小鼠诺如病毒分离鉴定及病毒衣壳蛋白基因序列分析

目的了解广东地区小鼠诺如病毒（murine norovirus,MNV）的分子遗传特征和进化来源。方法采用小鼠巨噬细胞系RAW264.7细胞对RT-PCR检测为阳性的小鼠样本进行病毒分离,通过细胞病变、RT-PCR、间接免疫荧光试验、测序方法对病毒分离株进行鉴定。应用RT-PCR技术针对15株MNV分离株的VP1基因的1626个核苷酸片段进行基因扩增,将扩增产物连接在pMD18-T载体后转化到大肠杆菌中进行克隆。通过氨苄青霉素平皿筛选,将鉴定为阳性的克隆菌进行核苷酸序列测定及序列分析。将这15株MNV分离株与从GenBank获得的19株MNV参考株进行序列比较分析,基于VP1基因的1626核苷酸片段构建系统发生进化树,一起进行分子流行病学研究。结果从80个小鼠样本中分离到了15株MNV病毒,通过细胞病变试验、RT-PCR试验、间接免疫荧光试验和测序分析鉴定确认分离到的病毒为MNV。序列分析结果显示MNV分离株的VP1蛋白基因全长均为1626个核苷酸,广东地区15株MNV分离株的核苷酸和氨基酸同源性分别在89.7%~100%和94.8%~100%之间,15株MNV分离株与其他19株MNV参考毒株核苷酸和氨基酸同源性分别在87.5%~92.9%和92.4%~98.2%之间。进化树分析表明来自设施A和设施D的13株病毒之间的亲缘关系较近,同属一个进化分支。来自设施B的ZD-1毒株和设施C的ZYY-163毒株与来自广东（K162）、日本（S7-P2、S7-PP3）、韩国（K4）和德国（Berlin/04/06/DE、Berlin/05/06/DE）同属另一个进化分支。结论成功分离到15株MNV病毒。遗传进化分析表明广东地区的MNV分离株来源并不相同,来自设施B和设施C的MNV分离株与国外分离株的亲缘关系较近,而来自设施A和设施D的13株MNV分离株可能是本地固有的毒株。

Isolation and identification of murine norovirus and genetic analysis of its capsid protein gene

Objective To isolate and identify murine norovirus（ MNV） from the infected mice breeding in four facilities in Guangdong Province. To elucidate molecular genetic features and evolutionary origin of the MNV isolates by analysing nucleotide sequence of MNV capsid protein（ VP1） gene. Methods Mouse macrophage cell line RAW264. 7 was used to isolate virus from the cecal contents of the infected mice tested by RT-PCR. Cytopathic effect（ CPE） assay,RTPCR technology,indirect immunofluorescence assay and nucleotide sequencing were performed to identify the MNV isolates. Fragments of 1626 nucleotides including VP1 genes from 15 MNV isolates were amplified by RT-PCR. The PCR products were ligated to pMD18-T vector and cloned to E. coli DH5α cells. By ampicillin plate selection,the positive clones were sequenced and analyzed. Based on the 1626 nucleotides of VP1 gene,the phylogenetic analysis was processed with 19 MNV strains downloaded from GenBank and 15 MNV isolates from Guangdong Province. Results 15 MNV isolates were obtained from 80 clinical samples. Cytopathic effect（ CPE） was observed by microscopy,the isolates were identified as MNV by RT-PCR assay,indirect immunofluorescence assay and nucleotide sequencing. The length of the VP1 gene was1628 nucleotides. 15 MNV isolates showed a nucleotide and amino acid similarity of 89. 7% to 100% and 94. 8% to100%,respectively. The homology of nucleotide and amino acid between 15 isolates and other MNV isolates which were registered in GenBank database were 87. 5% to 92. 9% and 92. 4% to 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 the same evolutional branch. The strains ZD-1（ isolated from mice of Facility B） and ZYY-163（ isolated from mice of Facility C） belonged to another evolutional branch,including also K162,S7-P2,S7-PP3,K4,Berlin /04 /06 / DE and Berlin /05 /06 / DE. Conclusions 15 MNV isolates are successfully isolated and identified. Evolutionary origin of the15 MNV isolates from Guangdong region are different. Two MNV isolates from Facility B and C show a close genetic relationship with the foreign MNV isolates,but 13 MNV isolates from Facility A and D are likely to be local strains already existing in Guangdong Province.