医学检验信息网 检验医学 2007-2-26 16:16:49

关键词: 流感病毒，H3N2亚型；基因，病毒 【摘要】 目的 了解近几年流感病毒在深圳地区活动的特点及甲3(H3N2)亚型毒株HA1基因演变概况。方法 病毒分离采用常规的鸡胚双腔接种，毒株检定用常量半加敏HI测定。新鲜收获含病毒粒的鸡胚尿囊液用来提取RNA，经逆转录合成cDNA，经聚合酶链反应(PCR)扩增，产物纯化，采用双脱氧链末端终止法进行核苷酸序列测定。结果 近几年来深圳地区流感活动概况与全国情况相一致：在人群中仍同时流行H3N2，H1N1亚型和乙型毒株，当甲型毒株活动减弱时，乙型毒株活动就增强，反之，甲型毒株增强时，乙型毒株就减弱。随着时间的推移，H3N2亚型毒株HA1基因不断地发生点突变，这种突变严重受人群免疫压力所影响，1996年的毒株与1995的毒株相比，不仅氨基酸替换点中多数是位于抗原决定簇区或受体结合部位上，并增加两个糖基化位点，故导致H3N2毒株於1996年活动明显增强。结论 近来在深圳地区人群中仍同时流行着H3N2，H1N1亚型和乙型流感病毒。然而，不同年其优势毒株是不一样的。1996年H3N2毒株活动增强是由于其HA1区氨基酸序列发生替换所造成。 The feature of influenza virus activity and genetic characterization of HA1 gene of influenza A H3N2 viruses isolated from 1994～1997 in Shenzhen

CHENG Xiaowen, LI Liangcheng, HE Jianfan.

(Municipal Hygiene and Anticepidemic Station of Shenzhen, Shenzhen 580020)

【Abstract】 Objective To understand the feature of influenza virus activity and genetic characterization of HA1 gene of influenza A H3N2 viruses isolated in recently years in Shenzhen.Methods The specimens were inoculated into amniotic and allantoic cavities of the embryonated chicken eggs for viral isolation. The isolates were typed with HI test. Virion RNA extracted from allantoic fluid by Rneasy RNA extraction kit was used for RT-PCR amplification. Afterward, the product of PCR was purified with purification kit. RNA sequence analysis was performed by Sanger dideoxy-mediated nucleotide chain termination method.Results Influenza A H3N2, H1N1 and B viruses were cocirculating in men in Shenzhen in recent years. However, the extent of their activities was not the same in a single year. The influenza B virus was the predominant strain in 1994 and 1997, but the predominant strain was H3N2 virus in 1996. The genetic analysis showed that the substitution of amino acids occured year by year and was mainly located at antigenic determinants or receptor binding site on HA1 domain of H3N2 viruses.Conclusion Influenza A H3N2, H1N1 and B viruses were still co-circulating in men in Shenzhen in recent years, the predominant strain of influenza virus was different in different years. An increase of influenza A H3N2 virus activity in 1996 was due to the substitution of amino acids sequences on viral HA1 domain. 【Key words】 Influenza A(H3N2) virus Gene, virus 我们对1994年以来流感病毒在深圳地区人群中活动概况进行分析，从分子生物学角度了解近几年来甲3(H3N2)亚型毒株HA1基因演变概况，尤其想弄清它于1996年活动明显加强的分子生物学基础。 1 材料和方法 1.1 标本采集及处理 1994～1997年，每周从深圳市某一监测医院采集类流感患者咽拭子5～10份，按常规加双抗处理，然后，按常规每份标本双腔接种9～10日龄鸡胚3只，35℃孵育72 h，收获，检查血凝活性。 1.2 分离物鉴定 凡所收获的羊水或尿液红细胞凝集阳性的滴定其HA滴度，然后用红细胞凝集抑制法(HI)进行初步鉴定(鉴定血清来自国家流感中心)。HA滴度过低者经鸡胚传代再进行鉴定。最后将阳性标本送家国流感中心进行复核鉴定。 1.3 病毒 A/深圳/203/95(H3N2)，A/深圳/160/96(H3N2)和A/深圳/223/97(H3N2)由深圳市卫生防疫站分离。国际代表株A/武汉/395/95(H3N2)毒株的HA1基因及其所编码的氨基酸序列由国家流感中心提供。深圳和武汉以下由WH和SZ表示之。 1.4 病毒RNA的提取

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