实验用小型猪乙型脑炎病毒的分离鉴定

Isolation and identification of Japanese encephalitis virus in the experimental minipigs

目的了解小型猪感染乙型脑炎病毒株的特点。方法猪脑组织经过处理,接种BHK21细胞,进行病毒培养、间接免疫荧光试验、中和试验、电镜观察,及通过逆转录-聚合酶链反应(RT-PCR)扩增新分离毒株E区段和PrM区段核苷酸序列,测序后进行基因型鉴定。结果 25只猪脑组织接种BHK_(21)细胞,有3个组织处理液能使BHK21细胞发生圆缩、集聚等病变;3个组织接种的BHK_(21)病变细胞滴片进行免疫荧光试验,均产生强绿色荧光反应;中和实验结果显示3株新分离株中和效价均为1∶64;电镜下可见直径40nm左右球型病毒粒子;RT-PCR产物测序结果与疫苗株E区段核苷酸比较同源性均为95%;3株新分离株均为GIII型乙脑病毒。结论实验表明小型猪场存在乙型脑炎病毒感染,为GIII型乙型脑炎病毒。

Objective To understand the characteristics of minipigs infected with Japanese encephalitis virus（JEV）.Methods After the brain tissues were treated,the pig brain tissue treatment solution was inoculated with BHK_（21） cells.Then,virus culture,indirect immunofluorescence assay,neutralization test,electron microscopic observation,and reverse transcription-polymerase chain reaction（RT-PCR） amplification of the new isolate E segment and Pr M segment nucleotide sequence were performed and the genotype was identified.Results BHK_（21） cells were inoculated into 25 pigbrain tissues.Among them,three tissue-treated fluid couldinduce shrinkage and aggregation of BHK_（21） cells,and immunofluorescence staining showed strong green fluorescence response.The results of neutralization test showed that the neutralization titer of these three new isolates was 1：64,and the size of the virus particles was about 40 nm under the electron microscope.The homology of both RT-PCR product sequencing results and E-segment of vaccine strain were 95%.Three new isolates were type GIII JEV.Conclusion The results ofthisstudydemonstrate that there is G III type Japanese encephalitis virus infection in the minipig farm.