phosphorylated form of the GSTtagged N-tail domain (GST-Nt) as a substrate and conducted in vitro kinase assay using 293 or COS7 cell lysate. We demonstrated that these cell lysate phosphorylated the GST-Nt successfully. This phosphorylation signal was not derived from GST-tag. Conversely, these cell lysate failed to phosphorylate the GST-Nt in the presence of c-jun N-terminal kinase (JNK) inhibitor (SP60025). We also showed that the JNK inhibitor suppressed the phosphorylation of transiently expressed full-length N protein in COS7 cells. Using the specific antibodies against phosphorylated S479 or S50, we demonstrated that JNK inhibitor was able to block both of the phosphorylation modification, suggesting that S479 and S50 sites were phosphorylated by the same protein kinase. These findings are helpful in identifying a responsible kinase that phosphorylates S479 and S50 of N protein and also in understanding the mechanism regulating phosphorylation of the N protein. 47

148 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P054 DISCOVERY AND DETECTION OF EBOV-ENCODED MICRORNA-LIKE MOLECULES IN INFECTED CELLS AND NON-HUMAN PRIMATES Ashley L. Silvia *, 2, Anthony Griffiths, 2 Texas Biomedical Research Institute, 2 UT Health, San Antonio, United States Abstract: MicroRNAs (mirnas) are important regulators of various cellular processes and can be used as biomarkers for disease. Some viruses have been shown to encode mirnas that may act using similar mechanisms as host mirnas to affect viral or host transcripts. Several reports have predicted Ebola virus (EBOV) encoded mirna-like molecules using computer-based algorithms, however, studies to detect EBOV-encoded mirnas during infection have been limited to one study using human serum. Using small RNA deep sequencing, we detected both novel and previously predicted EBOVencoded mirna-like molecules in various infected cell lines 26 hours post infection. Using the sequencing data, we designed custom qpcr TaqMan mirna assays to quantify expression in various cell lines at, 6, and 26 hours post infection and in sera from EBOV-infected non-human primates (NHPs). Future experiments will aim to address the biogenesis and potential role of these molecules during EBOV infection. 48

149 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P055 STRUCTURAL CHARACTERIZATION OF RSV PHOSPHOPROTEIN Christophe Cardone *, Nelson Pereira, Safa Lassoued, Charles-Adrien Richard 2, Jenna Fix 2, Marie Galloux 2, Jean- François Eléouët 2, Christina Sizun CNRS, ICSN, Gif-sur-Yvette Cedex, 2 INRA, VIM, Jouy-en-Josas, France Abstract: In Mononegavirales, the phosphoprotein (P) is the main co-factor of the viral RNA polymerase. It positions the polymerase complex onto its template, a ribonucleoprotein complex formed of genomic RNA and the associated nucleoprotein. P proteins recruit viral and cellular proteins to the viral RNA dependent RNA polymerase (RdRp) complex. The mechanisms of action of these proteins are still not fully understood. In particular they often elude structural characterization, owing to extensive structural disorder. Among Mononegavirales, human Respiratory Syncytial Virus (hrsv), of the Pneumoviridae family, is the main viral cause of lower respiratory tract illness worldwide. We have undertaken a structural investigation of hrsv P and of its interactions by using nuclear magnetic resonance spectroscopy, an atomic scale tool well adapted to study highly dynamic proteins. hrsv P is a 24-residue protein with a short ~40 residue oligomerization domain (OD), flanked by large intrinsically disordered regions (IDRs) at its N-and C-termini. On closer inspection, NMR shows that these IDRs are very heterogeneous. Almost stable C-terminal helices are formed downstream of the OD. Very weak helical propensity is observed in two N-terminal regions. All transient helices as well as the OD mediate transient internal long-range contacts. These likely contribute to overall compaction of P in the absence of a defined tertiary structure, protection against proteolysis and/or unspecific contacts. Indeed, these transient secondary structure elements in hrsv P provide protein binding sites that are specifically recognized by RdRp proteins, like the hrsv transcription antitermination factor M2-. Very recently we found that a transient extended region next to the M2- binding site was recognized by the cellular phosphatase PP. The proximity of both sites in P tetramers explains how P-dependent dephosphorylation of M2- by PP can take place. 49

150 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P056 DETECTION AND CHARACTERISATION OF INFLUENZA VIRUS RNA POLYMERASE DIMERS USING BIMOLECULAR FLUORESCENCE COMPLEMENTATION (BIFC) Alex Walker *, Haitian Fan, David Bauer, Ervin Fodor Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom Abstract: Influenza virus encodes a heterotrimeric RNA-dependent RNA polymerase (RdRP), composed of subunits PB, PB2 and PA. The RdRP carries out both transcription and replication of the viral RNA genome segments in the context of ribonucleoproteins (RNPs). Replication of negative-sense viral RNA is a two-step process, progressing via a positive-sense complementary RNA intermediate. The mechanism of viral genome replication is mostly unknown, though there are multiple reports indicating RdRP-RdRP interactions may be central for the process. Purified RdRPs from human and avian influenza A viruses both form dimers of heterotrimers in solution. Using a combination of X-ray crystallography and SAXS analysis our group has identified the interface involved in RdRP dimerization, which is primarily located on the PA C-terminal domain. We establish a bimolecular fluorescence complementation (BiFC) assay to monitor intermolecular interactions between RdRPs in cells expressing viral RNPs. Using this system we confirm the existence of RdRP dimers in the context of actively replicating RNPs. Mutating amino acid residues at the identified dimer interface causes loss dimerization and inhibition of RNA replication in minigenome assays. These data suggest that dimerisation of RdRP via the PA C-terminal domain is important for replication of the viral RNA genome. 50

151 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P057 THE CONNECTOR DOMAIN OF VESICULAR STOMATITIS VIRUS LARGE PROTEIN IS AN ACCEPTOR SITE FOR PHOSPHOPROTEIN BINDING Joseph Gould *, Shihong Qiu, Qiao Shang, Peter Prevelige, Chad Petit 2, Todd Green Microbiology, 2 Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, United States Abstract: Vesicular stomatitis virus (VSV) is an archetypical negative strand virus with a simple molecular biology. In addition to its importance as a model system for the study of Mononegavirales, VSV has been explored as a potential oncolytic agent and as a vaccine platform. VSV encodes for a large (L) protein, which supplies all catalytic activities for viral RNA synthesis and transcript modification, and phospho- (P) protein, a multifunctional molecular chaperone and cofactor. In order for viral mrna and genomic RNA synthesis to take place, an interaction between the L and P proteins must occur. Despite the importance of L-P interaction(s), the interface or interfaces at which they occur have not been described. Using structural and biophysical methods, we report here that the connector domain of VSV L, which previously had no assigned functional significance, is an acceptor site for P-protein binding. We offer a model of the connector-phosphoprotein interface, supplemented by functional information from targeted disruption of the interaction in a minigenome reporter system. Given the extensive conservation of large and phospho- proteins in Mononegavirales, we assert this discovery has implications for studies of negative strand viral RNA synthesis as a whole. 5

152 EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P058 MEASLES VIRUS PHOSPHOPROTEIN MULTIMERIZATION DOMAIN: COILED-COIL FEATURES AND FUNCTION Antoine SCHRAMM *, Louis-Marie BLOYET 2, Carine LAZERT 2, Maggy HOLOGNE 3, Olivier WALKER 3, Denis GERLIER 2, Sonia LONGHI CNRS UMR 7257, Aix Marseille University, Architecture et Fonction des Macromolécules Biologiques (AFMB), Marseille, 2 CIRI INSERM U, CNRS UMR5307, University Lyon, ENS Lyon, 3 CIRI INSERM U, CNRS UMR5308, University Lyon, ENS Lyon, Univ Lyon, Lyon, France Abstract: Measles virus (MeV) phosphoprotein (P) plays a central role in viral transcription and replication. Understanding the relationships between sequence, structure, dynamics and function of P is crucial to draw a consistent mechanistic model for viral transcription and replication. This 54 kda protein is prevalently intrinsically disordered with the notable exception of a small 3-helix bundle (termed X domain, XD) and a coiled-coil multimerization domain (PMD). Coiled-coils consist in multiple a-helices packed together in a fiber shape that results from a repeated seven residues motif (heptad). MeV PMD is tetrameric and features a stammer, i.e. a three-residues insertion between two heptad repeats. This induces the formation of a 30 helix and hence to a local distortion referred to as kink. To achieve further insights onto the roles played by PMD, PMD variants were characterized in cellula, in vitro and in silico in order to understand how PMD may influence the polymerase cofactor activity of P. Biochemical and biophysical characterization made use of circular dichroism, X-ray crystallography and molecular dynamic simulations. The introduction of a proline causes the 30 helix to shift with no concurrent functional impact on transcription and replication. In parallel, we investigated the coiled-coil cohesion by substituting buried residues with residues of varying hydrophobicity. The variants possess the same oligomeric state as that of the wild-type P protein but exhibit different stabilities and activities in a minigenome assay. 52

153 NSV 208, Verona Abstract Book EXPRESSING AND MULTIPLYING viral gene expression Abstract final identifier: P059 COMBINED PROTEOMICS AND TRANSCRIPTOMICS SUGGESTS THAT M MRNA SPLICING INFLUENCES IAV HOST SPECIFICITY Boris Bogdanow *, 2, Anne Sadewasser, Gudrun Heins, Immanuel Husic 2, Katharina Paki, Barbara Vetter 3, Xi Wang 4, Jingyi Hou 4, Wei Chen 4, Lüder Wiebusch 3, Thorsten Wolff, Matthias Selbach 2 Unit 7, Robert-Koch-Institute, 2 Proteome Dynamics, Max-Delbrück-Center, 3 Laboratory for Pediatric Molecular Biology, Charité University Medicine, 4 BIMSB, Max-Delbrück-Center, Berlin, Germany Abstract: A successful Influenza A virus (IAV) infection requires that viral proteins are synthesized at the right time in the right order and correct proportions. Many avian IAV strains lack adaptation to the human host, which leads to abortive nonproductive infections in human cell lines. Here we used transcriptomic and proteomic methods to assess the regulation of viral and host mrna and protein production comparatively for an avian (H3N2/A/Mallard) and a human (H3N2/A/Panama) influenza isolate. First, we monitored protein synthesis profiles in human A549 cells in 4 h intervals over a 6 h period. We observed the expected overall shut-down in host protein synthesis for both strains, which was more pronounced at later intervals. Cluster analyses revealed subsets of host proteins that escaped the shut-down, including proteins involved in antiviral response, immune response and translation. Concomitantly, synthesis of viral proteins was potently induced and showed striking strain differences for the M, HA and NA proteins. mrna levels obtained via RNA-seq indicate that the differences in protein production between the two strains could largely be explained by differences at the mrna level. The impairment of the avian strain to produce high amounts of M was linked to an increased intensity of M mrna splicing. Experiments using eukaryotic expression vectors containing the coding sequence of the M segment indicate that the 3' splice site is responsible for strain-specific control of M mrna splicing. We then integrated the avian segment 7 3 splice site into the Panama strain. This mutant virus replicated to lower titers and produced lower levels of M mrna and protein than the wildtype. This suggests that control of M mrna splicing by the 3 splice site influences IAV host specificity. 53

154 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P060 MXA-DEPENDENT INFLAMMASOME RESTRICTS INFLUENZA A VIRUS INFECTION IN RESPIRATORY EPITHELIAL CELLS SangJoon Lee, Peter Staeheli 2, Kyosuke Nagata 3, Atsushi Kawaguchi * 3 PhD Program in Human Biology, University of Tsukuba, Tsukuba, Japan, 2 Institute of Virology, University Medical Center Freiburg, Freiburg, Germany, 3 Faculty of Medicine, University of Tsukuba, Tsukuba, Japan Abstract: Inflammasomes are cytosolic molecular complexes that typically consist of ASC, caspase-, and cytoplasmic pathogen recognition receptors (PRRs) such as NOD-like receptor family proteins. Previous studies demonstrated that influenza A virus (IAV) infection triggers NLRP3-mediated inflammasome in immune cells. However, NLRP3 is not expressed in respiratory epithelial cells, although an inflammasome is formed upon IAV infection. To identify the novel respiratory epithelium-specific inflammasome receptor, we performed the high-content shrna library screening and LC-MS proteomics analysis with human respiratory epithelial cells. Using two different screening systems, we identified human myxovirus resistance protein (MxA) as a novel inflammasome receptor in respiratory epithelial cells that specifically forms inflammasome complexes with ASC and caspase- upon viral infection. The secretion of IL-β upon IAV infection was reduced by MxA knockdown. The reduction of IL-β secretion by MxA knockdown was complemented by NLRP3 expression in respiratory epithelial cells. In vivo analysis using hmxa-transgenic mice revealed that the rapid activation of the MxA inflammasome in bronchiolar epithelial cells represses virus spread from the bronchioles to distal alveolar regions. Our study highlights a novel function of MxA that is required for inflammasome activation in the respiratory epithelium after IAV infection which enhances virus resistance. 54

155 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P06 CHARACTERIZATION OF VIRUS - HOST INTERACTION DYNAMICS WITHIN THE RESPIRATORY EPITHELIUM Ronald Dijkman *, 2, Melle Holwerda, 2, Hulda Jonsdottir, 2, Volker Thiel, 2 Federal Food Safety and Veterinary Office, Institute of Virology & Immunology, Bern and Mittelhäusern, 2 Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty of the University of Bern, Bern, Switzerland Abstract: The respiratory epithelium is the main entry port of respiratory pathogens and serves as an important barrier to infection. Within the respiratory epithelium the innate immune system plays a major protective role as the first line of defence. However, the dynamics of the innate immune response towards respiratory pathogens at the main entry port is not well understood. To characterize these complex dynamics,we established transgenic primary human Airway Epithelial Cell (haec) cultures harbouring a reporter cassette with either fluorescent or enzymatic reporter proteins under the control of a promoter element known to be induced during the intermediate (e.g. IFNB) or late (e.g. MxA) stage of the interferon (IFN) response. Besides phenotypical comparison, the functional comparison of these reporter haecs with naïve haecs using exogenous stimuli revealed similar characteristics. Furthermore, infection experiments with Influenza A virus HN (pdm2009) wildtype, or a NS mutant, demonstrated no significant differences of viral kinetics between reporter or naïve haecs. We confirmed that pdmhn does not evoke a pronounced IFN response during infection, in contrast to the pdmhn NS mutant, suggesting that pdmhn is well equipped to evade the first line of defence at the main entry port. Most importantly, the reporter haecs allow for active monitoring of the innate immune response, providing a robust method to gain detailed knowledge on virus host interaction dynamics within the respiratory epithelium. 55

156 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P062 NUCLEAR RESIDENT RIG-I SENSES INFLUENZA A VIRUS REPLICATION MOUNTING AN ANTIVIRAL RESPONSE Guanqun Liu, Yao Lu, Qiang Liu, Yan Zhou * University of Saskatchewan, Saskatoon, Canada Abstract: The spatiotemporal detection of influenza A virus (IAV) by RIG-I is puzzling; as a rare nuclear-replicating RNA virus, IAV conceals its genome replication in the nucleus potentially limiting the access of cytoplasmic RNA sensors. Here, we report the existence of nuclear RIG-I under homeostatic conditions sensing IAV nuclear replication. We demonstrate an intimate association of RIG-I activation with nuclear viral RNA accumulation, and specific nuclear RIG-I staining unless circumvented by the introduction of a nuclear export signal. A nuclear-localized RIG-I efficiently mediated a type I interferon response via the canonical signaling axis, and exhibited greater signaling capacity following the recognition of viral replication. Reconstitution of cells with nuclear RIG-I exerted exclusive sensing and restriction of IAV, but not of cytoplasmicreplicating Sendai virus. These results demonstrate for the first time, the sensing of a RNA virus by RIG-I within the nucleus and implicate a previously unrecognized subcellular milieu for RLR sensing. 56

157 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P063 SPECIFICITY AND FUNCTIONAL INTERPLAY BETWEEN INFLUENZA VIRUS PA-X AND NS SHUTOFF ACTIVITY Toru Takimoto *, Chutikarn Chaimayo, Megan Dunagan Microbiology & Immunology, University of Rochester Medical Center, Rochester, United States Abstract: Influenza A viruses modulate host antiviral responses to promote viral growth and pathogenicity. Through viral PA-X and NS proteins, the virus is capable of suppressing host protein synthesis, termed host shutoff. Although both proteins are known to induce general shutoff, specificity of target genes and their functional interplay in mediating host shutoff are not fully elucidated. In this study, we generated four recombinant influenza A/California/04/2009 (phn) viruses containing mutations affecting the expression of active PA-X and NS. We then analyzed viral growth, the kinetics of viral RNA and protein synthesis, host shutoff function, and specific mrna targets of these mutants. Our results show that PA-X is the major protein that affects general host protein expression. Intriguingly, our RNA-Seq data from infected human airway A549 cells indicate that active NS specifically targets host mrnas related to interferon (IFN) and cytokine signaling pathways. Specificity of target mrnas was less evident in PA-X, although it preferentially degraded transcripts from genes associated with cellular protein metabolism and protein repair. Interestingly, in the presence of active NS, PA-X also degraded viral mrnas, especially NS segments. The virus expressing active NS with reduced amount of PA-X most efficiently suppressed antiviral and innate immune responses in human cells, indicating the need for influenza virus to optimize the contribution of these two shutoff proteins to circumvent the host environment for optimum growth. 57

158 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P064 THE AIRWAY EPITHELIUM MAINTAINS THE BARRIER FUNCTION AFTER INFLUENZA VIRUS INFECTION DESPITE THE EXTENSIVE LOSS OF CILIATED CELLS Georg Herrler *, Nai-Huei Wu, Wei Yang, Peter Valentin-Weigand 2 Virology, 2 Microbiology, Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany Abstract: We established an air-liquid interface (ALI) culture system to analyze the infection of differentiated airway epithelial cells by influenza viruses. Porcine ALI-cultures were sensitive to infection by swine and human influenza viruses. Release of virus at a high level was observed for up to eight dpi. Infection was characterized by a dramatic change of the epithelium. A large number of ciliated cells were lost due to virus-induced apoptosis. As a consequence, the thickness of the epithelial layer was reduced. However, the epithelial cell layer remained intact and there was no decrease of the transepithelial electrical resistance (TEER) and the tight junction (TJ) network was not destroyed. Our findings are explained by the regeneration of epithelial cells to compensate for the loss of ciliated cells. Basal cells had started to differentiate into specialized cells. The early differentiation process was sufficient to maintain the barrier function as indicated by the TJ network and the TEER. However, the differentiation process had not yet proceeded to the generation of ciliated cells. During differentiation, the epithelial cells showed different surface properties as compared to well-differentiated cells. The latter cells were characterized by the presence of a2,6-linked sialic acid, whereas basal cells mainly contained a2,3-linked sialic acid. Lectin staining indicated that both linkage types are present on the surface of regenerating cells. Because of the different surface markers, regenerating and well-differentiated airway epithelial cells may have different susceptibilities to infection by viral and bacterial pathogens Taken together, the ALI culture system allows to analyze the regeneration of airway epithelial cells after influenza virus infection. These cells will be valuable to study viral-viral and viral-bacterial co-infections. 58

159 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P065 INSIGHTS INTO TETHERIN COUNTERACTION BY THE EBOLA VIRUS GLYCOPROTEIN Markus Hoffmann, Mariana González-Hernández, Inga Nehlmeier, Constantin Brinkmann, Verena Krähling 2, Laura Behner 2, Anna-Sophie Moldenhauer, Nadine Krüger 3, Julia Nehls 4, Michael Schindler 4, Andrea Maisner 2, Stephan Becker 2, Stefan Pöhlmann * Infection Biology Unit, German Primate Center, Göttingen, 2 Institute of Virology, Philipps-University Marburg, Marburg, 3 Institute of Virology, University of Veterinary Medicine Hannover, Hannover, 4 Institute of Medical Virology and Epidemiology of Viral Diseases, University Hospital Tübingen, Tübingen, Germany Abstract: Tetherin is an interferon (IFN) induced host cell factor that can inhibit release of progeny virions from infected cells. The Ebola virus glycoprotein (EBOV-GP) counteracts tetherin in transfected cells but the determinants in GP that control tetherin antagonism are incompletely understood. Moreover, it is unclear whether tetherin counteraction occurs in infected cells and contributes to viral spread. Here, we show that residues in the receptor binding domain of GP and a GXXXA motif in the transmembrane domain (TMD) of GP are required for counteraction of human tetherin. In addition, we provide the first evidence that GP can antagonize human tetherin in the context of an infectious, replication-competent EBOV surrogate chimeric VSV encoding EBOV-GP. Finally, we found that tetherin of fruit bats, the EBOV reservoir, is largely resistant to counteraction by EBOV-GP, at least when expressed at high levels, and that tetherin expression is IFNinducible in fruit bat cells. More importantly, we discovered that tetherin expression is critical for efficient IFN-mediated inhibition of EBOV and Nipah virus (NiV) infection of fruit bat cells. Collectively, our results identify determinants in GP that are required for counteraction of human tetherin and provide evidence that counteraction may promote viral spread in infected cells. Moreover, our findings indicate that tetherin might be important for IFN-dependent control of EBOV and NiV infection of fruit bats, the natural reservoir. 59

160 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P066 INFLUENZA VIRUS MOUNTS A TWO-PRONGED ATTACK ON RNA POLYMERASE II TRANSCRIPTION David L. V. Bauer *, Michael Tellier, Mónica Martínez-Alonso, Takayuki Nojima, Nick J. Proudfoot, Shona Murphy, Ervin Fodor Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom Abstract: Influenza virus intimately associates with host RNA Polymerase II (Pol II) and mrna processing machinery. Here, we use mammalian nascent elongating transcript sequencing (mnet-seq) to examine Pol II behavior during viral infection. We show that influenza virus executes a two-pronged attack on host transcription. First, cleavage of nascent host transcripts by the viral polymerase causes Pol II to terminate prematurely at the start of genes. Second, virus-induced cellular stress leads to a catastrophic failure of Pol II termination at poly(a) sites, with transcription often continuing for tens of kilobases. Defective Pol II termination occurs independently of the ability of the viral NS protein to interfere with host mrna processing. Instead, this termination defect is a common effect of diverse cellular stresses and underlies the production of previously-reported downstream-of-gene transcripts (DoGs). Our work has implications for understanding not only host-virus interactions, but also fundamental aspects of mammalian transcription. Graphical Abstract: 60

161 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P067 FLYING UNDER THE (R)ADAR: ADAR-P50 EDITING COUNTERACTS INTRINSIC IMMUNITY ACTIVATION BY SELF AND VIRAL DOUBLE STRANDED RNA Christian K. Pfaller *, Ryan C. Donohue, 2, Stepan Nersisyan 3, 4, Leonid Brodsky 4, Roberto Cattaneo, 2 Molecular Medicine, 2 Mayo Clinic Graduate School of Biomedical Sciences, MAYO CLINIC, Rochester, United States, 3 Lomonosov Moscow State University, Moscow, Russian Federation, 4 Tauber Bioinformatics Research Center, University of Haifa, Haifa, Israel Abstract: Adenosine deaminase acting on RNA (ADAR) edits and destabilizes double-strand RNA (dsrna). This prevents translational shutdown through PKR, but favors viral replication. To understand how, we generated ADAR- knockout cells, and cells expressing either nuclear ADAR p0, or interferon-inducible cytoplasmic ADAR p50. We show here that ADAR primarily edits Alu elements embedded in opposite polarity in 3 untranslated regions (UTRs) of more than hundred polymerase II transcripts. Through fine mapping of A-to-I editing events in RNA deep sequencing analyses, we determined editing scores for each position in these long UTRs, and validated the editing patterns in primary human samples. Edited elements form extensive dsrna structures conserved in primates, and even in rodents, confirming their broad biological relevance. They activate intrinsic immune responses in ADAR-deficient cells, inhibiting viral replication. Reconstitution with catalytically active ADAR p50 suppresses autoimmunity and rescues replication of measles virus (MeV), but not that of a mutant generating excess dsrna, which activates PKR. In contrast, catalytically inactive ADAR p50 has modest effects. We also analyzed editing of dsrna structures in MeV genomes, concluding that these structures underlie the same type of editing as cellular dsrna. Thus, our data indicate that intrinsic immunity recognizes self and foreign dsrna through the same ADAR-dependent mechanism. By interfering with the detection of low amounts of dsrna, ADAR increases tolerance to autoimmunity but weakens the antiviral response. 6

162 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P068 GLOBAL SIRNA SCREEN IN HUMAN MACROPHAGES IDENTIFY TBCD5 AS A NOVEL CELLULAR RESTRICTION FACTOR FOR INFLUENZA A VIRUS REPLICATION Laura Martin-Sancho *, Ariel Rodriguez-Frandsen, Shashank Tripathi 2, Maite Sanchez-Aparicio 2, Judd Hultquist 3, David Jimenez-Morales 3, Paul De Jesus, Max Chang 4, Hong Moulton 5, David Stein 5, Chris Benner 4, Megan Shaw 2, Nevan Krogan 3, Adolfo Garcia-Sastre 2, Sumit Chanda Infectious and Inflammatory Center, SBP Medical Discovery Institute, La Jolla, 2 Microbiology, Icahn School of Medicine at Mount Sinai, New York, 3 Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, 4 School of Medicine, University of California San Diego, La Jolla, 5 Department of Biomedical Sciences, Oregon State University, Corvallis, United States Abstract: Influenza A virus (IAV) is an important respiratory pathogen. Current treatments to combat influenza infection are suboptimal and new strategies are required. Targeting cellular factors that naturally prevent influenza infection represents a powerful alternative approach, since these will benefit from broad spectrum activities and are likely to confer a higher barrier to viral resistance. Using genome-wide sirna screening in human macrophages, we successfully identified 04 novel restriction factors that affect the replication of various IAV strains differing in pathogenesis. This data was integrated with global transcriptomic and proteomic datasets generated using the same experimental settings, to identify those host factors that are regulated upon IAV infection and that modulate their responses through physical interactions. Among the identified host factors, we focused on the GTPase-activating protein TBCD5. TBCD5 negative effect on viral replication was validated using CRISPR KO cells and in vivo models. Additionally, we detected co-localization of TBCD5 and M2 in the perinuclear region of IAV-infected cells. Critically, these meta-analyses provide comprehensive maps of cellular factors and pathways that regulate IAV infection, and that have the potential to serve as the basis to develop new strategies to combat IAV infection. 62

163 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P069 INTERFERENCE BEFORE INTERFERON: CHARACTERIZATION OF INTERFERON SIGNALING INDEPENDENT ANTIVIRAL INNATE IMMUNITY AGAINST INFLUENZA A VIRUSES Shashank Tripathi *, 2, Sumit Chanda 3, Adolfo Garcia-Sastre, 2 Microbiology, 2 Global Health and Emerging Pathogen Institute, Icahn School of Medicine at Mount Sinai, New York, 3 Immunity and Pathogens Program, Sanford Burnham Prebys Medical Discovery Institute, San Diego, United States Abstract: The mammalian antiviral innate immune response begins with the detection of viral pathogen associated molecular patterns by host pattern recognition receptors, which leads to activation of Interferon Regulatory Factors (IRFs), which transcribe Interferons (IFN), which in turn activate the JAK-STAT pathway and leads to expression of a battery of antiviral genes collectively called as Interferon Inducible Genes (ISGs). Among IRFs, IRF3 is the key driver of IFN induction. In this study we examined the cellular transcriptome induced by IRF3 in absence of downstream IFN signaling. For this we overexpressed constitutively active form of IRF3 in STAT -/- mouse fibroblasts and performed expression profiling using Illumina microarray. Results indicated that IRF3 induces a large set of ISGs, even in the absence of downstream IFN signaling. Importantly, this also resulted in the antiviral response demonstrated by reduced Influenza A virus (IAV) replication in STAT-/- cells overexpressing IRF3. We compiled additional similar gene lists from published studies, which included genes induced by IRF, IRF3 and IRF7 in STAT-/- or IFNAR-/- background, and combined them with IRF3 overexpression dataset. From the resulting combined list, we tested the anti-iav activity of 58 genes, on a lentiviral overexpression setup with a Luciferase reporter-iav. This resulted in the identification of a set of anti-iav genes which manifest antiviral activity in absence of IFN signaling. Further characterization of the underlying mechanisms of novel anti-iav factors identified in this study and validation of their independence from IFN signaling is being carried out presently. 63

164 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P070 COMPARATIVE TRANSCRIPTOMICS REVEAL THAT ANTIVIRAL GENE EXPRESSION IN THE EGYPTIAN ROUSETTE BAT IS ANTAGONIZED IN VITRO BY MARBURG VIRUS INFECTION Jonathan C. Guito *, Catherine E. Arnold 2, Louis A. Altamura 3, Elyse Nagle 2, Sean Lovett 2, Mariano Sanchez-Lockhart 2, Jonathan S. Towner, Gustavo Palacios 2 Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, 2 Center for Genome Sciences, 3 Diagnostic Systems Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, United States Abstract: Marburg virus (MARV) causes severe human disease and is responsible for several large, sporadic outbreaks in sub-saharan Africa. Recently, the Egyptian rousette bat, Rousettus aegyptiacus (ERB), was identified as a MARV reservoir host. When infected by MARV, these bats develop a productive infection with viremia and shedding but without overt disease, which current thinking hypothesizes is due to unique ERB antiviral responses. In humans, an insufficient interferon (IFN) response early in infection may be linked to fatal outcomes, likely due to MARV IFN antagonist activity. Unfortunately, investigative efforts into ERB immune responses in vitro or in vivo have been hampered by a lack of species-specific reagents. Helpfully, the newly-annotated ERB genome and transcriptome can now be used to study immune gene function on a transcriptional level. Employing two platforms, RNA-seq and NanoString ncounter, we assessed antiviral responses in a MARV-infected ERB cell line. Strikingly, both platforms showed that MARV suppressed the IFN response in ERB cells, while an IFN antagonist-impaired MARV mutant significantly stimulated responses, as previously reported for human cells. Interestingly, despite evolutionary expansion of IFN loci in ERBs, we saw almost no induction of IFNs by either virus, but did observe high basal expression for several key response genes. Our findings suggest that, in vitro, antiviral gene upregulation unlikely determines ERB resistance to MARV infection, but that a putative role may exist for an IFNindependent, constitutively-transcribed antiviral system. 64

165 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P07 TRANSPOSON-MEDIATED ACTIVATION SCREENING IN HUMAN CELLS IDENTIFIES THE CLASS II TRANSACTIVATOR AS A RESTRICTION FACTOR FOR EBOLA Anna Bruchez *, Ky Sha, Joshua Johnson 2, Li Chen 3, Gene Olinger 2, Lynda Stuart, 4, Adam Lacy-Hulbert, 5 Immunology Research Program, BENAROYA RESEARCH INSTITUTE, Seattle, 2 Integrated Research Facility, NIAID, Fort Detrick, 3 Center for Cancer Research, Massachusetts General Hospital, Boston, 4 Discovery and Translational Sciences, Bill and Melinda Gates Foundation, 5 Immunology, University of Washington, Seattle, United States Abstract: Ebola virus causes sporadic outbreaks of severe hemorrhagic fever. Recent outbreaks have exposed our limited understanding of cellular mechanisms to counteract this virus. Viral infections can be controlled by restriction factors, which although poorly defined, represent potential targets for host-directed anti-viral therapies. We employed a forward genetic approach to identify host genes conferring resistance to viral infection. In this system, cells are mutagenized by widespread, near-random transposon insertion, which is capable of either up- or downregulating genes depending on orientation and location. Candidate resistance genes are identified by sequencing of transposon insertion sites in cells that survive challenge with cytotoxic virus. We used this approach to identify genes involved in resistance to recombinant vesicular stomatitis virus bearing the Ebola virus glycoprotein. Validating our screen, we found that transposon-mediated inactivation of NPC, the Ebolavirus entry receptor, conferred resistance. We also identified genes that conferred resistance when overexpressed. These included the MHC class II transactivator (CIITA), which was a potent host restriction factor, increasing cellular resistance over,000 fold. CIITA induces resistance through transcription of host target genes, including CD74. These in turn trigger reorganization of the endo-lysosomal pathway, directing Ebola glycoprotein containing virions into the intraluminal vesicles of multi-vesicular bodies. These data implicate CIITA and CD74 in an intrinsic host defense mechanism against infection, independent from their roles in antigen presentation. Furthermore, these findings provide evidence for the power of transposon-mediated gene-activation as a screening strategy able to reveal roles for genes and pathways that elude conventional screening approaches. 65

166 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P072 PARAINFLUENZA VIRUS 5 (PIV5) P PROTEIN IS MODIFIED IN RESPONSE TO IFNΑ, AND THIS INHIBITS PIV5 INFECTION Jelena Andrejeva *, Steve Goodbourn 2, Richard Randall 3, David Hughes 3 School of Biology, UNIVERSITY OF ST. ANDREWS, SCOTLAND, UK, St. Andrews, 2 St. George's University of London, Institute of Infection and Immunity, London, 3 School of Biology, University of St. Andrews, St. Andrews, United Kingdom Abstract: The ubiquitin-like protein (Ubl) ISG5 is strongly induced by type I interferon (IFNα/β) and is critical for regulating how cells respond to infections. Like other Ubls, it can be covalently attached to target proteins in a process known as ISGylation, and in many cases, modification of viral proteins forms part of the antiviral response. Here we show that the parainfluenza virus 5 (PIV5) P protein (a component of the viral polymerase complex) is modified in response to IFNα, and this inhibits PIV5 infection. ISGylation of P requires the E3 ligase HERC5, and a lack of HERC5 alleviates inhibition of PIV5 infection. We also show that P is the only PIV5 protein that is modified, suggesting that ISGylation specifically inhibits the PIV5 polymerase complex. ISG5 only modifies a minority of the P proteome ; as paramyxovirus P proteins function as tetramers, we hypothesise that only a small fraction of modified P is sufficient for inhibition. This aligns well with the dominant-negative function that has recently emerged for the antiviral function of ISGylation. Consequently, ISGylation blocks the formation of a competent ribonucleoprotein complex, leading to an inhibition of virus transcription and replication. 66

167 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P073 STRUCTURAL BASIS OF IMPORTIN ALPHA SPECIFICITY FOR HENIPAVIRUS W PROTEINS Megan R. Edwards *, Kate M. Smith 2, Sofiya Tsimbalyuk 2, David Aragão 3, Jade K. Forwood 2, Christopher F. Basler Center for Microbial Pathogenesis, Georgia State University, Atlanta, United States, 2 School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, 3 Australian Synchrotron, Australian Synchrotron, Melbourne, Australia Abstract: Nucleocytoplasmic trafficking of proteins is important for a number of cellular processes, including viral infection. However, how cargo bearing classical nuclear localization signals (NLSs) can selectively utilize a subset of importin alpha nuclear transport proteins present in human cells remains unclear. To address this question, we studied the Hendra (HeV) and Nipah (NiV) virus W proteins. HeV and NiV, members of the family Paramyxoviridae, are recently emerged, highly lethal zoonotic pathogens. The non-segmented, negative-sense RNA genome encodes for nine-proteins, including the W protein, expressed from the P gene through mrna editing. W plays a role in controlling the inflammatory response to NiV infection in ferret models. It also inhibits responses to interferon by STAT tyrosine phosphorylation and inhibits signalling through TLR3, IKKε and TBK, likely through inhibition of IRF3. Notably, W localizes to the cell nucleus, through the specific interaction of its classical NLS with the Qip- subfamily of importin alpha nuclear transporters, importin α3 and importin α4. To gain insight into the basis of this specificity, we combined structural, biophysical and molecular approaches. X-ray crystal structures between HeV/NiV W and importin α3 or importin α (Rch- subfamily) identified differential positioning of the armadillo (ARM)-repeat domains 7 and 8 in the importins that allows for a more extensive interface between W and importin α3. Co-immunoprecipitation assays using mutagenesis and chimeras of importin α3/importin α confirmed the basis of specificity is present in the C-terminal ARM-repeat domains. These findings explain how W selectively uses Qip- transporters and, together with a previously defined mechanism for isoform specificity, suggest that differential importin alpha usage may depend on how the NLS is positioned in the cargo. 67

168 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P074 PUUMALA HANTAVIRUS INFECTION MODULATES THE CIRCADIAN CLOCK Agnieszka Szemiel *, Brian Willett MRC - University of Glasgow Centre for Virus Research, University of Glasgow, Glasgow, United Kingdom Abstract: The circadian clock synchronises host physiology in approx. 24 h oscillations. These processes are controlled by the central oscillator located in the hypothalamus, and peripheral oscillators present in organs, tissues, and most cell types. At the molecular level, circadian timekeeping system is regulated by transcriptional feedback loops involving two activators, CLOCK and BMAL transcription factors; which promote expression of their repressors PER and CRY proteins. Circadian disruption has been associated with multiple diseases like cancer, neurodegeneration, metabolic syndrome, cardiovascular diseases and recently with susceptibility to viral infections. Puumala hantavirus (PUUV), from the family Hantaviridae (order Bunyavirales) causes haemorrhagic fever with renal syndrome, and it is the most common rodent-borne pathogen in Europe. As a hantavirus that causes relatively mild symptoms, it is an excellent model to study infections caused by highly pathogenic hantaviruses, like Hantaan or Seoul. Our study investigated the interplay between PUUV infection and the circadian clock machinery in cell culture. We demonstrate that PUUV infection in BHK2 cells is enhanced when the host circadian rhythm is activated. This enhancement is abolished when the clock machinery is inhibited. Our investigation showed that PUUV infection slows down the oscillation of PER mrna, therefore lengthening the circadian cycle in BHK2 cells. In addition, this function can be attributed to PUUV N protein, as transient expression of PUUV N protein alters the oscillation pattern of PER protein. Our results suggest that PUUV infection modulates the molecular circadian clock to facilitate its own replication. 68

169 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P075 HUR RESTRICTS EBOLA VIRUS REPLICATION Kristina L. Schierhorn *, Rui P. Galao, Harry Wilson, Stuart Neil, Chad Swanson Infectious Diseases, King's College London, London, United Kingdom Abstract: Ebola virus (EBOV) is a nonsegmented, negative-strand RNA virus that replicates in the cytoplasm of infected cells. The presence of viral RNA can trigger antiviral mechanisms, mostly involving RNA-binding proteins (RBPs) which can lead to the formation of stress granules (SGs). These are non-membrane-bound cytosolic organelles that assemble due to different stress conditions, such as viral infection, and have been shown to directly or indirectly inhibit viral replication. To identify cellular RBPs that regulate EBOV replication, we performed an overexpression screen with candidate RBPs that localise to SGs. Using a transcription- and replication-competent virus-like particle (trvlp) system, we identified five proteins which inhibit EBOV trvlp propagation at least 0-fold. All these proteins presented a dose-dependent inhibition which cannot be overcome by high trvlp input. To determine if the endogenous proteins inhibit trvlp replication, we generated single cell clone knockout cell lines using CRISPR genome editing. Using these knockout cell lines as producer as well as target cells for trvlps, we identified HuR as a potential antiviral factor targeting EBOV propagation. To map the antiviral effect to the viral life cycle, we examined the impact of overexpression on viral transcription and replication. Strand-specific quantitative RT-PCR revealed a block of transcription as well as replication of the viral genome in the target cells. This suggests HuR inhibits EBOV trvlp propagation at an early step in the viral life cycle. We are currently investigating the exact mechanism how HuR inhibits EBOV in the trvlp system. 69

170 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P076 TERMINAL DEPTH SINGLE-MOLECULE SEQUENCING OF CAPPED TRANSCRIPTS REVEALS HOST-PATHOGEN DYNAMICS IN HUMAN MACROPHAGES. Sara Clohisey *, Nicolas Bertin 2, Helen Wise 3, Andru Tomiou 4, Piero Carninci 5, Yoshihide Hayashizaki 5, David Hume 6, Paul Digard 3, J. Kenneth Baillie Genetics and Genomics, The Roslin Institute, Edinburgh, United Kingdom, 2 Division of Genomic Technologies, RIKEN, Edinburgh, Japan, 3 Infection and Immunity, 4 Developmental Biology, The Roslin Institute, Edinburgh, United Kingdom, 5 Division of Genomic Technologies, RIKEN, Yokohama, Japan, 6 Developemental Biology, The Roslin Institute, Edinburgh, United Kingdom Abstract: Immune-regulatory actions of macrophages in the lung determine the severity of pathology in influenza. In order to obtain parallel observations of both host and influenza mrna production, we used cap analysis of gene expression (CAGE) to sequence all capped RNA species, at terminal depth, from influenza-infected primary human macrophages from multiple donors at multiple timepoints. Exploiting both the 5 and 3 non-coding regions in viral RNA, we directly observe the dynamics of viral mrna production over 24 hours. Influenza viruses replicate by snatching 5 capped mrna sequences from host mrnas from abundant transcripts. By sequencing these transcripts from the 5 end, we have identified the genomic origin of these snatched sequences, revealing that non-codingrnas are significantly over-represented compared to their abundance. Additionally, pathway analysis on identified sources of cap-snatched transcripts allows us to speculate on the targets of this process. Finally, by comprehensively polling host gene expression, we not only recapitulate canonical antiviral signalling pathways, but also identify numerous host transcripts with highly-specific expression in influenza-treated macrophages. 70

171 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P077 IS THE PATHOGENICITY OF SFTSV DETERMINED BY VIRAL NSS PROTEIN? Rokusuke Yoshikawa *, 2, Jiro Yasuda, 2 Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, 2 National Research Center for Control and Prevention of Infectious Diseases (CCPID), Nagasaki University, Nagasaki, Japan Abstract: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by SFTS phlebovirus (SFTSV), which is a novel phebovirus of the Phenuiviridae. SFTSV was first isolated in China and subsequently identified in South Korea and Japan. SFTSV is pathogenic to human, while immunocompetent adult mice infected with SFTSV never show apparent symptom. However, mice lacking interferon (IFN)-a/b receptor are highly susceptible to SFTSV infection and result in death. We found that mice deficient for the signal transducer and activator of transcription 2 (STAT2) which is the key factor of type I IFN signaling pathway are also highly susceptible to SFTSV infection. It suggests that innate immunity depending on STAT2 inhibits the efficient replication of SFTSV in mice. Recently, NSs of SFTSV has been reported to inhibit the type I IFN response through sequestration of human STAT2 protein in viral cytoplasmic inclusion bodies. In this study, we examined anti-stat2 activity of NSs in human and mice and proposed a hypothesis that the inhibitory activity of NSs to STA2 is associated with the difference in susceptibility to SFTSV between human and mice. Reporter assay revealed that type I IFN signaling was antagonized by NSs in human cells, while NSs did not inhibit the signaling in mouse cells. Moreover, NSs suppressed the phosphorylation of human STAT2 protein. In contrast, phosphorylation of murine STAT2 was not inhibited by NSs. In addition, co-immunoprecipitation assay revealed that NSs bound to human STAT2, but not murine STAT2. Our results imply that the activity of NSs to antagonize STAT2 may be attributed to the pathogenesis of SFTSV in human. 7

172 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P078 REGULATION OF CELL INTRINSIC IMMUNITY BY THE TRANSCRIPTIONAL REPRESSOR CAPICUA Julianna Han *, Jasmine Perez, Cindy Chen, Yan Li 2, Asiel Benitez 3, Matheswaran Kandasamy, Yoontae Lee 4, Jorge Andrade 2, Benjamin tenoever 3, Balaji Manicassamy Microbiology, 2 Center for Research Informatics, The University of Chicago, Chicago, 3 Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States, 4 Life Sciences, Pohang University of Science and Technology, Pohang, Korea, Republic Of Abstract: Host innate immune pathways are important for sensing viruses and mounting robust responses against viral infections. However, negative regulation is critical to prevent erroneous antiviral responses and to ramp down these pathways after clearance of viral infection. Through a genome-wide CRISPR/Cas9 screen to discover host factors critical for influenza virus replication, we have identified capicua (CIC) as a novel host factor critical for regulating intracellular antiviral responses. CIC is a highly conserved DNA binding repressor and mutations in CIC lead to neuropathogenesis and cancer progression. However, the role of CIC in viral infection remains unknown. In CIC knockout cells (KO), we observed higher expression of interferon and interferon stimulated genes. Additionally, replication of viruses from diverse families were highly attenuated in CIC KO cells due to the heightened antiviral status in these cells. In contrast, ectopic expression of CIC repressed antiviral gene promoter reporter constructs containing CIC binding sites. Interestingly, CIC protein and mrna levels were downregulated during influenza virus infection. Thus, our studies have identified a novel function of CIC in modulating cell intrinsic immunity. We hypothesize that under steady-state conditions CIC represses the transcription of antiviral genes, and upon viral infection, CIC is downregulated to facilitate robust expression of antiviral genes. Our future investigations will reveal a mechanism by which CIC regulates the intracellular antiviral state and may have significant implications in our understanding of host-pathogen evolution. Graphical Abstract: 72

173 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P079 ACUTE PLASMODIUM INFECTION PROMOTES RESISTANCE TO EBOLA VIRUS VIA TYPE IMMUNITY IN MACROPHAGES Kai Rogers *, Rahul Vijay, Chester J. Joyner 2, Mary R. Galinski 2, Noah S. Butler, Wendy J. Maury Microbiology and Immunology, University of Iowa, Iowa City, 2 Emory Vaccine Center, Emory University, Atlanta, United States Abstract: Ebola virus (EBOV) outbreaks occur sporadically in Africa with enormous fatality rates. Individuals are often infected with other endemic pathogens, but consequences of co-infections are understudied. During the epidemic, a significant number of EBOV patients were co-infected with P. falciparum when admitted to Ebola treatment units. Currently, there is no consensus regarding how malaria affects EBOV infection, with studies suggesting better or worse outcomes in co-infection patients. Here, we investigated the effect of pre-existing malaria on EBOV challenge using a model virus, recombinant VSV bearing EBOV glycoprotein (EBOV/rVSV). Interferon a/b receptor knock out mice were infected with Plasmodium yoelii (Py) and subsequently challenged with EBOV/rVSV. Acute infection with Py protected against lethal virus challenge. Further, mice were protected against EBOV challenge for weeks after resolution of malarial disease, indicating that innate host responses to Py rendered mice resistant to EBOV. Protection against EBOV was linked to IFNγ-mediated M polarization of peritoneal macrophages (pmacs) in Py-infected mice. Serum from acutely Py infected mice also reduced EBOV/rVSV infection of pmacs in an IFNγ-dependent manner. Similarly, human macrophages treated with serum from rhesus macaques acutely infected with P. cynomolgiwere protected against EBOV/rVSV. Protection was abolished by neutralization of IFNg. Finally, Py-infected mice lacking the IFNg receptor were not protected from EBOV/rVSV, yet their serum, containing IFNg, protected wild-type pmacs. These experiments support the hypothesis that acute malaria infection protects against EBOV by production of IFNγ, which in turn elicits an M state in macrophages. 73

174 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P080 RECOGNITION OF INFLUENZA A VIRUS BY DNA SENSORS Miyu Moriyama *, Takeshi Ichinohe Division of Viral Infection, UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: Influenza A virus is the etiological agent of a highly contagious acute respiratory disease that causes epidemics and considerable mortality annually. The recognition of influenza virus plays a key role not only in limiting virus replication and inflammatory responses at early stages of infection but also in initiating and orchestrating virus-specific adaptive immune response. It has become increasingly evident that influenza viral infection is recognized by at least three classes of pattern-recognition receptors, including TLR-7, the retinoic acid inducible gene-i (RIG-I) and nucleotide-binding domain and leucine-rich-repeat-containing protein 3 (NLRP3), a member of the Nod-like receptor family. In contrast, the role of DNA sensing pathway in recognition of influenza virus remains to be defined. Here we show that recombinant influenza virus lacking the NS gene (ΔNS virus) stimulates translocation of mitochondrial DNA (mtdna) into the cytosol and elicits stimulator of interferon genes (STING)-dependent innate antiviral immunity. Release of mtdna into the cytosol dependent on MAVS and mitochondrial permeability transition. The STING-dependent antiviral signaling was amplified by bystander cells via gap junction. Our results show a mechanism by which influenza virus activates STING pathway and provide insight into the role of DNA sensing pathway in recognition of influenza virus. 74

175 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P08 REGULATION OF CELL DEATH BY THE EBOLAVIRUS VP24 PROTEIN Diego Cantoni *, Jeremy S. Rossman School of Biosciences, University of Kent, Canterbury, United Kingdom Abstract: Ebolavirus (EBOV), a member of the filoviridae family, is a highly pathogenic virus which causes cell death through a variety of mechanisms, including as as-yet-undefined necrosis pathway. Here we have investigated the pathway and mechanisms of EBOV necrotic cell death and define a crucial role of VP24 in regulating cell death signalling pathways. EBOV VP24 is a 25 amino acid protein that has been shown previously to counteract antiviral defences and increase stability of the viral nucleocapsid. Using a combination of biochemical and microscopy-based assays, we have investigated the role of VP24 in the regulation of cell death following the induction of different signalling pathways, including stimulation by tumour necrosis factor alpha. We find that VP24 interacts with several mitochondrial proteins, including VDAC and plays a crucial role in the upregulation of necroptosis and the downregulation of apoptosis, thus regulating the pathway of cell death following EBOV infection. 75

176 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P083 SANDFLY FEVER SICILIAN VIRUS NSS SPECIFICALLY TARGETS IRF3 TO INHIBIT TYPE I INTERFERON INDUCTION Jennifer Wuerth *, Matthias Habjan 2, Andreas Pichlmair 3, Giulio Superti-Furga 4, Friedemann Weber Justus-Liebig University, Giessen, 2 Max Planck Institute of Biochemistry, Martinsried, 3 Technical University of Munich, Munich, Germany, 4 Center for Molecular Medicine, Vienna, Austria Abstract: Different members of the Phlebovirus genus exhibit different degrees of virulence. Rift Valley fever virus (RVFV), for example, causes severe symptoms, whereas Sandfly fever Sicilian virus (SFSV) is much milder. The major virulence factor of phleboviruses is the non-structural protein NSs, an antagonist of the type I interferon (IFN) system. Whereas RVFV NSs is known to entirely shut off cellular transcription by targeted sequestration and destruction of host factors, the function of SFSV NSs has remained elusive. Here, we show that SFSV NSs inhibits the transcriptional induction of type I IFN. Applying tandem affinity purification and mass spectrometry (Pichlmair et al. Nature 202), we identified IFN regulatory factor 3 (IRF3) as host interactor. Coimmunoprecipitation and reporter assays confirmed IRF3 as interactor and functional target of SFSV NSs: SFSV NSs alone abrogated IRF3-dependent promoter activity induced via MAVS, TRIF, TBK, or constitutively active IRF3. However, neither phosphorylation, dimerization nor nuclear accumulation of IRF3 were affected. Instead, SFSV NSs was found to act by obstructing the DNA-binding domain of IRF3, thereby hindering enhanceosome formation. To our knowledge, this is the first report of an RNA virus factor that directly disrupts the binding of activated IRF3 to the IFN promoter. Thus, SFSV NSs is different from the NSs of highly virulent RVFV, as it does not induce a general host transcription block but instead specifically targets IRF3-driven gene expression. Although these viruses are highly related, their NSs proteins display remarkably diverse strategies of counteracting the IFN system, probably contributing to their respective virulence levels. 76

177 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P084 IDENTIFICATION OF TWO NEW POLYPEPTIDES FROM SEGMENT 2 OF IAV THAT MODULATE THE TYPE I INTERFERON RESPONSE Rute M. Pinto *, Helen Wise, Liliane M. W. Chung, Marlynne Quigg-Nicol, Samantha Lycett, Gregory Heikel 2, Gracjan Michlewski 2, Bernadette Dutia, Paul Digard The Roslin Institute, 2 Wellcome Centre for Cell Biology, The University of Edinburgh, Edinburgh, United Kingdom Abstract: Alternative translation events that produce additional viral polypeptides have been shown to be significant in influenza A virus (IAV) biology. IAV segment 2 is a pathogenicity determinant, known to encode PB, PB-F2 and PB-N40 proteins, starting from AUG codons, 4 and 5 respectively. We investigate the expression of two additional polypeptides, arising from translation initiation at AUGs 0 and of segment 2. These codons are highly conserved in IAV and direct the translation of two N-terminally truncated versions of the primary PB product (PB-N92 and -N respectively). Mutation of AUGs 0 or (M92V, MV) in the background of PR8 had minor effects on virus replication kinetics although the viruses displayed elevated levels of IRF3 phosphorylation and type-i IFN secretion compared to WT virus. However, simultaneous mutation of AUGs 0 and severely decreased viral fitness, despite the full-length mutant PB possessing normal genome transcription and replication activity. Similar patterns of defective replication and elevated innate signalling were seen when mutating AUGs 0 and/or in other mammalian virus isolates. The propagation deficit of the ΔAUG0, mutant recovered in IFN-deficient models, including in ovo in pre-day-8 embryonated eggs, in vitro in IFN a/β receptor (IFNAR) knockout bone marrow-derived macrophages and in vivo in IFNAR -/- mouse lungs. Moreover, expression of PB- N92 or N polypeptides blocked poly I:C-induced activation of IFN-β and ISRE promoters in transfected cells. In vitro, knockout of TRIM25 did not increase growth of the mutant viruses, but the addition of a TBK/IKKε inhibitor did. In conclusion, segment 2 expresses previously undescribed N-terminally truncated versions of PB which play a role in antagonising the host IFN response, most likely independently of RIG-I/TRIM25, but upstream of IRF3 phosphorylation. 77

178 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P085 EARLY HOST RESPONSES OF HUMAN PRIMARY LUNG MICROVASCULAR ENDOTHELIAL CELLS AND RESTING MONOCYTES TO BLACK CREEK CANAL ORTHOHANTAVIRUS (BCCV) Evan P. Williams, Elizabeth A. Fitzpatrick, Colleen B. Jonsson * Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, United States Abstract: Hantaviruses are single-stranded, negative-sense, trisegmented RNA viruses that persistently infect species within the Rodentia, Eulipotyphla and Chiroptera. Of these, only those viruses harbored by rodents cause disease in humans. Infection begins with inhalation of virus particles into the lung and trafficking to the lung microvascular endothelial cells (L-MVEC). Studies suggest that monocytes may also become infected at some point during infection. Since monocytes are one of the first responders to infection, it is possible that these immune cells may become infected upon arrival; however, this is not known. We hypothesized that the addition of monocytes to infected-lmvec would elevate the standing proinflammatory response. We asked how host gene expression would be modulated following BCCV infection of L-MVEC or monocytes or L-MVEC and monocytes following BCCV infection from 24 to 96 hours with time-points every 2 hours. QuantiGene probes were used to measure the kinetics of genes representing antiviral, proinflammatory, anti-inflammatory, metabolic pathways. As expected in L-MVEC, anti-inflammatory genes were not upregulated in any experimental group while proinflammatory genes (e.g., IL8, NFKB IFNA/B) were upregulated. Surprisingly, upon addition of monocytes, the proinflammatory genes of the L-MVEC were suppressed. Notably, ITGAM was highly upregulated in L-MVEC-monocytes (presumably in the monocytes); however, a number of genes (e.g., TNF, TLR7, and ILRN) were not affected. In experiments with monocytes alone, IFNG was upregulated along with IDO, IL5, PPARG and TNF. In conclusion, in contrast to our original hypothesis, monocytes plus L-MVEC did not amplify the proinflammatory response suggesting that monocytes may contribute to evasion of the innate immune response. 78

179 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P086 TWO CONSERVED AMINO ACIDS WITHIN THE NSS PROTEIN OF SFTS VIRUS ARE ESSENTIAL FOR ANTI- INTERFERON ACTIVITY Takeshi Ichinohe *, Miyu Moriyama Department of Virology, UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: Severe fever with thrombocytopenia syndrome (SFTS) is a newly emerging infec tious disease caused by the SFTS virus (SFTSV). The nonstructural protein (NSs) of SFTSV sequesters TANK-binding kinase (TBK) into NSsinduced cytopl asmic structures to inhibit the phosphorylation and nuclear translocation of interferon regulatory factor 3 (IRF3) and subsequent interferon beta (IFN-β) production. Although the C-terminal region of SFTSV NSs (NSs66-249) has been linked to the formation of NSs-induced cytoplasmic structures and inhibition of host IFN-β responses, the role of the N-terminal region in antagonizing host antiviral responses remains to be defined. Herein, we demonstrate that two conserved amino acids at positions 2 and 23 in the SFTSV NSs protein are essential for suppression of IRF3 phosphorylation and IFN-β mrna expression following infection with recombinant influenza virus lacking the NS gene. Surprisingly, formation of SFTSV NSs-induced cytoplasmic structures is not essential for inhibition of host antiviral responses. Rather, association between SFTSV NSs and TBK is required for suppression of mitochondrial antiviral signalling protein (MAVS)-mediated activation of IFN-β promoter activity. Our findings strongly implicate the N-terminal region of SFTSV NSs in the inhibition of host antiviral immunity, and will aid the development of novel therapeutic strategies to treat SFTSV infection and associated diseases. 79

180 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P088 ANATOMY OF CYTOTOXIC T LYMPHOCYTE RESPONSE DURING THE EBOLA OUTBREAK IN WEST AFRICA Saori Sakabe *, Kristian Andersen, Michael B. Oldstone, Brian M. Sullivan of Immunology and Microbial Sciences, The Scripps Research Institute, La Jolla, United States Abstract: Ebola virus (EBOV) causes severe febrile disease in humans and large scale outbreak occurred in West Africa in , resulting in 28,66 cases and,30 deaths. EBOV-vaccinated non-human primates are protected from lethal challenge of EBOV if their CD8+ T cell response is operational (Sullivan et al., Nat Med, 20). Passive transfer of high titered anti EBOV antibodies failed to protect in the absence of CD8 T cells. This data and observations that T cell responses are observed in humans surviving acute viral hemorrhagic fevers indicate that T cells play a key role in the disease severity. We defined the Ebola viruses-specific cytotoxic T lymphocyte (CTL) response in 3 Ebola survivors living in Sierra Leone to seven of the eight Ebola full-length proteins (GP, sgp, NP, VP24, VP30, VP35, and VP40), and aa truncations of these proteins by using recombinant single cycle VSVs library encoding EBOV proteins. Our results indicated a minor of (only 0) of the 27 individuals (37%) showed CD8+ CTL response to GP or sgp. By contrast 26/27 (96%) showed response to NP, 8% to VP24, 74% to VP40, 44% to VP35, and no response to VP30. Further, we mapped various novel CD8+ CTL epitopes derived from EBOV proteins and their.hla restriction. Our data suggest that an EBOV vaccine designed to include not only the Ebola GP but also NP would be more effective to elicit both cellular and humoral responses in most individuals than a vaccine designed just include the Ebola GP which may elicit good humoral but limited CTL responses. This research is funded by NIH Contract HHSN C under BAA-NIAID-DAIT-NIHAI

181 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P089 THE OVARIAN TUMOR (OTU) DOMAIN OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS (CCHFV) INFLUENCES THE POLYMERASE RDRP ACTIVITY, BUT DOES NOT COUNTERACT INNATE IMMUNITY Stéphanie Devignot *, Ali Mirazimi 2, Friedemann Weber Institute for Virology, FB0 Veterinary medicine, Giessen, Germany, 2 Public Health Agency, Solna, Sweden Abstract: The Crimean-Congo Hemorrhagic fever virus (CCHFV) polymerase L encodes an Ovarian Tumor (OTU) domain, with deubiquitination and deisgylation activities. Studies based on the ectopic expression of the OTU domain showed an inhibition of both NFκB and type I interferon (IFN) induction. Hence, the OTU domain has been suggested to take part in immune evasion. Since the OTU domain is small compared to the full-length polymerase (69 versus 3549 amino acids), we asked whether the OTU domain retains the same activities in the context of the entire, RdRp active L. Using reporter assays, we confirmed that the ectopic OTU domain inhibits both NFκB and IFN induction, but this effect was lost in the context of the full-length L. Then, we used the minireplicon (Bergeron et al., JVI, 200) and the transcriptionallycompetent virus-like particle (tc-vlp) (Devignot et al., JVI, 205) systems to assess the role of the OTU domain on the RdRp activity. While the OTU domain is not required in the minireplicon system, only few tc-vlps could be produced with an OTU-inactive L. However, both wild-type and OTU-deficient polymerases are equally sensitive to IFN. Moreover, neither trans-complementation with wild-type OTU domain, nor blocking IFN signalling were able to rescue OTU-inactive mutant tc-vlp production. This suggests distinct trans- and cis- activities of the OTU domain. Lastly, and very surprisingly, we could rescue tc-vlp production over the wild-type polymerase levels, by over-expressing a conjugable Interferon Stimulated Gene 5 (ISG5). To conclude, the role of the CCHFV OTU domain regarding innate immune response needs further careful investigation, which should be made in the context of the full-length polymerase. 8

182 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P090 NIPAH VIRUS C PROTEIN INHIBITS INFLAMMATORY CYTOKINE INDUCTION BY INTERACTION WITH PHOSPHATASE 2A INHIBITOR. Ryo Horie, Misako Yoneda, Shotaro Uchida, Asuka Yoshida *, Hiroki Sato, Chieko Kai Laboratory Animal Research Center, THE INSTITUTE OF MEDICAL SCIENCE, THE UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: Nipah virus (NiV) is a member of the genus Henipavirus, which emerged in Malaysia in 998. NiV causes severe encephalitis in human with high mortality. We had previously reported that the NiV nonstructural C protein (NiV-C) plays a key role in its severe pathogenecity. Recently, it is becoming clear that NiV-C regulates proinflammatory response, however the mechanism is still unclarified. In present study, we isolated human proteins binding to the NiV-C using an affinity purification method. Inhibitor of serine/threonine protein phosphatase 2A (I2PP2A) was identified as a NiV-C binding protein among them. It has been reported that PP2A interacts with numerous kinases including the MAPK proteins, which related pathways regulate proinflammatory cytokine production. In the cells stably expressing C protein, total cellular PP2A activity was significantly increased and induction of proinflammatory cytokines, IL-8 and CXCL2, by poly(i:c) treatment were suppressed. These results suggest that interaction between NiV-C and I2PP2A results in increase of PP2A activity and inhibition of proinflammatory cytokine induction through MAPK pathway. 82

183 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P09 MX REQUIREMENTS FOR INFLUENZA A VIRUS RESTRICTION Olivier Moncorgé *, Joe McKellar, Wendy Barclay 2, Caroline Goujon IRIM, CNRS, MONTPELLIER, France, 2 Department of Medicine, Imperial College London, LONDON, United Kingdom Abstract: Type interferons (IFNs) are produced by infected cells upon detection of pathogenic agents and are the first line of defence against viral infections. IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs), both in infected and neighbouring cells. The products of these ISGs in turn induce in cells a potent antiviral state, capable of limiting viral replication. The dynamin-like, high-molecular weight GTPases MX and MX2 play a significant role in the IFN-induced inhibition of viral replication. Human MX is a restriction factor of broad antiviral activity, able to inhibit influenza A virus and a great diversity of RNA and DNA viruses at different stages of their life cycles. Human MX2 is notably able to inhibit HIV- and some primate lentiviruses. Although the antiviral activity of human MX has been studied extensively, the molecular mechanism of action remains largely unsolved. MX and MX2 are 63% identical at the amino acid level, share a similar domain organization and their crystal structures are almost practically superimposable. Taking advantage of chimeras between MX and MX2 in which their different domains have been swapped as well as point mutants, we have notably identified a new motif required for influenza A restriction by MX. Importantly, some MX/MX2 chimeric proteins are highly active against influenza A viruses but not in the context of minireplicon assays. Additional ongoing efforts to better characterize MX s requirement for influenza A restriction and mechanism of action will be presented. 83

184 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P092 UBIQUITINATION OF TOSCANA VIRUS NSS UNDERMINES ITS STABILITY AND HAS A ROLE IN RIG-I DEGRADATION Claudia Gandolfo *, Gianni Gori Savellini, Shibily Prathyumnan, Maria G. Cusi Medical Biotechnologies, University of Siena, Siena, Italy Abstract: Toscana Virus (TOSV) is a Phlebovirus (Phenuiviridae family) responsible for central nervous system (CNS) injury in humans. Although TOSV pathogenicity is largely unclear, its non structural protein (NSs), when over-expressed, inhibits host innate immunity, silencing the IFN-β pathway. This antagonistic effect is due to the interaction with RIG-I, by addressing the latter to proteasome degradation upon NSs ubiquitination. Some lysine residues of NSs targeted for ubiquitination were identified by theoretical prediction and mutated to arginine. K50R and K54R point mutations restored RIG-I activation. Indeed, besides the accumulation of RIG-I in cells co-transfected with NSs mutants, an activation of IFN promoter was observed by luciferase assay. Unexpectedly, K04R and K09R mutations, although not determining RIG-I degradation, showed an activation of the luciferase expression. These results suggested a dual function mediated by NSs protein on both RIG-I degradation and interferon activation. In parallel, the effects on NSs stability was evaluated for all of these mutants, determining a considerable increase of protein accumulation in the cytoplasm. Mass-spectrometry analysis performed on wt-nss confirmed that lysine at 04, 09, and 54 position are linked to K48-polyubiquitin chains. Unfortunately, K50 ubiquitination was not confirmed, due to the undetectable size of the peptide generated during sample preparation. Taken together, these results provide, for the first time, evidence that Toscana virus NSs is ubiquitinated, opening a new insight on NSs function. Moreover, it is probable that TOSV NSs could exert an E3 ubiquitin ligase activity either on RIG-I and NSs itself, suggesting an important role of this protein among Phleboviruses. 84

185 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P093 THE GLYCOPROTEIN OF VESICULAR STOMATITIS VIRUS IS A TETHERIN ANTAGONIST IN TRANSFECTED BUT NOT INFECTED CELLS Markus Hoffmann *, Constantin Brinkmann, Anastasia Lübke, Inga Nehlmeier, Annika Krämer-Kühl 2, Michael Winkler, Stefan Pöhlmann Infection Biology Unit, Deutsches Primatenzentrum GmbH Leibniz-Institut für Primatenforschung, Göttingen, 2 Poultry Viral Vaccine R&D, Boehringer Ingelheim Veterinary Research Center GmbH & Co. KG, Hannover, Germany Abstract: Vesicular stomatitis virus (VSV) is a prototype member of the Rhabdoviridae family and used as a model virus for various scientific endeavors. VSV release from infected cells is inhibited by the interferon-inducible antiviral host cell factor tetherin (BST-2, CD37). Since several viruses encode tetherin antagonists the present study investigated whether residual VSV spread in tetherin-positive cells is also promoted by a virus-encoded tetherin antagonist. Here, we show that the VSV glycoprotein (VSV-G) acts as a tetherin antagonist in transfected cells, but does so with reduced efficiency as compared to the Vpu protein of human immunodeficiency virus. Tetherin antagonism by VSV-G did not rely on alteration of tetherin expression and could further be linked to a glycine-x-x-x-glycine (GXXXG) motif within the transmembrane domain of VSV-G. However, when the GXXXG motif was investigated in the context of replicationcompetent VSV, disruption of this motif had no impact on tetherin sensitivity of VSV spread and thus, no evidence for a tetherin-antagonizing activity of VSV-G in infected cells could be detected. In sum, we identified VSV-G as a tetherin antagonist in transfected cells but could not provide evidence for a contribution of tetherin antagonism to spread of authentic VSV. 85

186 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P094 HUMAN PARAINFLUENZA VIRUS TYPE 2 V PROTEIN INDUCES FILAMENTOUS ACTIN FORMATION Keisuke Ohta *, Yusuke Matsumoto, Natsuko Yumine, Masato Tsurudome 2, Machiko Nishio Department of Microbiology, Wakayama Medical University, Wakayama, 2 Department of Microbiology and Molecular Genetics, Mie University Graduate School of Medicine, Tsu, Japan Abstract: The growth of human parainfluenza virus type 2 (hpiv-2) is promoted by RhoA activation, through the binding of its V and P proteins with Graf (Ohta et al., 206, J. Virol.). However, it remains unknown how RhoA activation affects hpiv-2 growth. Here, we focused on actin reorganization that is regulated by RhoA activation. We have found that hpiv-2 infection induced filamentous actin (F-actin) formation, and that cytochalasin D, an actin polymerization inhibitor, reduced hpiv-2 growth. To investigate whether RhoA-induced F-actin formation affects hpiv-2 growth, wild type (wt) RhoA- or dominant negative (DN) RhoA-expressing HEK293 cells were established. Overexpression of wt RhoA caused an apparent F-actin formation even in mock-infected cells, and promoted hpiv-2 growth. In contrast, hpiv-2 growth was inhibited in DN RhoA-expressing cells where F-actin formation was not observed. Immunoprecipitation studies revealed that hpiv-2 V protein bound to inactive DN RhoA but not to wt and active RhoA, while P protein bound to none of these RhoAs. We also found that mutation of the Trp residues within the C-terminal region of V protein lost the capacity to bind DN RhoA. Infection with recombinant hpiv-2 carrying this Trp-mutated V did not cause F-actin formation. Furthermore, F-actin formation was observed in HEK293 cells constitutively expressing wt V, but not Trp-mutated V. The interaction between V protein and inactive RhoA is important for F-actin formation. This interaction might enhance the exchange of GDP for GTP, thereby converting inactive to active. The analysis of this possibility is now in progress. 86

187 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P095 REGULATION OF HAZARA VIRUS GROWTH THROUGH APOPTOSIS INHIBITION BY VIRAL NUCLEOPROTEIN Takashi Nouchi, Yusuke Matsumoto *, Keisuke Ohta, Machiko Nishio Department of Microbiology, School of Medicine, Wakayama Medical University, Wakayama, Japan Abstract: Hazara virus (HAZV) is closely related to Crimean-Congo hemorrhagic fever virus (CCHFV) but is nonpathogenic to humans. Since HAZV was first isolated in 954, the biological characteristics of this virus, particularly its behavior within cultured cells, have not been well studied, despite the importance of HAZV as a surrogate model for CCHFV. Nucleoprotein (N), the main component of viral ribonucleoprotein complex and the most abundant protein in the virion, is believed to play a pivotal role in the virus lifecycle. The generation of a series of anti-hazv N monoclonal antibodies were used to directly examine the involvement of this protein in viral growth. HAZV infection was found to induce severe apoptosis, which was further characterized by DNA ladders and elevated caspase-3/7 activity. HAZV titers initially increased in culture cells, but rapidly turned to decline after reaching the peak titer. HAZV particles were found to be very unstable in the culture medium at 37 o C, suggesting that progeny virions tends to lose infectivity. We also found that HAZV N inhibits the apoptosis, which supports the maintenance of viral infectivity. The contrary effects of induction and inhibition of apoptosis are considered to be an important property of this virus lifecycle. 87

188 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P096 MEASLES VIRUS INFECTION TRIGGERS CGAS-DEPENDENT ANTIVIRAL RESPONSES. Hiroki Sato *, Fusako Ikeda, Miho Hoshi, Misako Yoneda, Chieko Kai Laboratory Animal Research Center, INSTITUTE OF MEDICAL SCIENCE, THE UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: In mammalian cells, intracellular RNA produced by RNA viruses are recognized by RIG-I or MDA5, and cytosolic DNA viruses are sensed by cgas, in principal, and trigger a series of downstream host innate antiviral signaling. Interestingly, recent reports revealed that endogenous cytoplasmic DNA such as mitochondrial DNA (mtdna) released from mitochondria by cellular stresses can also activates cgas pathway. Our previous report indicated that measles virus (MeV) induces rapid antiviral responses, and proceeding of the infection causes comprehensive downregulation of host gene expressions including genes encoding mitochondrial proteins in an epithelial cells. In the present study, we found that increased amount of mtdna in the cytosol was detected accompanied by proceeding of the infection. Furthermore, we revealed that the cytosolic mtdna was captured by cgas in the infected cells, and knockdown of cgas suppressed IFN-β expression after MeV infection similarly to knockdown of MAVS which mediates RIG-I/MDA5 signaling. Our study proposes two-step induction of antiviral responses by MeV infection; intracellular RNA sensors are activated directly at early phase, and prolonged infection induces mtdna-activated cgas pathway for full innate control of MeV. 88

189 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P097 EBOLA VIRUS INHIBITS CELLULAR NRF2-DEPENDENT ANTIOXIDANT SIGNALING PATHWAY VIA EXPRESSION OF STRUCTURAL PROTEIN VP24. Valentina Volchkova *, Kirill Nemirov, Mathieu Mateo, St Patrick Reid, Audrey Page, Nicolas Danet, Viktor Volchkov BMPV, CIRI, Lyon, France Abstract: Virus replication in cells is often associated with release of reactive oxygen species and oxidative stress. Transcription factor Nrf2 (Nuclear factor erythroid-related factor-2) plays a central role in cellular defence against oxidative stress conditions. During stress, Nrf2 is translocated into the nucleus and activates a panoply of genes implicated in cell protection but also in the regulation of oxidative stress responses. Deregulated inflammatory response is a hallmark of Ebola virus (EBOV) pathogenesis. Inflammation and oxidative stress are known to be interrelated. In this study we investigate whether EBOV infection affects Nrf2 dependent anti-oxidative cell responses. We demonstrate that: (i) expression of Nrf2-dependent genes is impaired in EBOV infected cells, (ii) this is mediated by VP24 and that (iii) VP24 prevents Nrf2 transport into the nucleus, which is essential for Nrf2 function. Moreover, we show that a VP24 mutant defective in binding with karyopherin alpha (KPNA) lacks the ability to prevent Nrf2 nuclear transport, suggesting that these two VP24 functions are some way interdependent. Using reverse genetics for EBOV we demonstrate that an EBOV mutant defective in binding of VP24 and KPNA is not capable to antagonize IFN signaling. Remarkably, this EBOV mutant is severely attenuated in Vero E6 cells albeit that this cell line is known to be defective for IFN production. The data obtained support the notion that impairment of Nrf2-dependent anti-oxidative pathway may play a role in productive virus replication but may also facilitate virus induced pro-inflammatory responses, inflicting additional damage to the host and thus contributing to high pathogenicity of infection. 89

190 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P098 SMALL RNAS DERIVED FROM AN ENDOGENOUS BORNAVIRUS ELEMENT IN MICE SUPPRESS EXPRESSION OF A REPORTER WITH BORNA DISEASE VIRUS SEQUENCES IN GC2 CELLS Tomoyuki Honda *, 2, Xiaoshu Liu, Bea C. Garcia, Nicholas F. Parrish, Keizo Tomonaga Kyoto University, Kyoto, 2 Osaka Univeristy, Osaka, Japan Abstract: Endogenous bornavirus-like nucleoprotein (EBLN) is a fossil record of an ancient bornavirus infection in vertebrate genomes and homologous to the nucleoprotein (N) gene of current bornaviruses, such as Borna disease virus (BoDV). Recently, we have found that EBLNs give rise to small RNAs, which appear to be PIWI-interacting RNAs (pirnas), in the adult mouse testis. pirnas associate with their partner PIWI proteins and silence their target sequences. Species with EBLNs seem to have resistance to the current bornavirus infection, leading to reasonable doubt about whether EBLNderived small RNAs might target the viral N sequences. In this study, therefore, we evaluated this possibility using a mouse spermatocyte cell line GC-2spd (GC2), which reportedly has functional pirna pathway. We first confirmed that EBLNderived small RNAs were loaded onto the MIWI protein. When we transduced a mouse EBLN-derived synthesized small RNA of 26 nt into GC2 cells, the small RNA significantly suppressed expression of GFP reporter containing its target sequence of N gene, suggesting that EBLN-derived small RNAs have a potential to silence the target N sequence. We also found that BoDV transcription was upregulated by downregulation of Myb, a transcription factor for the pirna loci. Collectively, EBLN-derived small RNAs may function as pirnas to silence bornavirus transcripts in the mouse germline cells. 90

191 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P00 THE NSS PROTEIN FROM THE ANDES VIRUS INHIBITS THE TYPE I IFN RESPONSE PATHWAY Jorge Vera Otarola *, Loretto Solis, Karla Pino, Marcelo Lopez Lastra Laboratorio de Virología Molecular, Instituto Milenio de Inmunología e Inmunoterapia, Centro de Investigaciones Médicas, Departamento de Enfermedades Infecciosas e Inmunología Pediátrica, Escuela de Medicina, Pontificia Universidad Católica De Chile, Santiago, Chile Abstract: Andes virus (ANDV) is a rodent-borne hantavirus member of the Bunyaviridae family of viruses. ANDV is endemic in Argentina and Chile and is the major etiological agent of hantavirus cardiopulmonary syndrome (HCPS) in south America. ANDV features a tripartite genome consisting of three negative polarity single-stranded RNA segments designated large (L), medium(m) and small (S), packed into helical nucleocapsids.the L and M messenger RNAs (mrnas) encode the RNA polymerase and a glycoproteinprecursor that is co-translationally processed to yield two envelopeglycoproteins (Gc and Gn), respectively. The SmRNA encodes the nucleocapsid (N) protein and the NSs protein from an overlapping (+) open reading frame. At early stages of infection ANDV inhibits the cellular type I IFN response, through a yet not fully understood mechanism. The ANDV Gn and N proteins have been shown to inhibit the type I IFN pathway by blocking its signaling at level of TANK-binding kinase (TBK). TBK is involved in the activation of NFkB and IRF-3 which in turns triggers transcription of the IFN genes. Here, we use an assay in which the luciferase reporter is under the control of IFN-beta promoter to demonstrate that the ANDV-NSs protein blocks the IFN-beta activation pathway. Thus, findings reveal a role for the ANDV-NSs protein in the evasion of the cellular innate immunity response. Work supported by FONDECYT Iniciación 506, CONICYT-PIA ACT408 and P09/06-F, Iniciativa Científica Milenio del Ministerio de Economía, Fomento y Turismo de Chile. 9

192 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P0 INFLUENZA A VIRUS INFECTION TRIGGERS PYROPTOSIS AND APOPTOSIS OF RESPIRATORY EPITHELIAL CELLS THROUGH TYPE I IFN SIGNALING PATHWAY IN A MUTUALLY EXCLUSIVE MANNER Sangjoon Lee *, Kyosuke Nagata 2, Atsushi Kawaguchi, 2 Ph.D. Program in Human Biology, School of Integrative and Global Majors, 2 Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan Abstract: Respiratory epithelial cell death by influenza A virus (IAV) infection is responsible for induction of inflammatory responses. Previous studies suggest that apoptosis is a major cell death pathway triggered by IAV infection in cultured epithelial cells isolated from malignant tumors. However, apoptotic cells are rapidly phagocytized without inflammatory responses. Thus, the exact cell death mechanism responsible for inflammatory responses by IAV infection is still unclear. Here we found that IAV infection induced apoptosis and pyroptosis in normal or precancerous human bronchial epithelial cells. Apoptosis was induced at early phases of infection, but the cell death pathway was shifted to pyroptosis at late phases of infection. We also found that the type I IFN-mediated JAK-STAT signaling pathway promotes the switch from apoptosis to pyroptosis by inhibiting apoptosis possibly through the induced expression of Bcl-xL anti-apoptotic gene. Further, the inhibition of JAK- STAT signaling repressed pyroptosis, but enhanced apoptosis in infected respiratory epithelial cells. Taken together, type I IFN signaling pathway plays an important role to induce pyroptosis but represses apoptosis in the respiratory epithelial cells to initiate pro-inflammatory responses against IAV infection. 92

193 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P02 DEVELOPMENT OF AN EBOLA VIRUS INFECTION MODEL USING IPSC-DERIVED HEPATOCYTES Whitney Manhart *, 2, 3, Steve Mazur 4, Ignacio Caballero 2, Liliana Mancio Silva 5, John G. Bernbaum 4, Sangeeta Bhatia 5, Reed F. Johnson 6, Elke Mühlberger, 3, Gustavo Mostoslavsky 2, 7 National Emerging Infectious Disease Laboratory (NEIDL), Boston University, 2 Center for Regenerative Medicine (CReM), Boston University and Boston Medical Center, 3 Microbiology, Boston University School of Medicine, Boston, 4 Division of Clinical Research, Integrated Research Facility, National Institute for Allergy and Infectious Disease, National Institute of Health, Frederick, 5 Institute for Medical Engineering and Science, Massachusetts Institute of Technology, Cambridge, 6 Emerging Viral Pathogens Section, Laboratory of Immunoregulation, Division of Intramural Research, National Institute for Allergy and Infectious Disease, National Institute of Health, Frederick, 7 Section of Gastroenterology, Department of Medicine, Boston University School of Medicine, Boston, United States Abstract: Background Ebola virus (EBOV) infection causes a severe disease in humans which causes liver damage. It is thought that hepatocytes are a key site of viral replication in vivo and potentially a promising target for therapeutic interventions. Primary liver cells are difficult to grow in vitro, and not all animal models recapitulate liver pathology. The goal is to use induced pluripotent stem cell (ipsc)-derived hepatocytes to develop a disease-relevant in-vitro infection platform for modeling EBOV pathogenesis. Methods Hepatocytes were differentiated from human ipscs using a published, directed differentiation protocol. ipsc-derived hepatocytes were characterized using flow cytometry, intracellular staining, qrt-pcr, and functional assays. ipsc-derived hepatocytes, primary human hepatocytes (PHH), and immortalized hepatic carcinoma cells (Huh7) were infected with EBOV at an MOI of 3 and harvested for analysis at and 4 dpi. Infected cells were analyzed by immunofluorescence, electron microscopy, and 3 RNA-seq (DGE, Broad Institute). Results ipsc-derived hepatocytes express mature hepatic markers and key hepatic enzymes are active. All three cell platforms supported EBOV infection as indicated by the presence of cytoplasmic viral inclusions. ipsc-derived hepatocytes contained significant amounts of replicating and nascent virions as shown by electron microscopy. ipsc-derived hepatocytes and PHHs were less susceptible to EBOV infection compared to Huh7s. Using RNA-seq data, ipsc-derived hepatocytes and PHHs respond more similarly to infection compared to Huh7 cells, and ipsc-derived hepatocytes express an interferon signature upon infection that is not observed in Huh7 cells. Conclusions ipsc-derived hepatocytes are a suitable in vitro model for EBOV infection of hepatocytes. The model can be used to better understand the molecular mechanisms leading to liver damage during human EBOV infection. 93

194 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P03 KNOCKING ON THE NUCLEAR DOOR: RIFT VALLEY FEVER VIRUS VIRULENCE FACTOR NSS TARGETS NUCLEAR-CYTOPLASMIC TRANSPORT Simone Lau *, Matthias Habjan 2, Giulio Superti-Furga 3, Andreas Pichlmair 2, Friedemann Weber Justus-Liebig University, Giessen, 2 Max-Planck Institute of Biochemistry, Martinsried/Munich, Germany, 3 CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria Abstract: Rift Valley fever virus (RVFV, genus Phlebovirus, family Phenuiviridae) causes large epizootics and epidemics among livestock and humans in Africa. Its main virulence factor NSs is known to inhibit the transcriptional upregulation of interferons (IFNs) by attacking the general transcription factor IIH (TFIIH) via subunit sequestration and proteasomal degradation. However, being such a strong IFN antagonist, NSs may engage additional strategies to manipulate the host cell. Accordingly, we observed that NSs induces a complete retention of poly(a) RNAs in the nucleus, as previously reported by Copeland et al. (Copeland et al., J.Virol 203 Nov, 87(2):659-69). Moreover, our mass spectrometry analysis of NSsinteracting host cell factors suggested a direct interaction with the mrna export factors Nup98 and RaeI, albeit verification by less sensitive methods have failed so far. However, using immunofluorescence analysis, we detected a disruption of the nuclear pore complex and an NSs-dependent displacement of specific nucleoporins. Our current investigations focus on the question whether RVFV also impairs protein transport. Overall, our results suggest that RVFV, which replicates in the cytoplasm and has mrnas lacking the prototypical poly(a) tail, constrains the nuclear export of host cell mrnas and possibly the transport of proteins by directly targeting nucleoporins. 94

195 NSV 208, Verona Abstract Book SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P04 SINGLE DOMAIN ANTIBODIES TO PROBE THE ANTIVIRAL FUNCTION OF INTERFERON-INDUCED MX PROTEINS Eline Soetens *, 2, Jan Spitaels, 2, Xavier Saelens, 2 Center for Medical Biotechnology, VIB, 2 Department for Biomedical Molecular Biology, Ghent University, Ghent, Belgium Abstract: For centuries, our immune system has been trapped in an evolutionary arms race with viruses. This relationship has shaped our immune defenses and led to the evolution of several specialized anti-viral proteins. An example of these are the Mx proteins; large GTPases that are part of the antiviral response induced by type I and type III interferons, and thus belong to the first line of defense against viral infections. Their importance is emphasized by their presence in almost all vertebrates and their ability to counteract a wide range of viruses. The human MxA and mouse Mx proteins confer resistance against influenza viruses, by blocking entry into the nucleus and transcription and replication, respectively. Despite the importance of these proteins, little is known about their antiinfluenza mode of action. Therefore, we aim to elucidate the interactions between human and mouse Mx proteins and influenza vrnps by using single domain antibodies (VHHs) as tools. We have isolated and sequenced 20 VHHs directed against human MxA and 20 against mouse Mx. These VHHs have been purified from the culture medium of Pichia pastoris transformants and are currently being characterized functionally for their capacity to interfere with the in vitro GTPase activity of MxA and Mx. We have also purified the vrnp complexes from an H7N7 influenza virus that is highly sensitive to Mx. In the near future, we also aim to visualize the interaction between Mx proteins and IAV by high resolution microscopy techniques, using directly labeled VHHs as imaging tools. Overall, our findings will lead to novel insights in the mechanism of IAV restriction of human and murine Mx proteins. 95

196 SUPPRESSING AND CONQUERING antiviral defenses and their suppression Abstract final identifier: P05 INVOLVEMENT OF ADAR2 IN BORNA DISEASE VIRUS INFECTION Mako Yanai *, 2, Shohei Kojima, 2, Nadine Gillich 3, Masayuki Horie, 4, Akiko Makino, 2, Keizo Tomonaga, 2 Lab. of RNA Viruses, 2 Graduate School of Biostudies, KYOTO UNIVERSITY, Kyoto, Japan, 3 Institute of Virology, University Medical Center, Freiburg, Germany, 4 Hakubi Center, Kyoto, Japan Abstract: Borna disease virus (BoDV) is, a non-segmented, negative-strand RNA virus, establishes a persistent infection in the cellular nucleus without cytopathic effect. Our previous study revealed that recombinant BoDV harboring certain foreign gene in the genome underwent A-to-G substitution only in the foreign gene, suggesting that the A-to-I editing catalyzed by adenosine deaminase acting on RNA (ADAR) may be involved in BoDV replication. Here, therefore, we evaluated the involvement of ADARs, ADAR and ADAR2, in BoDV life cycle. To assess the significance of ADARs on BoDV infection, we infected ADAR- or ADAR2- knockdown cells with BoDV, and the viral replication was monitored. The ADAR- or ADAR2-knockdown was shown to significantly decrease the infection efficiency at the early phase of BoDV infection, compared to the control cells. Next, we evaluated the number of days required to reach almost 00% infection within the cultured cells. While the control and ADAR-knockdown cells took 20 and 6 days to expand the infection in the cultures, respectively, the ADAR2-knockdown cells required 40 days to establish the persistent infection. These observations suggested that ADAR2 plays important role both in the initial infection and expansion of BoDV. To clarify the mechanisms of how ADAR2 affects BoDV infection, we performed microarray analysis and identified 65 differentially expressed genes between control and ADAR2-knockdown cells. To verify the result of microarray analysis, sirna screening was conducted with 50 genes, whose expression levels were elevated in ADAR2- knockdown cells and identified a number of genes associated with the deficiency of BoDV infection by knockdown of ADAR2. 96

197 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P07 CHARACTERIZATION OF THE H SUBTYPE-SPECIFIC NONCODING REGIONS OF INFLUENZA A VIRUSES IN REGULATING HA SEGMENT VIRION INCORPORATION Tao Deng *, Yue Xiao Institute of Pathogen Biology,Chinese Academy of Medical Sciences, Beijing, China Abstract: We previously reported that the noncoding region at both the 3 and 5 ends of the HA segments of influenza A viruses, are subtype-specific and vary significantly in sequence and length. Our study of the substitutions of the H subtypespecific NCR (ssncr) with H-H7 and H9 ssncrs indicated that both the length and particular nucleotide(s) of the HA ssncr might be involved in regulating HA segment virion incorporation. In this report, we further characterized the length and sequence requirements of the H ssncr in determining H segment packaging in the WSN (HN) rescue system. In terms of the length requirement, we used serial 3 and 5 end H ssncr truncation mutants with progressively reduced length. We described the differential effects of the length at the two ends of the H ssncr. In terms of the sequence requirement, we first used serial H ssncr mutants in which we replaced 3 and 5 H ssncrs by random sequences with the wildtype length. We found that big sequence variations at both ends showed distinct HA packaging efficiencies. We then further performed Packaging Sequence Selection Assay (PSSA) with H ssncr libraries containing random nucleotides in the H ssncr. The results suggested that both the high A/U content and the specific locations of the A/U in the H ssncr play important roles in regulating the H segment packaging efficiencies. We concluded that the H segment packaging efficiency may not be determined by a single nucleotide or specific successive nucleotides but by discrete nucleotides with low G/C content in the H ssncr. Therefore, our experiments further illustrated detailed characteristics of the H ssncr in regulating the H segment incorporation efficiency. 97

198 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P08 THE COMPLEXITY OF THE N-GLYCOSYLATIONS NEAR SITE SA ON THE HA OF THE INFLUENZA A MODULATE ITS ANTIGENIC PROPERTY AND THE AVIDITY FOR ITS RECEPTOR Rafael A. Medina *, 2, Gabriel Guajardo-Contreras 2, Richard Cadagan, Adolfo Garcia-Sastre Microbiology, Icahn School of Medicine at Mount Sinai, New York, United States, 2 Pediatric Infectious Diseases and Immunology, PONTIFICIA UNIVERSIDAD CATOLICA DE CHILE, Santiago, Chile Abstract: Antigenic drift is a key process that incorporates mutations that become fixed in the surface of the Hemagglutinin (HA) immunodominant glycoprotein of Influenza A Virus (IAV). HA also recognizes the sialic acid receptors in the host cells. One strategy used by IAVs to evade pre-existing immunity is to add N-glycosylation sites, in the globular head of HA. Glycosylation at residue 44 within site Sa of the 2009 HN can induce a polyclonal response capable of neutralizing other glycosylated HN variants. This glycosylation is also very effective at shielding this antigenic site. We hypothesized that the complexity of glycosylation 44 modulates the antigenic properties of HA and it has an effect in viral fitness. We made recombinant viruses and soluble HA proteins harboring variants of the A/Netherlands/602/09 HN virus HA containing glycosylations at sites 44, 42, 72, and We also generated either high-mannose or complex glycosylations, by growing these viruses in the presence or absence of the mannosidase inhibitor, kifunensine. Analyzes indicated a higher molecular weight of the HAs containing the 44 glycosylation in both, the recombinant viruses and soluble proteins, implying that this position allows the assembly of longer glycans compared to glycosylations in other positions. Reducing the length of the glycans increased the avidity of HA for its receptor, suggesting that glycosylations near the receptor-binding site can have an impact on viral infection. Additionally, infection of mice with viruses with complex glycosylated HAs induced more broadly neutralizing and higher titers antibodies as compared to the high-mannose glycosylated HAs viruses. These results provide new insights of the biological relevance of the N-glycosylations near site Sa and their role in antigenicity and viral fitness. 98

199 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P0 ASSEMBLY AND SPREAD OF NIPAH VIRUS ENCODING A MATRIX PROTEIN WITH A DEFECTIVE NUCLEAR EXPORT SIGNAL Marc Ringel *, Laura Behner, Anja Heiner, Lucie Sauerhering, Larissa Kolesnikova, Andrea Maisner PHILIPPS-UNIVERSITY MARBURG, INSTITUTE OF VIROLOGY, Marburg, Germany Abstract: The NiV matrix protein (M) plays a major role in virus assembly by mediating the contact between viral RNPs and surface glycoproteins. As many paramyxoviral matrix proteins, M has been shown to undergo a nuclear transit before becoming assembly competent (Pentecost et al., 205). So, aside of its role in cytoplasmic virus assembly, M might play a role in the nucleus. However, the influence of nuclear M expression on host cell functions or virus replication is mostly unclear yet. To determine if nuclear M expression affects the propagation of an assembly-defective NiV, we wanted to characterize NiV replication in the presence of an assembly-defective M protein that is readily expressed in the nucleus. For this, we generated a recombinant NiV encoding an M protein with a disrupted nuclear export signal (rniv-mnesmut). Immunofluorescence analysis confirmed that mutant M was predominantly expressed in the nucleus. Though some MNESmut colocalized with cytoplasmic inclusions, it was not found in inclusion bodies associated with the plasma membrane. Corroborating the expected assembly defect, cell-free virus titers and the relative particle infectivity of rniv-mnesmut were decreased by about 00 to 000-fold compared to NiV wildtype. Furthermore, cells infected with rniv-mnesmut showed an increased syncytia sizes suggesting that MNESmut is not only assembly defective but also fails to downregulate cell-cell fusion mediated by cell surface expressed NiV glycoproteins. Side by side analysis of rniv-mnesmut with rniv M did not reveal any differences in replication rates and particle infectivity suggesting that growth kinetics of an assembly-defective NiV is neither supported nor counteracted if M protein is functionally expressed in the nucleus. 99

200 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P TYROSINE 32 OF INFLUENZA A VIRUS MATRIX PROTEIN IS ESSENTIAL FOR EFFICIENT VIRAL GENOME PACKAGING AND PARTICLE ASSEMBLY Angeles Mecate-Zambrano, Darisuren Anhlan, André Schreiber, Lilo Greune 2, Stephanie Grothe 3, Ludmilla Wixler, Alexander Schmidt 2, Klaus Langer 3, Tianlai Shi 4, Stephan Ludwig, Yvonne Börgeling * Institute of Virology (IVM), 2 Institute of Infectiology, 3 Institute of Pharmaceutical Technology and Biopharmacy, University of Muenster, Muenster, Germany, 4 Immunology, Inflammation and Infectious Diseases (I3) DTA, Roche Pharma Research and Early Development, Roche Innovation Center, Basel, Switzerland Abstract: Influenza is a zoonotic disease with pandemic potential. The limited protection provided by current antivirals emphasizes the urgent need for a deeper understanding of the virus life cycle to develop novel therapeutics with a broad spectrum. The highly conserved matrix protein (M) is a master regulator of the virus life cycle and essential for particle morphogenesis. Its multifunctionality qualifies M as a promising target for combating influenza, since it provides the opportunity to potentially block infections at various stages. M tyrosine 32 (Y32) was previously suggested to be essential for virus fitness as viruses carrying a mutation at this site could not be rescued. Based on overexpression data, it was hypothesized that this might be due to defective nuclear entry of M. In the present study, we were able to rescue a virus mutant carrying an alanine at Y32 allowing for analysis of the role of this tyrosine during genuine infection. WSN M Y32A showed strongly decreased viral replication compared to wild type. While we did not detect any reduced nuclear import, coarse M protein clusters were observed at the plasma membrane in late stages of infection. Deeper characterization showed random defects in viral genome packaging, resulting in an increased production of non-infectious progeny. Furthermore, while general M Y32A association to membranes was not altered, resistance to detergent solubilization from membrane fractions was decreased. This suggests a potential defect in M recruitment to IAV assembly sites in lipid raft domains, which resulted in a diminished structural stability of viral progeny and the presence of filamentous particles. These findings indicate that M Y32 is crucial at late stages of IAV replication, and that efficient particle assembly including genome packaging is triggered by Y32 of the M protein. 200

201 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P2 ARENAVIRUS EXIT, UBIQUITIN, AND THE ESCRT PATHWAY Christopher Ziegler *, Loan Dang, Emily Bruce, Bryan Ballif 2, Jason Botten Medicine, 2 Biology, UNIVERSITY OF VERMONT, Burlington, United States Abstract: Viral late domains are used by many viruses to recruit the cellular endosomal sorting complex required for transport (ESCRT) pathway to mediate membrane scission during viral budding. Unlike the P(S/T)AP and YPXL late domains, which interact directly with the ESCRT proteins Tsg0 and ALIX, the molecular linkage connecting the PPXY late domain to ESCRT proteins is unclear. The mammarenavirus lymphocytic choriomeningitis virus (LCMV) matrix protein, Z, contains only one late domain, PPXY. We recently showed that this domain in Z and the ESCRT pathway are required for the release of defective interfering (DI) particles but not infectious virus. To better understand the molecular mechanism driving this process, affinity purification-mass spectrometry was used to identify host proteins that interact with the Z proteins of LCMV and Lassa virus. Nedd4 family E3 ubiquitin ligases were common host partners between the two Z proteins and this interaction with LCMV Z required an intact PPXY. We demonstrated that these ligases directly ubiquitinate LCMV Z and have mapped the specific lysine residues modified. We are currently testing recombinant LCMVs containing mutations at these particular lysine residues for their ability to produce both infectious virus and DI particles and these findings will be discussed. 20

202 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P3 THE IQGAP FAMILY PROTEINS IN MARBURG VIRUS INFECTION Olga Dolnik *, Susanne Berghöfer, Stephan Becker Institut für Virologie, PHILIPPS-UNIVERSITY MARBURG, Marburg, Germany Abstract: IQGAPs are evolutionary conserved proteins involved in the regulation of many cellular processes including cytokinesis, cell migration and proliferation, intracellular signaling, vesicle trafficking and cytoskeletal dynamics. Humans express three isoforms IQGAP, IQGAP2 and IQGAP3. IQGAP is expressed ubiquitously, whereas IQGAP2 is mainly expressed in the liver and IQGAP3 in the brain. We could show earlier that IQGAP forms a comet-like structure at the rear end of Marburg virus (MARV) nucleocapsids during their transport in infected Huh-7 cells and sirna down-regulation of IQGAP resulted in decreased MARV release. To further characterize the role of IQGAP in MARV infection we analyzed the release of infectious MARV particles from IQGAP knockout HAP cells. We observed almost no differences or even slightly increased titers in comparison to the parenteral HAP cells. Interestingly, an upregulation of IQGAP2 expression was detected in the IQGAP knockout cells in comparison to the HAP cell line, suggesting redundant usage of IQGAP2 for efficient virus release. Accordingly, MARV release from a IQGAP2 knockout cell line was slightly reduced and expression of IQGAP was elevated. We then analyzed the expression of IQGAP3 in the HAP cells and detected IQGAP3 being expressed and recruited into the MARV-induced inclusion bodies. Further analyses revealed elevated IQGAP3 expression levels in a IQGAP and IQGAP2 double knock-out cell line, suggesting that all the three human IQGAP proteins are involved in MARV release. To determine the importance of the IQGAP protein family in MARV infection we generate a triple-knock-out cell line for all three IQGAP proteins (IQGAP triple- ) using the CRISPR/Cas9 methodology. MARV infected IQGAP triple- cells are currently analyzed for virus release and nucleocapsid transport. 202

203 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P4 THE VRNA-VRNA INTERACTIONS IMPORTANT FOR HA VRNA PACKAGING OF THE INFLUENZA A VIRUS Sho Miyamoto *, Yukiko Muramoto, Keiko Shindo, Jamie L. Gilmore, Masahiro Nakano, Takeshi Noda Laboratory of Ultrastructural Virology, Institute for frontier life and medical sciences, Kyoto University, Kyoto, Japan Abstract: The influenza A virus genome is composed of eight single-stranded negative-sense RNA segments (vrnas). The eight vrnas are selectively packaged into progeny virions in a regular arrangement. For efficient vrna packaging, each vrna possesses the segment-specific packaging signal sequence located in the noncoding and terminal coding regions of both 3 and 5 ends. The incorporation of eight different vrnas evokes specific interactions among the vrnas via the packaging signals. However, vrna-vrna interactions involved in the selective genome packaging remains largely uncertain at the nucleotide level. Here, to identify vrna(s) that potentially interact with HA vrna during genome packaging, we generated several mutant influenza viruses (A/WSN/33) possessing silent mutations in the packaging signal of HA vrna. Amongst them, one mutant virus, which showed a significant reduction in viral replication, was serially passaged in cultured cells until the replication efficiency was restored. RT-qPCR analysis showed that the mutant virus had a specific defect in HA vrna incorporation. After the passages, the mutant virus obtained point mutations in the packaging signals of HA and PB2 vrnas, both of which synergistically contribute to efficient virus replication and HA vrna packaging. Furthermore, a direct RNA-RNA interaction between HA and PB2 vrnas was confirmed in vitro. Overall, our data suggest that direct interactions of HA vrna with PB2 vrna are involved in the HA vrna packaging into influenza virions. 203

204 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P5 ANALYSIS OF HIGHER ORDER RNA STRUCTURES IN THE INFLUENZA A VIRUS GENOME Bernadeta Dadonaite *, Ervin Fodor, Alain Laederach 2, David Bauer Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom, 2 Department of Biology, University of North Carolina, Chapel Hill, United States Abstract: Influenza A viruses (IAVs) are respiratory pathogens that constitute a major threat to human health. IAVs have negative sense, segmented, single stranded RNA genomes, which are maintained in viral ribonucleoprotein complexes (vrnps). Each vrnp consists of the viral RNA (vrna), viral polymerase and an oligomer of the viral nucleoprotein (NP). However, little is known about how the IAV RNA genome is structured inside virions. In this study we have used multiple high-throughput sequencing methods to analyse native secondary RNA structures in the context of vrnps inside IAV virions. Using selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) we show that secondary RNA structures form in the context of vrnps. Different viral RNA segments have unique secondary RNA structures, suggesting an uneven distribution of NP along the vrna. In addition, using sequencing of psoralen crosslinked, ligated, and selected hybrids (SPLASH), we show that IAV maintains a redundant network of inter-vrnp RNA interactions. Mutation of nucleotides involved in these interactions resulted in nonviable or attenuated influenza viruses with vrnp packaging defects. Further research into the vrna interaction networks present in different influenza strains may lead to improved understanding of the molecular mechanisms governing the reassortment of influenza viruses. 204

205 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P6 THE INFLUENZA VIRUS M2 PROTEIN CYTOPLASMIC TAIL INTERACTS WITH HOST PROTEINS TO FACILITATE VIRAL MORPHOGENESIS. Matthew Badham *, Jeremy S. Rossman School of Biosciences, UNIVERSITY OF KENT, Canterbury, United Kingdom Abstract: Influenza A virus (IAV) is an unusual pathogen in that it is pleomorphic, capable of forming virions ranging in shape from spherical to filamentous. Despite decades of research on IAV, much remains unknown about the formation of filamentous IAV and their role in the viral replication cycle. Hijacking of the host cell systems not only for replication but also for subversion of defence mechanisms is crucial for efficient proliferation of progeny virus. The 97 amino acid protein M2 possesses ion channel activity, necessary for acidification of the interior of the virus during initial infection and enabling uncoating of the viral RNP. However, the cytoplasmic tail of M2 (M2-CT) is known to also undergo interactions with multiple cellular proteins during virus assembly and budding, affecting the morphology of budding virions. It was recently found that the M2-CT is essential for the IAV evasion of autophagy and for filamentous virion formation. The M2-CT binds and sequesters LC3, a small ubiquitin-like protein and key autophagy regulator, thus attenuating the progression of autophagy and facilitating viral morphogenesis though an unknown mechanism. In this work we use mass spectrometry, confocal microscopy and biochemical assays to further characterise the interactions between the M2-CT and the small ubiquitin-like proteins (including LC3) and define their affect on virus morphology. We find that IAV does not form filamentous virions in the absence of the M2-CT and that interaction between the M2-CT and several small ubiquitin-like proteins are essential for viral morphogenesis, though many of these interactions are expendable for virus replication in vitro. 205

206 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P7 EVIDENCE FOR A LINK BETWEEN BUDDING MORPHOLOGY AND TRANSMISSION OF AVIAN INFLUENZA A VIRUSES Carina Conceicao *, Helen Wise 2, Nikki Smith, Lita Murphy, Seema Jasim, Samantha Lycett, Anamika Mishra 3, Ashwin Raut 3, Dave Burt, Darrell Kapczynski 4, Munir Iqbal 5, Lonneke Vervelde, Paul Digard Infection and Immunity, The Roslin Institute, University of Edinburgh, Roslin, 2 Institute of Genetics and Molecular Medicine, Heriot Watt University, Edinburgh, United Kingdom, 3 ICAR-NIHSAD, NIH, Bhopal, India, 4 Exotic & Emerging Avian Viral Diseases, USDA, Athens, United States, 5 Avian influenza, The Pirbright Institute, Pirbright, United Kingdom Abstract: Pleomorphic virus particles are a characteristic of influenza A virus (IAV), which can produce spherical virions of 00nm in diameter and filaments up to 30µm long. The majority of non-laboratory adapted mammalian IAV strains are thought to be filamentous however avian IAV budding morphology remains largely uncharacterised. To investigate this, 22 avian viruses representing the major clades found from phylogenetic analysis of segment 7 were characterized for budding morphology, either as virus isolates and/or as 7: segment 7 reassortants in an PR8 backbone. The majority of viruses produced filaments up to 0µm but a sizeable minority were non-filamentous. Budding phenotype did not correlate with any particular subtype of virus or clade. However, the filamentous phenotype was common in duck viruses but less common in chicken viruses. Previous rules determined for M sequence polymorphisms affecting virion shape in mammalian strains were not confirmed, but mutagenesis of closely related strains with differing budding morphologies identified positions 59, 69 and 234 as IAV strain-dependent determinants. Furthermore, statistical analysis of the galliforme and anseriforme strains characterized here coupled with bioinformatic analysis of all avian segment 7s identified Ile at position 234 as significantly overrepresented in chicken IAVs and underrepresented in duck. Chicken viruses with Ile at this position produced spherical virions, while filamentous duck strains possessed Leu. Initial analyses of in vivo transmission experiments in chickens with the normally spherical A/chicken/Pakistan/UDL-0/08 and its filamentous mutant showed significantly raised buccal shedding of the latter from two sets of contact-infected birds. These results suggest that filamentous viruses are more common in ducks than chickens and the filamentous trait may be associated with enhanced shedding. 206

207 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P8 CHARACTERIZATION OF THE MOLECULAR MECHANISMS UNDERLYING INTERACTION OF LASSA VIRUS Z PROTEIN WITH THE HOST FACTOR KIF3A Sarah Fehling *, Annika I. Meyer, Birthe Ehlert, Shuzo Urata 2, Jiro Yasuda 2, Thomas Strecker Institute of Virology, PHILIPPS UNIVERSITY MARBURG, Marburg, Germany, 2 Department of Emerging Infectious Diseases, Nagasaki University, Nagasaki-shi, Japan Abstract: Lassa virus (LASV), a member of the Arenaviridae family, exits its host cell by budding from the plasma membrane, a process that is driven by the viral matrix protein Z. We have shown earlier that the microtubule-dependent motor protein KIF3A mediates the intracellular transport of Z protein. However, the molecular details of Z-KIF3A interaction remained elusive. KIF3A is characterized by an N-terminal motor domain, a forkhead-associated (FHA) domain, and five predicted coiled-coil domains. To identify domains within KIF3A that are important for interaction with Z protein, we generated a series of KIF3A deletion constructs. Biochemical and microscopic analyses pinpointed the Z-KIF3A binding interface to a C-terminally located coiled-coil motif and adjacent amino acids, while the FHA domain is dispensable for interaction. Our results describe a novel interaction domain for KIF3A-mediated cargo transport, which is distinct to previously defined cargo binding sites. To shed light on the interaction domain within Z protein, we also used a series of Z deletion constructs. Co-immunoprecipitation analyses and co-localization studies of Z protein mutants with KIF3A revealed a region spanning amino acids 3 to 50 within the highly conserved RING domain of Z that is critical for binding. The precise mapping of the binding sites within Z protein and KIF3A will allow the development of therapeutic strategies using inhibitors that target Z-KIF3A interaction. 207

208 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P9 ACCESSIBILITY ASSESSMENT OF THE TERMINAL REGION OF INFLUENZA A VIRUS GENOME SEGMENT BY FLUORESCENCE IN SITU HYBRIDIZATION Fumitaka Momose *, Yuko Morikawa Kitasato Institute for Life Sciences, KITASATO UNIVERSITY, Tokyo, Japan Abstract: The genome of influenza A virus consists of eight vrna segments and forms vrnp complexes. It is widely accepted that the segments selectively assemble, and one set of the segments is packaged into a virion. The packaging signals of each vrna segment have been narrowed to both terminal regions by reverse genetic analyses using deletion and substitution mutants. However, the core nucleotide sequences essential for the recognition and interaction of the segments remain to be elucidated. It is possible that the core sequences would be covered if a segment assembled with others by base pairing or a mediating factor. In this study, fluorescence in situ hybridization (FISH) was applied to assess the accessibility of the 5' terminal region of the eighth segment (Seg. 8). Each cdna probe binding within the 5' terminal region of Seg. 8 has a complementary sequence (ca 25 bases) overlapping each other, followed by a common detection sequence. MDCK cells were infected with A/Puerto Rico/8/34 strain and were subjected to FISH at six hours post infection. The maintenance of vrnp structure was confirmed using the anti-np mab6a5 preferentially binding to NP in an RNP complex. Fluorescent signals of cdna probes bound to vrna were intensified and detected with a fluorescence microscope. When hybridization was performed in the presence of 3% formamide, the hybridization efficiencies of three cdna probes targeting 37-62, 62-86, and bases of 5' terminal region were low. No correlation of the hybridization efficiency with the Tm values or the GC contents of complementary sequences suggested that the low efficiencies of the three probes were not due to poor probe characteristics but rather were due to the competition with other segments or factors. We supposed that the 5' terminal selective packaging signal of the Seg. 8 had been split into multiple regions. 208

209 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P20 DETAILED FUNCTIONAL MAPPING OF THE PARAMYXOVIRUS ACCESSORY PROTEINS IN VIRAL INFECTION Ryoko Kawabata, Asuka Yoshida 2, Takemasa Sakaguchi, Takashi Irie * Department of Virology, Institute of Biomedical & Health Sciences, Hiroshima University, Hiroshima, 2 Laboratory Animal Research Center, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Abstract: Among Mononegavirales, paramyxoviruses are unique in producing more than one polypeptide species (C and/or V) from the P gene, in addition to the P protein. The C and V proteins are known as accessory proteins because they are not essential for minimal viral growth in cultured cells. However, they play a variety of important roles in viral growth and pathogenicity in vitro and in vivo. Indeed, a growing number of functions of the accessory proteins have been revealed, such as counteracting host innate immunity, avoiding host detection of infection, inhibiting apoptosis, induction of necroptosis, regulating polarized viral RNA synthesis, and promoting viral assembly and budding. These functions can be assessed by generating recombinant viruses in which the accessory proteins are knocked out. However, due to overlaps of their open reading frames, a more detailed evaluation of their functions during actual viral infection is severely limited by the difficulties in introducing amino acid substitutions only into the accessory proteins without any alterations within the P protein. To avoid this restriction, we generated recombinant Sendai viruses (rsevs) in which the C or V protein was expressed from an independent cistron introduced between the HN and L genes, not from the P gene. Both of the viruses replicated as well as the parental wild-type virus. Based on this rsev system, a series of rsevs possessing five alanine substitutions throughout the Y2 (3-204 amino acid [aa]) region of the C protein, 204 amino acids long, were generated and examined in terms of their effect on viral replication as well as the other known functions of the C protein. These results show, for the first time, a detailed functional map of the SeV C protein in an actual viral infection. 209

210 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P2 INFLUENCE OF NUCLEAR LOCALIZATION SITES IN HENDRA VIRUS MATRIX PROTEIN ON INTERACTION WITH ANP32B AND VIRUS LIKE PARTICLE FORMATION Maria Günther *, Stefan Finke Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany Abstract: In addition to the essential role of paramyxovirus matrix (M) proteins in assembly and budding of the cytoplasmically replicating viruses at the plasma membrane, many M proteins are also transported into the nucleus. For some paramyxoviruses, interactions with nuclear host factors have been described and host manipulatory functions are assumed. M protein of Hendra virus (HeV) comprises a bipartite nuclear localization signal (NLS) and interaction with acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) has been shown. Notably, nuclear import of henipaviral Ms is not only considered to be important for host manipulation but also for virus budding. The NLS contains a lysine residue that is mono-ubiquitinylated in the nucleus and nuclear mono-ubiquitinylation has been shown to be involved as a trigger for nuclear export of M. Since also budding activity at the plasma membrane was affected by mutations at that position, it has been suggested that nucleo-cytoplasmic trafficking and monoubiquitinylation are requirements for virus budding at the plasma membrane. In order to map interaction sites for ANP32B, we performed Ala scanning mutagenesis on HeV M and screened for effects of ANP32B on intracellular localization of M and co-purification with ANP32B. Here we focus on NLS-mutants with amino acid exchanges in the bipartite NLS of HeV M. Immunofluorescence analyses showed that mutations in one part of the NLS led to the loss of nuclear localization, whereas plasma membrane localization was not affected. First budding assays suggest the release of virus like particles (VLPs) after expression of the NLS mutant. These data indicate that nuclear shuttling and mono-ubiquitinylation in the nucleus are mechanistically not directly linked. 20

211 NSV 208, Verona Abstract Book BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P22 SURFACE-EXPOSED LYSINE RESIDUES OF THE CDV MATRIX PROTEIN CONTRIBUTE TO MEMBRANE ASSOCIATION AND BUDDING ACTIVITY Nicole Kadzioch *, 2, Matthieu Gast, 2, Philippe Plattet 2 Graduate School for Cellular and Biomedical Sciences, University of Bern, 2 Division of Experimental Clinical Research, University of Bern, Vetsuisse Faculty, Bern, Switzerland Abstract: The Matrix (M) protein of Morbilliviruses coordinates viral packaging, membrane budding and particle formation. Two monomers presumably assemble to form dimer and higher oligomers. The current model of cell-exit postulates that M associates with lipids to ultimately trigger membrane budding at the plasma membrane. Additionally, recent data revealed that ubiquitination of a lysine residue within Nipah Virus M, likely mediated within the nucleus, played a key role in viral particle production. Based on a homology model of Canine Distemper Virus (CDV) M, surface-exposed positively charged lysine (K) residues locating within M-CTD were selected for substitutions. Indeed, lysines are well-known to potentially interact with the negatively charged groups of the phospholipid heads. The selected lysines were mutated into either arginine (R) or glutamic acid (E), and the derivative M-mutants submitted to assays investigating Virus-Like-Particles (VLPs) production (biochemical analyses) and cellular localization (immunofluorescence). Preliminary data indicated that the conservative mutations K227R, K228R, K265R and K268R did not affect budding and the plasma membrane-targeting capacity of M. In contrast, the non-conservative M-variants K227E, K228E, K265E and K268E were deficient in budding and exhibited a clear cytosolic phenotype. Remarkably, substituting residue K240 of M led to opposite profiles: while mutant M-K240R was inefficient in VLPs production, M-K240E remained fully functional, although both M proteins were properly localized at the cell periphery. Since it was reported that Morbillivirus M-K240 undergoes ubiquitination, the functional impacts of several substitutions at this position is currently under investigation. In summary, our preliminary data already revealed a potential role of some lysine residues in membrane association and budding activity. 2

212 BUILDING AND ESCAPING viral assembly and budding Abstract final identifier: P23 INVESTIGATION OF Α-HELICES OF CANINE DISTEMPER VIRUS MATRIX PROTEIN IN OLIGOMERIZATION AND MEMBRANE BUDDING ACTIVITY Matthieu Gast *, 2, Nicole Kadzioch, 2, Philippe Plattet 2 Graduate School for Cellular and Biomedical Sciences, University of Bern, 2 Division of Experimental Clinical Research, University of Bern Vetuisse Faculty, Bern, Switzerland Abstract: The Canine Distemper Virus (CDV), related to the human Measles virus, induces devastating epidemics in numerous wild species. It is supposed that specific antiviral compounds associated with vaccination campaigns may allow a better control of ongoing epidemics. CDV Matrix (M) protein orchestrates viral assembly and budding of progeny virions at the plasma membrane. Two M-protomers, composed of N- and C-terminal domains (NTD and CTD), likely assemble in a head-to-tail fashion, via a large dimeric binding interface, to form dimers. Interestingly, M-NTD and -CTD contain surface exposed α-helices (α2 and α9, respectively) that are proposed to be involved in dimer-dimer and higher oligomers formation. To investigate a potential role of high M-oligomers assembly in membrane budding and Virus-Like-Particles (VLPs) formation, we substituted multiple residues mapping within the critical α2 and α9 helices. Production of VLPs was assessed by biochemical analyses as well as a newly developed very sensitive NanoLuc Luciferase (NLuc)-based M-budding assay, which enables direct detection of VLPs in the cell supernatant. Surprisingly, among the various mutations introduced, two non-conservative mutants (M-E89R [α2] and M-L239D [α9]) exhibited strong impairments in VLPs formation using both assays, while displaying unaltered cell periphery accumulation. Other mutants were characterized by wild type-like profiles in localization and budding activity. Dimerization and ubiquitination propensities as well as membrane association capacity of the two identified budding-deficient M-mutants (E89R and L239D) are currently under investigation. Overall, these preliminary data suggest an important role of two specific amino acids locating within the α-helices of CDV-M in controlling membrane egress activity by, perhaps, regulating high oligomers assembly at the plasma membrane. 22

213 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P24 A RECOMBINANT MEASLES VIRUS BLIND TO SLAM IS A PROMISING CANDIDATE FOR SCIRRHOUS GASTRIC CANCER THERAPY Chieko Kai *, Koichiro Shoji, Hyun-jeong Kwon, Akihiro Sugai, Kazuyoshi Yanagihara 2, Hideki Yamaguchi 3, Tomoko Nakanishi, Hiroki Sato, Tomoko Fujiyuki, Misako Yoneda Laboratory Animal Research Center, The Institute of Medical Science, THE UNIVERSITY OF TOKYO, Tokyo, 2 Exploratory Oncology & Clinical Trial Center, National Cancer Center, Chiba, 3 Department of Cancer Cell Research, Sasaki Institute, Tokyo, Japan Abstract: Virotherapy using oncolytic virus is expected as a novel therapy for cancer. We previously generated a recombinant measles virus (rmv-slamblind), which is unable to use SLAM as its principal receptor and lost the MV pathogenicity. We have reported that it infects and kills cancer cells derived from breast, lung, colorectal, and pancreatic cancers dependently on expression of the virus receptor molecule, Nectin-4. (Gene Therapy, 202; Oncotarget, 205; Scientific Reports, 206; Cancer Science, 206). Gastric cancer is the third most common cause of cancer-related death in the world. Especially, scirrhous gastric cancer (SGC) has a very poor prognosis, and thus novel therapies are desired. In this study, we examined applicability of rmv- SLAMblind for SGC. Nectin-4 expression was observed in approximately 70% of SGC cell lines, and showed severe cytotoxic activity to SGC cell lines highly expressing nectin-4. Intratumoral administration of rmv-slamblind to subcutaneous xenograft mouse model with a SGC cell line remarkably suppressed the tumor growth. Furthermore, rmv- SLAMblind intraperitoneally inoculated into peritoneal dissemination xenograft model targeted scattered tumors in the peritoneal cavity, and significantly suppressed the tumor growth and clinical symptoms. These results suggest that rmv-slamblind is a promising candidate as a therapeutic agent for treatment of scirrhous gastric cancer, particularly with peritoneal dissemination. 23

214 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P25 RIFT VALLEY FEVER VIRUS INFECTION OF PREGNANT SPRAGUE-DAWLEY RATS RESULTS IN FETAL INFECTION AND DEMISE Amy Hartman *, Cynthia McMillen Center for Vaccine Research, UNIVERSITY OF PITTSBURGH, Pittsburgh, United States Abstract: Rift Valley fever is an important emerging zoonotic disease in Africa and the Saudi Arabian peninsula. Domesticated livestock are higly susceptible to severe disease when infected with RVFV, and death of fetuses in pregnant animals is extraordinarily high. The effect of RVFV infection on human pregnancy is less clear. Two cases of vertical transmission of RVFV have been described in humans and one cross-sectional study reported a 4-fold increased risk of miscarriage in RVFV+ women. Here, we sought to determine the effect of RVFV in pregnant rats with the goal of developing a reliable rodent model of congenital RVF. Pregnant immunocompetent Sprague-Dawley rats at early (embryonic day 5; ED5) and late (embryonic day 4; ED4) gestational stages were subcutaneously infected with pathogenic RVFV. Pregnant E4 dams with no clinical signs had widespread virus detected in the spleen, liver, lung, brain, uterus, and ovaries at five days post-infection. Some pups were stillborn, and surviving 0-day old pups from these mothers retained 3-log more viral RNA than maternal tissue despite grossly normal physiology of the pups. In infected ED5 rats, virus preferentially homed to the placenta and fetal tissue, in which 000x more virus was detected than in maternal tissue. Potential readsorption and hemorrhage of the fetuses was evident. This study is the first of its kind to develop a rodent model of congenital RVF and further explore the potential for RVFV to cause congenital disease in humans. This rodent model can serve as a tool to screen antiviral therapies and vaccinations targeting RVFV. 24

215 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P26 GENOME-WIDE ANALYSES OF FILOVIRUS-HOST CELL PROTEIN-PROTEIN INTERACTIONS Douglas J. LaCount *, Veronica J. Heintz, James P. Connell, Venkatesh Sivanandam, Aditi Kesari, Ling Wang, Olena Shtanko 2, Ann Reyes 2, Robert A. Davey 2 Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, 2 Department of Virology and Immunology, Texas Biomedical Research Institute, San Antonio, United States Abstract: Ebola (EBOV) and Marburgvirus (MARV) cause potentially lethal hemorrhagic fevers in humans characterized by uncontrolled viremia, a systemic pro-inflammatory response, and multi-organ failure. Recent outbreaks highlight the critical need to better understand the interactions of these viruses with host cells. Using a combination of genome-wide yeast two-hybrid (Y2H) screens and sirna knockdowns, we are identifying critical virus-human protein-protein interactions. We performed 432 Y2H library screens with 90 filovirus bait constructs against three cdna libraries. We identified 96 unique interactions between filovirus and cellular proteins, of which 365 were reproduced in at least two independent screens. One hundred and two interactions were shared between EBOV and MARV. Enrichment analyses revealed that VP30 targets proteins involved in RNA metabolism, many of which localize to stress granules. We screened 66 cellular proteins that bound to either EBOV or MARV VP30 in sirna knock down experiments followed by live virus infection. We observed a higher percentage of the cellular proteins identified in the Y2H screens affected virus replication than previously observed in experiments with randomly selected sirnas. Two distinct phenotypes were observed. Although most sirnas that affected virus replication caused a decrease, eight sets of sirnas, including those targeting RBBP6, increased replication. This suggests these cellular factors normally function to inhibit virus replication. Studies to ascertain the mechanism by which virus replication is affected are underway. This study represents the largest effort to date to compare the host factors targeted by EBOV and MARV. 25

216 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P27 INFLUENZA VIRUSES AND PARAMYXOVIRUSES ENHANCE THE ADHERENCE AND INVASION PROPERTIES OF STREPTOCOCCI BY A SIALIC ACID-DEPENDENT INTERACTION WITH THE CAPSULAR POLYSACCHARIDE Georg Herrler *, Fandan Meng, Jie Tong, Nai-Huei Wu, Yuguang Fu, Nadine Krüger, Maren Seitz 2, Peter Valentin- Weigand 2 Virology, 2 Microbiology, Stiftung Tierärztliche Hochschule Hannover, Hannover, Germany Abstract: Viral-bacterial co-infections may result in more severe disease than the respective mono-infections. The underlying mechanisms varies among different pathogens. We report a mechanism involving the interaction of viral hemagglutinins with sialic acids of encapsulated streptococci. The porcine pathogen Streptococcus suis that has a zoonotic potential contains a2,6-linked sialic acid that is recognized by porcine and human influenza viruses. Using precision-cut lung slices (PCLS) as a model for differentiated airway epithelial cells we found that hemagglutinins expressed on the surface of virus-infected cells interact with S. suis resulting in enhanced bacterial adherence. Late in infection, when the airway epithelium is destroyed, the porcine pathogen adhered and invaded subepithelial cells independent of the capsular sialic acid. Group B streptococci (GBS, S. agalactiae), a human pathogen, contains a similar capsular polysaccharide differing only in the linkage type of the terminal sialic acid. GBS contains a2,3-linked sialic acid which is recognized by avian influenza viruses. Adherence to and invasion of airway cells was greatly enhanced when the cells were preinfected by avian influenza viruses. Co-infections by GBS and avian influenza viruses may be rare events. However, for some paramyxoviruses, e.g. human parainfluenzavirus 3 (HPIV3) and mumps virus (MuV) it has been shown that they use a2,3-linked sialic acid as a receptor determinant. We found that adherence to and invasion of airway cells is enhanced when the cells express the HN proteins of either HPIV3 or MuV on the surface. Taken together, bacterial pathogens containing capsular sialic acid residues may use the interaction with virus-infected cells to enhance their adherence and invasion properties which my result in a more severe disease. 26

217 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P28 SEX-DEPENDENT HOST RESPONSES ASSOCIATED WITH EBOLA VIRUS PATHOGENICITY Atsushi Okumura, Friederike Feldmann 2, Elaine Haddock 2, David Threadgill 3, Heinz Feldmann 2, Angela Rasmussen * Center for Infection and Immunity, Columbia University, New York, 2 Laboratory of Virology, Rocky Mountain Laboratories, NIAID/NIH, Hamilton, 3 Veterinary Pathobiology, Texas A&M University, College Station, United States Abstract: The sexes differ in susceptibility to viral infection and subsequent disease pathogenesis. During the West African Ebola virus disease (EVD) outbreak, female patients had a significantly lower risk of death compared to male patients (p<0.00), despite similar numbers of exposures to sick individuals. Genetic and physiological differences between the sexes may thus dictate host responses to Ebola virus (EBOV) infection that determine disease severity and outcome. We used the Collaborative Cross (CC) mouse model to investigate the relationship between sex and pathogenicity in the context of multiple EVD phenotypes. The CC is a panel of genetically diverse mice that develop background-dependent distinct disease presentations. Both standard CC mice and F mice bred from intercrossed CC lines are susceptible to three major outcomes analogous to different presentations of human EVD: survival, lethal EVD, and lethal EVD with hemorrhagic syndrome. Of 9 lines screened for sex specific outcomes, we observed stark differences (in which all the animals of one sex survived while the other sex succumbed to lethal disease) in 2 lines, and measurable sex-specific disease features in 74% of the lines tested, including body weight, viral titers in liver and spleen, kinetics of disease progression, and survival rate. We then used RNAseq to develop sex-specific transcriptomic profiles associated with disease severity and outcome. We identified a network of differentially expressed testosterone-dependent genes related to inflammation, shock, vascular remodeling, and apoptosis that were strongly upregulated in male mice with lethal disease, indicating that distinct sexspecific host responses underlie pathogenesis. We are currently expanding these efforts to identify sex-specific host responses that determine disease severity, as well as performing more detailed studies of sex-dependent mechanisms of pathogenicity. 27

218 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P30 WHOLE GENOME CRISPR/CAS9-KO SCREEN REVEALS PROTEIN KINASE R (PKR) AS THE KEY PLAYER IN RABIES VIRUS CYTOTOXICITY Alexander Ghanem *, Alexandru A. Hennrich, Maximilian F. Eizinger, Karl-Klaus Conzelmann MAX V PETTENKOFER-INSTITUTE AND GENE CENTER, LMU MUNICH, Munich, Germany Abstract: Wildtype neurotropic rabies rhabdovirus (RABV) is typically a non-cytolytic "stealth" virus, while cell cultureadapted attenuated vaccine strains like SAD eventually kill infected cells. Understanding of the differential mechanisms behind RABV cytotoxicity is crucial for the development of vaccines, oncolytic viruses, and rabies vectors for neuronal tracing. To identify cellular factors mediating RABV cytotoxicity, we performed a pooled genetic CRISPR/Cas9 KO-screen. Human HEK293T cells expressing Cas9 were transduced with a genome-wide lentiviral sgrna library and infected with cytotoxic SAD. Genomic DNA from surviving cells was isolated and analyzed by NGS for enriched sgrna sequences. Notably, in cells surviving SAD infection, only sgrnas targeting a single gene (EIF2AK2) encoding protein kinase R (PKR) were highly enriched. Independent knock-out of the EIF2AK2 gene rendered HEK293T and A549 cells resistant to SADmediated cell death. Knock-out of the PKR-activating PACT, or of RIG-I and MAVS, which are instrumental in sensing RABV 5 -triphosphate RNAs and inducing interferon, further illustrate that PKR is the sole RNA sensor mediating death in SADinfected cells. While infection of wt HEK293T cells with SAD and cytotoxic versions of SAD (SAD TR) strongly activated PKR and caused phosphorylation of eif2α, a biologically selected non-cytotoxic SAD version (SAD Le) did not. Nevertheless, cytotoxic viruses produce more IFN-inducing RNAs than persistent variants. We are currently investigating the contribution of different viral RNA species to the activation of cellular RNA sensors in different cell lines, including stemcell derived neurons. 28

219 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P3 FUNCTIONAL ANALYSIS OF A CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS GENOME FROM TICKS IDENTIFIES A GLYCOPROTEIN VARIANT THAT POORLY INFECTS HUMAN CELLS Brian Hua *, Florine Scholte, Valerie Biewener 2, Marco Marklewitz 2, Christian Drosten 2, Stuart Nichol, Christina Spiropoulou, Sandra Junglen 2, Éric Bergeron Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, GA, United States, 2 Institute of Virology, Charité Universitätsmedizin Berlin, Berlin, Germany Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a widespread emerging pathogen that can cause severe human disease with case mortality rates up to 30%. CCHFV is a tick-borne virus exhibiting extensive genomic diversity across strains with highly divergent strains known to co-circulate in the same geographical area. It is not fully understood how these differences in genomic sequence regulate virus activity especially in the context of virus transmission to humans. To further investigate CCHFV genomic contribution in the infection of human cells, we obtained the full genomic sequence of a CCHFV strain directly from a tick captured in Malko Tarnovo (MT), Bulgaria. The CCHFV MT strain is closely related to the Greek AP92 strain, which is known to exhibit widespread dissemination but relatively infrequent detection in clinical disease. Using the CCHFV MT genome sequence, we employed reverse-genetics approaches to generate virus-like particles (VLPs) and a plasmid set to rescue recombinant MT CCHFV. Attempts to generate VLPs with the MT glycoprotein precursor (GPC) uniquely resulted in attenuated reporter activity as compared to VLPs generated with the GPCs of strains representing all other CCHFV phylogenetic clades. Consistently, we rescued virus with the MT S and L genomic segments but failed to rescue virus containing the MT M (GPC-encoding) genomic segment. Analysis of chimeric and point mutant GPC constructs revealed that a single amino acid in the MT Gc glycoprotein region, Gly6, is responsible for poor MT GPC activity in human cells. Our study warrants further functional analyses of CCHFV sequences from tick reservoirs to better understand CCHFV transmission and disease association. 29

220 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P32 TISSUE TROPISM AND DISSEMINATION OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS IN IFNAR KNOCKOUT MICE VISUALIZED BY IN SITU FLUORESCENT IMAGING Stephen R. Welch *, Florine E. Scholte, Jana M. Ritter 2, JoAnn D. Coleman-McCray, Jessica R. Harmon, Sherif R. Zaki 2, Stuart T. Nichol, Eric Bergeron, Jessica R. Spengler, Christina F. Spiropoulou VSPB, 2 IDPB, Centers for Disease Control and Prevention, Atlanta, United States Abstract: Animal models of disease are a vital tool for understanding viral pathogenesis and immunity. The use of reporter viruses in these models allows for direct visualization and identification of infected tissues and cells in the host. Here, we investigated a Crimean-Congo hemorrhagic fever virus (CCHFV, strain IbAr0200) expressing the ZsGreen (ZsG) fluorescent protein (0200/ZsG) in the IFNAR knockout mouse model. We also evaluated in vivo a reporter mutant virus in which the non-structural protein NSm ORF was removed from the M segment (0200ΔNSm/ZsG). While functions have been ascribed to the NSm proteins of other species of the Bunyavirales family, nothing is known about the role CCHFV NSm plays during infection. We followed infection by assessing survival, viral loads, and histopathology, and imaged tissues expressing ZsG in situ. We found that disease phenotype and pathology were comparable between the reporter 0200/ZsG virus and wild-type 0200 CCHFV, both showing uniform lethality within 5 6 days. We also found that CCHFV NSm was dispensable both in vitro and in vivo, but the virus lacking the NSm protein exhibited an attenuate phenotype. Disease onset was delayed in mice infected with 0200ΔNSm/ZsG, and of the 5 animals survived infection. In situ visualization confirmed the highly hepatotropic nature of CCHFV, and revealed viral tropism towards previously overlooked tissues and organs. Overall, the lack of attenuation of the 0200/ZsG reporter virus, and the ease of directly visualizing infected tissue and cells, will facilitate further investigations into virus-host dynamics of CCHFV infection in vivo. 220

221 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P33 HUMAN IMMUNE RESPONSES TO LASSA VIRUS INFECTION IN NIGERIA Lisa Oestereich *, 2, Emily Speranza 3, 4, David M. Wozniak, 2, Julia R. Port, 2, Sabrina Bockholt, 2, Elisa Pallasch, 2, Jonas Müller 2, Danny Asogun 5, Ephraim Ogbaini-Emovon 5, Meike Pahlmann, 2, John H. Connor 3, Cesar Muñoz-Fontela, 2, Stephan Günther, 2 Virology, German Center for Infection Research (DZIF), 2 Virology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 3 Department of Microbiology, Bioinformatics Program, Boston University School of Medicine, Boston, 4 Laboratory of Virology, RML NIH, Hamilton, United States, 5 Institute for Lassa Fever Research and Control, Irrua Specialist Teaching Hospital, Irrua, Nigeria Abstract: Lassa virus (LASV) is a zoonotic pathogen that is endemic in several West African countries. It causes annual outbreaks of Lassa fever (LF) with a case fatality rate of 20-30% in hospitalized patients. LF is on the R&D Blueprint list of the World Health Organization. The pathogenesis of LF is poorly understood, although deregulated host responses presumably play a key role. However a better understanding about the underlaying mechanisms is urgently needed in order to identify currently licensed drugs that might have a beneficial effect on the course of the infection. Together with cooperation partners from the Irrua Specialist Teaching Hospital (ISTH) in Irrua, located in a hyper-endemic LF zone in Nigeria, we investigate the host response in LF. Between 204 and 208 more than 000 samples belonging to more than 300 patients were collected and analyzed. The analyses include serology for viral proteins, virus titer determination, clinical chemistry, hematology, soluble cytokine and chemokine analysis and multiparameter flow cytometry. Here we summarize the data gathered from the immuno phenotyping and the result of a principal component analysis to identify key biomarkers that can predict the outcome of the infection. 22

222 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P34 IDENTIFICATION OF ESCAPE MUTANTS OF EQUINE INFLUENZA USING POLYCLONAL SERA AND MONOCLONAL ANTIBODIES: A POTENTIAL ANTIGENIC DRIFT WITH AN IMPACT ON VACCINE EFFICACY Shadia Omar *, Neil Bryant, Debra Elton virology, Animal Health Trust, Newmarket, United Kingdom Abstract: Equine influenza virus (EIV) is known to antigenic drift similar to other Influenza Viruses which causes vaccine breakout when vaccinated horses are infected with some escape mutants that is significantly distant from the vaccine strains. Antigenic drift occurs by multiple amino acid substitutions in the surface protein haemagglutinin (HA). HA is responsible for initiating entry into upper respiratory tract epithelial cells which involves multiple HAs binding to sialic acids on cells surface, therefore, HA is the main target for neutralizing antibodies and the mutation will enable the escape mutants to evade the antibodies. We attempted to predict future variants of EIV by carrying out serial passage of EIV vaccine recommended strains A/equine/Richmond//07, A/equine/South Africa/04/03 in eggs using polyclonal ferret and equine sera. This methodology has been used to map the antigenic sites of human influenza viruses, including H3N2, but has not been applied to influenza viruses from other mammals. We also made a panel of 50 recombinant viruses with additional point mutations in antigenic sites that have occurred naturally in field isolates, using reverse genetics and site-directed mutagenesis, to test the effect of substitutions in the antigenic sites on their own and in combination with additional changes, in order to determine how many changes and of what type are most likely to make an antigenic difference for EIV. The reassortants and recombinant viruses described above were tested by haemagglutination-inhibition assays. We identified single amino acid substitution at residue K56N in antigenic site B and we found that mutations at positions 59, 72 and 89 have altered antigenicity. These methods will improve the current selection process for vaccine strains. 222

223 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P35 IDENTIFICATION OF AMINO ACIDS THAT ENHANCE VIRUS REPLICATION IN MAMMALIAN HOSTS IN THE PB2 AND PA PROTEINS OF A HIGHLY PATHOGENIC H7N9 INFLUENZA VIRUS ISOLATED FROM A HUMAN Seiya Yamayoshi *, Maki Kiso, Atsuhiro Yasuhara, Mutsumi Ito, Yuelong Shu 2, Yoshihiro Kawaoka, 3, 4 Division of Virology, INSTITUTE OF MEDICAL SCIENCE, UNIVERSITY OF TOKYO, Tokyo, Japan, 2 Sun Yat-Sen University, Shenzhen, China, 3 University of Wisconsin-Madison, Madison, Wisconsin, United States, 4 International Research Center for Infectious Diseases, INSTITUTE OF MEDICAL SCIENCE, UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: Highly pathogenic H7N9 viruses emerged in late 206 and infected humans. The highly pathogenic H7N9 human isolate A/Guangdong/7SF003/206 (GD/3) possesses HA with human-type-receptor preference and NA with inhibitor resistance, and transmits among ferrets via respiratory droplets. GD/3 also shows high replication capability and high virulence in mice. To understand the role of the viral polymerase complex in the high pathogenicity of GD/3, we investigated its polymerase activity, growth kinetics, and pathogenicity by using a mutagenesis approach. We found that arginine at position 482 and valine at position 588 of PB2 and arginine at position 497 of PA individually enhanced viral polymerase activity in human cells, upregulated virus propagation in human cells, and contributed to efficient replication and high virulence in mice. In combination, these mutations had additive effects. PB2-482R and PB2-588V are located at the cap-binding domain and near PB2-627, respectively, whereas PA-482R is not exposed on the protein surface. These findings suggest that PB2-588V is likely involved in ANP32A-dependent high polymerase activity in mammalian hosts (ANP32A is a key host protein for PB2-627K-dependent high polymerase activity). Our data further suggest that PB2-482R and PA-497R contribute to enhanced polymerase activity in other ways. Our findings indicate that the presence of multiple replication-enhancing mutations in a single highly pathogenic H7N9 virus isolate may contribute to its high virulence in mammalian hosts. 223

224 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P36 COMPARATIVE IN VITRO AND IN VIVO ANALYSIS OF SWINE-ORIGIN HN AND HN2 INFLUENZA VIRUSES ISOLATED FROM HUMAN CASES BETWEEN 20 AND 206. Joanna Pulit-Penaloza *, Jessica A. Belser, Claudia Pappas, Xiangjie Sun, Nicole Brock, Hui Zeng, Terrence M. Tumpey, Taronna R. Maines CENTERS FOR DISEASE CONTROL AND PREVENTION, Atlanta, United States Abstract: Influenza A virus is a continuously evolving respiratory pathogen. Endemic in swine, H and H3 subtype viruses sporadically cause human infection, and are termed variant viruses. As each zoonotic infection represents an opportunity for human adaptation, the emergence of a transmissible influenza virus to which there is little or no pre-existing immunity is an ongoing threat to public health. Recently isolated H variant viruses were shown to display great genetic diversity and in many instances were antigenically different from human vaccine strains. We utilized in vitro and in vivo approaches to provide extensive characterization of HN and HN2 variant viruses isolated since the 2009 pandemic. A majority of the tested variant viruses emerged as a result of reassortment between HN 2009 pandemic and diverse swine triple reassortant viruses and showed similarities in molecular markers in the HA and polymerase genes with the 2009 pandemic viruses. The H variant viruses were well adapted to replicate in a human respiratory cell line and the respiratory tracts of mice and ferrets. However, the variant viruses had HA activation ph thresholds closer to that of most North American swine isolates rather than viruses that had adapted to humans. Consistent with what was previously observed for swine isolates, all of the tested variant influenza viruses were capable of efficient transmission between co-housed ferrets, but the ability to efficiently transmit via respiratory droplets differed between strains. This investigation highlights the need to closely monitor variant viruses for additional molecular changes that could facilitate efficient transmission between humans. 224

225 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P37 ONCOLYTIC RECOMBINANT MEASLES VIRUS IS A CANDIDATE THERAPEUTIC AGENT FOR REFRACTORY BREAST CANCER Tomoko Fujiyuki *, Yosuke Amagai, Koichiro Shoji, Akihiro Sugai, Mutsumi Awano, Hiroki Sato, Misako Yoneda, Chieko Kai Laboratory Animal Research Center, The Institute of Medical Science, THE UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: Oncolytic virotherapy is a new approach for cancer treatment. A recombinant measles virus (rmv-slamblind) that we have previously generated lost its affinity to a principal receptor of MV, SLAM, and thus lost the MV pathogenicity, whereas it is able to use Nectin-4 on cancer cells. We have previously demonstrated that rmv-slamblind infected several breast cancer cell lines using Nectin-4 and showed antitumor activity. Triple-negative breast cancer (TNBC) is known to be aggressive and often relapses with poor prognosis more than other types of breast cancer. Owing to resistance to both hormone and trastuzumab treatments, TNBC often causes recurrence and metastasis. Thus, novel effective therapies for TNBC are needed. In this study, we examined whether rmv-slamblind is effective for TNBC. We examined expression level of nectin-4 on the surface of TNBC cell lines with flow cytometry, and found that it was expressed on the surface of 75% of the analyzed cell lines. By inoculating the cells with rmv-slamblind, rmv-slamblind infected the nectin-4-expressing TNBC cell lines, and showed cytotoxicity in vitro. Furthermore, we examined anti-tumor effect of rmv-slamblind in vivo by using mouse xenograft models. Intratumoral administration of the virus suppressed tumor growth. To further assess the effectiveness of rmv-slamblind treatment for metastatic cancer, we administered rmv- SLAMblind expressing luciferase via intravenous route. In vivo imaging indicated that the virus replicated selectively in the tumor. In addition, the tumor growth was significantly suppressed. These results suggest that rmv-slamblind is a promising candidate as a therapeutic agent for TNBC. We are advancing translational research of rmv-slamblind. 225

226 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P38 SPECIFIC IMMUNOLOGICAL SIGNATURE DEPENDING ON THE DISEASE OUTCOME IN EBOLA VIRUS-INFECTED PATIENTS FROM MACENTA (GUINEA) Stephanie Reynard *, 2, Emilie Gloaguen 3, Cedric Laouénan 3, Jimmy Mullaert 3, Natalia Pietrosemoli 4, Hugo Varet 4, Justine Schaeffer, 2, Sylvain Baize, 2 Unité de Biologie des Infections Virales Emergentes, Institut Pasteur, 2 Centre International de Recherche en Infectiologie, Lyon, 3 Département d Epidemiologie, Biostatistiques et Recherche Clinique, Inserm, 4 Centre de bioinformatique, biostatistique et biologie intégrative, Institut Pasteur, Paris, France Abstract: Ebola virus (EBOV), responsible for the West African epidemic in , causes severe hemorrhagic syndrome. The pathogenic events leading to a fatal outcome or to survival remain poorly understood. Samples from 75 EBOV-infected patients and 5 febrile controls admitted in an Ebola Treatment Center (Macenta, Guinea) but who did not receive any specific EBOV therapy, were analyzed to characterize virological and immunological parameters. Transcriptomic analyses were performed on RNA extracted from leucocytes (GeneChip Human ST 2.0, Affymetrix) and 96 analytes were detected in plasma samples using a multiplex assay (Luminex) to investigate the course of the immune response. Viral loads and specific antibody responses were also measured. The transcriptomic analysis revealed that similar immunological pathways were activated for fatal and non-fatal outcomes. However, the protein levels for immunological parameters were consistently different depending on the issue. Survivors presented moderated and controlled cytokine levels associated with an antibody response and a moderated viral load, while fatal outcomes were characterized by an inefficient antibody response and an excessive immunological response unable to control viral dissemination. Moreover, our data revealed a strong correlation between the cytokine profiles from fatal issues and the immunological patterns described in sepsis. In addition, we identified 8 cytokines as robust markers for the disease outcome that could be used for predictive diagnosis and to manage the sepsis-like syndrome. 226

227 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P39 MEASLES VIRUS V PROTEIN CONTRIBUTES TO THE TRANSPORT OF NUCLEOPROTEIN COMPLEXES AND EFFICIENT VIRAL ASSEMBLY IN NEURONAL CELLS Shotaro Uchida *, Hiroki Sato, Tetsuro Arai, Fusako Ikeda, Chieko Kai, Misako Yoneda Laboratory animal research center, Institute of Medical Science, The University of Tokyo, Tokyo, Japan Abstract: Measles virus (MV) is a causative agent of measles, but in rare cases, it also causes fatal neurological disease such as subacute sclerosing panencephalitis and measles inclusion body encephalitis. It has been reported that V protein is critical for its neurovirulence, but its function has not been understood. In this study, we have established a live imaging system for directly observing the transport of nucleoprotein complexes (NCs) of MV, and found that V protein is required for the Rab-dependent transport of NCs in neuronal cells, which was required for the efficient viral assembly. To understand the molecular mechanism of V protein-dependent transport of NCs, we analyzed host binding proteins, and found that V protein directly binds to the microtubule to intermediate the association of microtubules and NCs. Then, we rescued mutant rmvs whose V proteins lost the affinities to microtubule. The transport of NCs of the mutant viruses were ablated, and they were significantly attenuated in a mouse brain. Our results suggest the novel function of MV V protein which contributes to its neurovirulence. 227

228 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P40 EVIDENCE OF VIRAL PERSISTENCE IN CYNOMOLGUS MACAQUES THAT SURVIVE PARENTERAL EXPOSURE WITH LASSA FEVER VIRUS Kathleen Cashman *, Eric Wilkinson, Xiankun Zeng 2, Jeremy Bearss 3, Paul Facemire 3, Todd Bell 3, Connie Schmaljohn 4 Virology Division, THE GENEVA FOUNDATION / USAMRIID, 2 Pathology Division, USAMRIID, 3 Pathology Division, 4 Office of the Chief Scientists, USAMRIID, Fort Detrick, United States Abstract: Lassa virus (LASV) is the etiological agent of Lassa fever (LF), a rodent-borne, severe, and often fatal hemorrhagic fever disease endemic to regions of Central and West Africa. Our laboratory is engaged in studies to elucidate the disease progression and pathogenesis of LF in nonhuman primates (NHP), as well as to develop and test pre- and postexposure medical countermeasures against infection. Typical LF in NHP occurs in two phases: ) an acute phase, in which NHP develop severe disease signs that sometimes require humane euthanasia approximately 2-8 days post-exposure; and/or 2) a chronic phase in which NHP experience severe disease signs in the expected window, but do not succumb. NHP that experience the chronic disease phase rarely recover. Chronically ill NHP clear LASV from the blood by 2 days post-exposure as measured by plaque assay. Samples of testicular tissue collected from a chronically ill male NHP stained positive for LASV antigen 45 days post-exposure as measured by conventional immunohistochemical (IHC) staining methods, but all other tissues collected from chronically ill NHP were negative for the presence of LASV antigen by study terminus (days 30-45). In addition, two of five chronically ill NHP developed sudden onset sensorineural hearing loss by the end of the studies, a known consequence of LF in approximately 30% of recovering human patients. NHP remained moderately to severely ill, without improvement, until the study endpoints, despite the lack of measurable LASV in serum or tissues. We sought a more sensitive method of identifying LASV in tissues and employed a novel in-situ hybridization method in which LASV-specific nucleic acid probes were used to re-stain tissues in the chronically ill NHP. Using this new method, we were able to identify the presence of LASV antigen in a number of different IHC-negative tissues. 228

229 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P4 IN SITU DEMONSTRATION OF RIFT VALLEY FEVER DISSEMINATION WITHIN CULEX PIPIENS MOSQUITOES Nicholas Johnson *, Sarah Lumley 2, Luis Hernandez-Triana, Dan Horton 3, Maria Del Mar Fernandez de Marco, Laura Hunter 2, Kirsty Emory 2, Dan Hicks 4, Jolyon Medlock 5, Roger Hewson 2, Anthony Fooks, Alejandro Nunez 4 Virology, Animal and Plant Health Agency, Addlestone, 2 Virology and Pathogenesis Group, Public Health England, Porton Down, 3 School of Veterinary Medicine, University of Surrey, Guildford, 4 Histology, Animal and Plant Health Agency, Addlestone, 5 Entomology, Public Health England, Porton Down, United Kingdom Abstract: Rift Valley fever phlebovirus (Family Phenuiviridae, Genus Phlebovirus - RVFV) is a zoonotic pathogen of veterinary and medical importance causing a haemorrhagic disease in humans. One of the main vector species is Culex pipiens, an abundant species across Africa and Europe. To investigate the competence of temperate populations of this species we have developed in situ hybridisation probes (RNA Scope) to detect the distribution of RVFV RNA. Culex pipiens mosquitoes (strain Caldbeck) were fed on blood containing RVFV (Strain ZH50, 0 7 pfu/ml) and maintained at 25 o C. An infection rate of 27% (n = 5) was observed at 7 days. In parallel, whole mosquitoes were fixed in 0% buffered formalin and embedded in paraffin. Sections were stained either with a bacterial DapB probe (negative control), mosquito GAPDH probe (positive control) or a RVFV probe directed to the nucleoprotein. Sections were counterstained with haematoxylin. Non-infected mosquitoes showed no staining with the exception of non-specific staining of the corneal lens in all sections. Infected sections showed extensive staining throughout body tissues with particularly strong staining in the basal membrane of the midgut and proventriculus. Extensive dissemination of virus within the head and thorax including tissues such as the ganglion, leg and Johnstone's organ. No staining was observed within the ovarian follicles. This study demonstrates the application of in situ detection of virus RNA to investigate RVFV dissemination within a key vector species. It also suggests that the proventriculus, the region of the oesophagus immediately anterior to the midgut is highly infected and could be a site of virus egress from the mosquito gut. Secondary sites of replication were evident throughout the thorax and likely precede infection of the salivary glands prior to potential transmission. 229

230 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P42 EUROPEAN SUBGROUP C AVIAN METAPNEUMOVIRUS: PRELIMINARY EXPERIMENTAL INVESTIGATIONS ON THE ROLE OF THE SH PROTEIN IN PATHOGENICITY FOR MUSCOVY DUCKS Nathan Szerman, Chantal Allée, Evelyne Lemaitre, Céline Courtillon, Michel Amelot 2, David Courtois 2, Clive Naylor 3, Aurélie Leroux 4, Pierrick Lucas 4, Yannick Blanchard 4, Nicolas Eterradossi, Paul Brown * VIPAC, 2 SELEAC, ANSES, Ploufragan, France, 3 Infection Biology, Infection and Global Health, Liverpool, United Kingdom, 4 GVB, ANSES, Ploufragan, France Abstract: Subgroup C Avian metapneumoviruses (AMPV-C) have the broadest avian host range of the four described AMPV subgroups (A, B, C and D) although two distinct lineages have been observed; one in galliformes (viruses isolated from turkeys) and the other in palmipeds (viruses isolated from ducks). AMPV-Cs also have a closer genetic relationship with human Metapneumoviruses. These properties of AMPV-C together with the fact that target species are available as experimental models makes it a key virus in the genus metapneumovirus for studying host tropism and its molecular basis. In the current study the first reverse genetics system for an AMPV C virus from palmiped lineage was developed and used to rescue viruses in which the small hydrophobic (SH) protein or just its ectodomain had been exchanged for those of AMPV-C galliform lineage. NGS analyses revealed that the rescued viruses consisted of a mixed virus stock combining SH modified viruses with correct full length genomes others with one or two nucleotide insertions resulting in a truncated attachment glycoprotein G ORF. Experimental infections of SPF ducks with these SH modified viruses consistently failed to produce clinical signs, as opposed to the unmodified recombinant AMPV-C duck virus, despite similar levels of viral RNA production in the trachea and choanal cleft. These preliminary results demonstrated that the phenotype of a pathogenic AMPV-C duck virus was modified in SPF Muscovy ducks when its full SH ORF or just the ectodomain portion was modified. However it must be considered that SH modified viruses containing G ORF sequence modifications were also present in the rescued virus inoculum. Future and ongoing studies are aimed at assessing G mutations in the pathogenic recombinant AMPV-C copy and at obtaining recombinant SH swap viruses with an intact G ORF. 230

231 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P43 INFLUENZA A VIRUS INFECTION PROMOTES LOCAL AND SYSTEMIC SUPER-INFECTION BY WEAKENING THE NATURAL SHIELD OF COMMENSAL BACTERIA Soner Yildiz *, Beryl Mazel-Sanchez, Damien Baud, Patrice Francois, Mirco Schmolke Department of Microbiology and Molecular Medicine, University of Geneva, CMU, Geneva, Switzerland Abstract: Microbiota integrity is essential for a growing number of physiological processes. Importantly, commensal microbes provide a natural shield against invading bacterial pathogens. We recently demonstrated that acute viral infection of the respiratory tract can dramatically alter the composition and abundance of systemic microbiota in a transient fashion. In the respiratory tract we identify a commensal E. coli, increasing in titer specifically in lungs of influenza A virus (IAV) infected animals. Associated with virus induced lung commensal dysbiosis was an increased sensitivity to S. pneumoniae super-infection. Remarkably, exogenously applied E. coli itself was able to promote bacterial super-infection with S. pneumoniae, in absence of IAV infection. In the small intestine, we found transient change in composition of microbiota, as well as quantitative depletion of total bacteria, at both genomic and cellular level after IAV infection. In parallel to depletion of bacterial content, IAV induced disruption of mucus integrity in the small intestine, which in turn increased the risk of pathogen S. typhimurium invasion in a mouse model. Our data suggest that either qualitative or quantitative changes in microbiota as a consequence of an acute IAV infection could increase the risk of bacterial super-infection, by reducing weakening this natural shield of commensals. 23

232 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P44 AMINO ACID SUBSTITUTIONS IN THE PB2 PROTEIN OF AN H5N AVIAN INFLUENZA VIRUS ENHANCE ITS REPLICATION EFFICIENCY AND PATHOGENICITY IN MAMMALIAN HOSTS. Takeaki Imamura *, Shinya Yamada, Kiyoko Iwatsuki-Horimoto, Reviany V. Nidom 2, Setyarina Indrasari 2, Kuncoro P. Santoso 2, 3, Chairul.A Nidom 2, 3, Yoshihiro Kawaoka, 4 Division of Virology, The Institute o Medical Science, The University of Tokyo, Tokyo, Japan, 2 AIRC Laboratory, Professor Nidom Foundation, 3 Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya, Indonesia, 4 Influenza Research Institute, School of Veterinary Medicine, Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, Wisconsin, United States Abstract: Highly pathogenic H5N avian influenza viruses have been circulating in Indonesia since 2004, and human cases have been reported since Characterization of highly pathogenic H5N avian influenza viruses isolated in Indonesia revealed two phylogenetically related strains belonging to Clade 2..3: A/chicken/South Kalimantan/UT52/200 (UT52) and A/chicken/South Sulawesi/UT54/200 (UT54). Compared to UT54, UT52 showed higher replication efficiency in cultured human lung epithelial cells (A549 cells) at 33 C and higher pathogenicity in BALB/c mice. Reassortant and mutant viruses of UT52 and UT54 were generated by reverse genetics, and characterization of these viruses revealed that unique amino acids in the polymerase binding protein 2 (PB2) of UT52 conferred its higher replication efficiency and pathogenicity. These findings are beneficial for assessing the risk of avian-to-human transmission of highly pathogenic H5N avian influenza field isolates. 232

233 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P45 LOW-PATHOGENIC AVIAN INFLUENZA VIRUSES HIJACK HOST S CENTRAL METABOLIC PATHWAYS Léa Meyer *, Olivier Leymarie, Christophe Chevalier, Evelyne Esnault 2, Marco Moroldo, Bruno Da Costa, Sonia Georgeault 3, Philippe Roingeard 3, Bernard Delmas, Pascale Quéré 2, Ronan Le Goffic INRA, Jouy-en-Josas, 2 INRA, Nouzilly, 3 Université François Rabelais et CHRU de Tours, Tours, France Abstract: Low-pathogenic influenza viruses are a major cause of infections in poultry farms. Close proximity of the chickens due to holding conditions promotes aerosol transmission of the virus, thus making lung epithelial cells a primary target. We aimed at characterizing the mechanisms ruling low-pathogenic influenza virus infection of a chicken lung epithelial cell line, CLEC23. We used a transcriptomic microarray approach to uncover the cell mechanisms that were affected upon infection. We found that viral infection induced a characteristic signature of immune response, involving pathways such as IL-6, IL-8, and STAT3. Noteworthy we could not detect changes in interferon-stimulated pathways. Unexpectedly we found a strong metabolic signature at the transcriptomic level with the induction of oxidative phosphorylation (OxPhos), a central metabolic pathway. The mitochondrial chromosome codes proteins involved in this pathway, presumably as a means to fine-tune OxPhos activity. Viral infection upregulated every gene of the mitochondrial chromosome. We validated our data by quantitative Polymerase Chain Reaction then quantified OxPhos s final product, Adenosine Triphosphate (ATP), and observed enhanced production upon viral infection. Viral replication was downregulated in the presence of an OxPhos inhibitor, oligomycin, indicating the virus relies on enhanced ATP production for optimal replication. Viruses have evolved ways to deflect central metabolism to promote their cycle. Influenza infection represents a cost of up to 0% of a cell s energy pool. Our work thus uncovers a reliance of avian low-pathogenic influenza viruses on OxPhos. 233

234 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P46 A DONOR-MATCHED HUMANIZED MOUSE MODEL REVEALS A CHIEF ROLE OF THE APC-T CELL INTERFACE IN EBOLA VIRUS DISEASE Monika Rottstegge *, 2, Estefania Rodriguez-Burgos 3, Paula Ruibal 4, Lisa Oestereich 2, 5, Elisa Pallasch 5, César Muñoz- Fontela, 2 Virus Immunology, Bernhard-Nocht-Institute, 2 Partner Site Hamburg, German Center for Infection Research, 3 Heinrich- Pette-Institute, Hamburg, Germany, 4 Leiden University Medical Center, Leiden, Netherlands, 5 Bernhard-Nocht-Institute, Hamburg, Germany Abstract: During the recent Ebola virus disease (EVD) outbreak in West Africa ( ), research from our laboratory as well as from other colleagues underscored the role of dendritic cells (DC) and T cells on EVD immunity. These studies showed that proper DC and T-cell-mediated responses were critical for virus clearance and survival. To further explore the relevance of this immune cell interaction, we have developed a humanized mouse model based on transplantation of donormatched peripheral blood APCs and T cells into HLA-transgenic NSG mice (NSG PBL-Dm ). Here we show that transplantation of donor-matched DCs and T cells in these mice is sufficient to render them highly susceptible to EBOV infection. Mice showed high viremia, weight loss, gastrointestinal bleeding, and hematuria, and therefore reproduced important features of human EVD. Of note, some pathological features including time to death depended on the donor, which suggested that these mice may reproduce to some extent interindividual differences to EBOV infection. We speculate that this model, which is easy and fast to generate, will facilitate future studies on immunogenetics, antigen presentation and T-cell biology in the context of filovirus and perhaps other human infections. 234

235 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P47 LYMPHOCYTE HOMING SIGNATURES IN ACUTE LASSA VIRUS INFECTION Julia Port *, 2, David Wozniak, 2, Yemisi Ighodalo 3, Jennifer Oyakhilome 3, Rita Esumeh 3, Jonas Müller 2, Stephan Günther, 2, Lisa Oestereich, 2, Cesar Muñoz-Fontela, 2 German Center for Infection Research (DZIF), 2 Bernhard Nocht Institut for Tropical Medicine, Hamburg, Germany, 3 Irrua Specialist Teaching Hospital, Irrua, Nigeria Abstract: Lassa virus (LASV), the etiological agent of Lassa fever, is endemic in several West African countries and causes around 300,000 cases annually with 5,000-6,000 deaths. Transmission occurs seasonally, mostly through direct or indirect contact with the reservoir host species, the multimammate rat Mastomys natalensis. Human to human transmission may also occur, in particular in nosocomial settings. Here we summarize data gathered in hyper-endemic areas in Nigeria from a hospital-based acute Lassa fever patient cohort (January 207 to March 208). Patient data on behavior patterns linked to LASV exposure and likely transmission scenarios was collected. We gathered data on cross-sectional and longitudinal T-lymphocyte signatures of tissue homing, which is thought to be associated with the site of antigen encounter. Flow cytometry was used to describe homing signatures by characterization of activated and LASV specific T cells expressing either a skin (CLA, CCR4), an intestinal (beta 7 integrin, CD49d) or an inflamed mucosal tissue and respiratory (CCR3, CD29, CD49a) specific pattern of chemokine receptors and adhesion molecules. Preliminary data indicate distinct homing signatures in activated T cells which are consistent with exposure through respiratory and intestinal mucosae. We speculate that this approach will provide valuable information on T cell homing behavior during acute LASV infection, which can clarify the overall role of T cells in Lassa fever pathogenicity. Placing this data into the context of the epidemiological background could help determine the more prevalent transmission routes, which has implications for public health policies. 235

236 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P48 MMP9 ASSOCIATES WITH ENDOTHELIAL GLYCOCALYX DEGRADATION DURING HEMORRHAGIC FEVER WITH RENAL SYNDROME Gregory Rankin, Julia Wigren Byström 2, Rasmus Gustafsson 3, Magnus Hansson 4, Therese Thunberg 2, Clas Ahlm 2, Anne- Marie Connolly-Andersen * 2 Department of Public Health and Clinical Medicine, 2 Department of Clinical Microbiology, Umeå University, Umeå, 3 Dept. of Clinical Neuroscience, 4 Dept. of Clinical Chemistry, Karolinska Institutet, Stockholm, Sweden Abstract: Haemorrhagic fever with renal syndrome (HFRS) is characterized by fever, hypotension, vascular leakage, thrombocytopenia and renal failure. HFRS in Sweden is caused by the Puumala hantavirus. It is spread by viral-infested droppings from bank voles. The health care system has little to offer these patients since there is no antiviral treatment and no vaccine available. We previously showed that a marker of endothelial glycocalyx degradation (syndecan-) was associated with disease severity and disseminated intravascular coagulation during HFRS. We analysed the levels of other endothelial glycocalyx (GCX) degradation markers (heparan sulfate, soluble thrombomodulin), potential "sheddase": Matrix metalloproteinase 9 (MMP9) and a neutrophil activation/tissue damage marker (neutrophil gelatinase-associated lipocalin, NGAL) in patient plasma collected consecutively following disease onset. We used the generalized estimating equation to analyse the association between endothelial GCX degradation, MMP9, neutrophil activation/tissue damage and HFRS disease outcome (needing oxygen, blood component transfusion, intensive care and renal damage). A total of 44 HFRS patients were included in this study. The levels of MMP9 was significantly associated with endothelial GCX degradation. Neutrophil activation/tissue damage (NGAL) was also significantly associated with MMP9 and endothelial GCX degradation. In addition, endothelial GCX degradation was significantly associated with HFRS disease outcome. Neutrophil activation leading to release of MMP9 could be partly responsible for endothelial GCX degradation during HFRS. 236

237 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P49 HANTAVIRUS RECEPTORS EXPRESSION AND DISTRIBUTION IN A LETHAL PULMONARY SYNDROME ANIMAL MODEL Amelina A. Albornoz *, Rebecca Brocato 2, Carola Otth 3, Jay Hooper 2, Nicole Tischler Molecular Virology, Fundación Ciencia y Vida, Santiago, Chile, 2 Virology, US Army Medical Research Institute of Infectious Diseases, Maryland, United States, 3 Virology, Universidad Austral de Chile, Valdivia, Chile Abstract: Hantaviruses are enveloped viruses which belong to the Hantaviridae family. They are known to cause hemorrhagic fever with renal syndrome (HFRS) and the highly lethal hantavirus pulmonary syndrome (HPS) in humans. Both diseases are related with an increased vascular permeability; yet the mechanisms leading to pathogenesis are not well understood. Among them, the usage of different cell entry factors has been described. Receptors such as the b3 integrin subunit and the receptor for the globular head domain of complement Cq (gcqr) have been shown to be used by pathogenic hantaviruses in vitro. In this work, we analyze the expression of b3 integrin and gcqr in an animal model for HPS. For this purpose, we performed qpcr and immunohistochemical studies of lungs and kidneys (as control) at 8 and 2 days post-infection (d.p.i.) with Andes hantavirus. In control and 8 d.p.i animals, both receptors are localized in pulmonary veins while at 2 d.p.i. moribund animals, blood vessel staining of the receptors is weaker. Interestingly, the qpcr results show that the expression of b3 integrin and gcqr are, in general, reduced in lungs of 8-2 d.p.i. animals, even though the histology of the 8 d.p.i. tissue appears normal with no signs of disease. The decrease of receptors mrna levels in the lungs at 8 d.p.i. seems to be highly specific since infected kidneys of the same animals show similar receptor mrna levels compared to controls. Together, these results suggest that the expression of receptors may be an important step involved in vascular permeability and subsequent pathogenesis in the lungs of the HPS model. FUNDING: CONICYT by grants FONDECYT 8799 and AFB

238 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P50 BIOMARKER ANALYSIS OF HUMAN EBOLA VIRUS DISEASE UNDERSCORES THE ROLE OF TISSUE INTEGRITY IN SURVIVAL Romy Kerber *, 2, Ralf Krumkamp 2, 3, Misa Korva 4, Toni Rieger, 2, Stephanie Wurr, 2, Sophie Duraffour, 2, Lisa Oestereich, 2, Martin Gabriel, 2, Jürgen May 2, 3, Tatjana Avšič Županc 4, César Muñoz-Fontela 2, 5, Stephan Günther, 2 Virology, 2 German Centre for Infection Research (DZIF), 3 Epidemiology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany, 4 Institute of Microbiology & Immunology, University of Ljubljana, Ljubljana, Slovenia, 5 Virus Immunology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany Abstract: Background. The largest epidemic of Ebola virus disease (EVD) occurred between in West Africa. Despite the fact that EVD is a severe viral infection with high case fatality rates (CFR) still little is known about putative immune correlates of outcome. Methods. Cross-sectional as well as longitudinal data on the expression of 54 biomarkers, measured in plasma samples from 80 hospitalized EVD patients, was obtained. Principal component analysis (PCA) was used to summarize the overall expression pattern of biomarkers in immune response. Results. Circulating pro-inflammatory cytokines and chemokines as well as markers of endothelial dysfunction and disseminated intravascular coagulation were highly expressed in EVD patients with fatal outcome. In contrast, biomarkers of gut integrity and T cell response were elevated in survivors. Overall expression profiles revealed no clear differences between fatalities and survivors. Conclusion. Endothelial integrity, gastric tissue protection and T-cell immunity play an important role in EVD survival. 238

239 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P5 CYTOKINE RESPONSE IN BUNYAVIRUS HEMORRHAGIC FEVERS Misa Korva *, Katarina Resman Rus, Ana Saksida, Miša Pavletič, Petra Bogovič 2, Xhevat Jakupi 3, Isme Humoli 4, Jusuf Dedushaj 4, Franc Strle 2, Tatjana Avšič Županc Virology, University of Ljubljana, Faculty of Medicine, Institute of Microbiology and Immunology, 2 Department of Infectious Diseases, University Medical Center Ljubljana, Ljubljana, Slovenia, 3 Department of Microbiology, 4 Department of Epidemiology, National Institute of Public Health of Kosovo, Pristina, - Abstract: Hemorrhagic fever with renal syndrome (HFRS) and Crimean-Congo hemorrhagic fever (CCHF) are important viral hemorrhagic fevers, especially in the Balkans. Infection with Dobrava or Puumala virus and CCHF virus can vary from a mild, nonspecific febrile illness, to a severe disease with a fatal outcome. The pathogenesis of both diseases is poorly understood, but it has been suggested that host s immune mechanism is an important factor in optimizing survival rate. Thus, the aim of our study was to investigate cytokine response in patients with VHF to expose possible biomarkers for clinical outcome. For cytokine analysis acute serum samples (first 7 days of illness) were collected from 00 HFRS patients infected with DOBV or PUUV, 70 CCHFV patients and 30 healthy controls. Using Human Cytokine/Chemokine Panel (Milliplex) we have examined 20 cytokines: IL-α, IL-b, IL-RA, IL-4, IL-5, IL-6, IL-0, IL-2p40, IL-2p70, IP-0, IFNα2, IFNγ, GM-CSF, GROα, scd40l, MCP-, MCP-3, MIP-α, MIP-b and TNFα. In comparison to the control group patient with HFRS or CCHF had significantly increased levels of IL-4, IL-6, IL-0, IL-2p70, IP-0, INFγ, TNFα, GM-CSF, MCP-3, and MIP-b. Interestingly, HFRS patients had higher concentrations of serum MIP-α, MIP-b, which promote activation of macrophages and NK cells. Also, HFRS patients had higher concentrations of IFNγ, where in CCHF patients significantly higher concentrations of IFNα were measured, suggesting activation of different inflammation pathways. Patients with fatal outcome had significantly elevated concentrations of IL-6, IFNα2 and MIP-α, while GRO, chemokine related to activation of neutrophils and basophils, was downregulated. Our study supports the hypothesis that the major players in the immunopathogenesis of CCHF and HFRS are pro-inflammatory cytokines, which mediate vascular dysfunction, disseminated intravascular coagulation, organ failure, and shock. 239

240 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P52 MOLECULAR CHARACTERIZATION OF PB-F2-MEDIATED PATHOLOGY IN INFLUENZA MOUSE MODEL OF INFECTION C Chevalier *, F Jamme 2, O Leymarie, B Da Costa, P Maisonnasse, M Réfrégiers 2, B Delmas, R Le Goffic VIM, INRA, Jouy-en-Josas, 2 DISCO, Synchrotron SOLEIL, St-Aubin, France Abstract: PB-F2 is a virulence factor of influenza A virus (IAV) known to increase viral pathogenicity in mammalian host. PB-F2 is an intrinsically disordered protein displaying a propensity to form amyloid-like fibers in IAV-infected cells. Using synchrotron Fourier-transform infrared (FTIR) spectroscopy, we previously evidenced the presence of PB-F2 fibers in IAVinfected cells and assigned an IR β-aggregated signature at the single-cell level. Using DUV (Deep Ultraviolet) microscopy and taking advantage of the high content of tryptophan residues in the sequence of PB-F2 (5/90 aa), we showed that the increase of the autofluorescent signal recorded in IAV-infected cells can be correlated with the IR detection of β-aggregates. Here, we used FT-IR and DUV microscopies to prove the presence of PB-F2 fibers in IAV-infected mice. Mice were infected with a wild-type IAV and its PB-F2 knockout mutant and monitored at different time post-infection. DUV microscopy was used to map the presence of PB-F2 β-aggregates within slices of lung tissues of IAV-infected mice. IR spectra were recorded in the regions of interest and subjected to multivariate analysis revealing the presence of β-aggregated structures in mice infected with PB-F2-expressing IAV. In order to study the correlation between PB-F2 structure and inflammatory response, NF-KB luciferase transgenic mice were intranasally instilled with monomeric, fibrillated or C- and N-terminal domains of recombinant PB-F2. Our results clearly show the pro-inflammatory effect of fibrillated PB-F2 compared to monomeric and non-fibrillated forms. It is noteworthy that only the N-terminal part of PB-F2, unable to fibrillate, does not provoke any inflammation. Thus, the PB-F2-induced inflammation is tightly correlated with sequence and oligomerization status of the protein. Graphical Abstract: 240

241 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P53 RECOMBINANT GENOTYPE G MUMPS VIRUS EXPRESSING ENHANCED GREEN FLUORESCENT PROTEIN EFFICIENTLY REPLICATES IN PRIMARY HUMAN CELLS AND IS VIRULENT IN COTTON RATS Linda J. Rennick *, Sham Nambulli, Connor G.G. Bamford 2, Steven Rubin 3, Nicholas A. Crossland 4, W. Paul Duprex Department of Microbiology and National Emerging Infectious Diseases Laboratories, Boston University School of Medicine, Boston, United States, 2 School of Medicine, Dentistry and Biomedical Sciences, Queen s University of Belfast, Belfast, United Kingdom, 3 Food and Drug Administration, Center for Biologics Evaluation and Research, Silver Spring, 4 National Emerging Infectious Diseases Laboratories, Boston University School of Medicine, Boston, United States Abstract: Mumps, caused by mumps virus (MuV), is a disease characterized by painful inflammatory symptoms, such as parotitis and orchitis. In many cases there is also central nervous system infection leading to meningitis or encephalitis. The mechanism of MuV infection and pathogenesis in humans are not well understood. This information is critical for the design and testing of live, rationally attenuated MuV vaccines, which are necessary due to the global resurgence of mumps (including in highly vaccinated populations) and the discontinued use of some MuV vaccines outside the U.S. due to insufficient attenuation and safety concerns. In this study, a recombinant (r) MuV (rmuv G09 ) was generated based on a consensus genotype G genomic sequence obtained directly from an unpassaged clinical specimen from a patient infected with MuV during the 2009 outbreak in New York City. An additional transcription unit encoding enhanced green fluorescent protein (EGFP) was introduced between the V/phosphoprotein and matrix genes (position 3) of the genome to generate rmuv G09 EGFP(3). Infection of polarised epithelial cell lines and primary differentiated normal human bronchial epithelial cells showed that this virus could infect, and be released, from both ciliated and non-ciliated cells via their apical and basolateral surfaces. Intranasal inoculation of cotton rats with rmuv G09 EGFP(3) led to both upper and lower respiratory tract infection with spread in lungs and virus isolation from lung tissue and bronchoalveolar lavage samples. The animals seroconverted by 2 days post-infection. In vivo tracking of infected cells by detection of EGFP fluorescence is an efficient means of identifying primary sites of infection and sites of secondary spread which are critical elements towards gaining an understanding of MuV pathogenesis. 24

242 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P54 THE HIGHLY PATHOGENIC AVIAN INFLUENZA H5N A/CHICKEN/FRANCE/5069A/205 PRESENTS IN VITRO PROPAGATION CHARACTERISTICS CONSISTENT WITH ITS PREDICTED TROPISM FOR AVIAN SPECIES Pascale Massin *, Cécile Guillou-Cloarec, Claire Martenot, Eric Niqueux, Audrey Schmitz, François-Xavier Briand, Chantal Allee, Carole Guillemoto, Marie-Odile Lebras, Aurélie Le Prioux, Katell Ogor, Nicolas Eterradossi VIPAC, ANSES, Ploufragan, France Abstract: Avian influenza A viruses (AI) are a major threat to animal and public health. Since 997, several highly pathogenic (HP) H5N avian viruses directly transmitted from poultry to humans caused numerous humans deaths and had a considerable economic impact on poultry market with high mortality rate and massive preventive culling. Surveillance and study of AI H5 viruses are essential to improve knowledge on its persistence, transmission and evolution. During 205/206, a HP-AI outbreak occurred in Southwestern France. Different subtypes circulated including a HP-H5N: A/chicken/France/5069a/205. Genomic analysis indicated that the virus does not possess the set of determinants known to promote the transmission to humans. However, a number of determinants are present as in other contemporary H5-AI viruses. To assess the absence of zoonotic potential of the HP-H5N 5069a virus, some host restriction determinants described in the literature have been analyzed: (i) cell receptor-specificity; (ii) temperature sensitivity of viral replication related to primary infection sites: 33 C in human upper respiratory tract and 37 C or 40 C in avian respiratory or digestive tracts, respectively. The receptor binding assay using synthetic sialylglycopolymers showed that the virus preferentially recognizes avian receptor as other avian viruses used as controls. The temperature sensitivity was analyzed through plaque phenotype and multiplication/genomic replication kinetics on mammalian cells (Madin-Darby Canine Kidney cells). A delay in genomic replication and multiplication was observed at 33 C as compared to 37 C. To conclude, our results suggest that HP-H5N A/chicken/France/5069a/205 has an avian phenotype in vitro in accordance with the initial in silico predictions based on genomic markers. 242

243 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P55 ADAPTIVE MUTATIONS IN INFLUENZA A/CALIFORNIA/07/2009 ENHANCE POLYMERASE ACTIVITY AND INFECTIOUS VIRION PRODUCTION Patrick Slaine *, Cara MacRae 2, Mariel kleer, Emily Lamoureux 3, Sarah McAlpine 4, Michelle Warhuus 4, André Comeau 3, Craig McCormick, Todd Hatchette 4, Denys Khaperskyy microbiology and immunology, Dalhousie university, halifax, 2 The Hospital for Sick Children, University Health Network, Toronto, 3 CGEB-Integrated Microbiome Resource (IMR) and Department of Pharmacology, Dalhousie University, 4 Department of Pathology and Laboratory Medicine, Nova Scotia Health Authority (NSHA), halifax, Canada Abstract: Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(HN) (a.k.a. CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic- (PB) T56A and F740L, and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering these three adaptive PB and PA mutations into the parental CA/07 strain, we demonstrated that adaptive mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions, and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation. 243

244 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P56 3D IMAGING OF VIRUS INFECTIONS IN SOLVENT-CLEARED ORGANS Luca Zaeck *, Madlin Potratz, Stefan Finke FRIEDRICH-LOEFFLER-INSTITUT, Greifswald - Insel Riems, Germany Abstract: The visualization of infection events in tissues and organs using immunolabeling is a key method of modern infection biology. The ability to observe and study the distribution, tropism, and abundance of pathogens inside of organ samples provides pivotal data on disease development and progression. Until recently, immunolabeling was mostly restricted to thin sections of paraffin-embedded or frozen samples. Because of the limited 2D image plane provided by thin sections, crucial information on the complex structure of respective organs as well as on both the compartment and the surrounding cellular context of the infection environment is lost. Consequently, distinct assertions on topics like epithelial barrier function, infiltration of cells to the site of infection, or directed virus spread in vivo can prove difficult. The introduction of a new solvent-based tissue-clearing technique [] and its successor udisco (ultimate 3D imaging of solvent-cleared organs) [2] as well as the implementation of an applicable immunostaining protocol [3] now provide an efficient tool to study high-volume image stacks of infected organs. Here, we applied udisco to both brain and lung tissue samples from animals infected with rabies virus and swine influenza virus, respectively. Confocal laser scanning microscopy paired with custombuilt, 3D-printed imaging chambers enabled us to obtain high-resolution image stacks of organ slices as thick as mm in order to gain further insights into the infection environment of respective target tissues. 244

245 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P57 HUMAN B-CELLS ARE HIGHLY SUSCEPTIBLE TO IN VITRO AND IN VIVO MEASLES VIRUS INFECTION Brigitta M. Laksono *, Rory D. de Vries, Christina Grosserichter-Wagener 2, Eline G. Visser 3, Pieter L. Fraaij 3, Wilhemina L. Ruijs 4, Marion P. Koopmans, Menno C. van Zelm 2, Albert D. Osterhaus, Rik L. de Swart Viroscience, 2 Immunology, 3 Paediatrics, Erasmus MC, Rotterdam, 4 Centre for Infectious Disease Control, National Institute for Public Health and the Environment (RIVM), Bilthoven, Netherlands Abstract: Measles is characterised by transient immune suppression. Studies in non-human primates showed that measles virus (MV) preferentially infects memory T-cells, which express higher level of the cellular receptor CD50 than naive T- cells. Based on these findings, we hypothesised that MV causes immune amnesia by infecting and depleting memory lymphocytes. Since limited information was available about CD50 expression and susceptibility of B-cells to MV infection, we investigated the susceptibility of human B-cell subsets to in vitro MV infection and demonstrated that they were more susceptible and permissive to infection than T-cells. To investigate whether infection of B-cells could be observed in naturally infected measles patients, we performed an observational cohort study in unvaccinated children during a measles outbreak in the Dutch Orthodox Protestant community in 203. We collected single blood samples from acute measles patients or paired blood samples from healthy children before and after measles. Detection of MV-infected cells in peripheral blood mononuclear cells (PBMC) by intracellular staining of the MV nucleoprotein confirmed that MV infected B-cells during prodromal measles. Staining of PBMC isolated from paired blood samples demonstrated a significant reduction in peripheral memory B-cells after measles. Altogether, our data strongly indicate that B-cells are highly susceptible to in vitro and in vivo MV infection and the loss of pre-existing memory lymphocytes contributes to measles-induced immune suppression. 245

246 DAMAGING AND SPREADING pathogenesis Abstract final identifier: P58 USE OF REVERSE GENETICS TECHNIQUE TO STUDY THE EARLY PATHOGENESIS OF PESTE DES PETITS RUMINANTS VIRUS Muneeswaran Selvaraj *, Mana Mahapatra, Pippa Hawes, Ricahrd Kock 2, Satya Parida Livestock viral disease, The Pirbright Institute, Woking, 2 Pathobiology and Population Sciences, Royal Veterinary College, London, United Kingdom Abstract: Across the developing world, Peste-des-petits ruminants virus (PPRV), places a huge disease burden on agriculture, in particular affecting small ruminant production and in turn increasing poverty in many developing countries. The current understanding of PPRV pathogenesis has been mainly derived from the closely related rinderpest virus (RPV). There are few studies that have focused on the late stages of pathogenesis of PPRV in the field and very little is known about the processes underlying the early stages of pathogenesis. It is believed that PPRV replicates mainly in the epithelial cells of the respiratory and gastro-intestinal tracts before disseminating throughout the host. We hypothesize that PPRV infects immune cells of the respiratory mucosa, but not respiratory epithelial cells and then migrates to the tonsil and local lymphoid organs for primary replication, after which virus enters the general circulation and secondary replication occurs in the epithelium of respiratory and gastro-intestinal tracts. The application of reverse genetics techniques provides a tool to gain a better understanding of the molecular factors underlying virus host range and pathogenesis. Recently we have established reverse genetics system for PPRV and using this we have engineered a GFP tagged PPR virus (rmorrocco 2008 GFP). Further, in the biosafety containment, we have infected targeted animals (goats) with this GFP tagged virus and following this virus in the body of infected goats in 6 hourly interval we could demonstrate that the virus primarily replicates inside the pharyngeal and palatine tonsils and then causes viremia and secondary replication. 246

247 NSV 208, Verona Abstract Book DAMAGING AND SPREADING pathogenesis Abstract final identifier: P59 ENTRY AND RELEASE OF LASSA VIRUS IN WELL-DIFFERENTIATED PRIMARY BRONCHIAL EPITHELIAL CELLS Helena Müller *, Sarah K. Fehling, Thomas Strecker Institute of Virology, PHILIPPS UNIVERSITY MARBURG, Marburg, Germany Abstract: Lassa virus (LASV), a member of the family Arenaviridae, is a highly pathogenic hemorrhagic fever virus that can cause severe systemic infections in humans. The primary reservoir is the multimammate rat Mastomys natalensis. Humans are primarily infected through mucosal exposure to virus-containing aerosols of rodent excreta. To advance our knowledge on the molecular mechanisms underlying LASV replication in the respiratory tract, we established well-differentiated primary cultures of human bronchial epithelial cells (HBEpC) grown under air-liquid interface conditions that closely mimic the bronchial epithelium in vivo. Our major findings were: (i) HBEpCs fully supported the entire lifecycle of LASV infection. (ii) LASV can infect polarized bronchial epithelial cells via the apical or basolateral membrane, while progeny virus particles are released predominantly from the apical surface. In vivo, such apical virus shedding from infected bronchial epithelia might support virus transmission via airway secretions. In summary, HBEpC represent a useful cell culture model system for the detailed analysis of LASV-host interactions in the respiratory tract. 247

248 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P60 THE PAN-EBOLAVIRUS THERAPEUTIC COCKTAIL MBP34 DEMONSTRATES UNIVERSAL PROTECTION FROM PATHOGENIC EBOLAVIRUSES. Zachary Bornholdt *, Andrew S. Herbert 2, Chad E. Mire 3, Shihua He 4, Robert W. Cross 3, Dafna M. Abelson, Joan B. Geisbert 3, Anna Z. Wec 5, 6, Krystal N. Agans 3, Bronwyn M. Gunn 7, Rebekah M. James 2, Marc Antoine de La Vega 4, Do Kim, Eileen Goodwin 6, Galit Alter 7, Laura M. Walker 6, Kartik Chandran 5, Larry Zeitlin, Thomas W. Geisbert 3, Xiangguo Qiu 4, John M. Dye 2 Mapp Biopharmaceutical, Inc., San Diego, CA, 2 USAMRIID, Fort Detrick, MD, 3 UTMB, Galveston, TX, United States, 4 PHAC, Winnipeg, Manitoba, Canada, 5 Albert Einstein College of Medicine, Bronx, NY, 6 Adimab, LLC, Lebanon, NH, 7 Ragon Institute, Cambridge, MA, United States Abstract: The Ebola virus (EBOV) outbreak highlighted the absence of available medical countermeasures for filoviruses. Importantly, one vaccine and one therapeutic (ZMapp ) demonstrated clinical efficacy during the outbreak. However, both products are specific for EBOV and have shown no efficacy against Sudan (SUDV) or Bundibugyo (BDBV) ebolavirus which together have caused nine sporadic and unpredictable deadly outbreaks responsible for 645 infections and 289 deaths since Our approach to address this unmet need was to develop a therapeutic based on fully human monoclonal antibodies derived from a single survivor of the EBOV/Makona outbreak. A thorough down-selection was performed on a 349 mab panel involving in vitro analyses and in vivo testing across multiple animal models for EBOV, SUDV and BDBV. The down selection resulted in two mabs being combined into the pan-ebolavirus immunotherapeutic cocktail, MBP34. MBP34 has since proven to be extremely potent against EBOV, SUDV, and BDBV providing protection from EBOV, SUDV and BDBV challenge in non-human primates (NHPs) when administered therapeutically once symptoms were apparent. MBP34 is the first product to demonstrate protective efficacy post-infection with a single dose against every major pathogenic ebolavirus species in NHPs. 248

249 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P6 ENHANCEMENT OF DNA VACCINES FOR EBOLA VIRUS WITH AN IMPROVED PLASMID DESIGN John J. Suschak, Charles J. Shoemaker, Lesley C. Dupuy, James A. Williams 2, Connie Schmaljohn * USAMRIID, Frederick, MD, 2 Nature Technology Corporation, Lincoln, NE, United States Abstract: We previously demonstrated that a DNA vaccine expressing the codon-optimized Ebola virus (EBOV) glycoprotein (GP) gene provides protective immunity to mice and nonhuman primates. To determine if modifications to the plasmid backbone could improve immune responses to EBOV in mice, we constructed EBOV GP DNA vaccines using minimalized Nanoplasmid expression vectors that are smaller than traditional DNA vaccine plasmids, have improved uptake, and persist longer in transfected cells. Mice were vaccinated by intramuscular injection of our standard pwrg7077- based EBOV GP DNA vaccine, an EBOV GP nanoplasmid construct, or EBOV GP nanoplasmids designed to improve innate immune responses by co-expressing a retinoic acid-inducible gene (RIG-I) agonist or the RIG-I agonist and CpG RNA. Additionally, we used the nanoplasmid vectors to launch EBOV GP virus-like particles (VLP) endogenously within the host. We then measured the immunogenicity and protective efficacy of each of these constructs in the vaccinated mice. While the DNA-launched VLPs did not improve immunogenicity, mice vaccinated with the nanoplasmid vaccine developed increased anti-ebov GP IgG and neutralizing antibody responses as compared to the traditional pwrg7077-based vaccine and had significantly increased populations of EBOV-specific IFN-γ + and IL-2 + T cells. Humoral and cellular immune responses were further boosted by vaccination with the nanoplasmids co-expressing the innate immune agonist genes. Challenge with mouse-adapted EBOV demonstrated that both the EBOV nanoplasmid and DNA-launched VLP vaccines elicited protective immunity in mice. Our results suggest that the nanoplasmid vectors can improve the immunogenicity of EBOV DNA vaccines. 249

250 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P62 THE EFFECTS OF OUTSIDE TEMPERATURE IN THE INDUCTION OF ADAPTIVE IMMUNITY TO INFLUENZA VIRUS INFECTION Takeshi Ichinohe *, Miyu Moriyama Department of Virology, UNIVERSITY OF TOKYO, Tokyo, Japan Abstract: Climate change and global warming increasingly pose a severe problem to public health, as illustrated by the recent outbreaks involving Zika fever, West Nile fever, and malaria. Although climate change may expand the geographical distribution of several vector-borne diseases, the effects of outside temperature in host defense to viral infection in vivo are largely unknown. Here, we demonstrate that warm exposure of mice at 36 C impaired the virus-specific CD8 T cells and antibody responses against influenza virus infection. In contrast, IgG antibody responses remained intact in warm-exposed mice after intraperitoneal infection with Zika virus or sever fever with thrombocytopenia syndrome (SFTS) virus, or subcutaneous vaccination with inactivated influenza virus. Following influenza virus infection, the warm-exposed mice failed to stimulate inflammasome-dependent cytokine secretion compared with room temperature (RT) or cold-exposed groups. Although the warm-exposed mice did not change commensal microbiota composition, they decreased their food intake and body weight by 0% and increased autophagy in the lung tissue. Induction of autophagy in RT-exposed mice by 24 hoursstarvation or rapamycin treatment severely impaired virus-specific CD8 T cells and antibody responses following respiratory influenza virus infection. By using parabiotic mice joining control and starvation mice, we show that signals from ad libitumfed mice restored the virus-specific CD8 T cell responses in starvation mice. These findings uncover an unexpected mechanism by which outside temperature alters virus-specific adaptive immune responses. 250

251 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P64 INSIGHTS INTO ANTIGENICITY OF INFLUENZA A(H3N2) VIRUS, L Gubareva *, V Mishin, P Jorquera, H Nguyen, A Chesnokov, B Mann, J Barnes, R Garten, J Katz, D Wentworth CDC, Atlanta, United States Abstract: Influenza A(H3N2) viruses evade immunity by rapidly acquiring changes in the hemagglutinin (HA). Some changes alter HA receptor-binding, precluding the use of HI assay for antigenic analysis of many recent viruses. Hence, next-generation sequencing has become a leading tool for monitoring virus evolution and neutralization assays have supplemented HI antigenic analysis. As existing neutralization assays have limited throughput, alternative methods to ascertain antigenic relatedness between co-circulating viruses and vaccines are desirable. We developed a high content imaging-based neutralization test (HINT) that limits artifacts of antigenic analysis caused by virus culturing. Reference antisera were raised by infecting ferrets with clinical specimens containing viruses representing antigenic groups of recent vaccines. Antigenic relatedness was evaluated using clinical specimens and MDCK-SIAT isolates. Antigenic cartography methods were applied. The data showed that gain of a glycan at 58, rather than F59Y, was a major determinant for the antigenic transition from A/TX/50/202-like to A/HK/480/204-like viruses. Glycan loss at 58 can occur upon culturing and it reduces the distance from A/TX/50/202, while increasing the distance from A/HK/480/204. Multiple introductions of F93S (site B) were associated with the increased distance from A/SW/ /203 and A/TX/50/202. Since the summer of 207, viruses carrying T35K and/or I92T have been detected in different countries; some viruses showed reduced neutralization (>4-fold) by anti-a/hk/480/204-like virus serum, an early sign of antigenic transition. Our data demonstrate that HINT is a valuable new approach for antigenic analysis of A(H3N2) viruses. 25

252 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P65 IDENTIFICATION OF ESSENTIAL HOST FACTORS FOR EBOLA VIRUS RNA SYNTHESIS USING A GENOME-WIDE SIRNA SCREEN Scott Martin, Abhilash Chiramel 2, Marie Schmidt 3, Yu-Chi Chen, Nadia Whitt, Ari Watt 2, Eric Dunham 2, Kyle Shifflett 2, Shelby Traeger 2, Anne Leske 4, Eugen Buehler, Cynthia Martellaro 2, Janine Brandt 3, Lisa Wendt 3, Sonja Best 2, Jürgen Stech 3, Stefan Finke 3, Angela Römer-Oberdörfer 3, Allison Groseth 4, Heinz Feldmann 2, Thomas Hoenen * 2, 3 Division of Preclinical Innovation, NIH, Bethesda MD, 2 Laboratory of Virology, NIH, Hamilton MT, United States, 3 Institute for Molecular Virology and Cell Biology, 4 Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, Riems, Germany Abstract: Ebola virus (EBOV) causes a severe hemorrhagic fever, with no licensed treatment currently available. Antivirals targeting host factors are advantageous since they may target a wider range of viruses, including newly emerging ones, and since development of resistance is unlikely. However, systematic approaches to screen for host factors important for EBOV are hampered by the need to work with this virus at biosafety level 4 (BSL4). In order to identify such EBOV-interacting host factors, we performed a genome-wide sirna screen against 2,566 human genes to assess their activity in viral RNA synthesis. As a platform for screening and subsequent hit characterization we used minigenome-based life cycle modelling systems, including transcription-and-replication competent virus-like particle (trvlp) systems. In trvlp systems minigenomes are packaged into VLPs that can infect target cells, but are biologically restricted to cells expressing multiple viral proteins in trans, providing a way to model virtually the entire EBOV life cycle (i.e. particle entry, RNA synthesis, and progeny particle production) over multiple infectious cycles under BSL conditions. Identified hits were confirmed using infectious EBOV. Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for RNA synthesis of EBOV. Targeting this pathway with an FDA and EMA-approved small molecule inhibitor showed antiviral activity against EBOV in both life cycle modelling systems and experiments with infectious EBOV, and also showed activity against some other non-segmented negative-sense RNA viruses (NNSVs). This work demonstrates the power of life cycle modelling systems for conducting large-scale host factor screens for BSL4 viruses, and provides a data set for future analysis examining the role every human gene in EBOV RNA synthesis. 252

253 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P66 CHARACTERIZATION OF INFLUENZA VIRUS CLINICAL ISOLATES OBTAINED DURING UMIFENOVIR CLINICAL STUDY ARBITR Irina Leneva *, Irina Falynskova, Nailya Makhmudova, Artem Poromov, Svetlana Yatsyshina 2, Victor Maleev 2 I.Mechnikov Research Institute for Vaccines and Sera, 2 Central Research Institute for Epidemiology, Moscow, Russian Federation Abstract: Oral drug umifenovir (Arbidol) is licensed and widely used in Russia for treatment and prophylaxis of influenza A and B infections. We investigated susceptibility of influenza viruses isolated from patients pre- and during administration of umifenovir in phase IV clinical trial ARBITR. In addition, we examined the susceptibility of a panel of reference and NA inhibitor-resistant viruses and their sensitive counterparts to umifenovir. Umifenovir inhibited replication of all reference human influenza A and B viruses that circulated in seasons. The wild-type influenza viruses and their oseltamivir-resistant mutants were susceptible to umifenovir at similar levels. All 8 isolates obtained before and during therapy with umifenovir in ELISA-cell assay were equally sensitive to umifenovir with IC50 falling in the range of 7.0 to 2,5 ug/ml and similar to IC50 previously observed for laboratory and clinical isolates. Matched isolates of two patients from whom we were able to obtain day 3, 5 and 7 samples were chosen for sequence analysis. No amino acid changes in HA that had previously been identified in vitro as being involved with reduction of susceptibility to umifenovir were observed. None of the viruses isolated before and during therapy with umifenovir displayed reduced susceptibility to NA inhibitors and no mutations that led to an amino acid substitution in the NA were found in studied samples. Umifenovir is effective against influenza viruses of season and oseltamivir-resistant influenza viruses. No umifenovir-resistance has emerged during therapy of acute influenza infection. Umifenovir administration did not affect the susceptibility of influenza viruses to NA inhibitors. 253

254 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P67 DEVELOPMENT AND APPLICATION OF ARENAVIRUS LIFECYCLE MODELLING SYSTEMS Anne Leske, Eric Dunham 2, Irke Waßmann, Kevin Schnepel, Kyle Schifflett 2, Ari Watt 2, Heinz Feldmann 2, Thomas Hoenen 2, 3, Allison Groseth *, 2 Junior Research Group Arenavirus Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany, 2 Laboratory of Virology, National Institutes of Health, Hamilton, MT, United States, 3 Institute for Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Greifswald - Insel Riems, Germany Abstract: Lifecycle modelling systems, which make use of miniature genome analogues (minigenomes) to recapitulate all or part of the viral lifecycle, are valuable tools for studying virus biology. If adequately optimized, they also provide robust systems for screening antiviral compounds and host cell factors regulating virus infection, and for mechanism of action studies. However, despite early successes developing arenavirus minigenomes, little subsequent effort has been focused on refining and optimizing these systems to make them suitable for such applications. To achieve this we developed both fluorescent and luciferase-based Junín and Tacaribe virus minigenomes, which form the basis for corresponding minigenome systems (to model replication and transcription) and transcription and replication-competent virus-like particle (trvlp) systems (which also model budding and virus entry). All these systems can be safely used under BSL conditions and thus open up access to technologies and expertise not usually available under BSL4 conditions. In particular, we have applied these tools for drug library screening, where we identified AVN-944 as a novel inhibitor of arenavirus RNA synthesis, as well as for a limited sirna screen to look for pro- and antiviral factors that are either unique or shared with other hemorrhagic fever viruses. Most recently we have also analysed neutralization of trvlps containing foreign arenavirus glycoproteins by serum from human Candid# vaccinees, the results of which suggest a lack of robust cross-neutralization between even closely related species. Overall our findings highlight the broad utility of arenavirus lifecycle modelling systems for a variety of applications. 254

255 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P68 VACUOLAR ATPASE INHIBITING NANOPARTICLES EXHIBIT POTENT HOST-TARGETED ANTIVIRAL ACTIVITY AGAINST INFLUENZA VIRUSES You-Ting Chen, Che-Ming Hu 2, Hui-Wen Chen * Department of Veterinary Medicine, National Taiwan University, 2 Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan Abstract: Conventional treatments against influenza virus infections are designed to target viral proteins. However, the emergence of new influenza viral strains carrying drug-resistant mutations that can outpace the development of pathogentargeting antivirals presents a major clinical challenge. Two vacuolar ATPase (v-atpase) inhibitors, diphyllin, and bafilomycin, were previously identified with a broad-spectrum antiviral activity. However, their poor water solubility and their potential adverse effect may limit its clinical application. The central objective of this project is to generate a novel antiinfluenza therapeutic strategy, integrating nanoparticle technology to enhance host-targeting antiviral delivery towards improved drug safety and efficacy. In this study, we have prepared a nanoformulation of diphyllin and bafilomycin, which the size of both nanoparticles were measured 200 nm by the dynamic light scattering and transmission electron microscopy. The drug encapsulation of diphyllin and bafilomycin were analyzed by the high-performance liquid chromatography. The nanoparticles showed to have efficient intracellular delivery in multiple cell lines including Fcwf-4, ARPE-9, and MH-S cells. The nanoformulation of diphyllin also exhibited lower cytotoxicity as compared to the free drugs in MDCK and MH-S cells. Furthermore, the nanoparticles demonstrated prominent anti-influenza activity against HN and H3N2 in vitro. Upon the investigation of in vivo safety study, diphyllin nanoparticles were found well-tolerated in mice, from which the body weight was monitored and blood chemistry parameters were evaluated following intravenous or intranasal administration. Collectively, this work highlights the nanoformulation of v-atpase blocking compounds as the potential effective hosttargeted treatment against influenza. 255

256 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P69 A VSV-MEASLES CHIMERIC VIRUS CAN TARGET NECTIN-4 POSITIVE BREAST CANCER TUMORS IN AN IMMUNE COMPETENT MOUSE AND IS ENHANCED BY TRANSIENT INHIBITION OF ANTI-VIRAL INNATE IMMUNITY Christopher D. Richardson *, Gary Sisson, Natalie Mishreky, Angelita Alcos Microbiology & Immunology/Pediatrics, DALHOUSIE UNIVERSITY, Halifax, Canada Abstract: Our laboratory previously discovered that the tumor marker Nectin 4 (PVRL4) is the epithelial cell receptor for measles virus, and is expressed on many adenocarcinomas. Breast cancer cells expressing Nectin 4 can be infected with measles virus or a vesicular stomatitis virus (VSV) hybrid virus containing the H and F proteins of measles virus. Tumors can be readily infected with measles and VSV-measles viruses that express either green fluorescent protein (egfp) or firefly luciferase (Luc) reporter genes. Histological analysis of MD468 or patient derived triple negative tumors showed that they expressed egfp reporter and viral proteins. Although the viruses replicate in the tumors engrafted onto immune deficient mice, the infectious agents were not oncolytic and only slowed tumor progression. To test the contribution of the host immune system, mouse 4T and 4T-Nectin 4 tumors were produced in immune competent BALB/C mice. The highly conserved Nectin-4 protein increased growth and metastasis to produce aggressive 300 cubic mm mouse breast tumors within 6 days. 4T and 4T-Nectin 4 tumors on xenographic (NIH III nude) and syngeneic (BALB/C) mice were injected with measles and VSV-measles viruses and monitored over 24 days. The growth of 4T-Nectin 4 tumors in the immune competent BALB/C mice was slowed by intra-tumoral injection of measles virus or VSV-measles chimeric virus. Tumor regression with the VSV-measles chimeric virus was further enhanced using either BX795 or vanadate chemical inhibitors, which transiently inhibited anti-viral innate immunity. Infection of tumors was documented using confocal immune microscopy. We concluded that the anti-tumor properties of measles and VSV-chimeric viruses are dependent on both the innate and adaptive host immune systems. 256

257 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P70 ENHANCING THE IMMUNOGENICITY AND ATTENUATION OF RESPIRATORY SYNCYTIAL VIRUS VACCINE CANDIDATES BY ALTERING NS FUNCTION Michael Teng *, Kim Tran, Andrew McGill, Olivia Harder 2, Stefan Niewiesk 2 Internal Medicine, UNIVERSITY OF SOUTH FLORIDA, Tampa, 2 Veterinary Biosciences, Ohio State University, Columbus, United States Abstract: Development of live-attenuated vaccines for respiratory syncytial virus (RSV) has been hampered by identifying candidates that are sufficiently attenuated but retain immunogenicity. One issue is that natural RSV infection itself only induces short-lived immune responses, resulting in recurrent infections. In addition, the likely target population for a liveattenuated vaccine are infants who do not have a completely developed immune system. Therefore, a highly attenuated, but strongly immunogenic, vaccine would be desirable. RSV encodes a small nonstructural protein (NS) that has multiple functions in the viral life cycle. NS inhibits production type I interferon (IFN) responses via inhibition of the RIG-I/MAVS pathway and plays a role in viral RNA synthesis. the IFN antagonism and viral replication functions of NS are independent functions of NS and could be segregated by mutagenesis. Decreasing the IFN antagonism of NS would allow for a liveattenuated virus that has increased immunogenicity due to the immunostimulatory properties of type I IFNs. Thus, we reasoned that the IFN antagonism and viral replication functions of NS are independent functions of NS and could be segregated by mutagenesis. Through this process, we have identified specific regions and residues in the N- and C-termini of NS that are required for each function. Recombinant RSV (rrsv) encoding NS point deletion mutations displayed increased IFN production and enhanced replication compared with NS deletion rrsv. In addition, these viruses are attenuated and immunogenic in cotton rats. Our studies have allowed us to develop a menu of NS mutations that we can use to independently regulate IFN antagonism and viral replication to tune potential vaccine candidates for immunogenicity and attenuation. 257

258 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P7 FAVIPIRAVIR BUT NOT RIBAVIRIN IS EFFECTIVE AGAINST TWO STRAINS OF CRIMEAN CONGO HEMORRHAGIC FEVER IN MICE David Hawman *, Elaine Haddock, Kimberly Meade-White, Kyle Rosenke, Takashi Komeno 2, Yousuke Furuta 2, Brian Gowen 3, Heinz Feldmann NIH/NIAD, Hamilton, United States, 2 Research Laboratories, Toyama Chemical, Toyama, Japan, 3 Utah State University, Logan, United States Abstract: Crimean-Congo hemorrhagic fever virus (CCHFV) is a negative sense RNA virus in the Orthonairovirus genus. CCHFV is widely distributed through Africa, Eastern Europe and the Middle East. Infected humans exhibit non-specific symptoms such as fever and myalgia and then progress into the hemorrhagic phase. Case-fatality rates can be as high as 30%. Ribavirin is recommended by the WHO for treatment of CCHFV but its efficacy in humans and animal models is inconclusive. Favipiravir is a promising antiviral for CCHFV and has shown efficacy against CCHFV in a mouse model. To reconcile the inconsistent data on the efficacy of ribavirin and test these antivirals against another isolate of CCHFV, we tested ribavirin and favipiravir against the prototypical CCHFV strain 0200 and the clinical isolate Hoti in our IFNAR -/- mouse model. Against either strain ribavirin treatment was unable to prevent terminal disease in infected mice. In contrast, favipiravir treatment protected 00% of 0200-infected mice even when started as late as 24 hours prior to terminal disease. Against strain Hoti, favipiravir treatment had clinical benefit when started as late as 6 days post-infection, a time when mice are exhibiting severe disease. Interestingly, in two favipiravir-treated mice recrudescence of CCHFV was observed weeks post-infection and several tissues collected from survivors five weeks-post infection showed persistence of CCHFV. Nevertheless, these data suggest that favipiravir is a potent antiviral against CCHFV. We plan to further evaluate favipiravir against CCHFV in our recently developed non-human primate model. This research was supported by the NIAID Intramural Research Program. 258

259 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P72 SCALABLE REPLICON-PARTICLE VACCINE PROTECTS AGAINST LETHAL LASSA VIRUS INFECTION IN THE GUINEA PIG MODEL Markus Kainulainen *, Jessica Spengler, Stephen Welch, JoAnn Coleman-McCray, Jessica Harmon, John Klena, Stuart Nichol, César Albariño, Christina Spiropoulou Viral Special Pathogens Branch, Centers for Disease Control and Prevention, Atlanta, United States Abstract: Lassa fever is an arenaviral zoonosis that causes a substantial number of human infections in West Africa every year. While Nigeria, Sierra Leone, Guinea and Liberia have long been considered Lassa-endemic countries, evidence for a wider distribution is accumulating. Despite the disease burden, there are so far no approved vaccines to prevent Lassa virus infections. To generate a vaccine candidate with scalability and efficacy benefits typical of live attenuated vaccines, we have used our Lassa reverse genetics system to remove the essential glycoprotein precursor gene from the virus genome, and expressed the glycoproteins in a stable Vero cell line instead. The system produces virus replicon particles (VRPs) that replicate in the first cells encountered, but do not spread due to lack of de novo virus glycoprotein synthesis. We have shown that a single subcutaneous vaccination with the VRPs protects guinea pigs from lethality and clinical signs after challenge with Lassa virus strain Josiah. No residual viral RNA was detected in the vaccinated animals at the end of the experiment. Currently, we are focusing our efforts on further refining and scaling up the VRP production in order to perform future studies in the nonhuman primate Lassa animal model. 259

260 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P73 REMOVAL OF THE N-GLYCOSYLATION SEQUON AT POSITION N6 LOCATED IN P27 OF THE RESPIRATORY SYNCYTIAL VIRUS FUSION PROTEIN ELICITS ENHANCED ANTIBODY RESPONSES AFTER DNA IMMUNIZATION Annelies Leemans *, Marlies Boeren, Winke Van der Gucht, Isabel Pintelon 2, Kenny Roose 3, Bert Schepens 3, Xavier Saelens 3, Dalan Bailey 4, Wim Martinet 5, Guy Caljon, Louis Maes, Paul Cos, Peter Delputte Laboratory of Microbiology, Parasitology and Hygiene, University of Antwerp, Wilrijk, Belgium, 2 Laboratory of Cell Biology and Histology, University of Antwerp, Wilrijk, Belgium, 3 Medical Biotechnology Center, VIB, Ghent University, Ghent, Belgium, 4 The Pirbright Institute, Surrey, United Kingdom, 5 Laboratory of Physiopharmacology, University of Antwerp, Wilrijk, Belgium Abstract: Prevention of severe lower respiratory tract infections in infants caused by the human respiratory syncytial virus (hrsv) remains a major public health priority and development of a prophylactic vaccine is very important in the control of RSV disease burden. Currently, the major focus of vaccine development relies on the RSV fusion (F) protein since it is the main target protein for neutralizing antibodies induced by natural infection. RSV F is highly conserved among RSV strains and has 5 N-glycosylation sites that are located in the F2 subunit (N27 and N70), the F subunit (N500) and the p27 peptide (N6 and N26). To our knowledge, improvements can be made in the understanding of the role of N-glycosylation in the immunogenicity of the RSV F protein. Expression vectors encoding RSV F glycosylation mutants were developed by replacement of the asparagine (N) codon by a glutamine (Q) codon. To study the effect of the loss of one or more N- glycosylation sites on RSV F immunogenicity, BALB/c mice were immunized with the different constructs encoding the RSV F glycomutants. In comparison with F WT DNA immunized mice, higher neutralizing titers were observed following immunization with F N6Q DNA. Upon subsequent RSV challenge, lower viral RNA levels were detected in the F N6Q DNA immunized mice compared with the levels in mice that had been immunized with F WT DNA. Since p27 is assumed to be post-translationally released from the mature RSV F protein after cleavage and thus not present on the mature RSV F protein, it remains to be elucidated how deletion of this N-sequon can contribute to enhanced antibody responses and protection upon challenge. These findings provide new insights to improve the immunogenicity of RSV F in potential vaccine candidates. 260

261 NSV 208, Verona Abstract Book FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P74 RIBAVIRIN HAS A DEMONSTRABLE EFFECT ON CRIMEAN-CONGO HEMORRHAGIC FEVER VIRAL POPULATIONS AND VIRAL LOAD DURING PATIENT TREATMENT Nicole Espy *, Unai Perez-Sautu, Eva Ramírez de Arellano 2, Anabel Negredo 2, Michael Wiley, 3, Sina Bavari, Marta Diaz Menendez 4, Maria Paz Sanchez-Seco 2, Gustavo Palacios United States Army Research Institute of Infectious Diseases, Frederick, United States, 2 Arbovirus and Imported Viral Diseases Unit, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain, 3 College of Public Health, University of Nebraska Medical Center, Omaha, United States, 4 High Level Isolation Unit, Hospital Universitario La Paz- Carlos III, Madrid, Spain Abstract: Next-generation sequencing of viral populations allow researchers to gather evidence of a nucleoside analogue s mechanism of action in vivo. The use of the guanosine analog ribavirin to treat infections of Crimean-Congo Hemorrhagic Fever virus (CCHFV) has been controversial based on uncertainties on its antiviral efficacy in clinical case studies. We studied the effect of ribavirin treatment on viral populations in a recent case. We collected plasma samples taken from a CCHFV-infected patient before, during, and after a five day regimen of ribavirin, in which CCHF viral load dropped during ribavirin treatment. Viral RNA extracted from these longitudinal samples was deep sequenced to measure subclonal diversity and specific base changes that occurred due to ribavirin treatment. Coverage in each sample trended with viral load, and subclonal diversity increased in each viral segment during treatment. Base changes consisted primarily of T to C, G to A, and C to T transitions and of indels that accumulated preferentially in the CCHFV L segment. These data demonstrate the mutagenic effect of ribavirin on CCHFV in vivo. Although the results are based on a single case, the paucity of other clinical data makes this result relevant for the clinical management of CCHFV-infected patients. Further, this study demonstrates that genomic analysis of viral populations can provide a clinical demonstration of nucleoside analogue activity. 26

262 FIGHTING AND RESPONDING antivirals and vaccines Abstract final identifier: P75 DEVELOPMENT OF BROAD-SPECTRUM PEPTIDOMIMETIC INHIBITORS OF PARAMYXOVIRAL FUSION Victor Outlaw *, Ross Cheloha, Matteo Porotto 2, 3, Sam Gellman, Anne Moscona2, 3, 4, 5 Department of Chemistry, University of Wisconsin, Madison, 2 Department of Pediatrics, 3 Center for Host-Pathogen Interaction, 4 Department of Physiology & Cellular Biophysics, 5 Department of Microbiology & Immunology, Columbia University Medical Center, New York, United States Abstract: The respiratory pathogens parainfluenza (HPIV3) and respiratory syncytial (RSV) negatively impact global child and elderly health, and effective clinical treatment options are lacking. Viral fusion has emerged as a compelling target for the development of novel antiviral therapeutics. Recently, peptides were reported that inhibit fusion by several related members of the Paramyxoviridae and Pneumoviridae families. While the fusion mechanism is conserved, the individual fusion (F) proteins possess distinct primary sequences; therefore, the ability of a single peptide to disrupt the fusion machinery within multiple viruses is intriguing. Using these inhibitors as a template, we have adopted a three-pronged approach to develop analogs with increased efficacy and enhanced resistance to enzymatic degradation. Protease resistance could improve efficacy by prolonging in vivo half-life; we predicted that beta-amino acid residues would enhance stability. First, to identify the interactions that underlie the broad-spectrum activity of the peptides, we structurally and biophysically characterized inhibitors bound to each distinct viral target. X-ray structures revealed six-helix bundle complexes and exposed key interactions that comprise each unique protein:inhibitor interface. Second, we used structureguided design principles to incorporate beta residues into the inhibitor sequence to create foldamers that structurally and functionally mimic the diverse secondary structures. These peptide mimics maintain potent activity against viral fusion but exhibit increased proteolytic stability. Finally, we created peptide-lipid conjugates to target the inhibitors to their sites of action at the host membrane and thereby increase potency. These agents combine potent inhibition of viral fusion with proteolytic resistance to lay a foundation for broad-spectrum antiviral therapeutics. Graphical Abstract: 262