Development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase PCR assay to detect murine norovirus 1 infection in mice

Abstract

Murine norovirus 1 (MNV-1) is a newly recognized pathogen of mice that causes lethal infection in mice deficient in components of the innate immune response but not in wild-type 129 mice. In this study, in vitro-propagated MNV-1 was used as antigen to develop a multiplexed fluorescent immunoassay (MFI) to detect antibodies to MNV-1 in infected mice. The MNV-1 MFI was 100% specific and 100% sensitive in detecting anti-MNV-1 antibody in sera from experimentally infected mice. Testing of a large number of mouse serum samples (n = 12,639) submitted from contemporary laboratory mouse colonies in the United States and Canada revealed that 22.1% of these sera contained antibodies to MNV-1, indicating infection with MNV-1 is widespread in research mice. In addition, a reverse transcriptase PCR primer pair with a sensitivity of 25 virus copies was developed and used to demonstrate that MNV-1 RNA could be detected in the spleen, mesenteric lymph node, and jejunum from some experimentally infected mice 5 weeks postinoculation. These diagnostic assays provide the necessary tools to define the MNV-1 infection status of research mice and to aid in the establishment of laboratory mouse colonies free of MNV-1 infection.

FIG. 1.

Transmission electron micrograph of negatively stained murine norovirus 1 particles (arrows) from cesium chloride-purified supernatants of infected RAW 264.7 cells grown in suspension culture.

FIG. 2.

The mean MNV-1 MFI fluorescence values of sera collected weekly from mice experimentally inoculated with MNV-1 by oral gavage that seroconverted (n = 7) and sham-inoculated control mice (n = 5). Mice inoculated with MNV-1 showed a steady increase in MFI fluorescence values over time. An MFI fluorescence value of 175 corresponds to a 95% test sensitivity, while an MFI fluorescence value of 600 corresponds to a 95% test specificity (dashed lines). These fluorescence values were used as thresholds so that MFI fluorescence values <175 were considered negative for anti-MNV-1 antibody, values from 175 to 600 were considered intermediate and possibly containing anti-MNV-1 antibody, and values >600 were considered positive for anti-MNV-1 antibody. Error bars represent the standard errors of the mean.

FIG. 3.

Western blot analysis of serum samples from mice 5 weeks after oral inoculation with MNV-1-infected lysates (lanes 6 to 15) or control mice sham inoculated with uninfected RAW 264.7 lysates (lanes 1 to 5). Samples containing anti-MNV-1 antibody (lanes 6 to 12) displayed a band approximately 59 kDa in size (arrow) representing the capsid protein. Control mice and the three mice that did not seroconvert after oral inoculation with MNV-1 (lanes 13 to 15) did not display any specific bands by Western blot analysis.

FIG. 4.

Ethidium bromide-stained agarose gel of MNV-1 RT-PCR products of log-fold serial dilutions of RNA extracted from cesium chloride-purified MNV-1-infected cell cultures. A 318-bp PCR product was generated with as little as 100 ag of RNA, representative of approximately 25 virus copies based on the estimated viral genome molecular mass of 2,366,090 Da per virion.