摘要
〔目的〕掌握南方口岸蚊媒携带病毒的本底资料,为蚊传疾病的预防控制工作提供依据。〔方法〕采用电动吸蚊器人工法和捕蚊磁场自动法采集南方5省口岸各类蚊虫。采集到的蚊类超低温送至实验室,研磨处理后用荧光PCR方法检测登革病毒、乙脑病毒、黄热病毒、西尼罗病毒、基孔肯雅病毒等重要蚊媒病毒,结果阳性的标本进一步进行PCR扩增和核苷酸序列测定分析;蚊标本研磨液同时用C6/36细胞进行虫媒病毒分离培养,出现细胞病变后分别用黄病毒科、甲病毒科各自的通用引物进行鉴定;对未能鉴定的未知病毒进一步用随机PCR方法进行扩增、克隆、序列测定、Blast搜索。〔结果〕从南方5省口岸采集到各类蚊虫12575只,鉴定后共分成254组。各组标本经荧光PCR方法检测,结果登革病毒、黄热病毒、西尼罗病毒、基孔肯雅病毒均为阴性;检测到2份福建省来源三带喙库蚊的标本乙脑病毒核酸阳性,经乙脑病毒E基因引物PCR扩增、测序分析证实为GⅠ型病毒。254份标本经C6/36细胞分离培养出现42份细胞病变,用黄病毒科、甲病毒通用引物PCR扩增,均未得到特异片段。选取1份典型病变的细胞培养物进行随机PCR鉴定,结果发现了1种潜伏于C6/36细胞中的浓核病毒。〔结论〕南方5省口岸蚊媒中可能未携带登革病毒、黄热病毒、西尼罗病毒、基孔肯雅病毒等重要蚊媒病毒,只有少量蚊虫携带乙脑病毒,蚊虫体内检测到的GⅠ型乙脑病毒属于福建省首次发现,出现病变的C6/36细胞可能是由自身潜伏的1种C6/36细胞浓核病毒引起。
Objective To know about the background data of mosquito-borne viruses at ports of South China in order to provide evidence for the prevention and control of mosquitoes transmitted diseases. Method An automatic method of Mosquito Magnet apparatus and handwork method of electronic mosquito capture device were selected to collect mosquito samples in South China. And the mosquitoes were sent to the laboratory in a ultra-low temperature. After grinding the mosquitoes, several important mosquito-borne viruses including dengue virus, Japancses encephalitis virus, yellow fever virus, West Nile virus and Chikungunya virus were detected by real-time PCR assay. A further study of PCR amplification and sequence analysis to the positive samples were performed. At the same time, a cell of C6/36 was used for Arbovirus isolation from the mosquito grinding samples. The cell samples with cytopathie effect were then subjected to RT-PCR assay to detect the flavivirus and alphavirus by using the universal primers specific to Flaviviridae flavivirus and Togaviridae alphavirus, respectively. The unknown virus that couldn＂ t be identified by the universal primers mentioned above was amplified by random PCR amplification, then the PCR products wer subjected to TA cloning, sequencing and Blast searching in C, eubank. Result 12 575 mosquitoes were collected from the ports in five Provinces of South China. The mosquitoes were divided into 254 groups after identification. Detecting by real-time PCR assay. All the virus including Dengue virus, yellow fever virus West Nile virus and Chikungunya virus showed negative results except for 2 positive results with Japanese encephalitis virus nucleic acid detecting. The 2 Japanese encephalitis virus positive samples were sourced from Culex tritaeniorhynchus collected from Fujian Province. The genotype of Japanese encephalitis virus was confirmed as G Ⅰ by PCR amplification and sequence analysis of the E gene. Virus isolate assay showed that eytopathie effect of 42 samples can be observed in C6/36 cell. And the RT-PCR assay to detect the flaviviru8 and alphavirus showed a result that no corresponding genetic fragments were amplified. One of the cell samples with typical eytopathie effect was selected to identify the unknown virus by random PCR amplification. A kind of densovirus hidden in C6/36 was found. Conclusion The mosquitoes collected at ports in five Provinces of the South China may not carry dengue virus, Chikungunya virus, West Nile virus and yellow fever virus, only a very low number of mosquitoes carried Japanese encephalitis virus. The genotype Ⅰ Japanese encephalitis virus carried with mosquito was found for the first time in Fujian Province. And the cytopathic effect obversed in C6/36 cell culture may be caused by a kind of Aedes albopietus C6/36 cell densovirus hidden in C6/36 cells.
出处
《中国国境卫生检疫杂志》
CAS
2009年第3期181-186,共6页
Chinese Journal of Frontier Health and Quarantine
基金
国家质量监督检验检疫总局科研基金项目(2005IK079)
