Murine norovirus (MNV-1) exposure in vitro to the purine nucleoside analog Ribavirin increases quasispecies diversity.

Ribavirin is a pharmaceutical antiviral used for the treatment of RNA virus infections including norovirus, hepatitis C virus, hepatitis E virus, Lassa virus, respiratory syncytial virus, and rhinovirus. Despite the drug's history and documented efficacy, the antiviral mechanism of Ribavirin remains unclear. Mechanisms proposed include depletion of the intracellular GTP pool, immunomodulatory effects, induction of error catastrophe, inhibition of viral polymerase activity, and/or inhibition of viral capping. In the present study, we leveraged deep sequencing data to demonstrate that Ribavirin increases murine norovirus (MNV-1) viral diversity. By serial passaging MNV-1 in RAW 264.7 cells for twenty generations in the presence of Ribavirin, we demonstrated statistically significant increases in both the number of unique haplotypes and the average pairwise difference (APD). Based on statistically significant differences in the probability of nucleotide mutations based on Roche 454 sequencing, we also demonstrated that single nucleotide substitutions are increased in the presence of Ribavirin. Finally, we demonstrated Ribavirin's impact on statistically significantly reducing the relative proportion of the dominant sequence within the quasispecies.

Newly isolated mAbs broaden the neutralizing epitope in murine norovirus.

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 C'D' 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.

Murine norovirus: propagation, quantification, and genetic manipulation.

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.

Flexibility in surface-exposed loops in a virus capsid mediates escape from antibody neutralization.

UNLABELLED: 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. IMPORTANCE: Human 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.

Diversity and relationships of cocirculating modern human rotaviruses revealed using large-scale comparative genomics.

Group A rotaviruses (RVs) are 11-segmented, double-stranded RNA viruses and are primary causes of gastroenteritis in young children. Despite their medical relevance, the genetic diversity of modern human RVs is poorly understood, and the impact of vaccine use on circulating strains remains unknown. In this study, we report the complete genome sequence analysis of 58 RVs isolated from children with severe diarrhea and/or vomiting at Vanderbilt University Medical Center (VUMC) in Nashville, TN, during the years spanning community vaccine implementation (2005 to 2009). The RVs analyzed include 36 G1P[8], 18 G3P[8], and 4 G12P[8] Wa-like genogroup 1 strains with VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5/6 genotype constellations of I1-R1-C1-M1-A1-N1-T1-E1-H1. By constructing phylogenetic trees, we identified 2 to 5 subgenotype alleles for each gene. The results show evidence of intragenogroup gene reassortment among the cocirculating strains. However, several isolates from different seasons maintained identical allele constellations, consistent with the notion that certain RV clades persisted in the community. By comparing the genes of VUMC RVs to those of other archival and contemporary RV strains for which sequences are available, we defined phylogenetic lineages and verified that the diversity of the strains analyzed in this study reflects that seen in other regions of the world. Importantly, the VP4 and VP7 proteins encoded by VUMC RVs and other contemporary strains show amino acid changes in or near neutralization domains, which might reflect antigenic drift of the virus. Thus, this large-scale, comparative genomic study of modern human RVs provides significant insight into how this pathogen evolves during its spread in the community.

Vaccine-derived NSP2 segment in rotaviruses from vaccinated children with gastroenteritis in Nicaragua.

Rotavirus (RV) vaccination programs have been established in several countries using the human-attenuated G1P[8] monovalent vaccine Rotarix (GlaxoSmithKline) and/or the human-bovine reassortant G1, G2, G3, G4, P[8] pentavalent vaccine RotaTeq (Merck). The efficacy of both vaccines is high (∼90%) in developed countries, but can be remarkably lower in developing countries. For example, a vaccine efficacy against severe diarrhea of only 58% was observed in a 2007-2009 Nicaraguan study using RotaTeq. To gain insight into the significant level of vaccine failure in this country, we sequenced the genomes of RVs recovered from vaccinated Nicaraguan children with gastroenteritis. The results revealed that all had genotype specificities typical for human RVs (11 G1P[8], 1 G3P[8]) and that the sequences and antigenic epitopes of the outer capsid proteins (VP4 and VP7) of these viruses were similar to those reported for RVs isolated elsewhere in the world. As expected, nine of the G1P[8] viruses and the single G3P[8] virus had genome constellations typical of human G1P[8] and G3P[8] RVs: G1/3-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. However, two of the G1P[8] viruses had atypical constellations, G1-P[8]-I1-R1-C1-M1-A1-N2-T1-E1-H1, due to the presence of a genotype-2 NSP2 (N2) gene. The sequence of the N2 NSP2 gene was identical to the bovine N2 NSP2 gene of RotaTeq, indicating that the two atypical viruses originated via reassortment of human G1P[8] RVs with RotaTeq viruses. Together, our data suggest that the high level of vaccine failure in Nicaraguan is probably not due to antigenic drift of commonly circulating virus strains nor the emergence of new antigenetically distinct virus strains. Furthermore, our data suggest that the widespread use of the RotaTeq vaccine has led to the introduction of vaccine genes into circulating human RVs.

Complete genome sequence analysis of candidate human rotavirus vaccine strains RV3 and 116E.

Rotaviruses (RVs) cause severe gastroenteritis in infants and young children; yet, several strains have been isolated from newborns showing no signs of clinical illness. Two of these neonatal strains, RV3 (G3P[6]) and 116E (G9P[11]), are currently being developed as live-attenuated vaccines. In this study, we sequenced the eleven-segmented double-stranded RNA genomes of cell culture-adapted RV3 and 116E and compared their genes and protein products to those of other RVs. Using amino acid alignments and structural predictions, we identified residues of RV3 or 116E that may contribute to attenuation or influence vaccine efficacy. We also discovered residues of the VP4 attachment protein that correlate with the capacity of some P[6] strains, including RV3, to infect newborns versus older infants. The results of this study enhance our understanding of the molecular determinants of RV3 and 116E attenuation and are expected to aid in the ongoing development of these vaccine candidates.