Development Of Swine JEV ELISA Antibody Detection Kit And Isolation And Identification Of JEV From Swine

Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is one of the vector-borne zoonotic diseases. when infected by JEV, sows and boars may be caused reproductive failure, and the newborn piglets may be show encephalitis. JEV not only infect pigs but also infect the human beings. Pig is the most important host of Japanese encephalitis virus. Monitoring and preventing the occurrence of the pig infected with JEV was of great significance for JE prevention and control. Therefore, a rapid and accurate diagnosis and effectively safety vaccine are prerequisites for prevention and control of the disease. Herein, our researches focused on swine Japanese encephalitis virus ELISA for detection antibodies and isolation and identification of Japanese encephalitis virus from swine, the details are as follows:1. The development of the swine Japanese encephalitis virus ELISA antibody detection kit.The purified and inactivated JEV SA14-14-2strains are as coating antigen. Through the phalanx titration method we determined the best antigen coating concentration and serum dilution were0.2μg per well and1:40, respectively. The optimum reaction conditions of the test are follows. The coating solution is the carbonate buffer, which pH value is9.6and molar concentration is0.05mol/L, blocking solution is one phosphate buffer containing0.5%bovine serum albumin and0.5%Tween20, the preferred binding reaction time of antigen and the serum is30minutes, the optimal dilution of the secondary antibody is1:5000, and the optimum reaction time for HRP secondary antibody was30minutes, substrates reaction time is10minutes.It proves good specificity for the kit by detecting the serums of swine foot-and-mouth disease, swine fever, swine circovirus type2disease, porcine reproductive and respiratory syndrome, porcine parvovirus, porcine pseudorabies’ positive serum. The quality control serum test results showed a well susceptibility to our kit. The intra repeat coefficient of variation were between0.60%to9.88%, and the batch coefficient of variation were in the range of1.29%to10.85%. Our kit can be kept for at least10months at2～8℃. Compare to the Latex agglutination test for swine Japanese encephalitis virus antibody detection kit, the total coincidence rate was86.81%.2. Isolation and Identification of Japanese encephalitis virus from swineHere we report a JEV strain named GX1209isolated from a piglet brain tissue in Guangxi province in september2012. The result of RT-PCR suggests the tissue sample is positive for JEV.The complete genome sequence of GX1209is10965nucleotide (nt) in lenth, with a GC content of53.5%. Phylogenetic analysis was conducted and GX1209belongs to genotype Ⅰ. GX1209has a close relationship with XJ69isolated from Xinjiang Province, China, which amino acid homology is99%. Compare to the vaccine strains SA14, SA14-14-2, P3, the amino acid homologies is98%,97%,98%, respectively. And compare to the strains isolated from culex tritaeniorhynchus in Guangxi province, BL06-50, BL06-54, GX0523/44, GX0519, all the amino acid homologies are99%. Through intracerebral inoculation of Kunming suckling mice to proliferate GX1209strains, the titer is1×107PFU/mL by plaque forming units (PFU) test.