The Molecular Epidemiology Investigation Of Japanese Encephalitis Virus In Sichuan

Japanese encephalitis is a serious mosquito mediator zoonosis caused by the Japanese encephalitis virus (JEV) which belongs Flavivirus. Japanese encephalitis can cause the serious lesion of the central nervous system of human. The swine is main increases host of Japanese encephalitis virus, procine Japanese encephalitis is closely related to Japanese encephalitis of human, so the prevention of procine Japanese encephalitis is not only reduce the negative impacts of pig-raising, but also the important approach to protect human from Japanese encephalitis. Sichuan Province is the high incidence provinces of Japanese encephalitis in China, and in recent years, the incidence rate is from2/100000to10/100000.In this study, a RT-PCR method of rapid diagnosis of JEV was established. A molecular epidemiology investigation of JEV in Sichuan was done through the RT-PCR method to know the molecular characteristics and variations of some JEV strains in partial area of Sichuan Province. This study can facilitate the prevention of Japanese encephalitis in Sichuan Province, and boost the research of molecular characteristics of JEV and development of DNA vaccine of JEV.1. Establishment of molecular epidemiology investigation method for porcine Japanese encephalitisIn this study, a RT-PCR method of rapid diagnosis of JEV was established There are31Japanese encephalitis virus strains from the GenBank were referenced to determine the target sequences of detection, and A pair of primers was designed according to nucleotides240-993of SA14-14-2vaccine strain. Optimize the reaction conditions by adjusting concentration of reagents, temperature of annealing and time of extension and so on. Classical swine fever virus, Porcine reproductive and respiratory syndrome virus and procine rotavirus were set to be the control groups in the test of RT-PCR detection and the result indicated this RT-PCR method of rapid diagnosis had a high specificity.The result of sensitivity test of RT-PCR detection indicated the The lowest concentration of RNA template can be detected was122ng/mL. So this diagnosis method had the high specificity and sensitivity and it can be applied in rapid clinical diagnosis of prcine Japanese encephalitis and molecular epidemiology investigation of Japanese encephalitis virus.2. The molecular epidemiology investigation of Japanese encephalitis virus in SichuanThere are53pig abortion stillbirth and mosquito samples were collected from27pig farms in Sichuan province.The area of collection include14regions such as Chendu, Ya’ an, Luzhou, Pengzhou, Dujiangyan, Suining, Nanchong, Guang’an, Guangyuan, Bazhong, Dazhou, Neijiang, Deyang which including a total of21counties. The samples were sorted with ther sources and detected respectively by the RT-PCR method which was established in this study.The result of RT-PCR detection indicated there were9positive samples of JEV respectively from where Nanchong, Luzhou, Pengzhou, Dujiangyan, Guang’an, Ya’an. And8of those9positive samples derived from culex, the other one derived from brain tissue of pig abortion stillbirth. The9PrM genes and6E genes of the9positive samples were cloned and plused a JEV strain isolated by our laboratory formerly, all of which were sequenced. The homology of the nucleotide and amino acids sequence of PrM gene and E gene between the9virus strains and the vaccine strain SA14-14-2were high, and the phylogenetic tree analysis on PrM-C gene nucleotide sequence indicated all of9virus strains were the genotype Ⅲ of Japanese encephalitis virus, and they were obvious regional because their hereditary features were most similar to the isolates in Sichuan in the past.