Diversity of murine norovirus strains isolated from asymptomatic mice of different genetic backgrounds within a single U.S. research institute

Abstract

Antibody prevalence studies in laboratory mice indicate that murine norovirus (MNV) infections are common, but the natural history of these viruses has not been fully established. This study examined the extent of genetic diversity of murine noroviruses isolated from healthy laboratory mice housed in multiple animal facilities within a single, large research institute- the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (NIAID-NIH) in Bethesda, Maryland, U.S. Ten distinct murine norovirus strains were isolated from various tissues and feces of asymptomatic wild type sentinel mice as well as asymptomatic immunodeficient (RAG 2(-/-)) mice. The NIH MNV isolates showed little cytopathic effect in permissive RAW264.7 cells in early passages, but all isolates examined could be adapted to efficient growth in cell culture by serial passage. The viruses, although closely related in genome sequence, were distinguishable from each other according to facility location, likely due to the introduction of new viruses into each facility from separate sources or vendors at different times. Our study indicates that the murine noroviruses are widespread in these animal facilities, despite rigorous guidelines for animal care and maintenance.

Figure 1. Sequence analysis of NIH MNV strains.

Genome organization of prototype strain MNV-1 and ORF1 polyprotein cleavage sites in comparison with the predicted cleavage sites in the ORF1 polyprotein of the NIH murine norovirus strains. Dipeptide recognition sites that differ from MNV-1 are indicated in bold type.

Figure 2. Phylogenetic analysis of NIH MNV viral genomes in comparison with representative viruses previously published.

Phylogenetic relationships were inferred using Neighbor-joining method, with the evolutionary distances computed by the Tamura-Nei method. The statistical support for tree nodes was evaluated by bootstrap analysis (500 replicates) and values higher than 65 are shown above the corresponding branches. The strains in this analysis and their GenBank accession numbers are shown in the Supplemental Table (Table S1).

Figure 3. Serial passage of MNV NIH-2409 in RAW264.7 cells.

A. Immunofluorescence assay of RAW264.7 cells inoculated directly with a lymph node homogenate from mouse. Medium was removed after 7 days, cells were fixed with methanol, and viral antigen expression was detected with anti-MNV-1 VP1 serum. Representative positive MNV antigen expression is indicated by white arrows. B. Mock-infected RAW264.7 cells incubated with same serum. C. First plaque purification of NIH MNV-2409 following P3 in RAW264.7 cells as described in Material and Methods. D. Plaque morphology after two additional plaque purifications in RAW264.7 cells (3XPP virus stock).