Preliminary study on the infection of mouse Norovirus and its effect on immune function in Chongqing

Abstract:

Objective：To establish a real-time fluorescent quantitative RT-PCR method to survey murine Norovirus（MNV） infection in laboratory mice in Chongqing，and explore the effect on immune function. Methods：①A pair of specific primers were designed targeting MNV GenBank genomic sequence，the specificity，sensitivity，repeatability and stability of the assay were evaluated，and comparison test of 123 clinical samples were carried out with two laboratory animal testing institutions to establish reliable real-time RT-PCR assay for MNV detection. ②The detection of MNV in different intestinal excretions（caecum contents，fresh feces，and excreted feces for 24 hours） was compared by real-time quan－titative RT-PCR. ③Real-time quantitative RT-PCR was used to investigate the infection of MNV in laboratory mice in Chongqing. ④Six to eight week-old BABL/c-nu mice were randomly di－vided into 3 groups（n=3 for each group）：control，MNV natural infection for 30 d，MNV infection for 60 d. Serum，liver lung，spleen，and colon were collected，the pathological changes of various organs were observed by HE staining；and tumor necrosis factor alpha（TNF-α） and interferon gamma（IFN-γ） levels in serum，tissue homogenates of spleen and colon were detected by enzyme linked immunosorbent assay（ELISA）. Results：①To established real-time RT-PCR assay could specifically detect MNV and had no cross reactions with mouse other virus. The sensitivity and repeatability were also excellent and the coincidence degree of comparison test in 123 clinical samples is as high as 98%. ②The detection results of MNV in fresh feces and caecum contents were completely consistent，excrement for 24 h in vitro can also accurately reflect MNV infection of mice in the cage. ③MNV infection rate in Chongqing is 65% to 85%，and no strain differences（68.3% vs. 70.6%， χ2=0.116，P=0.733），but genetically modified mice were more susceptible to MNV than normal mice（78% vs. 64%， χ2=6.434，P=0.011）. ④Compared with Control group，BABL/c-nu mice showed that inflam－matory cell infiltration and the obvious pathological changes in liver，lung，spleen and colon after natural infection with MNV，and the levels of TNF-α，IFN-γ in colon were significantly increased（F=21.682，P=0.002；F=77.223，P=0.000，respectively）. Conclusion：The established real-time fluorescent quantitative RT-PCR method is reliable and can monitor the infection of MNV through the stool samples of mice，MNV infection rate of mice in Chongqing is high and the infection in BABL/c-nu mice has interfered with the exper－imental results. Therefore，it is necessary to enhance the animal breeding management to reduce the risk of MNV infection in mice.