Establishment And Evaluation Of Real-time PCR And ELISA For Detection Of West Nile Virus

West Nile virus(WNV) is a member of the Japanese encephalitis (JE) virus group of the genus Flavivirus, family Flaviviridae. Infection of WNV is clearly an emerging and significant public health problem. It causes infectious disease in human and other animals featured by fever and encephalitis. The outbreak of WNV encephalitis in the United States in 1999 and subsequent epidemic in North America in the following years have caused great concerns. Moreover, with the development of the epidemic, there are more and more concerns on the transmitting routes in addition to mosquito bites, such as blood transfusion, organ transplantation and vertical transmission.Since there is no proven therapy for WN encephalitis in humans or animals, nor any human vaccine to prevent WNV infection, preventative public health measures are of primary importance in the control of transmission of WNV. Effective controlling transmission of WNV requires rapid and sensitive assaying systems.Real time quantitative PCR is used routinely for the high-throughput diagnosis of pathogens, such as WNV. In this study we developed and validated a less expensive assay with the intercalating dye SYBR green I . The SYBR green-based assay was as sensitive as the TaqMan assay for WNV.An indirect ELISA developed for assaying WNV antibody in infectious and suspicious samples from humans and animals. Detection of WNV antibody with ELISA has the advantage of a simple, rapid in vitro procedure, and the same time, the detecting method does not require the extensive support facilities needed for conventional methods of virus isolation. Another important aspect of this assay is the stability of the noninfectious viral protein, which is safety for assaying. Our research work includes the following:1. Establishment and Evaluation of Real-time PCR for Detection ofWNVA rapid real-time polymerase chain reaction (RT-PCR) for detection of WNV was established in this study. Primers were designed according to capsid protein gene with Primer Premier5.0. In response, we developed and validated a less expensive assay with the intercalating dye SYBR green I . Amplifying curve showed that this method could successfully amplify WNV gene, nevertheless reference Japanese encephalitis and blank control were all negative. 10-fold dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assaying system could detect 10~2 Copies/μL WNV gene. The newly-built real-time PCR has high sensitivity, good specificity, reliable stability, so it has potential to apply in inspection and quarantine of WNV.2. Expression and Identification of WNV E DomainⅢin Drosophila S2 Cell LinesThe envelope protein is a major determinant of tropism and the primary target of virus-neutralizing antibody. In particular, domainIII of envelope is the major protective antigen of WNV, which can induce the production of neutralizing antibodies to neutralize the infectivity of the virus. This study constructed a expressed vector(pMT-DIII). pMT-DIII was transfected into S2 cells. S2- DIII cell which could permanently express recombinant DIII protein was established. The assessment of purity and speciality of WNV E DIII used SDS-PAGE and Western blotting.3. Establishment and Evaluation of Antibody ELISA Assaying System of WNVWe adapted an indirect Ab enzyme-linked assay to facilitate studies of WNV and evaluated its application. Serological diagnosis of WNV infection is complicated by extensive antigenic cross-reactivity with other closely related virus, such as JEV and BVDV. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WNV surveillance in our country. Here we describe a recombinant, S2 cell expressed antigen equivalent to structural domain III of the WNV envelope protein that has allowed clear discrimination of antibody responses to WNV from other related virus in indirect enzyme-linked assays using standardized control antiserum and some samples. This recombinant antigen has great potential to become the antigen of choice and will facilitate the standardization of reagents and implementation of WNV surveillance in our country.