| Japanese encephalitis virus (JEV, family Flaviviridae, genus Flavivirus) is lipid-enveloped virus containing a single-stranded positive-sense RNA genome, which can cause an acute disease of central neutral system, Japanese encephalitis (JE). JE was described in Japan from the 1870s onwards, since then the disease apparently spread across Asia to affect most of China and the Asian subcontinent, all of southeast Asia, and the Pacific Rim, reaching northern Australia in 1998. Now it is distributed in many areas of Asia and parts of Oceania, with 50,000 human cases and 10,000 deaths reported annually and up to 50% of survivors are left with severe neuropsychiatric sequelae.JEV is maintained in a transmission cycle between amplifier swine and vector mosquitoes. And it can also cause severe reproductive failure leading to abortion, still-birth and mummification of fetus in sows and to orchitis in boars, severely influence the expanding of swine population and causing huge economic loss. Now JEV, porcine reproductive and respiratory syndrome virus(PRRSV), porcine parvovirus(PPV), and pseudorabies virus(PRV) are the most important virus which lead to reproductive failure in pig industry in this country, all these disease have the similar syndrome, and the existence of cross and combined infection make it necessary to establish a rapid and specific diagnosis method. Recently the widespread vaccination has controlled JEV to a certain extent, but also complicated the serodiagnosis and epidemiological study since antibodies are induced by either infection or vaccination.The envelope protein(E protein) is the major structural protein that makes up most of the surface of the flavivirus particle and entails a putative receptor-binding domain, numerous neutralization epitopes, and virulence determinants. The domain III of E protein(DIII) independently folded as a unique Ig-like conformational structure presented on the viral surface, is a major target for neutralizing antibodies, so it could act as a good diagnostic antigen.Nonstructural protein NS1 is also one important antigen of JEV, which has the soluble complement-fixing activity; NS1-specific antibodies have been demonstrated to provide protective immunity against JE virus in mouse. Since NS1 is secreted only from infected mammalian cells, it is considered the most suitable antigen to differentiate infection from inactivated vaccine.According to published sequence of the JEV SA 14-14-2 strain, unique primers for DIII and NS1 were designed, and genes were amplified by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pMD18-T vector, then inserted into the downstream of T7 promoter of expression plasmid, pET-32a(DIII), and pET-28a(NS1). After induction by IPTG, the fusion proteins were expressed in E. coli BL21. SDS-PAGE and Western-blot analysis showed that the purified fusion proteins could both react with pig serum containing antibody against JEV.The DIII-ELISA and NS1-ELISA were established by using the recombinant proteins as antigens, and all reaction conditions of the indirect-ELISA were explored, and optimized, under the conditions P/N>5. The results were negative when the sera contained antibodies against PRRSV,PPV and PRV were detected by the optimal procedures. In the reproducibility tests the CVs of detected samples in different wells of one plate were less than 7%.280 swine serum samples were detected JEV-antibody by using this developed indirect DIII-ELISA and the cellular neutralization test. It was found that the specificity and sensitivity of the developed DIII-ELISA were 89.4% and 91.8% respectively, the coincidence rate was 87.9%.409 swine serum samples were detected by DIII-ELISA, 142 were positive. And NS1-ELISA result is comparable with DIII-ELISA. It shows both methods are feasible to detect antibody to JEV. Since the immuno-backgroud of the detected serum was unknown, it needs further studies to confirm the potential application of NS1-ELISA to differentiate infection from vaccination.
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