媒介动物(蚊、猪)体内黄病毒分子流行病学调查研究

〔目的〕通过对珠海注册养猪场及珠海口岸地区的蚊虫体内黄病毒的带毒率、宿主动物(猪群)及其人群进行血清黄病毒类(日本脑炎、登革热等)抗体水平的调查研究,为控制黄病毒在人群中的流行提供科学依据。〔方法〕采集养猪场猪血清、养猪场和口岸蚊标本、养猪场从业人员、入境的东南亚船员和城区部分发热病人血清,采用酶联免疫吸附试验(ELISA)法分别检测猪群和人群血清中的日本脑炎、登革热抗体;使用紫外灯诱蚊法在养猪场和口岸捕捉蚊类,经鉴定种类后,按20只蚊一组制成蚊悬液后,采用细胞培养、逆转录-聚合酶链反应(RT-PCR)以及荧光定量PCR技术检测蚊体内日本脑炎病毒、登革和西尼罗病毒等黄病毒的携带情况。〔结果〕1.猪血清日本脑炎病毒IgG抗体:结果已发表于《中国国境卫生检疫杂志》2007年第3期《珠海市部分注册猪场日本脑炎调查研究》。2.蚊媒带毒情况检测:2005年1月～2006年12月共捕获的2763只成蚊,经鉴定隶属4属8种,其中白纹伊蚊所占比例最高(32.75%),其次为致倦库蚊(26.06%)、三带喙库蚊(25.30%)、海滨库蚊(7.75%)、中华按蚊(3.84%)、骚扰阿蚊(3.18%),其他(常型曼蚊和巨型阿蚊等)(1.12%);在132组蚊研磨液的病毒细胞培养中,有7份标本出现CPE病变,使用黄病毒通用引物,RT-PCR反应能扩增出相应的阳性条带,其中白纹伊蚊组阳性3份,致倦库蚊组阳性3份,三带喙库蚊组阳性1份,但用日本脑炎病毒、登革病毒Ⅰ～Ⅳ型和西尼罗病毒等特异性引物,未能扩增出相应的阳性条带,说明可能存在着其它黄病毒类的病毒感染;采用荧光定量PCR法,对蚊悬液和细胞培养液日本脑炎、登革和西尼罗病毒进行检测,从1组海滨库蚊标本中检出日本脑炎病毒核酸阳性,但强度较弱,这与珠海地区是乙脑低发地区相一致。3.人群登革热和日本脑炎血清抗体检测:从东南亚的入境船员、本地发热病人和养猪场从业人员等血清标本511份中,登革病毒抗体IgG总阳性率为7.24%,其中以东南亚的入境船员的阳性率最高,为12.76%,没有检出登革病毒抗体IgM和日本脑炎IgM抗体。〔结论〕珠海地区存在日本脑炎的主要传播媒介,并且在蚊媒中携带有日本脑炎病毒;蚊标本细胞培养出现细胞病变和RT-PCR扩增出黄病毒基因的特异性片段,提示在捕获蚊标本中可能存在着其他病毒感染的可能;虽然养猪场猪群日本脑炎感染率不高,但存在着日本脑炎病毒的隐性感染或曾经感染过,提示不能够放松对乙型脑炎的预防控制工作,应采取积极的预防措施,防止人群和猪场日本脑炎的发生与流行。此外,研究结果表明东南亚船员有较高的登革热感染率,因此,在登革热流行期间,我国口岸应加强对来自东南亚国家交通工具员工和旅客的登革热的监测,以及有可能藏带、孳生蚊虫的废旧物品、集装箱等的检验检疫工作,以防止登革热的传入与流行。

Molecule Epidemiologic Study for Vector-Born (Mosquito and Pig) Flavivirus in Zhuhai

Objective To investigate Flavivirus in mosquito, serum antibody of Flavivirus (Japanese encephalitis, Dengue fever and so on) in host animal (pig) and human in Zhuhai hoggeries and Zhuhai port, hereby to provide scientifical evidences for strengthening monitoring, preventing and controlling the epidemic of Flavivirus. Methods Pig sera from hoggeries, human sera from Southeast Asian crew, fever patients in the urban city and workers in hoggeries were collected to detect serum antibodies for Japanese encephalitis virus (JEV) and Dengue virus (DV) by ELISA; mosquitoes were captured at 4 hoggeries and ports in Zhuhai from January 2005 to December 2006. Mosquitoes samples were sorted, identified to species, and were ground to make suspension which performed on groups containing 20 female mosquitoes per sample. RNA of virus were extracted directly from mosquito suspension, or after suspension was inoculated into cells of Aedes albopictus C6/36s and cultured. RT-PCR and Real-Time RT-PCR methods was carried out to detect JE, DV and West Nile virus (WNV) in mosquitoes. Results 1. Positive rate of Anti-JEV IgG antibodies in pigs: Data had been published in Chinese Frontier Health Quarantine Jun.2007,Vol 30,No.3 (Sero- epidemiological Survey on Japanese Encephalitis at Pig Farms Registered in Zhuhai). 2. Flavivirus in mosquitoes: 2 763 mosquitoes were captured together during January 2005 to December 2006. They belonged to 4 genera and 8 species, of which Aedes albopictus were the most dominant species, accouting for 32.75%, next was Culex fatigans (26.06%), Culex tritaeniorhynchus (25.30%), Culex Sitiens (7.75%), Anopheles Sinensis (3.84%), Armigeres subalbatus (3.18%), other mosquito (1.12%). 132 pools of mosquito suspensions were conducted by cell culture, 7 samples presented CPE pathological change. Culture supernatants were collected for the RT-PCR using Flavivirus universal primers, and 7 samples with CPE were amplified the amplicons produced the expected bands of 835 bp, among them, 3 cases were positive for the Aedes albopictus groups, 3 cases were positive for Culex fatigans and 1 cases was positive for Culex tritaeniorhynchus pools, further using the specific primers of JEV, DEN 1-4 serotype and WNV, no positive bands were amplified by two-step PCR amplification, which was posibly proved other Flavivirus infection. Real-time RT-PCR methods was also used to detect JEV, DV and WNV in mosquito suspensions and culture supernatants, 1 positive sample was identified as a mild JEV from the Culex Sitiens pools in the hoggeries. It is accordant to the fact of low local epidemic JE in Zhuhai. 3. Humen serum antibody of DV and JEV: 511 serum samples from Southeast Asian crew, fever patients in the urban city and workers in hoggeries were detected by ELISA, total 7.24% of positive rate for anti-DV IgG antibodies, among them, with the highest positive rate of 12.76% in the entry crewman from Southeast Asia countries. While anti-IgM antibodies of JE and DV were negative in these peoples. Conclusion The main vectors of JEV existed in Zhuhai area, and there was a evidence that the JEV was existed in the mosquito vectors. CPE pathological change in the cell culture of mosquito' samples and the Flavivirus genus specific bands amplified by RT-PCR, suggested that other Flavivirus was possibly infected in mosquitoes, which was needed further studying and confirming. Although anti-JEV IgG antibodies in pigs was not very high, the natural JEV infection in pigs was occurred, the JEV transmission was still common in the natural environment in Zhuhai city, so the immunization of JE vaccine can not be neglected. In addition, the result of study indicates Southeast Asian crew had a higher infection rate of dengue fever, quarantine measure should be take to prevent from dengue fever transmission in the port.