Inherent characteristics predisposing to reassortment & mutation

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1 Viral characteristics of H5N1 influencing mutation/reassortment events with pandemic potential FAO-OIE-WHO Scientific Consultation on Avian Influenza at the Human-Animal Interface Ruben Donis, Ph.D. Influenza Division, NCIRD, CCID Centers for Disease Control and Prevention Atlanta, Georgia, United States Inherent characteristics predisposing to reassortment & mutation Genome Structure 8 Interchangeable RNA molecules: Reassortment Conservation of critical compatibility features in type A viruses RNA polymerase without proof-reading PB1 fidelity: frequent mutations Numerous envelope alleles: 16 HA and 9 NA Broad range of biological activities: host, tropism, shedding, immune evasion Short generation time Accelerate evolution, evade host defenses 1

2 Inherent characteristics predisposing to mutation & reassortment Viral host range Established in multiple avian and mammalian host species, including humans Multiple options for interspecies transmission and reassortment Multiple HA and NA alleles Reduced interference for co-infection Expand circulation of reassorted internal genes Viruses with broad host range: dual infection and reassortment Mixing vessel species such as swine Gap of knowledge: host range of animal influenza strains Mucosal infection Limited immunologic memory, favors immune evasion Repeat infections, co-infection, reassortment and mutation Inherent characteristics predisposing to mutation & reassortment Genome Structure Segmentation and reassortment Avian-Avian: in nature Avian-Mammalian: in nature and experimental RNA polymerase without proof-reading PB1 fidelity: frequent mutations 2

3 Avian-Avian Reassortment in Nature Obenauer et al North American AI isolates, n=169 Extensive gene reassortment HA and NA most mobile Linkage of segments Stressed the importance of protein sequences in the analysis: proteotypes H6 North American viruses (example): Linkage of HA and NA proteotypes Linkages of PB1-PA-M-NS and HA-NA-M Proteotype combinations persist over years Postulate that functional interactions responsible for linkage Hitchhiking effects not ruled out completely Obenauer, J. C., et al Large-scale sequence analysis of avian influenza isolates. Science 311: Finkelstein, D. B., et al Persistent host markers in pandemic and H5N1 influenza viruses. J Virol 81: Proteotypes Identify Associations Proteotype association between HA and M 3

4 Avian-Avian Reassortment Dugan et al (2008) North American AI isolates; n=167 Extremely frequent reassortment Little clear linkage among specific internal gene segments Tree incongruence index Dugan, V. G., et al The evolutionary genetics and emergence of avian influenza viruses in wild birds. PLoS Pathog 4:e H5N1 Eurasian Lineage: Extensive Reassortment with Avian Viruses China: Harbin, HKU and St. Jude studies Vietnam: Wan et al (2008) PLoS in press Vietnam 2007, clade 1-2 reassortant genotypes Conclusions: 1. High frequency reassortment between avian viruses 2. Contribution to host range expansion? Wen XF et al, Evolution of Highly Pathogenic H5N1 Avian Influenza Viruses in Vietnam between 2001 and PLoS One In Press 4

5 Reassortment Between Avian And Mammalian Viruses Natural Reassortment of swine, avian and human viruses Triple reassortant SIV: 1998 USA H3N2 Swine 3X reassortant HA NA PB2 PB1 PA NP M NS Experimental Reassortment avian H5N1 and human H3N2 Chen et al (2008) Chen, L. M., et al Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses. PLoS Pathog 4:e reassortant viruses H5N1 H3N2 HA NA PB2 PB1 PA NP M NS PFU/mL PFU/mL 63 combinations Reassortant Genotypes or or or or or or SYSTEMATIC H5N1 human/avian combinatorial 2 6 5

6 Replication in Cell Culture 20% 12% 63 45% >10 6 PFU/mL 22% >6 log10 >4 log10 > 2 log10 < 2 log10 Growth of 63 human-avian H5N1 reassortant viruses Virulence in mice (i.n. route) H5N1 HA NA PB2 PB1 PA NP M NS LD PFU Replication of 38 reassortant genotypes 53% 38 13% 8% 26% PFU PFU Lethality of >2.8 human-avian log10 >3.8 log10 H5N1 >4 log10 reassortant >5 log10 viruses 6

7 NP Gene Interactions Chen et al HA NA PB2 PB1 PA NP M NS Rescue efficiency 7.6 < <2.0 Plaque size (mm) 4-5 ND ND Conclusions: 1. Human NP needs cognate M and NS 2. RNP reconstitution not beneficial Polymerase Gene Interactions HA NA PB2 PB1 PA NP M NS Rescue Plaque size efficiency (mm) < Conclusions: 1. Avian PB2 prefers cognate PA 2. Functional interaction 7

8 Polymerase Gene Reassortment H5N1 HU/AV reassortant HA NA PB2 PB1 PA NP M NS Lowest mouse LD 50 among 38 reassortants Resemble 1957 and (in part) 1968 pandemic strains Conclusions: 1. Avian HA-NA may benefit from cognate PB1 in vivo 2. Contribution to host range expansion? Chen, L. M., et al Genetic compatibility and virulence of reassortants derived from contemporary avian H5N1 and human H3N2 influenza A viruses. PLoS Pathog 4:e Influenza Interactome vs. Reassortment Physical PPI: direct (D) or indirect (I) PB2 PB1 (F2) PA HA NP NA M1 (M2) NS (NS2) Functional () PB2 PB1(F2) PA HA NP NA 1 D 1 I D 1 3 D I I n 4 D D M1(M2) n (D) NS1(NS2) 2 Reassortment phenotype (Chen et al: avian/human) (Li et al: equine/human) Gap of knowledge: fitness loss/gain by reassortment Li, C., et al Compatibility among polymerase subunit proteins is a restricting factor in the reassortment between equine H7N7 and human H3N2 viruses. J Virol. In press. 8

9 Inherent characteristics predisposing to mutation & reassortment Genome Segmentation Reassortment RNA genome and polymerase lacking proof-reading PB1 fidelity: frequent mutations Critical role of selection Mutations: focus on HA Diverse selection forces drive evolution Receptor specificity, antigenic drift, etc are driven by the host Expansion of viral host range, increased risk for humans Mutations in the remaining 7 genes also influence host range Consequence of mutations depends on the subtype of the HA and strain variations in the RBS structure Examples of RBS changes in H9, H7 and H5 9

10 Stevens, J. et al., 2006, Nature Rev Microb 4(11): H5 Eurasian Lineage Stevens et al (2008) Wildtype A/Vietnam/1203/2004 A/Indonesia/5/2005 From Stevens, J., et al Recent avian H5N1 viruses exhibit increased propensity for acquiring human receptor specificity. J Mol Biol 381:

H7 HA North American & Eurasian Belser et al.

rhea/n Carolina/39482/1993 turkey/virginia/4529/2002 Netherlands /230/2003? Belser, J.

Contemporary North American influenza H7 viruses possess human receptor specificity:

H9 HA Eurasian Lineage Wan et al (2008) Emergence of avian viruses with changes at

quail/hk/88 guinea fowl/hk/wf10/99 Gap of knowledge: What are the selection forces for

11 H7 HA North American & Eurasian Belser et al. (2008) North American H7: mutations near receptor binding site? Eurasian H7:? rhea/n Carolina/39482/1993 turkey/virginia/4529/2002 Netherlands /230/2003? Belser, J. A., et al Contemporary North American influenza H7 viruses possess human receptor specificity: Implications for virus transmissibility. Proc Natl Acad Sci U S A 105: H9 HA Eurasian Lineage Wan et al (2008) Emergence of avian viruses with changes at position 226 of HA Gln to Leu change increases sialoside & reduces α2-3 binding quail/hk/88 guinea fowl/hk/wf10/99 Gap of knowledge: What are the selection forces for RBS switch? Gap of knowledge: RBS profiles for a broad range of viruses From: Wan, H., et al Replication and transmission of H9N2 influenza viruses in ferrets: evaluation of pandemic potential. PLoS ONE 3:e

12 RBS of Avian and Mammalian Viruses α2-3 Binding Gap of knowledge: sialoside receptor expression in different species Sialic Acid Structural Diversity Determine sialic acid receptor structures in upper and lower airway 12

Sialic Acids in Animals Acknowledgements CDC Limei Chen James Stevens Todd Davis Sara Jackson Yumi Matsuoka Amanda Balish Hong Zhou Laurie Kamimoto Cathy Smith Michael Shaw Joe Bresee

Surveillance Network David Swayne; USDA, ARS, SEPRL James Paulson, TSRI/CFG Ola Blixt, TSRI/CFG Ian Wilson, TSRI Gavin Smith, HKU Malik Pieris, HKU Hien Nguyen, NIHE, Vietnam Daniel Perez, U

Jude Kanta Subbarao, NIH Patrick Blair, NAMRU2 Ken Earhart, NAMRU3 Jeffrey Tjaden, NAMRU3 Armen Donabedian, BARDA Robin Robinson, BARDA Michael Perdue, BARDA Ben Schwartz, NIP Roland

13 Sialic Acids in Animals Acknowledgements CDC Limei Chen James Stevens Todd Davis Sara Jackson Yumi Matsuoka Amanda Balish Hong Zhou Laurie Kamimoto Cathy Smith Michael Shaw Joe Bresee Jacqueline Katz Alexander Klimov Ann Moen Tim Uyeki Terry Tumpey Tony Marfin Sharon Daves Dan Jernigan Nancy Cox SRP Core, ARB, IACUC MVVB, VSDB, IPB, Influenza Division Beyond CDC WHO GIP Surveillance Network David Swayne; USDA, ARS, SEPRL James Paulson, TSRI/CFG Ola Blixt, TSRI/CFG Ian Wilson, TSRI Gavin Smith, HKU Malik Pieris, HKU Hien Nguyen, NIHE, Vietnam Daniel Perez, U Maryland Richard Webby, St Jude CRH Aloke Chakraborty, NIV, India Tung Nguyen, NCVD, Vietnam Yuelong Shu, China CDC Catherine Gerdil, Sanofi Ervin Fodor, U Cambridge Erich Hoffmann, St. Jude Kanta Subbarao, NIH Patrick Blair, NAMRU2 Ken Earhart, NAMRU3 Jeffrey Tjaden, NAMRU3 Armen Donabedian, BARDA Robin Robinson, BARDA Michael Perdue, BARDA Ben Schwartz, NIP Roland Lewandowski, NIH Glycan microarrays were obtained from the Consortium for Functional Glycomics (funded by GM062116), and from the Centers for DiseaseControl that were produced for the CDC with the CFG glycan library. 13

Importance of 1918 virus reconstruction on current assessments to pandemic risk

Importance of 1918 virus reconstruction on current assessments to pandemic risk Jessica A Belser, PhD Immunology and Pathogenesis Branch Influenza Division Centers for Disease Control and Prevention National