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目的分离我国鼠诺如病毒(murine norovirus,MNV)(命名SH1603),并进行基因组序列测定和分析。方法利用RAW264.7细胞分离MNV SH1603,参考国外已发表的MNV4(DQ223043)基因组序列设计引物,经重叠的反转录PCR扩增全基因组,利用生物信息学软件对全基因组序列进行分析。结果 MNV SH1603引起RAW264.7细胞病变,其基因组全长7 238个核苷酸,包括4个开放阅读框(open reading frames,ORF),预测的ORF1、ORF2、ORF3、ORF4编码蛋白大小分别为186 000、58 000、22 000和23 000,其中ORF1和ORF2之间有14个核苷酸重叠,ORF2和ORF3之间有1个核苷酸重叠,ORF4包含在ORF2中。NCBI Blast比对发现,SH1603基因组与MNV4同源性最高(94%)。衣壳蛋白区的进化分析发现,SH1603与MNV4(DQ223043)和Berlin0606(EF531291)亲缘关系最近。衣壳蛋白区氨基酸比对发现,SH1603与MNV4比较,有4个位点突变,分别为aa211S→A、aa358I→V、aa404E→L、aa534V→A。结论已分离到1株MNV,与国外报道的MNV4具有高度的进化亲源关系,为我国进一步研究MNV流行和致病机制奠定了基础。
Objective To isolate Murine norovirus (MNV) (named SH1603) in our country and analyze the genomic sequences. Methods MNV SH1603 cells were isolated from RAW264.7 cells. The primers were designed according to the published sequence of MNV4 (DQ223043) in foreign countries. The whole genome was amplified by overlapped reverse transcription PCR and the whole genome sequence was analyzed by bioinformatics software. Results MNV SH1603 caused the pathological changes of RAW264.7 cells. The genome of the MNV SH1603 strain was 7 238 nucleotides in length, including 4 open reading frames (ORFs). The predicted ORF1, ORF2, ORF3 and ORF4 protein sizes were 186 000, 58 000, 22 000 and 23 000 with 14 nucleotide overlap between ORF1 and ORF2, 1 nucleotide overlap between ORF2 and ORF3, and ORF4 contained in ORF2. The NCBI Blast alignment revealed that the SH1603 genome has the highest homology to MNV4 (94%). The evolutionary analysis of the capsid protein region found that SH1603 has the closest genetic relationship with MNV4 (DQ223043) and Berlin0606 (EF531291). Amino acid alignment of the capsid protein region showed that there were 4 mutations in SH1603 and MNV4, which were aa211S → A, aa358I → V, aa404E → L and aa534V → A, respectively. Conclusion A MNV has been isolated and has a high degree of phylogenetic relationship with MNV4 reported in foreign countries, which laid the foundation for further study on the prevalence and pathogenesis of MNV in China.