Human norovirus culture in B cells

Jones, Melissa K.; Grau, Katrina R.; Costantini, Verónica; Kolawole, Abimbola O.; Graaf, Miranda de; Freiden, Pamela; Graves, Christina; Koopmans, Marion; Wallet, Shannon M.; Tibbetts, Scott A.; Schultz‐Cherry, Stacey; Wobus, Christiane E.; Vinjé, Jan; Karst, Stephanie M.

doi:10.1038/nprot.2015.121

Cited by 229 publications

(189 citation statements)

References 26 publications

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“…Available evidence supports this cell tropism during in vivo infections as well (9,(12)(13)(14). HuNoVs also infect cultured B cells (9,15), and available data suggest that they share cell tropism properties with MuNoV in vivo as well (recently reviewed in ref- erence 16). While MuNoV infection of immunocompetent mice fails to cause overt disease, MuNoVs do replicate along the intestinal tract and are spread via fecal-oral transmission, indicating that they share general pathogenic features with HuNoVs.…”

mentioning

confidence: 91%

Regulation of Norovirus Virulence by the VP1 Protruding Domain Correlates with B Cell Infection Efficiency

Zhu

Watanabe

Kirkpatrick

et al. 2016

J Virol

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Human noroviruses are a leading cause of gastroenteritis across the globe, but the pathogenic mechanisms responsible for disease are not well established. The availability of a murine norovirus model system provides the opportunity to elucidate viral and host determinants of virulence in a natural host. For example, previous studies have revealed that the protruding domain of the murine norovirus capsid protein VP1, specifically residue 296 of VP1, regulates virulent infection. We identified a panel of nonsynonymous mutations in the open reading frame 2 (ORF2) gene encoding VP1 that arose in persistently infected mice and tested whether these mutations conferred phenotypic changes to viral replication and virulence. Consistent with previous studies, we demonstrate that a glutamic acid at position 296 results in attenuation. For the first time, we also demonstrate that a lysine at this position is sufficient to confer virulence on an otherwise attenuated murine norovirus strain. Moreover, our studies reveal a direct correlation between the efficiency of viral replication in B cells and virulence. These data are especially striking because mutations causing reduced B cell replication and attenuation had minimal effects on the ability of the virus to replicate in macrophages. Thus, norovirus infection of B cells may directly contribute to disease outcome. IMPORTANCE Human noroviruses are a major global cause of disease, yet we know very little about their pathogenic mechanisms. The availability of a murine norovirus model system facilitates investigation of noroviruses in a natural host organism and the identification of viral and host determinants of pathogenesis. We have identified a panel of mutations arising in the viral capsid protein VP1 during persistent infection of mice. Our data reveal that the protruding domain of VP1 regulates the ability of the virus to replicate in B cells, and this directly correlates with virulence. Importantly, mutations impairing B cell infection had minimal effects on macrophage infection, revealing a potentially critical role for B cell infection in norovirus pathogenesis.H uman noroviruses (HuNoVs) are the major cause of nonbacterial gastroenteritis worldwide, causing rapid bouts of nausea, severe vomiting, and watery diarrhea in all age groups. They are the leading cause of foodborne disease globally and of severe childhood diarrhea in parts of the world where rotavirus vaccination is being implemented (1-3). Although HuNoV infection is typically self-limited in immunocompetent adults, more severe consequences can occur in risk groups, including the elderly, immunocompromised and transplant patients, and infants (4-6). Considering the huge disease burden attributable to HuNoV infections (7) and the tremendous economic loss associated with these infections (8), development of effective therapeutics and vaccines is highly warranted. However, HuNoV studies are greatly hampered by the lack of robust culturing systems and small-animal models. Although there have been exciti...

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confidence: 91%

Regulation of Norovirus Virulence by the VP1 Protruding Domain Correlates with B Cell Infection Efficiency

Zhu

Watanabe

Kirkpatrick

et al. 2016

J Virol

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“…A previous study (Jones et al , 2014) and accompanying protocols article (Jones et al , 2015) demonstrate infection of genogroup II genotype 4 (GII.4) HuNoVs in vitro in the B cell line BJAB in the presence and absence of synthetic H type histo-blood group antigen or Enterobacter cloacae . This infection can be prevented by UV treatment of the virus stock prior to infection.…”

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confidence: 94%

“…However, the genetic diversity between these viruses represents one challenge for HuNoV drug development. Thus, the recently reported successes in propagation of HuNoV in immunodeficient mice (Taube et al , 2013) and in a human B cell line (Jones et al , 2015; Jones et al , 2014) provide exciting tools for the evaluation of potential antiviral strategies and vaccine candidates directed against HuNoV. A number of anti-NoV strategies, including targeting essential viral proteins, have been described in recent years (for a recent review see (Thorne et al , 2016)).…”

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confidence: 99%

“…1B). BJAB cells were infected with unfiltered HuNoV-positive stool containing the indicated genome copy numbers of HuNoV GII.4 Sydney as described (Jones et al , 2015; Jones et al , 2014) without extra addition of synthetic H type histo-blood group antigen or Enterobacter cloacae . After removing the unbound virus inoculum, cells were resuspended in fresh media.…”

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confidence: 99%

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Inhibition of human norovirus by a viral polymerase inhibitor in the B cell culture system and in the mouse model

et al. 2016

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The recently developed human norovirus (HuNoV) B cell culture and mouse models hold promise for drug discovery and development but their suitability for antiviral studies has not been assessed. We demonstrate the inhibitory effect of the nucleoside analogue 2′-C-methylcytidine (2CMC) on HuNoV replication in the human B cell BJAB cell line and in Balb/c Rag/gamma chain-deficient (Rag-γc−/−) mice. These data suggest the applicability of both models for future study and development of antiviral drugs for the treatment of HuNoV infections.

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“…2,3) However, murine norovirus (MNV) can replicate efficiently in the murine macrophage cell line RAW264.7. MNV belongs to the genus Norovirus and shares common biological and molecular properties with human norovirus.…”

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confidence: 99%

Discovery and Synthesis of Heterocyclic Carboxamide Derivatives as Potent Anti-norovirus Agents

Ohba

Oka

Ando

et al. 2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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There is an urgent need for structurally novel anti-norovirus agents. In this study, we describe the synthesis, anti-norovirus activity, and structure-activity relationship (SAR) of a series of heterocyclic carboxamide derivatives. Heterocyclic carboxamide 1 (50% effective concentration (EC 50 ) 37 µM) was identified by our screening campaign using the cytopathic effect reduction assay. Initial SAR studies suggested the importance of halogen substituents on the heterocyclic scaffold and identified 3,5-di-boromo-thiophene derivative 2j (EC 50 24 µM) and 4,6-di-fluoro-benzothiazole derivative 3j (EC 50 5.6 µM) as more potent inhibitors than 1. Moreover, their hybrid compound, 3,5-di-bromo-thiophen-4,6-di-fluoro-benzothiazole 4b, showed the most potent anti-norovirus activity with a EC 50 value of 0.53 µM (70-fold more potent than 1). Further investigation suggested that 4b might inhibit intracellular viral replication or the late stage of viral infection.Key words norovirus; heterocyclic carboxamide; antiviral activity; murine norovirus Human norovirus causes acute nonbacterial gastroenteritis. Although the gastroenteritis symptoms are generally self-limiting, the elderly, infants, and immunocompromised individuals have a higher risk of mortality. Moreover, norovirus has a low infectious dose and spreads very easily from infected persons, contaminated food, water, or a contaminated environment. 1)Noroviruses are single-stranded, positive-sense RNA viruses that belong to the family Caliciviridae and genus Norovirus, and species Norwalk virus. The norovirus genome contains three open reading frames (ORFs): ORF1, ORF2, and ORF3. ORF1 encodes six or seven nonstructural proteins, including RNA-dependent RNA polymerase (RdRp) and 3C-like cysteine protease (3CLpro), responsible for viral replication. ORF2 and ORF3 encode the major and minor structural proteins, respectively. 1)Establishment of an efficient cell culture system for human norovirus is still challenging.2,3) However, murine norovirus (MNV) can replicate efficiently in the murine macrophage cell line RAW264.7. MNV belongs to the genus Norovirus and shares common biological and molecular properties with human norovirus.4,5) Therefore, it is frequently used for identifying antiviral compounds based on the cell culture assay. [6][7][8][9][10][11][12] Several anti-norovirus agents have been reported that target viral attachment/entry, viral protein translation, or viral replication. 7,8) The RdRp inhibitor used as an anti-hepatitis C virus agent 2′-C-methylcytidine (2′-CMC) 9,10) and anti-influenza agent favipiravir 11) also have anti-MNV activity, and the broad spectrum 3CLpro inhibitor, dipeptidyl inhibitor GC376 also inhibits MNV protease activity in vitro.12) Currently, there are no vaccines or drugs for clinical use, and there is an urgent need for new anti-norovirus compounds.We describe here the discovery and synthesis of anti-norovirus thienyl benzothiazolyl carboxamide compounds bearing multiple halogen substituents, and discuss their structure-activity ...

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Human norovirus culture in B cells

Cited by 229 publications

References 26 publications

Regulation of Norovirus Virulence by the VP1 Protruding Domain Correlates with B Cell Infection Efficiency

Regulation of Norovirus Virulence by the VP1 Protruding Domain Correlates with B Cell Infection Efficiency

Inhibition of human norovirus by a viral polymerase inhibitor in the B cell culture system and in the mouse model

Discovery and Synthesis of Heterocyclic Carboxamide Derivatives as Potent Anti-norovirus Agents

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