两株小鼠诺如病毒的分离与鉴定

摘要: 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pEASY-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( GenBank 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。

Isolation and Identification of Two Murine Norovirus Strains

Abstract: 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 macrophage cell line ＲAW264. 7 to isolate MNV from the rectal contents of the infected mice． Fragments of VP1genes from MNV isolates were amplified by ＲT-PCＲ and then ligated to pEASY-Blunt Simple CloningVector and cloned to Trans1-T1 chemically competent cells． 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 cells 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 VP1specific primers，we found the specific bands appeared in the CPE cells 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 S7-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．