Isolation and Identification of Murine Norovirus Virus and Genetic Analysis of the Capsid Protein Gene(VP1)of The Virus
Received:June 04, 2013  Revised:July 19, 2013
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KeyWord:Mouse norovirus (MNV); virus isolation and identification ; Genetic analysis
袁文 广东省实验动物监测所
张谱华 广东省实验动物监测所
王静 广东省实验动物监测所
刘香梅 广东省实验动物监测所
赵维波 广东省实验动物监测所
张钰 广东省实验动物监测所
黄韧 广东省实验动物监测所
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      Objective To isolate and identify Murine Norovirus Virus (MNV) from the infected mice breeding in four facilities in Guangdong Province. To illuminate molecular genetic features and evolutionary origin of MNV isolates by analysing nucleotide sequence of MNV capsid protein (VP1) gene. Methods RAW264.7 cell line was used to virus isolation from the cecal contents of the infected mice tested by RT-PCR. Cytopathic effect (CPE) assay, RT-PCR technology, indirect immunofluorescence assay and nucleotide sequencing was used to identified the MNV isolates. Fragments of 1626 nucleotides including VP1 genes from 15 MNV isolates were amplified by RT-PCR, the PCR products were ligated into pMD18-T vector and cloned to DH5α cell. By ampicillin plate selection, the positive clones were sequenced and analyzed. Based on the 1626 nucleotides of VP1 gene, the phylogenetic analyses were processed with 19 MNV strains down loaded from GenBank and 15 MNV isolates from Guangdong Province. Results 15 MNV isolates were isolated from 80 clinical samples. Cytopathic effect (CPE) was observed by microscopy, the isolates was identified as MNV by RT-PCR technology, indirect immunofluorescence assay and nucleotide sequencing. The length of the VP1 gene was 1628 nucleotides. 15 MNV isolates showed a nucleotide and amino acid similarity of 89.7~100% and 94.8~100% respectively. The homology of nucleotide and amino acid between 15 isolates and other MNV isolates which were registed in GenBank database were 87.5~92.9% and 92.4~98.2% respectively. Phylogenetic analysis showed that there was a close genetic relationship between strains isolated from mice of Facility A and Facility D, all the 13 isolates belonged to a the same evolutional branch. The strains ZD-1(isolated from mice of from Facility B )and strains ZYY-163(isolated from mice of from Facility C)belonged to an another evolutional branch, this branch also included K162, S7-P2, S7-PP3, K4, Berlin/04/06/DE and Berlin/05/06/DE. Conclusion 15 MNV isolates were isolated and identified successfully. Evolutionary origin of 15 MNV isolates from Guangdong region were different. Two MNV isolates from Facility B and Facility C showed a close genetic relationship with the foreign MNV isolates, but 13 MNV isolates from Facility A and Facility D are likely to be the local strains already existing in Guangdong Province.
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