海岛地区腹泻病例中诺如病毒基因序列分析

Analysis on gene sequence of Norovirus among diarrhea cases in Zhoushan islands

目的了解海岛地区病毒性腹泻病原中诺如病毒的流行状况,对优势流行株进行基因序列分析。方法对舟山市监测医院疑似病毒性腹泻粪便样本经酶联免疫吸附法(ELISA)筛选轮状病毒,对阴性样本采用逆转录聚合酶链反应(RT-PCR)扩增诺如病毒RNA聚合酶与主要衣壳蛋白基因,PCR扩增产物进行测序反应,序列结果与GenBank已公布的序列进行比对,测其同源性;并绘制成系统发生树进行进化分析。结果在35份诺如病毒扩增阳性样本中挑选病毒载量最高的Hu/Zhejiang/9/2006/CHN株进行测序,并与Hunter株、Farming Hill株等进行比对并绘制系统发生树。Hu/Zhejiang/9/2006/CHN株属于诺如病毒GⅡ-4群,与Hunter株和Lanzhou株形成的分支较接近,RNA聚合酶有部分序列与这2株完全相同。结论舟山市(海岛地区)病毒性腹泻病原中存在诺如GⅡ-4群,采用GDD基序分析有利于更好地发现新的变异株的存在,为病毒性腹泻的病原学和基因学研究提供依据。

Objective To understand the prevalence of Norovirus among viral diarrhea patients in Zhoushan islands and to analyze the gene sequence of dominant strains. Methods 150 stool samples of doubtful viral diarrhea patients from monitoring hospital in Zhoushan city were screened by enzyme linked immunosorbent assay （ELISA）. Gene RdRp and VP1 region of negative samples were amplified with inverse polymeras chain reaction （RT- PCR）. Sequencing reaction for PCR products was carried out and the results were compared with C, enBank to test the homology, phylogenetic trees were then drawn and analyzed. Resdts 25 rotavirus positive samples were screened by ELISA ; while among the remaining 125 negative samples, 35 norovirus positive samples were found. Hu/Zhejiang/9/2OO6/CHN strains had the highest viral load and were then sequenced, and the result showed that it belonged to G Ⅱ -4 group and was close to the branch of Hunter strain and Lanzhou strain. Conclusion There was G Ⅱ -4 group in viral diarrhea pathogens in Zhoushan islands. GDD motif analysis could help to find the new variants and to provide a more reliable basis for viral diarrhea etiology and genomics.