黑龙江地区鲤春病毒血症病毒的分离与基因型分析

对2015—2016年黑龙江不同地区的40个养殖场送检的鲤(Cyprinus carpio)进行鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的细胞培养分离、PCR鉴定、病毒滴度测定、病毒表面糖蛋白(glycoprotein,G)氨基酸序列聚类分析及基因分型研究。细胞培养结果显示,来自4个不同养殖场的鲤组织样本能够感染鲤上皮细胞(epithelioma papulosum cyprini,EPC)产生典型细胞病变(cytopathic effect,CPE),收集病毒悬液分别称为Shlj1~Shlj4。PCR鉴定结果表明,该4株病毒均为SVCV。病毒滴度实验测算出SVCV Shlj 1~Shlj 4的滴度分别为10~(6.28)、10~(6.88)、10~(7.57)和106.38 TCID50/mL。Shlj的糖蛋白基因核苷酸序列的聚类分析和遗传进化分析结果显示,Shlj 1~Shlj 4与Gen Bank收录的中国参考株A2、BJ0505-2和美国参考株USA、212364聚为一簇,同源性为98.4%~99.8%;Shlj 1~Shlj 4毒株之间的糖蛋白核苷酸序列相似性在98.6%~99.8%,其中Shlj 3与美国SCVC毒株USA、212364具有最高的核苷酸相似性(99.8%),Shlj 2与英国参考毒株880163具有最低的相似性(88.0%)。糖蛋白氨基酸序列比对结果显示,Shlj4中氨基酸突变最多,与另3个毒株差异较大。基因型分析结果显示,Shlj 1~Shlj 4均为基因Ia型。本研究结果表明,黑龙江地区2015—2016年间的SVCV检出率约为10%,并且来源于不同养殖场的病毒分离株的核酸序列呈现不同程度的差异,该结果进一步证明SVCV毒株在中国不同的鲤养殖环境中正在不断地进化。

Isolation and genotype analysis of spring viremia of carp virus strains from Heilongjiang Province in China

Spring viremia of carp virus (SVCV) was routinely isolated and identified from tissues of carp (Cyprinus carpio) from 40 carp farms in Heilongjiang Province by separation in cell culture and identification using polymerase chain reaction.SVCV isolates were subjected to virus titer determination,phylogenetic reconstruction based on their glycoprotein amino acid sequences and genotyping.Cell culture results showed that the isolates from four different farms infected Cyprinus carp epithelial cells and caused a cytopathic effect.The isolated SVCV strains were designated as Shlj 1-Shlj4 and were confirmed as SVCVs by polymerase chain reaction.The titers of SVCV Shlj1-Shlj 4 were 106.28,106.88,107.57,and 106.38 × the 50％ tissue culture infectious dose/mL,respectively.The results of the Shlj glycoprotein gene cluster analysis showed that Shlj 1-Shlj4 clustered with China strains A2 and BJ0505-2 as well as America strains USA and 212364.The nucleotide sequence similarities of Shlj 1-Shlj 4 with those previously reported SVCV strains were 98.4％-99.8％,and the similarities among Shlj 1-Shlj 4 were 98.6％-99.8％.The highest nucleotide sequence similarity (99.8％) was observed between Shlj3 and USA/212364,and Shlj2 had the lowest similarity (88.0％) with England reference strain 880163.The amino acid sequence alignment results showed that,among the four Shlj strains,the glycoproteins of Shlj 4 contained the most mutations.Genotype analysis showed that Shlj 1-Shlj 4 all belonged to genotype Ia.The study showed that the SVCV detection rate was about 10％ in Heilongjiang Province during 2015-2016,and the glycoprotein gene sequences of SVCV strains from different farms varied,indicating that SVCV strains are evolving in the Chinese fish farm environment.