黑龙江地区鲤春病毒血症病毒的分离与基因型分析
发布时间：2019-04-27 20:48
【摘要】：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。
[Abstract]:Cell culture, PCR identification, viral titer determination and surface glycoprotein (glycoprotein,) of Cyprinus Carpio (Cyprinus carpio) from 40 farms in different areas of Heilongjiang Province from 2015 to 2016 were carried out by culture and isolation of Cyprinus Carpio viremia virus (spring viremia of carp virus,SVCV), PCR identification, viral titer determination and surface glycoprotein (glycoprotein,). G) amino acid sequence cluster analysis and genotyping. The results of cell culture showed that the common carp tissue samples from four different farms could infect common carp epithelial cells (epithelioma papulosum cyprini,EPC) to produce typical cytopathic effects (cytopathic effect,CPE). The results of Shlj1~Shlj4.PCR identification showed that the virus suspensions were collected respectively. The four strains were all SVCV.. The viral titers of SVCV Shlj 1~Shlj-4 were determined to be 106.28,106.88,107.57and 106.38 TCID50/mL.Shlj respectively by cluster analysis and genetic evolution analysis. Shlj 1~Shlj 4 was clustered with Chinese reference strain A2, BJ0505 / 2 and American reference strain USA,212364, and the homology was 98.4% and 99.8%, respectively, compared with that of Chinese reference strain A2, BJ0505 / 2 and American reference strain USA,212364. The nucleotide sequence similarity of glycoprotein among Shlj 1~Shlj 4 strains was 98.6% and 99.8% respectively. Among them, Shlj 3 had the highest nucleotide similarity with American SCVC strain USA,212364 (99.8%). The lowest similarity was found between Shlj 2 and British reference strain 880163 (88.0%). The results of amino acid sequence alignment showed that amino acid mutation was the most in Shlj4, which was significantly different from that of the other three strains. Genotype analysis showed that all Shlj 1~Shlj-4 were genotype Ia. The results showed that the detection rate of SVCV in Heilongjiang region from 2015 to 2016 was about 10%, and the nucleic acid sequences of virus isolates from different farms showed different degrees of difference. The results further demonstrate that the SVCV strain is evolving continuously in different culture environments of Cyprinus Carpio in China.
【作者单位】： 中国水产科学研究院黑龙江水产研究所;上海海洋大学水产与生命学院;
【基金】：黑龙江省应用技术研究与开发计划项目(GA13B401)
【分类号】：S943
本文编号：2467299
[Abstract]:Cell culture, PCR identification, viral titer determination and surface glycoprotein (glycoprotein,) of Cyprinus Carpio (Cyprinus carpio) from 40 farms in different areas of Heilongjiang Province from 2015 to 2016 were carried out by culture and isolation of Cyprinus Carpio viremia virus (spring viremia of carp virus,SVCV), PCR identification, viral titer determination and surface glycoprotein (glycoprotein,). G) amino acid sequence cluster analysis and genotyping. The results of cell culture showed that the common carp tissue samples from four different farms could infect common carp epithelial cells (epithelioma papulosum cyprini,EPC) to produce typical cytopathic effects (cytopathic effect,CPE). The results of Shlj1~Shlj4.PCR identification showed that the virus suspensions were collected respectively. The four strains were all SVCV.. The viral titers of SVCV Shlj 1~Shlj-4 were determined to be 106.28,106.88,107.57and 106.38 TCID50/mL.Shlj respectively by cluster analysis and genetic evolution analysis. Shlj 1~Shlj 4 was clustered with Chinese reference strain A2, BJ0505 / 2 and American reference strain USA,212364, and the homology was 98.4% and 99.8%, respectively, compared with that of Chinese reference strain A2, BJ0505 / 2 and American reference strain USA,212364. The nucleotide sequence similarity of glycoprotein among Shlj 1~Shlj 4 strains was 98.6% and 99.8% respectively. Among them, Shlj 3 had the highest nucleotide similarity with American SCVC strain USA,212364 (99.8%). The lowest similarity was found between Shlj 2 and British reference strain 880163 (88.0%). The results of amino acid sequence alignment showed that amino acid mutation was the most in Shlj4, which was significantly different from that of the other three strains. Genotype analysis showed that all Shlj 1~Shlj-4 were genotype Ia. The results showed that the detection rate of SVCV in Heilongjiang region from 2015 to 2016 was about 10%, and the nucleic acid sequences of virus isolates from different farms showed different degrees of difference. The results further demonstrate that the SVCV strain is evolving continuously in different culture environments of Cyprinus Carpio in China.
【作者单位】： 中国水产科学研究院黑龙江水产研究所;上海海洋大学水产与生命学院;
【基金】：黑龙江省应用技术研究与开发计划项目(GA13B401)
【分类号】：S943
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