一株兔出血症病毒2型毒株的分离鉴定

Isolation and identification of a rabbit hemorrhagic virus type 2 strain

【背景】我国于2020年4月突发兔出血症病毒2型(Rabbit Hemorrhagic Disease Virus 2,RHDV2)疫情,严重威胁兔养殖业和生态平衡,而且目前国内对RHDV2的病原学以及遗传特征等基础研究匮乏。【目的】分离鉴定RHDV2毒株,对分离株进行全基因测序与遗传进化分析。【方法】对成都某兔场疑似RHDV2感染致死的家兔进行病理剖检,通过RT-qPCR检测和动物回归试验,分离鉴定得到RHDV2毒株,进一步进行全基因测序及遗传进化分析。【结果】病死兔剖检表现为各实质器官出血肿大,以心、肺、肝脏尤为明显,经RT-qPCR确诊为RHDV2,而且不存在其他病原混合感染,试验感染家兔可致相似病变。将分离株命名为SCCN03,其基因序列全长为7464 bp,与参考毒株(GenBank登录号为MN901451.1)一致性为99.21%。对比参考株氨基酸序列,分离株的非结构蛋白和结构蛋白氨基酸序列发生了多处错义突变,其中非结构蛋白p16和结构蛋白的几处错义突变可能与毒株变异有关。进化树显示毒株SCCN03属于GI.2基因型。【结论】分离鉴定出一株RHDV2毒株,获得其基因序列,丰富了RHDV2的全基因数据资料,为后续RHDV毒力相关研究和相关疫苗研发奠定了基础。

[Background]Rabbit hemorrhagic disease virus 2(RHDV2)was found in April 2020 in China,which seriously threatened the rabbit breeding industry and ecological balance.Besides,basic researches on the etiology and genetic characteristics of RHDV2 are still lacking in China.[Objective]To isolate and identify RHDV2 strain,and to conduct whole gene sequencing and genetic evolution analysis of the isolated strain.[Methods]We planned to conduct pathological dissection,RT-qPCR detection,and animal regression test to isolate and identify the RHDV2 strain in a rabbit farm suspected to have died from RHDV2 infection.[Results]Further whole gene sequencing and genetic evolution analysis were performed.The necropsies of dead rabbits showed hemorrhage and enlargement of all parenchymal organs,especially the heart,lung and liver.RHDV2 was confirmed by RT-qPCR,and there was no mixed infection with other pathogens.We found the infected rabbits could cause similar lesions.The isolates were named SCCN03,and the gene sequences is 7464 bp in length,which was about 99.21%consistent with the reference strain(GenBank accession number:MN901451.1).By comparing the amino acid sequence of the reference strain,many missense mutations occurred in the amino acid sequence of the non-structural proteins and structural proteins in the isolated strain,among which several missense mutations of the non-structural proteins P16 and structural proteins may be related to the variation of the virus strain.The evolutionary tree showed that SCCN03 belonged to GI.2 genotype.[Conclusion]One strain of RHDV2 was isolated and identified,and its gene sequence was obtained,which enriched the whole genetic data of RHDV2 and laid a foundation for subsequent studies on virulence of RHDV and related vaccine development.