两株小鼠诺如病毒的分离与鉴定

Isolation and Identification of Two Murine Norovirus Strains

目的了解江苏地区常用实验小鼠诺如病毒(murine norovirus,MNV)的感染情况及进化特征。方法在江苏地区抽取三个实验动物中心的实验小鼠,采集直肠及内容物样本,应用RT-PCR方法检测MNV ORF2特异性基因(547bp,MNV nt5104-5650)。阳性样本经RAW264.7细胞分离病毒,对MNV分离株的VP1基因进行扩增,将其连接在p EASY-Blunt载体上,转化至大肠杆菌Trans1-T1感受态细胞,通过Kanamycin抗性筛选,挑取菌落进行PCR鉴定,鉴定为阳性的克隆送检测定其VP1基因序列,构建系统发生进化树。结果共检测小鼠直肠内容物30份,检出MNV阳性样本4份,包括ICR小鼠(2/8)、BALB/c小鼠(1/12)和C57BL/6J小鼠(1/10),感染率为13.3%;RAW264.7细胞接毒盲传三代,两只ICR小鼠样品接种的细胞产生明显细胞病变(CPE),表现为细胞圆缩聚集,大部分脱落死亡,而C57BL/6J和BALB/c小鼠样本接种RAW264.7细胞无CPE;应用VP1特异引物经RT-PCR检测,表现CPE的细胞样本出现特异条带,测序结果显示分离到的2株MNVVP1核酸序列均为1626bp,与引物设计参考毒株S7-P2(Gen Bank登录号:AB435514)VP1基因大小相同,进化树图表显示检测到的2株MNV属同一独立小分支与参考株BJ10-2062、CR4最为相近。结论此次抽检的实验小鼠MNV感染率为13.3%(4/30),考虑可能在同一设施内传播。常用实验小鼠携带病毒的生物学意义有待进一步研究。

Objective The purpose of the study is to investigate the prevalence of murine norovirus （MNV） in Jiangsu province and analyze their genetic molecular characterization. Method Experimental mice were extracted from three experimental animal centers in Jiangsu area. Rectal samples from the experimental mice were tested for partial ORF2 region （547bp, MNV nt 5104-5650） of MNV using RT-PCR method. We used mouse maerophage cell line RAW264.7 to isolate MNV from the rectal contents of the infected mice. Fragments of VPlgenes from MNV isolates were amplified by RT-PCR and then ligated to pEASY-Blunt Simple CloningVector and cloned to Transl-T1 chemically competent ceils. By kanamycin lysogeny broth plate selection, the positive clones were sequenced and analyzed. Based on the VP1 gene, thephylogenetic analysis tree was created. Result 4 positive MNV isolates were obtained from 30 mice rectal samples, including ICR mice （2/8）, BALB/c mice （1/12） and C57BL/6Jmice （ 1/10）, infection rate was 13%. On infection, the RAW264.7 cell line inoculated with two ICR mice sample showed obvious cytopathic effects （CPE） after three genetations, as infected ceils became rounded and shrunken, with ultimate disintegration of the cell sheet. The RAW264.7 cell line inoculated with BALB/c and C57BL/6J mice samples did not exhibit CPE. Used VPlspecific primers, we found the specific bands appeared in the CPE ceils sample of two ICR mice by RT-PCR method. The sequencing results showed both of the complete VP1 gene of two MNV strains was 1626bp, which was the same length as that of the reference strain $7-P2 （GenBank accession number： AB435514） VP1 gene. Phylogenetic analysis showed that the two MNVs of this study were clustered into one group which was mostly related withBJ10-2062 and CR4. Conclusion The MNV infection rate was 13.3% （4/30） in the experimental mice of Jiangsu area. May be MNV transmitted within the breeding facilities. We need to further explore the biological significance of the experimental mice carrying MNV.