三带喙库蚊体内猪繁殖与呼吸综合征病毒的分离与鉴定

Isolation and identification of the porcine reproductive and respiratory syndrome virus(PRRSV)from Culex tritaeniorhynchus(Diptera:Culicidae)in pig farms

【目的】调查猪场蚊虫是否能携带猪繁殖与呼吸综合征(PRRS)病毒。【方法】采集发生PRRS疫情的3个养猪场蚊虫样本,采用RT-PCR方法检测PRRS病毒核酸,取阳性蚊虫样本接种Marc-145细胞进行病毒的分离培养,以间接免疫荧光抗体技术和分子克隆技术进行病毒的鉴定。【结果】养猪场内的蚊虫主要有三带喙库蚊Culex tritaeniorhychus、凶小库蚊Culexmodestus、中华按蚊Anopheles sinensis和骚扰阿蚊Armigeres obturbans,其中三带喙库蚊占86.76%;以PRRS病毒N基因引物进行扩增,三带喙库蚊样本呈现阳性反应,而其他蚊种均为阴性。在蚊虫接种的Marc-145细胞中可见细胞融合和空泡形成等细胞病变效应;用抗PRRS病毒N蛋白抗体和羊抗猪IgG(H+L)-FITC进行间接免疫荧光染色,感染细胞呈现黄绿色荧光;以NSP2基因引物进行RT-PCR扩增、克隆与测序,发现库蚊源病毒与相应猪场猪源病毒中相应基因的序列具有较高同源性。【结论】三带喙库蚊为猪舍优势蚊种,并能携带猪繁殖与呼吸综合征病毒。

[ Objective ] To determine whether the porcine reproductive and respiratory syndrome virus （PRRSV） can survive in mosquitoes in infected pig farms. [ Methods] The mosquito samples were collected from 3 pig farms infected by the porcine reproductive and respiratory syndrome （PRRS） and determined by RT-PCR method based on the primers of PRRSV N gene, then the PCR-positive mosquitoes were cultured in Mare-145 cells for isolation of PRRSV, which was identified by indirect immuno-fluorescence assay and sequence analysis of Nsp2 gene. [ Results ] At least 4 mosquito species, Culex tritaeniorhychus, Culex modestus, Anopheles sinensis and Armigeres obturbans were identified on 3 pig farms and C. tritaeniorhynchus was found to be the predominant species （86.76%）. By RT-PCR, PRRSV was detected in the samples of Culex tritaeniorhychus but none in other 3 mosquito species. The cell pathological effects such as cell fusion and cavitation were observed in Marc-145 cells inoculated by the samples of C. tritaeniorhynchus. The antigen of PRRSV in cultural cells was detected by indirect immunofluorescence assay. The PRRSV NSP2 gene, which was amplified and sequenced from infected moisquito cells, was highly homologous in nueleotide sequence with those from the infected pigs in same farm. [ Conclusion ] C. tritaeniorhynchus is the predominant species and PRRSV can survive in this mosquito.