Isolation, Identification and Molecular Characteristics of Japane Encephalitis Virus in Henan Province
|School||Henan Agricultural University|
|Course||Preventive Veterinary Medicine|
|Keywords||Japanese encephalitis virus RT-PCR PrM gene E gene molecular characteristics|
Japanese encephalitis virus(JEV),being a member of the family Flavivi -ridae, is usually transmitted among the human and animal through mosquitoes and then may cause acute viral encephalitic, neurological disease and sows reproductive disorders, The disease influences the development of animal husbandry especially pig industry, and bring serious threat to human health. Especially, it has rised significant impact on public health in China, due to approximately 80% cases live in China. The pig is the natural amplifier and reservoir host, therefore, swine can be the most important reservoir of JE. Hence, it is essential to prevent swine JE, which is useful for swine production as well as human health.A pairs of primers were designed according to the sequences of the JEV PrM-C gene published in GenBank. The genome of JEV vaccine strain was used as template to develop the RT-PCR method for detecting Japanese encephalitis virus. The 495bp specific fragment was obtained from the JEV vaccine strain RNA by this RT-PCR. The sensitivity of the method reached to 10 pg JEV-RNA. 136 mosquito samples collected from pig farms in Henan were detected by this RT-PCR. Among these samples, 6 samples were JEV positive. These results show that the established RT-PCR method for detecting JEV is a powerful tool for rapid diagnosis in infected earlier period of JE.In order to understand the ecological distribution of JEV in Henan province from July to September in 2009 and 2010, the 136 mosquito samples from 55 pig farmwere collected. Samples were chosen to inoculate Vero cells, suckling mice respectively for isolating JEV. The five isolated strains were confirmed as JEV by RT-PCR detection, AGP and virus infectivity titration (TCID50) and named as HN10-1 strain, HN10-2 strain, Shilipu strain, Guangshan strain and Luoshan strain respectively.The PrM and E genes of the two isolated strains were amplified by RT-PCR. The sequences of PrM and E gene were determined and analyzed with the corresponding sequences of 21 JEV strains published in GenBank. Phylogenetic analyses show that HN10-1 strain belong to genotypeⅠand the other strains belong to genotypeⅢ, which are most closely related to JEV vaccine strain SA14-14-2. There are a 99.0% and 97.4% homology of the nucleotide and amino acid between Guangshan strain and Luoshan strain in PrM gene, and 99.7 and 99.5% homology of the nucleotide and amino acid between Guangshan strain and Luoshan strain in E gene. The PrM gene of Guangshan strain homology of the nucleotide sequence and amino acid with JEV vaccine strain SA14-14-2 are 99.7% and 96.6%. And the E gene of Guangshan strain homology of the nucleotide sequence and amino acid with JEV vaccine strain SA14-14-2 are 99.3% and 98.7%. The PrM gene of Luoshan strain homology of the nucleotide sequence and amino acid with JEV vaccine strain SA14-14-2 are 98.7% and 99.1%. The E gene of Luoshan strain homology of the nucleotide sequence and amino acid with JEV vaccine strain SA14-14-2 were99.4% and 98.9%. Compared with PrM and E protein of the vaccine strain SA14-14-2, there are 1 and 5 different amino acids in Guangshan strain, and there are 3 and 3 different amino acids in Luoshan strain. There are no difference between the E138 and E176 of two strains amino acid and vaccine strain SA14-14-2, which shows that two strains maybe avirlent strains. And there will be more research about HN10-1 strain, HN10-2strain and Shilipu strain.