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.

New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A=B= and E=F= loops. Six mutations in the E=F= loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. IMPORTANCEHuman noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of the antibody structure into the virion/antibody complex identifies two conformations of the antibody binding domain of the viral capsid: one with a superior fit and the other with an inferior fit to the antibody. These data suggest a unique mode of antibody neutralization. In contrast to other viruses that largely escape antibody neutralization through direct disruption of the antibody-virus interface, we identify mutations that acted indirectly by limiting the conformation of the antibody binding loop in the viral capsid and drive the antibody binding domain into the conformation unable to be bound by the antibody.

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Here, we report the isolation and functional characterization of mAbs against two murine norovirus (MNV) strains, MNV-1 and WU20, which were isolated following oral infection of mice. The mAbs were screened for reactivity against the respective homologous and heterologous MNV strain by ELISA. Selected mAbs were of IgA, IgG1, IgG2a or IgG2b isotype and showed a range of Western blot reactivities from non-binding to strong binding, suggesting recognition of conformational and linear epitopes. Some of the anti-MNV-1 antibodies neutralized both MNV-1 and WU20 infections in culture and in mice, but none of the anti-WU20 mAbs neutralized either virus. The non-neutralizing anti-MNV-1 IgG2b antibody 5C4.10 was mapped to the S domain of the MNV-1 capsid, whilst the epitopes of the neutralizing anti-MNV-1 IgA antibodies 2D3.7 and 4F9.4 were mapped to the P domain. Generation of neutralization escape viruses showed that two mutations (V339I and D348E) in the C9D9 loop of the MNV-1 P domain mediated escape from mAb 2D3.7 and 4F9.4 neutralization. These findings broaden the known neutralizing epitopes of MNV to the main surface-exposed loops of the P domain. In addition, the current panel of antibodies provides valuable reagents for studying norovirus biology and development of diagnostic tools.