Isolation And Identification Of Japanese Encephalitis Virus In Henan Province And Genetic Analysis Of E Gene

Japanese Encephalitis (JE), caused by Japanese Encephalitis Virus (JEV) that transmitted by mosquitoes, is an acute viral encephalitic and neurologic disease of human and many animals. Swine is considered to be the main host and represents an important amplifier and reservoir for JEV. It is essential to prevent swine JE, which is useful for swine production as well as human health.JEV belongs to the family Flaviviridae, genus Flavivirus. Its genome contains a single-stranded, positive-sense RNA of approximately 11 kb in length.The single open reading frame contained in the genome is translated into a polyprotein which is cleaved into 3 structural proteins and 7 non-structural proteins.Among them, protein E, the major structural agent, plays a great role in virus attachment, fusion cellular tropism, viral virulence and introduction of protective immune response.In this study, we collected cerebrospinal fluid (CSF) of sow aborted fetus stillbirth from large pig farms in Henan province, and isolated virus by inoculating PK-15 cells.Three isolates were obtained and confirmed to be RNA virus.Primers were designed and synthesized according to the sequences of E gene published in GenBank. cDNA fragments were cloned by RT-PCR and sequenced. Compared with the sequence of E gene of vaccine SA14-14-2E, the nucleotide homology was more than 95.5%.Hence those isolates were confirmed to be JEV and named as CSF.BY-1,CSF.ZY-2,CSF.ZMD-3 respectively.E protein is a JEV membrane glycoprotein. It is an important structural protein and plays an important role in many functions. In this study we analyzed the sequence of nucleotide and amine acid of E protein to clarify the phylogenetic characteristics and molecular variation.Using the E genes of CSF.BY-1,CSF.ZY-2,CSF.ZMD-3 and other 18 reference strains, a phylogenetic tree was constructed by software Mega3.1 through the neighbor analysis and draw gene evolution tree on the basis of the sequences of nucleotide and amino acids respectively. The variation between nucleotide and amino acids of the isolates and control strains were analyzed by DNAstar and online software MultAlin. The results demonstrated CSF.BY-1,CSF.ZY-2,CSF.ZMD-3 belonged to gene typeâ…¢.The homologies of nucleotide sequences are 92.1%-95.9%. The homologies of amino acids sequences are 72.1%-77.3%.Nucleotide mutates in 19 sites, which results in 14 amino acids substitutions.The mutation sites of isolated strains are not the key sites of virulence, and have no significant effect to the virus virulence.