Core 1: Molecular Virology

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1 Core 1: Molecular Virology Co-Leads: Estes (BCM) Vinjé (CDC) Collaborators: Baylor College of Medicine (Atmar, Petrosino) Ohio State U. (Saif, Wang) Cincinnati Children s Hosp. (Jiang)

2 Core 1: Molecular Virology * Purpose: Develop improved methods to facilitate the study of foodborne viruses and to further elucidate the significance of viral foodborne disease * Activity 1.1: Develop a human norovirus (HuNoV) in vitro cultivation system * Activity 1.2: Validate alternate cultivable HuNoV surrogates * Activity 1.3: Identification of agents potentially associated with foodborne (viral) disease of unknown etiology * Activity 1.4: Develop mathematical models to predict HuNoV emergence and virulence

3 Task 1.1 (BCM) Goal A2empt HuNoV replica>on in primary cells isolated from human intes>nal >ssues Ra>onale: The single largest roadblock to all studies of HuNoVs is the lack of an efficient cul>va>on system. Background: HuNoV RNA is infec>ous in mammalian cells of human origin (Guix et al., 2007). Approach: Obtain biopsies or isolated primary enterocytes from secretor posi>ve individuals and test replica>on of NV or irradiated NV (neg control) Cytopathic effect Immunofluorescence RNA replication by RT-PCR Virus spread and production of infectious virus

4 Norwalk Virus (NV) (+) ORF1 ORF2 ORF3 Genomic RNA (~7.7 kb) AAA (n) (-) p48 NTPase p22 VPg Protease RNA pol (+) Subgenomic RNA (~2.4 kb) AAA (n) Cytoplasm VP1 Encapsidation Assembly VP2

5 Small Intestine Histopathology Following NV Infection Baseline 8 hpi 24 hpi 30 hpi (Estes, Unpublished)

6 NV RNA transfection into cells leads to a single cycle of viral replication and virus production 2 d p.t. 4 d p.t. Mix transfected cells with GFPexpressing cells > no virus spread 1d p.t. 2d p.t. 3d p.t. 5d p.t. Guix et al., 2007 Huh-7 cells

7 Conclusions -1 NV RNA can be infectious in human mammalian cells. However, fully permissive replication of NV does not occur. Need more fully permissive cells test nontransformed human intestinal cells - Human intestinal tissues - Human mini-guts from stem cells

8 Norovirus structural and nonstructural proteins can be detected in human intestinal tissue exposed to infectious norovirus Identification of the type of cell(s) expressing these viral proteins may help identify a permissive cells for cultivating virus Estes, Ajami, Atmar, unpublished data

9 Intes>nal organoids as a new preclinical model for enteric microbes Stem cell-derived Intestinal organoid Enterocyte Enterocyte (Sato et al., Nature, 2009)

10 Intes>nal organoids/enteroids as new preclinical models for enteric microbes (rotavirus example) villin NSP4 (RV) KineAcs: RNA replicaaon H9 Stem Cells (secretor +) + Rotavirus Organoids (cut) (Spence et al., Nature 470: ) 28+ days DAPI E-Cad (Finkbeiner et al, MBio, 2012) Finkbeiner SR, Zeng XL, Utama B, Atmar RL, Shroyer NF, Estes MK. Stem cell-derived human intestinal organoids as an infection model for rotaviruses. MBio :e Human Jejunem Tissue + Rotavirus E- Cad Muc- 2 DAPI NSP4 (RV) Enteroids (disperse) (Sato et al., Gastro 141: ) 14+ days

11 Human Intestinal Organoids and Enteroids are being Evaluated as Normal Human Mini-Guts that may support Norovirus Replication (Estes, unpublished data)

12 Replication of Norovirus in Human Intestinal Organoids Developed from Pluripotent Stem Cells Dongsheng Zhang 1, Weiming Zhong 1, Ming Xia 1, Pengwei Huang 1, Kyle W. McCracken 2, Jason R. Spence 2, Ming Tan 1, James M. Wells 2, and Xi Jiang 1 1 Division of Infectious Diseases, 2 Division of Developmental Biology, Cincinnati Children s Hospital Medical Center, Cincinnati, OH, USA

13 A typical human intestinal organoid with microvilli on the surface 3-D organoid Microvilli with carbohydrate 500 µm 100 nm The 3-D intestinal organoid consisted of a polarized, columnar epithelium. The enterocyte cell with brush border and microvilli.

14 Inoculation and assays of NoVs * Organoids from two human embryonic stem cell lines WA09 and WA01 were studied. * A stool sample pool containing six NoVs was used as an inoculum for infection of organoids. * The organoids were assays for NoVs by RT-PCR, immunostaining and electron microscopy examination at 24, 48 and 72 hours post inoculation. * The inoculated culture was also passed in fresh organoids for up to 10 times and assayed for replicating viruses by RT-PCR and sequencing following each passage.

15 Detection of NoV capsid antigens in organoids by immunofluorescent staining Anti-NoV / nuclei The sample was collected at 18 hours post infection Stained using anti-nv antibody Positive signals in epithelium cells by fluorescent microscopy

16 Activity 1.1. Cell culture adaptation of human noroviruses (HuNoVs) by using gnotobiotic (Gn) pig- or calf-passaged virus or human cells L.J. Saif, Q.Wang and KI Jung, The Ohio State University Obj 1. Passage different HuNoV strains in Gn pigs and calves and assess clinical signs, shedding titers and infectivity; identify factors to enhance HuNoV infectivity HuNoVs strains (GII.4, GII.12, GII.6 and GII.2) were inoculated and serially passaged (2 X) in Gn pigs and calves (3 X): - Fecal viral RNA shedding titer was up to 10^8 GE/mL - Shedding period of >17 days in pigs HuNoV antigens observed in cytoplasm or on surface of enterocytes, implying that fecal shedding is a result of virus replication in the intestine. Simvastatin treatment of Gn pigs before and after virus inoculation induced significantly earlier onset and longer duration of HuNoV fecal shedding, frequently with higher fecal HuNoV titers (Jung et al., PLoS ONE. 7(7):e41619). Jejunum of a Simvastatin + HuNoV -pig at PID 3. IHC results: NoV antigens (green stain; arrows) were in the cytoplasm or on the surface of enterocytes; nuclei were stained with blue-fluorescent DAPI.

17 Obj 2. Cell culture adaptation of original HuNoVs or Gn pigor calf-passaged HuNoVs in porcine, bovine or human cells Ø HuNoVs (GII.4, GII.12, GII.6 and GII.2) were inoculated and serially passaged into the following cells: Porcine jejunum cell line (IPEC-J2): - Detected viral capsid antigens in the cytoplasm in approximately 1% of IPEC-J2 cells (GII.12/HS206) Porcine duodenum primary cells (IPEC-D1) Human primary small intestinal cells (HIEC) Human embryonic intestinal cell line (INT-407) Human colorectal adenocarcinoma cell line (Caco-2) - No log 10 increase of viral RNA in above cell cultures. Similar to the effect of simvastatin on HuNoV infectivity in the Gn pig model, HuNoV RNA titers in supernatants or lysates of IPEC-J2 cells treated with simvastatin were increased (1.9-fold vs control) in trials using GII.12 HS206 strain (Takanashi, Wang, Saif et al. 2012, unpublished) HuNoV GII.12/HS206 antigen detection (in green) in IPEC-J2 cells and DAPI staining (in blue) for nuclei at 24 hpi.

18 Comparison of cultivable surrogate viruses of foodborne viruses Theresa Cromeans, Geun Woo Park, Jan Vinjé Division of Viral Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA

19 1.2 - Cultivable HuNoV Surrogate Viruses host genus target symptoms virus titer source Feline calicivirus Murine norovirus Tulane virus Aichi virus Porcine sapovirus feline Vesivirus Not enteric No diarrhea 108 ATCC murine Norovirus Not enteric No diarrhea 108 limited primates Recovirus enteric diarrhea 107 limited human Picornavirus enteric diarrhea 108 limited porcine Sapovirus enteric diarrhea 106 limited Low virus titer No robust plaque assay Theresa Cromeans, Geun Woo Park, Jan Vinjé

20 Characterization of Physicochemical Properties of Four NoV Surrogate Viruses Based on Infectivity

21 Conclusions-2 * MNV, Tulane virus, and Aichi virus were stable at ph 2. (similar to HuNoVs); FCV is very sensitive * MNV and FCV were more resistant than Tulane virus and Aichi virus to heat inactivation (20 minutes 56 C) * Aichi virus and FCV are more resistant than MNV and Tulane virus to alcohol (70%, room temperature, 1 min) * All viruses showed similar resistance to low concentration of chlorine (200 ppm).

22 Activity 1.2. Validation of tissue culture-adapted porcine sapovirus as an improved surrogate for human norovirus to assess viral stability and decontamination methods: comparisons with FCV and MNV Q Wang and L.J. Saif, The Ohio State University Like HuNoVs, SaV was stable at ph SaV and MNV had similar resistance, and both were more resistant than FCV to heat inactivation (56 C for 30 min or 2 hrs) SaV and MNV were less resistant than FCV to ethanol (60% and 70%, room temperature for 30 sec). SaV, MNV and FCV showed similar resistance to low concentrations (2.5 and 10 mg/l) of chlorine at room temperature and a short incubation time (1 min); after increasing the incubation time (30 min), MNV was more resistant than SaV and FCV to chlorine. SaV was more resistant than FCV and MNV to ultraviolet (UV) treatment. The higher stability of SaV than FCV to heat and acid, its higher resistance to UV than FCV and MNV and its replication in cell culture (with bile acids) make this enteropathogenic virus a promising surrogate for HuNoVs for in vivo and in vitro studies (Wang et al., Appl Environ Microbiol 78: ).

23 Key Outcomes: Molecular Virology Progress is being made towards cultivating human noroviruses l NoV infection of tissues ex vivo l Human intestinal organoids/enteroids l Gnotobiotic pigs and porcine cell lines l Further optimization of culture conditions and robust replication is needed l Meanwhile, novel surrogate viruses may be suitable to assess effectiveness of disinfection conditions

24 Questions?