Development of a reverse-genetics system for murine norovirus 3: Long-term persistence occurs in the caecum and colon

Section of Virology, Department of Medicine, Imperial College London, Norfolk Place, London, W2 1PG, UK.
Journal of General Virology (Impact Factor: 3.18). 04/2012; 93(Pt 7):1432-41. DOI: 10.1099/vir.0.042176-0
Source: PubMed


Human noroviruses (HuNoV) are a major cause of viral gastroenteritis worldwide, yet, due to the inability to propagate HuNoV in cell culture, murine norovirus (MNV) is typically used as a surrogate to study norovirus biology. MNV-3 represents an attractive strain to study norovirus infections in vivo because it establishes persistence in wild-type mice, yet causes symptoms resembling gastroenteritis in immune-compromised STAT1(-/-) mice. The lack of reverse-genetics approaches to recover genetically defined MNV-3 has limited further studies on the identification of viral sequences that contribute to persistence. Here we report the establishment of a combined DNA-based reverse-genetics and mouse-model system to study persistent MNV-3 infections in wild-type (C57BL/6) mice. Viral RNA and infectious virus were detected in faeces for at least 56 days after inoculation. Strikingly, the highest concentrations of viral RNA during persistence were detected in the caecum and colon, suggesting that viral persistence is maintained in these tissues. Possible adaptive changes arising during persistence in vivo appeared to accumulate in the minor capsid protein (VP2) and the viral polymerase (NS7), in contrast with adaptive mutations selected during cell-culture passages in RAW264.7 cells that appeared in the major capsid protein (VP1) and non-structural protein NS4. This system provides an attractive model that can be readily used to identify viral sequences that contribute to persistence in an immunocompetent host and to more acute infection in an immunocompromised host, providing new insights into the biology of norovirus infections.

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Available from: Armando Arias, Oct 16, 2014
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    • "MNV-1 efficiently replicates in cell culture although it shows limited virulence in wild-type mice. In contrast, MNV-3 typically produces lower yields in tissue culture, yet establishes long-term persistent infections in wild-type mice, being detected for at least 8 months after inoculation (Arias et al., 2012a; McFadden et al., 2013). Previous studies demonstrated that ribavirin and favipiravir elicit antiviral activity against MNV-1 and human norovirus (HuNoV) replicon in cell culture (Chang and George, 2007; Rocha-Pereira et al., 2012). "
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    ABSTRACT: Lethal mutagenesis has emerged as a novel potential therapeutic approach to treat viral infections. Several studies have demonstrated that increases in the high mutation rates inherent to RNA viruses lead to viral extinction in cell culture, but evidence during infections in vivo is limited. In this study, we show that the broad-range antiviral nucleoside favipiravir reduces viral load in vivo by exerting antiviral mutagenesis in a mouse model for norovirus infection. Increased mutation frequencies were observed in samples from treated mice and were accompanied with lower or in some cases undetectable levels of infectious virus in faeces and tissues. Viral RNA isolated from treated animals showed reduced infectivity, a feature of populations approaching extinction during antiviral mutagenesis. These results suggest that favipiravir can induce norovirus mutagenesis in vivo, which in some cases leads to virus extinction, providing a proof-of-principle for the use of favipiravir derivatives or mutagenic nucleosides in the clinical treatment of noroviruses. DOI:
    eLife Sciences 10/2014; 3. DOI:10.7554/eLife.03679 · 9.32 Impact Factor
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    • "MNV-1.CW3, MNV-3 Mouse (129SvEv, CD1, C57BL/6 RAG/STAT 1À/À; IFN receptor deficientÀ Oral Acute, $100% mortality 4–9 days after infection CW3—diarrhea, gastric bloating, weight loss, mortality, MNV-3—weight loss, fecal inconsistency Liver, spleen, intestine, MLN, lungs Not reported CW3—severe, acute necrosis, MNV-3—mild inflammation Yes Yes [31] [48] [49] [51] [52] [96] "
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    ABSTRACT: The development of antiviral strategies to treat or prevent norovirus infections is a pressing matter. Noroviruses are the number #1 cause of acute gastroenteritis, of foodborne illness, of sporadic gastroenteritis in all age groups and of severe acute gastroenteritis in children less than 5 years old seeking medical assistance [USA/CDC]. In developing countries, noroviruses are linked to significant mortality (̃200.000 children ˂5 years old). Noroviruses are a major culprit for the closure of hospital wards, and associated with increased hospitalization and mortality among the elderly. Transplant patients have significant risk of acquiring persistent norovirus gastroenteritis. Control and prevention strategies are limited to the use of disinfectants and hand sanitizers, whose efficacy is frequently insufficient. Hence, there is an ample need for antiviral treatment and prophylaxis of norovirus infections. The fact that only a handful of inhibitors of norovirus replication have been reported can largely be attributable to the hampering inability to cultivate human noroviruses in cell culture. The Norwalk replicon-bearing cells and the murine norovirus-infected cell lines are the available models to assess in vitro antiviral activity of compounds. Human noroviruses have been shown to replicate (to some extent) in mice, calves, gnotobiotic pigs, and chimpanzees. Infection of interferon-deficient mice with the murine norovirus results in virus-induced diarrhea. Here we review recent developments in understanding which norovirus proteins or host cell factors may serve as targets for inhibition of viral replication. Given the recent advances, significant progress in the search for antiviral strategies against norovirus infections is expected in the upcoming years.
    Biochemical Pharmacology 05/2014; 91(1). DOI:10.1016/j.bcp.2014.05.021 · 5.01 Impact Factor
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    • "For other strains (i.e. MNV-3) or viruses containing deleterious mutations affecting replication, longer incubation periods may be required for optimal yields (Arias, Bailey et al. 2012). [*Table 1 near here] 7. Freeze cells at -80°C. "
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    ABSTRACT: Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. It is the most common pathogen in biomedical research colonies. MNV is also related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Like the human noroviruses, MNV is an enteric virus that replicates in the intestine and is transmitted by the fecal-oral route. MNV replicates in murine macrophages and dendritic cells in cells in culture and in the murine host. This virus is often used to study mechanisms in norovirus biology, because human noroviruses are refractory to growth in cell culture. MNV combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Herein, we describe a panel of techniques that are commonly used to study MNV biology. Curr. Protoc. Microbiol 33:15K.2.1-15K.2.61. © 2014 by John Wiley & Sons, Inc.
    Current protocols in microbiology 05/2014; 33:15K.2.1-15K.2.61. DOI:10.1002/9780471729259.mc15k02s33
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