Isolation And Identification Of Two Japanese Encephalitis Virus From Swine And Coding Sequence Analysis

Japanese encephalitis is a zoonotic mosquito-borne viral disease caused by Japanese encephalitis virus (JEV) which is belongs to the genus Flavivirus of the family Flaviviridae. JEV infects a number of animal hosts, humans as the dead-end hosts. In addition to its public health importance, JEV also causes reproductive disturbance in swine which act as amplifying host, does great harm to the animal husbandry.There is a high incidence of JE in China, in recent years, animal husbandry development rapidly, especially the pig industry, increased the difficulty of JE monitoring and the risk of encephalitis occurr. In the present study, two JEV strains were isolated from aborted swine brain tissue. The biological characteristics were researched systematically. The coding region sequences of these two JEV strains were determined and compared with other JEV strains deposited in GenBank. This work provided a basis of molecular epidemiology, enriched the material for JE research, play an important role in prevention and control of JE in China, especially Sichuan Province.1. Isolation and identification of two Japanese encephalitis virus from swineTotal mosquito and aborted foetus simples were collected from diverse regions in Sichuan Province. Nucleic acid of JEV detected by reverse transcription-polymerase chain reaction (RT-PCR) for all simples. Positive samples inoculated 5-day-old suckling mouse by intracerebral inoculation. Neonatal rats behaved as depressed, poor feeding, seizures and became thin 4 days after inoculated with two batches of swine brain collected in yaan till 3 generations. Finally paralysed and dead at 5th day. The sick rat brains were grinded and centrifuged at 5000 rpm for 10 min and the supernatant fluid was cultured in BHK-21 showing cytopathic effect(CPE), CPE characterized by rounding, shrinkage and dislodgment from the growth surface. As passaged on BHK-21 cell, CPE appears regularly. CPE appears advanced, as 4th day CPE approached 90% after the 15th passage.Two isolates were confirmed as Japanese encephalitis virus (JEV) by agarose gel diffusion precipitation test, indirect immunofluorescence assay (IFA), reverse transcription polymerase chain reaction (RT-PCR) and virus neutralization test after investigating the physicochemical characterization. The results show:two strains of the virus are RNA virus, sensitive to ether, chloroform and trypsin, were inactivated under pH 3.0 conditions. Be inactivated 60℃ for 60min,100℃for 2min. RT-PCR can amplify specific fragments of JEV prM/C gene, agarose gel diffusion precipitation test, indirect immunofluorescence and micro-neutralization test showed a positive reaction. Two isolates were confirmed as JEV and named SCYA201201, SCYA201202.2 Cloning and analyze the coding sequenceCloning and sequencing coding sequence (CDS) of the two isolates,8 pairs of primers were designed to amplify 8 overlapping cDNA segments. CDS of SCYA201201 strain and SCYA201202 strains both containing 10 296 nt, encoding 3432 amino acids, and shared 99.9% nucleotide sequence identity and deduced amino acid sequence identity. The genetic relationship of SCYA201201 and SCYA201202 with other representative JEV strains from different areas of the world and diverse hosts, deposited in GenBank, were analyzed and phylogenetic trees based on nucleotide sequences of the E gene and C/prM were constructed. The tree was rooted using Dengue virus as the out-group virus. Both strains fell into genotypes GI. Coding sequence analysis show that, SCYA201201 had high nucleotide identity (≥97.7%) and amino acid (≥98.3%) identities with the other GI strains. Similarly, SCYA201202 shared high nucleotide identity (≥97.8%) and amino acid identity (≥ 98.4%) with the other GI strains. Two isolates were highly homologous, only three amino acid were different in all proteins. Both of the isolates, had a glutamate at the E138, an isoleucine at the E 176 and a Lysine at the E 279 of the E protein, show a strong toxicity neuronophagia molecular characteristics.3. Biological characteristics research preliminary of two JEV isolatesTo explore the suitable conditions of hemagglutination, used Sucrose-acetone extraction method to extract hemagglutinin of two isolates. The result shows that hemagglutinin hemagglutinate red blood cell of Pigeon at pH6.4 condition stably. Real-time RT-PCR method was used to research tissue tropism, viral replication trait of SCYA201201 strains, SA14-14-2 strains as a reference to compare. JEV SCYA201201 strains was detected in heart, liver, spleen, kidney, thymus and brain tissue.The data of viral replication in brain tissue suggest that SCYA201201 strains is neuorovirulence.