Research Of Japanese Encephalitis Pathogen Detection And Immune Prevention

Japanese encephalitis (JE) is an acute viral infection of the central nervous system, which is caused by Japanese encephalitis virus (JEV). JEV is active in many countries, not only damage on the hog industry, but also on human health. Japanese encephalitis can be detected through the clinical detection combine with epidemiological studies, final diagnosis require laboratory testing, like serological diagnosis and pathogen detection. At present, there is no effective treatment for Japanese encephalitis, except vaccination with kill-mosquito work.1. Development and Application of SYBR Green I Real-time RT-PCR for detection of Japanese encephalitis virusRespectively design three pairs of primers by taking conservative prM, NS1, and E gene fragments in Japanese encephalitis virus as templates. Select the best real-time fluorescence PCR primers basing on the model of SYBR Green I.,In order to establish a rapid and accurate real-time fluorescent PCR method for detection of Japanese encephalitis virus. Establish a standard curve by using the positive plasmid with correct sequence, and evaluate sensitivity, specificity and repeatability of the method.It’s proved that sensitivity of primer designed by using E protein nucleotide sequence of957-143lbp region as a template is higher than the other two. This method can only amplify signal from JEV-positive samples, do not cross-react with other pathogens; Melting temperature is86.2±0.5℃, The melting curve of PCR products appear only a single specific peak without primer-dimers; A good linear relationship between the1×103～1×109copies can be seen,with cforrelation0.999, amplification efficiency94.2%. The minimal copy of the positive standard is1000,which is103times sensitive compared to common PCR; Coefficient of variation in an between groups are0.53%～1.42%and1.16%; Testing16cases of clinical suspected materials of encephalitis B,we found that the positive rate is81.25%(13/16), amplified fragment of positive sample is475bp, with the same size of the expected. The SYBR Green I real-time fluorescence PCR established by this study is specific, highly throughputing, highly sensitive and rapid,it provide a new method for rapid detection of JEV and molecular epidemiological investigation.2. Comparison of porcine inactivated Japanese encephalitis vaccine with various adjuvants for immunogenicity and protective efficacy in miceIn this study, a swine source Japanese encephalitis virus strain(NJ2008) isolated from mosquito was used as stock virus for preparation of JEV inactivated vaccine. The valence of JEV-NJ2008is108-67TCID50/mL on BHK-21cell. The virus was inoculated in roller bottle covered with Vero cells, then the culture was collected, purified, concentrated and inactivated by0.2%formalin. The inactivated was given along with montanide ISA15AVG, alum, FIA and propoils as adjuvants, a live attenuated vaccine, SA14-14-2and PBS as comparison. The vaccines were evaluated in4to6week-old mice, and challenge experiment was carried out after second booster. The results showed that, JEV-NJ2008inactivated vaccine with ISA15AVG adjuvant stimulated better immune response in comparison with other adjuvants. It not only stimulated early immune response and produced a high level antibody, but can induce humoral-and cell-mediated immunity simultaneouly. The vaccine candidacy of JEV-NJ2008inactivated vaccine with ISA15AVG adjuvant was proposed.