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)

*  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  

Core 1: Molecular Virology

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

Norwalk Virus (NV)

VP1 Cytoplasm Encapsidation

Assembly

AAA(n)

AAA(n)

Genomic RNA (~7.7 kb)

Subgenomic RNA (~2.4 kb)

ORF1 ORF2 ORF3

p48 NTPase p22 VPg Protease RNA pol

VP2

(+)

(-)

(+)

24 hpi 30 hpi

Baseline 8 hpi

Small Intestine Histopathology Following NV Infection

(Estes, Unpublished)

NV RNA transfection into cells leads to a single cycle of viral replication and virus production

1d p.t. 2d p.t. 5d p.t. 3d p.t.

2 d p.t. 4 d p.t.

Huh-7 cells

Mix transfected cells with GFP-expressing cells > no virus spread

Guix et al., 2007

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

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Estes, Ajami, Atmar, unpublished data

Enterocyte  

(Sato et al., Nature, 2009)

Stem cell-derived Intestinal organoid

Enterocyte

Intes>nal  organoids  as  a  new  preclinical  model  for  enteric  microbes  

Intes>nal  organoids/enteroids  as  new  preclinical  models  for  enteric  microbes  (rotavirus  example)  

+

Organoids (cut) (Spence  et  al.,  Nature  470:105–109)

28+ days

Rotavirus

NSP4 (RV)

DAPI E-Cad

(Finkbeiner  et  al  ,  MBio,  2012)  

H9  Stem  Cells  (secretor  +)  

Human  Jejunem  Tissue  

E-­‐Cad      Muc-­‐2  DAPI    

                     Enteroids  (disperse)    (Sato  et  al.,  Gastro  141:1762-­‐1772)                                                        14+  days  

NSP4  (RV)  

villin  

Rotavirus

+  

KineAcs:  RNA  replicaAon  

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. 2012 3:e00159-12.

Human Intestinal Organoids and Enteroids are being Evaluated as Normal Human “Mini-Guts” that may support Norovirus Replication

(Estes, unpublished data)

Replication of Norovirus in Human Intestinal Organoids Developed

from Pluripotent Stem Cells

Dongsheng Zhang1, Weiming Zhong1, Ming Xia1, Pengwei Huang1, Kyle W. McCracken2, Jason R.

Spence2, Ming Tan1, James M. Wells2, and Xi Jiang1

1 Division of Infectious Diseases,

2 Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center,

Cincinnati, OH, USA

A typical human intestinal organoid with microvilli on the surface  

•  The 3-D intestinal organoid consisted of a polarized, columnar epithelium. •  The enterocyte cell with brush border and microvilli.

Microvilli with carbohydrate

100  nm  

3-D organoid

500  µm  

*  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.

Inoculation and assays of NoVs

Detection of NoV capsid antigens in organoids by immunofluorescent staining

•  The sample was collected at 18 hours post infection •  Stained using anti-NV antibody •  Positive signals in epithelium cells by fluorescent microscopy

Anti-NoV / nuclei

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

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.

•  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., 2012. PLoS ONE. 7(7):e41619).

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

Obj 2. Cell culture adaptation of original HuNoVs or Gn pig-

or calf-passaged HuNoVs in porcine, bovine or human 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 log10 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 .

Ø  HuNoVs (GII.4, GII.12, GII.6 and GII.2) were inoculated and serially passaged into the following cells:

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

1.2 - Cultivable HuNoV Surrogate Viruses

Feline calicivirus

feline Vesivirus Not enteric No diarrhea 108

ATCC

Murine norovirus

murine Norovirus Not enteric No diarrhea 108

limited

Tulane virus

primates Recovirus enteric diarrhea 107

limited

Aichi virus

human Picornavirus enteric diarrhea 108

limited

Porcine sapovirus

porcine Sapovirus enteric diarrhea 106

limited

Low virus titer No robust plaque assay

host genus target symptoms virus titer

source

Theresa Cromeans, Geun Woo Park, Jan Vinjé

Characterization of Physicochemical Properties of Four NoV Surrogate Viruses Based

on Infectivity

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).

• Like HuNoVs, SaV was stable at pH 3.0-8.0. • 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 78:3932-40).

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

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

Key Outcomes: Molecular Virology

Questions?