Isolation And Genome Sequence Of Murine Norovirus Strain SH1603 In China And Identification The Human Norovirus Infectious

Objective?1?To isolate murine norovirus?MNV??named SH1603?in China and analyze the genomic sequence.?2?In order to produce infectious human norovirus?HuNV?in vitro,this paper validates the sucess of an infectious clone of human norovirus.Methods?1?Mouse norovirus strain SH1603 was isolated by using RAW264.7 cells from mouse feces.PCR primers were designed according to the reference sequence of foreign published MNV4?DQ223043?.The whole genome was amplified by overlapping reverse transcription PCR and analyzed by bioinformatics software.?2?The infectious clone was transfected into BHK-T7 cells.Western Blotting was used to test the expression of capsid protein VP1.The level of HuNoV genome was detected with real-time PCR technique to identify the infectivity of infectious clone.Results?1?The murine norovirus strain SH1603 caused cytopathic effect in RAW264.7 cells.The genome of murine norovirus SH1603 is about 7238bp,including 4 open reading frames?ORFs?.The predicted encoding protein size of ORF1,ORF2,ORF3 and ORF4,is respectively 186KD,58KD,22KD and 23KD.Between ORF2 and ORF1,there are 14nt nucleotide overlap.Between ORF2 and ORF3,there are 1nt nucleotide overlap.ORF4 is contained in ORF2.By sequence analysis,murine norovirus isolates of SH1603 showed the highest homology in complete sequence with MNV4?DQ223043??94%identity?.Compared with MNV4?DQ223043?,according to the capsid protein amino acid alignment,there are four site mutations,namely aa₂₁₁S?A,aa₃₅₈I?V,aa₄₀₄E?L,aa₅₃₄V?A.?2?After the transfection of the recombinant plasmid into BHK-T7 cells,the slight cytopathic effect was observed.Western blotting detected the expression of capsid protein VP1.The resuLts of real-time PCR indicated that the amplification of HuNV genome along with the time of transfection.Conclusion?1?We isolated a mouse norovirus strain SH1603 from the faeces of mouse.The phylogenetic affinity of mouse norovirus is similar to MNV4.It establishes the foundation for the further study of epidemic pathogenic mechanisms of murine norovirus.?2?In this study,the HuNoV infectious clone was validated.The successfuL establishment of HuNV infectious clone provides an important tool for the study of molecuLar pathophysiology for HuNV.