Develepment And Application Of Laboratory Diagnostic Methods For Japanese Encephalitis

Japanese encephalitis (JE) is a viral disease of humans and animals, caused by Japanese encephalitis virus (JEV), a member of the family Flaviviridae. JE causes neurological illnesses in humans and leads to porcine reproductive obstacle. Pig is the spread host. Human can be infected by mosquitos. It is important to diagnoses and prevent of Japanese encephalitis for human and animals' health. Thereforhe following studies were carried out.1. Development of ELISA and ELISA Test Kit for Japanese encephalitis and application of ELISA Test KitJapanese encephalitis virus SA14-14-2 was propagated in chicken embryo. After virus purification, the suitable concentration of antigen was chosen through square matrix titration. 85 serum samples tested with HI and ELISA.. The positive coincidence rate is up to 86.2%. The specificity,sensitivity, reproducibility of this method, were determined. 1454 serum samples from 127 pigs herds were tested by this ELISA, 882 samples were tested positive ,the positive rate is 60.7%.2. Establishment and application of Japanese encephalitis virus RT-PCR Based on JEV genome, a pair of primers was designed for amplifying theconservative sequence of NS1 gene, clinical suspected cases were measured with RT-PCR . This method can detecte JEV antigen rapidly with high sensitivity and specificity. On this basis, 38 pathological material of inspected disease have been measured by ready samples. There are 13 copies positive samples among them. Positive rate is 39.5%. So RT-PCR method is an effective method detecteing JEV.3. Establishment and application of Japanese encephalitis virus E protein-ELISAE gene of Japanese encephalitis virus was expressed by E. coli BL21(DE3). After the conditions of expression were optimized and the inclusion bodies were purified, the re-natured protein was used as antigen to develop an indirect Enzyme-Linked Immunosorbent Assay(ELISA) for detecting antibody to Japanese encephalitis. The suitable concentration of antigen was determined by square matrix titration., and the specificity, sensitivity, reproducibility of this method have been determined. 97 copies of serum from two pig farms were tested in step with HI and E-ELISA. The coincidence rate is up to 95.8%, the difference between the two methods is notsignifcant. 97 copies of serum from two pig farms were tested in step with whole viral ELISA and E- ELISA and the agreement rate is up to 95.8%, 892 copies of cliniced serum were tested with E-ELISA, and 422 samples tested positive. Positive rate is 47.3%.