Infection Of Murine Norovirus And Expression Of Isolated Viruses In Shanghai Area

Murine norovirus (MNV) is the member of Calicivirus family, which is highly natural infected in mice, and it was originally discovered in immunodeficient mice. After infected with MNV, there is usually no obvious symptoms in the immunocompetent mice, but it can cause lethal infection in immunodeficient mice. MNV has genetic and antigenic diversity, and prone to mutation.So far, the reports about MNV is still relatively limited, its pathogenicity and impact on experimental studies is not entirely clear, and there is no definite infection investigation.In order to understand the natural infected situation of MNV in laboratory mice in Shanghai aera, we chose 319 SPF mice from commissioned testing units. Cecal contents and serum samples were collected. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect cecal contents by amplification of specific gene fragments in MNV. At the same time, ELISA (Enzyme linked immunosorbent assay, ELISA) were used to compare with nucleic acid detection methods. MNV can replicate in the mouse macrophage cell line RAW264.7 cells and produce cytopathic effect (CPE).Take advantage of this feature, the positive stool samples were diluted and filtered through 0.22μm membrane, inoculated into RAW264.7 cells, and then indentified by RT-PCR. The RNA geneome of MNV was divided into four open reading frames, and ORF2 encode the capsid protein VP1. Currently, many studies on MNV concerned more with VP1, based on the sequence of Guangzhou strain we designed a pair of primers for ORF2 to amplify the target fragment by RT-PCR.Then through cloning and prokaryotic expression, to observe whether produce proteins.There are 95 positive samples in 319 cecal contents by RT-PCR, and the positive rate is 29.78%. Among 180 serum samples,70 were positive by ELISA, and the povitive rate is 38.89%. After 5 times blind passage,RAW264.7 cells which were inoculated with positive virus performed obvious CPE in 72 hours. After 3 times of freezing and thawing, RT-PCR obtained a 187bp band, and sequencing results consistent with the sample source.ORF2 fragment was amplified by RT-PCR, and the sequence has 1626bp. Use the NCBI BLAST analysis software, the homology is above 87% with other MNV strains. In order to obtaine a lot of stable ORF2, it was cloned into pMD-18T vector. The plasmid and expression vector were connected after double digestion. The recombinant plasmid pET28a-ORF2 transformed into E. coli BL21 (DE3). After IPTG induction, nickel agarose affinity chromatography and anion exchange chromatography, obtained a fusion protein by SDS-PAGE electrophoresis, which about 60 KD.The results from the present study show that there is a high natural infection rate of MNV in laboratory mice in Shanghai aera. We also isolate the MNV by cell culture and get the VP1 through prokaryotic expression. As important expermental animals, we must strictly control the quality of mice, and strengthen management of laboratory mice,in order to avoid interference the results of animal experments because of its inherent pathogens.