广东省中山市登革Ⅰ型病毒的分离鉴定及E/NS1基因序列分析

Isolation and Identification of Dengue Virus I from Zhongshan City and Sequence Analysis of E/NS1 Gene

目的分离鉴定中山市2007年及2012年流行的登革病毒，从分子水平分析其地域来源和系统进化情况。方法用C6／36细胞分离10份2007年登革病毒特异性IgM抗体阳性血清和4份2012年登革病毒核酸阳性血清中的病毒，采用Realtime—PCR对毒株进行血清型鉴定，RT—PCR扩增毒株E／NSl基因并进行序列测定，分析其与不同区域流行株和国际标准株的同源性和系统进化情况。结果共分离鉴定到6株登革I型病毒，其中2株为2007年流行株，4株为2012年流行株，分别命名为2007ZSDenguel-2和2012ZSDenguel-4。2012ZSDenguel-2与柬埔寨、越南等东南亚国家近年流行的登革I型毒株相似性高（大于99．0％），进化距离最短（0．011～0．012）。2012ZSDengue3—4，2007Z—SDenguel-2与广东省珠海市2007年流行和新加坡、日本、泰国等亚洲国家近年流行的登革I型毒株相似性高（大于99．0％），进化距离最短（0．007～0．024）。中山流行株与登革I型国际标准株、非洲和美洲流行株相似性在90．0％～94．0％之间，进化距离较远（0．065--0．108）；与登革Ⅱ、Ⅲ、Ⅳ型国际标准株NGC、I-187、I-t241相似性约为68．0％、66．0％、71．0％，进化距离最远（0．369--0．505）。结论中山市2007年及2012年流行株为登革I型病毒，可能属于东南亚国家输入性毒株，但输入源头有所不同。

Objective To isolate and identify the epidemic strains of dengue virus in Zhongshan city in 2007 and 2012, and analyze their origins and phylogeny at the molecular level. Methods Dengue viruses from the sera of 10 patients with pos- itive dengue virus specific IgM antibody in 2007 and of 4 patients with positive dengue virus nucleic acid in 2012 were isolated with C6/36 cells. The serotypes were identified by real time - PCR. E/NS1 genes were amplified by RT - PCR and sequenced. The homology of the epidemic strains in Zhongshan city with those from different regions and with the international standard strains were compared. Their phylogeny was analyzed. Results Six dengue virus strains isolated were all identified as dengue virus I, among which 2 strains were from 2007 and named 2007ZSDenguel - 2 and 4 from 2012 and named 2012ZSDenguel - 4. The sequence similarities of the 2012ZSDenguei - 2 with the dengue virus I strains prevalent in Cambodia and Vietnam in recent years were higher than 99.0%, and the evolutionary distances were the shortest （0.011-0.012）. The sequence similarities of the 2012ZSDengue3- 4, 2007ZSDenguel- 2 with the dengue virus I strains prevalent in Zhuhai in 2007 and in Singapore, Ja- pan, Thailand in recent years were higher than 99.0 96, and the evolutionary distances were the shortest （0. 007 - 0. 024）. The sequence similarities of the strains in Zhongshan city with the dengue virus I international standard strains, the prevalent strains in Africa and America were between 90.0% - 94.0%, and the evolutionary distances were far （0. 065 - 0.＇108）. The sequence similarities with dengue virusⅡ、Ⅲ、Ⅳ international standard strains NGC, H87, H241 were about 68.0%, 66.0% and 71.0 %, and the evolutionary distances were the farthest （0. 369 - 0. 505）. Conclusions The epidemic strains of dengue virus in Zhongshan city in 2007 and 2012 are dengue virus type I. It could be imported strains from Southeast Asia but have different origins.