云南省部分地区猪圆环病毒2型的分离鉴定与进化分析

Isolation, identification and evolution analysis of Porcine circovirus 2 in partial areas of Yunnan Province

为了了解云南省部分地区猪圆环病毒2型(PCV-2)的基因型,试验采用PCR方法检测了从养殖场采集的临床症状表现为疑似断奶仔猪多系统衰竭综合征(PMWS)的组织病料,阳性病料经无菌处理后接种于PK-15细胞进行病毒分离培养,病毒液经PCR扩增、克隆和测序后进行间接免疫荧光检测,利用DNAStar软件比较分析全基因和ORF2基因序列,用MEGA 6.0软件绘制遗传进化树,MegAlign软件比较分析氨基酸序列,按照Karber法计算TCID_(50)。结果表明:试验成功分离到2株分离株,分别命名为DH-202005和WS-202009,2株分离株的目的片段大小约为549 bp,全基因大小约为1767 bp,感染2株分离株的PK-15细胞均可观察到特异性绿色荧光,2株分离株的全基因序列同源性为99.9%,与GenBank中不同国家和地区的22株PCV-2分离株的同源性为92.6%~99.8%,与疫苗株SH(HM038027)和LG(HM038034)的同源性分别为95.6%和95.5%,2株分离株的ORF2基因序列的同源性为99.9%,与GenBank中不同国家和地区的22株PCV-2分离株的同源性为84.0%~100%,与疫苗株SH(HM038027)同源性分别为94.0%和93.9%,与疫苗株LG(HM038034)的同源性分别为92.0%和91.9%,2株分离株在同一遗传进化分支上,与YN/QuJing-2018(MK405699)的遗传关系最近,都属于PCV-2d基因型,2株分离株与其他PCV-2d基因型毒株ORF2的氨基酸序列同源性较高,均编码234个氨基酸,与PCV-2a、PCV-2b基因型相比,在第234位多一个赖氨酸(K)残基,位于第86~91位点上的SNPLTV和第190~191/206/210位点上的TGID氨基酸序列都是典型的PCV-2d基因型序列,2株分离株第10代病毒的TCID_(50)分别为1×10^(5.0) TCID_(50)/0.1 mL和1×10^(4.5) TCID_(50)/0.1 mL。说明云南省存在PCV-2d基因型的流行,且分离株与国内外其他PCV-2毒株遗传关系较近。

In order to understand the genotype of Porcine circovirus 2(PCV-2)in partial areas of Yunnan Province,PCR was used to detect tissue samples collected from farms with clinical symptoms suspected to be multisystem failure syndrome(PMWS)of weaned piglets.The positive diseased materials were aseptically treated and then inoculated with PK-15 cells for virus isolation and culture.The virus liquid underwent indirect immunofluorescence detection after PCR amplification,cloning and sequencing.DNAStar software was used to compare and analyze gene sequences.MEGA 6.0 software plotted the genetic evolution tree.MegAlign software compared and analyzed the amino acid sequences.TCID_(50) was calculated by Karber method.The results showed that two strains were successfully isolated,named DH-202005 and WS-202009,respectively.The ORF2 gene fragment size of the two strains was about 549 bp,and the full length of the genes of the two isolates was about 1767 bp.Specific green fluorescence was observed in PK-15 cells infected with both isolated strains.The homology of the whole gene sequences of the two isolates strains was 99.9%.The homology was 92.6%-99.8%with 22 strains of PCV-2 from different countries and regions in GenBank,and 95.6%with vaccine strain SH(HM038027)and 95.5%with vaccine strain LG(HM038034),respectively.The homology of the ORF2 gene sequence of the two isolates strains was 99.9%,and 84.0%-100%with 22 strains of PCV-2 from different countries and regions in GenBank,and 94.0%and 93.9%with vaccine strain SH(HM038027),respectively;and 92.0%and 91.9%with vaccine strain LG(HM038034),respectively.The two strains were on the same genetic evolution branch,closest to YN/Qujing-2018(MK405699)strain,belonging to PCV-2 d type.The amino acid sequences of ORF2 of the two strains are highly homologous with other PCV-2 d strains,and both of them encoded 234 amino acids.Compared with PCV-2 a and PCV-2 b,there was one more lysine(K)residue at the 234 th position.SNPLTV at 86-91 position and TGID at 190-191/206/210 were typical PCV-2 d subtype sequences.The TCID_(50) of the 10 th generation of the two isolates were 1×10^(5.0) TCID_(50)/0.1 mL and 1×10^(4.5) TCID_(50)/0.1 mL,respectively.The results indicated that PCV-2 d was prevalent in Yunnan Province,and the genetic relationship between the isolated strain and other PCV-2 strains in the domestic and overseas was close.