Development Of Real-time RT-PCR Assay For Detection Of JEV And Preliminary Investigation In The Endocytosis Pathway Of JEV Entry Into BHK-21 Cells

Japanese Epidemic Encephalitis is a serious mosquito-borne disease caused by the Japanese encephalitis virus (JEV), which threatens the health of humans and livestock. Virus entry into the host cells is a critical step at the early phase of infection. The pathway of virus entry into host cells are differs from cell to cell. In the study, a real-time RT-PCR assay was developed for efficient detection of JEV, which was used to analysis the endocytic pathway followed by JEV to infect cell.1. Development a real-time RT-PCR assay for detection of JEVSpecific primers were designed from the nucleotide sequence of JEV (SA14-14-2) that was retrieved from GenBank. The standard plasmid was constructed by these primers. This assay was used to detect JEV standard plasmids with serial 10 fold dilutions, and the sensitivity can be up to 15 copies per reaction. The test of repetition was carried out with different concentration of standard plasmids between inter-and intra-assay, and the coefficient of variation (CV) was all below 5%. This indicated that the repetition of this method was good. Through amplification for JEV, HCV, PRRSV and others, the result showed that the method had high specificity to JEV. Used this method and traditional PCR to assay JEV in mosquito, it suggested that the rates of accordance of positive were 100%. However, positive assay rate using real-time RT-PCR was higher than traditional PCR. The correlation coefficient of standard curve was Y=-0.3007X+10.53, r2=0.997, and the circulatory efficiency of PCR was 96.53%. The study outcomes show that the method was fast, high sensitivity, good specificity and repeatability, and real-time detection. It could be used to isolate and identify JEV, detect infected and suspected cases, investigate detecting the JEV infection quantificationally, and study the molecular epidemiological.2. The endocytic pathway of JEV entry into BHK-21 cell.Cells were treated with Chlorpromazine, Nystatin and Cytochalasin D, and clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis were blocked. Then those treated cells infected with JEV. Real-time RT-PCR method was used in this research to estimate the intracellular virus copy-number at 1h,12h, and 24h after infection. Compared with control group untreated with drug, the intracellular virus copy numbers of three groups treated with drug were different degrees of decline. The change of the group treated with Chlorpromazine the was most obvious, and the intracellular virus copy number decreased by 59.58%,70.20%, 61.35% at 1h,12h,and 24h after infection. But the cytochalasin D group decreased by 23.59%,55.79%,30.14%, and the nystatin group decreased by 35.56%,58.10%, 34.40%. Results indicated that JEV could infect BHK-21 cells by clathrin-mediated endocytosis.