北京地区1株鸽圆环病毒全基因组测定及遗传进化分析

Genome Determination and Genetic Evolution Analysis of a Pigeon Circovirus Strain in Beijing

【目的】了解北京地区流行的鸽圆环病毒(Pigeon circovirus,PiCV)的基因组特征及变异规律。【方法】以3只发病鸽的肝脏和脾脏组织为模板,采用PCR技术检测病原。以检测阳性的肝脏组织DNA为模板,应用PCR技术分段扩增PiCV的全基因序列。应用DNAStar和Mega 7.0软件对扩增得到的全序列进行拼接和核苷酸序列比对,并构建系统进化树,对病毒基因组的2个开放阅读框分别进行核苷酸和氨基酸序列比对,并构建系统进化树。【结果】经PCR检测,3只病鸽中有1只病鸽的组织中检测到PiCV阳性,并未检出其他病毒。采用PCR分段扩增成功获得了1株PiCV的全基因组序列,命名为PiCV BJ,该病毒基因组大小为2034 bp,包含有2个主要的开放阅读框(ORFs),ORF-V1编码Rep蛋白,ORF-C1编码Cap蛋白。相似性比对结果显示,PiCV BJ株基因组序列与GenBank上登录的其他参考序列的核苷酸相似性在86.0%~97.0%之间,与2011年分离自波兰的PL53相似性为97.0%。遗传进化结果显示,PiCV BJ株与2014年分离自波兰的PL124在同一分支,亲缘关系较近;与其他禽源圆环病毒不在同一分支,亲缘关系较远。PiCV BJ株Cap基因的起始密码子为ATG,与2011年分离自比利时的11-08304株核苷酸、氨基酸序列相似性高达95.8%和85.2%;Rep基因与2011年分离自波兰的PL53相似性最高,核苷酸和氨基酸相似性分别高达94.6%和96.2%;Cap和Rep基因的进化树分析结果也显示,PiCV BJ株均与PL53和11-08304株在同一分支,亲缘关系较近,这与相似性分析的结果一致。【结论】PiCV BJ株来自国外,可能由赛鸽引种传入中国,提示在外部引种时要做好病毒监测。本研究丰富了PiCV的遗传学研究资料,为进一步探究PiCV的遗传变异及传播机制提供了参考依据,也为PiCV的防控提供了重要的理论基础。

【Objective】To understand the genome characteristics and variation of pigeon circovirus(PiCV)circulating in Beijing.【Method】Used the liver and spleen tissues of 3 diseased pigeons as templates,the pathogen was detected by PCR technology.Using the DNA of the positive liver tissue as a template,PCR technology was used to amplify the whole gene sequence of PiCV.DNAStar and Mega 7.0 softwares were used to splice and compare the nucleotide sequence of the amplified sequence,and construct evolutionary tree.The nucleotide sequence and amino acid sequence of the two open reading frames of the virus genome were compared,and the phylogenetic tree was constructed.【Result】The PCR test showed that PiCV positive was detected in the tissues of one of the three sick pigeons,but no other viruses were detected.The whole genome sequence of a PiCV strain was successfully obtained by PCR fragmented amplification,named PiCV BJ.The virus genome was 2034 bp in size and contained 2 main open reading frames(ORFs),ORF-V1 encodes the Rep protein,ORF-C1 encodes the Cap protein.The similarity comparison results showed that the nucleotide similarity between the genome sequence of PiCV BJ strain and other reference sequences registered on GenBank was 86.0%to 97.0%,and the homology with PL53 isolated from Poland in 2011 was 97.0%.The genetic evolution results showed that the PiCV BJ strain was in the same branch as PL124 isolated from Poland in 2014,and was closely related;It was not in the same branch with other avian circoviruses,and was relatively distant.The start codon of Cap gene of PiCV BJ strain was ATG,which had 95.8%and 85.2%nucleotide and amino acid similarity with 11-08304 strain isolated from Belgium in 2011.The Rep gene had 94.6%and 96.2%nucleotide and amino acid similarity with PL53 isolated from Poland in 2011.The similarity of nucleotide and amino acid was 94.6%and 96.2%,respectively.The phylogenetic tree analysis of Cap and Rep genes also showed that PiCV BJ strains was in the same branch as PL53 and 11-08304,which was close to the results of similarity analysis.【Conclusion】PiCV BJ strain was from abroad and might be introduced into China by racing pigeons,which suggested that virus monitoring should be done well in external introduction.This study enriched the genetic research data of PiCV,provided a reference basis for further exploring the genetic variation and transmission mechanism of PiCV,and also provided an important theoretical basis for the prevention and control of PiCV.