PRRSV HN-08株的分离鉴定及其部分生物学特性研究

Isolation,identification and some biological characteristics of PRRSV HN-08 strain

【目的】对2008年河南省猪呼吸与繁殖综合征病毒(PRRSV)流行株进行分离鉴定,并对其生物学特性进行研究,以了解免疫猪群发病的原因。【方法】从河南省猪场采集发病猪的血清和组织,采用Marc-145细胞对其中的PRRSV进行分离及初步鉴定。应用RT-PCR法对分离株进行扩增,并构建克隆载体,对阳性质粒测序后进行ORF5基因核苷酸及推导氨基酸序列分析,同时对分离毒株进行非免疫猪人工感染试验,确定其致病性。【结果】从发病猪组织和血清中分离到1株PRRSV(HN-08株),其可引起Marc-145细胞产生细胞病变(CPE),可在Marc-145细胞中稳定传代,能被PRRSV高免血清特异性中和;从人工感染发病猪组织和血清中均分离到PRRSV。RT-PCR得到与预期大小相符的特异片段,提交NCBI鉴定为HN-08株,属美洲型PRRSV。【结论】从河南省发病猪体内分离的HN-08株属美洲型PRRSV,对猪具有很强的致病力,可引起Marc-145细胞产生CPE,病毒滴度TCID50为105.1/mL。

[Objective] PRRSV epidemic strains of Henan Province were isolated and identified and its biological characteristics were studied in 2008 to understand the aetiological agent of immunized swine flocks. [Method] PRRSV was isolated and preliminarily identified from tissues and serum samples of pigs collected in Henan using Marc-145 cell. The isolates were amplified by RT PCR,and the clonal plamic were constructed and sequenced,then the nucleotide and deduced amino acid of ORF5 gene sequence were ana- lysed. The artificial infection test of non-immunization swine were carried out to determined the pathoge- nicity of the isolated strain. [Result] A PRRSV strain （HN-08 strain） was isolated from tissues and serum samples of pigs in Henan province,the HN-08 strain was able to induce CPE typical of PRRSV on MARC145 and stably propagated in vitro and it could be recognised by PRRSV-hyperimmune serum. PRRSV was isolated both in tissues and serum samples of artifically infected pigs. A special gene sequence was obtained by RT- PCR,matched with the expected result. The sequence analysis showed that HN-08 strain belongs to the North American type of PRRSV. [Conclusion] These results suggested that HN-08 strain isolated from infected pigs in Henan province belongs to the North American type of PRRSV,and the virus has a high pathogenicity to pigs,which was able to induce CPE typical of PRRSV on MARC-145 and the virus titers TCID50 was 105.1/mL.