摘要
西尼罗病毒(WNV)感染是一种经蚊虫传播,以鸟类为主要动物宿主的自然疫源性疾病。人被携带病毒的蚊虫叮咬后而感染,表现为西尼罗热或西尼罗脑炎。该病自1999年传入美国以来,已连续肆虐了六个流行季节,流行范围迅速扩大,感染人数逐年攀升,给美国社会造成了极大恐慌。目前,我国尚缺乏对WNV感染的系统研究。本研究应用免疫学与分子生物学技术,建立敏感、特异的WNV感染检测方法,并在我国开展WNV感染的流行病学调查。旨在了解WNV在我国的存在情况和潜在媒介蚊种及自然疫源地性质,为深入开展WNV相关研究和大规模疫源地调查奠定基础。
为证实引进的WNV毒株应用于相关研究的可行性,对病毒的致病性、细胞敏感性、形态学、免疫原性、分子生物学特征进行了探讨。结果表明:给乳鼠脑内接种WNV可致全部动物死亡;WNV可使Vero-E6细胞与C6/36细胞产生细胞病变效应(CPE),分别以细胞圆缩和细胞融合为主要特征;电镜下所见病毒体为有包膜、直径约30～50nm的球状颗粒;灭活病毒可诱导小鼠产生抗WNV抗体;采用RT-PCR对病毒C-prM区和E区部分基因片段进行扩增,在病毒培养液及感染乳鼠脑组织中均检测到目的核苷酸片段,经BLAST程序分析仅与WNV有较高的同源性。
进一步,对WNV和乙型脑炎病毒(JEV)的上述生物学性状进行了比较,结果显示:两种病毒在电镜下的形态、大小和对乳鼠的致病性相似;与WNV不同,JEV所致C6/36细胞CPE以细胞脱落为主要特征;WNV与JEV之间存在抗原交叉反应;用黄病毒通用引物可从WNV和JEV感染组织中检出病毒核酸,而用特异引物仅能扩增出相应病毒的核苷酸片段。
在WNV基因组C-prM区和E区设计3对特异引物,以病毒培养液为标本,
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.
引文
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