The Study On Diagnosis Methods And Epidemiological Survey Of West Nile Virus Infections

West Nile virus (WNV) infection is an endemic disease caused by WNV that is maintained in a natural transmission cycle involving mosquito vectors and bird reservoir hosts. Humans are recognized to be incidental host, and clinical symptoms associated with WNV infection include febrile illness or encephalitis. Since 1999, WNV was detected for the first time in the U.S.A., the virus rapidly extended its range and the virus-infected case was increasing during the next 4 years. The continuous outbreak of West Nile encephalitis has caused wide concern and great panic in North America, whereas there were few systemic WNV-related studies in China. In the present study, sensitive and specific methods for the detection of WNV infections were established using immunological and molecular biological techniques, and then primary epidemiological survey of the disease was conducted. The aim of this study was to understand the background information and potential vectors of WNV in China to lay a basis for the further studies.To demonstrate weather the WNV strain previously imported can be applied in the study with respect to WNV, the biological characteristics of the strain was investigated, mainly including pathogenicity, cell sensitivity, morphologic observation, immunogenic competence and molecular biological feature. The results showed that Suckling mice inoculated intracerebrally with the virus cultures were all, killed. WNV can induce the cytopathic effect (CPE) in Vero-E6 and C6/36 cells, which was characterized by cell shrinking and cell fusion, respectively. The morphologic observation showed that the virions were spheral particles with envelope and were approximately 30~50nm in diameter. Serum antibodies against the virus were detectable in BALB/c mice immunized with inactivated WNV. The target DNA fragments located in C-prM and E regions of the virus genome could be amplified by RT-PCR from both the virus cultures and brain tissues of infected suckling mice. The homology analysis with BLAST program indicated that the nucleoside sequences detected were only matched with WNV.Simultaneously, the comparison and discrimination of the biological characteristics between WNV and Japanese encephalitis virus (JEV) were also carried out. There were no significant differences in the pathogenicity to suckling mice, and shape and size of the virion between two viruses. In contrast with WNV, JEV-caused CPE was characterized by cell shedding. WNV and JEV were found to have antigenic cross-reaction. The viral RNA could be detected from the both WNV and JEV samples with universal primer set, but only nucleoside fragments of corresponding virus could be amplified when specific primers were used.Three sets of specific primers were designed in C-prM and E regions of the virus genome. The parameters for RT-PCR amplification were optimized using the template from the viral cultures. Then this method was used to detect WNV in infected brain tissues and simulated mosquito samples. The viral nucleotides were identified in two WNV samples. Nested PCR could remarkably increase the sensitivity up to 108~109-fold. On the other hand, indirect immunofluorescence assay (IFA) and enzyme linked immunosorbent assay (ELISA) using the whole viral proteins as antigen were developed for the detection of serum anti-WNV.Based on the above studies, mosquito and / or serum samples were collected from Beijing, Xinjiang and Yunnan. The results of serological survey showed that the prevalence of antibodies against flaviviruses, such as WNV and JEV, in Xinjiang population was significantly higher than that in recruit population who never lived in Xinjiang. There was certain immune cross-reaction between WNV and JEV. No WNV nucleotides or new isolate was found in mosquitoes sampled from Xinjiang and Beijing, while several suckling mice were observed to have encephalitis-liked symptoms when they were inoculated intracerebrally with mosquito samples collected from Yunnan.To establish a specific method for serological diagnosis of WNV infections, the gene encoding partial envelope (E) protein was cloned into pQE-30 plasmid and was then expressed in E. coli. Ml5 strain. As expected, the target protein was expressed and was purified. It could specially react with the serum from mice immunized with inactivated WNV in Western blotting. ELISA used the expressed E protein as antigen could discriminate anti-WNV positive serum not only from the negative control serum but also from anti-JEV positive serum.In conclusion, WNV strain investigated can be used for further WNV-related studies. The virus-caused CPE in C6/36 cell and detection of the viral RNA should be of utility in discrimination of WNV and JEV. RT-PCR assay established is a sensitive and specific method for the detection of WNV, and can be applied in epidemiological survey of WNV infections. The expressed E protein is useful for the specific serological diagnosis of WNV infections. More studies are needed to search for the competent evidences of WNV or other new mosquito-borne flaviviruses.