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(Estes, Unpublished)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Estes, Ajami, Atmar, unpublished data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?