2010–2015年华东地区猪圆环病毒2型的分子流行病学分析

Molecular epidemiological analysis of porcine circovirus type 2 in East China from 2010 to 2015

摘要:

[目的] 猪圆环病毒2型（PCV2）是严重危害世界养猪业的重要传染病，即猪圆环病毒相关疾病（PCVAD）的重要病原，其致病机制及流行规律尚未阐明。本研究对2010-2015年间采集自中国华东地区的病料进行PCV2检测，以探讨中国华东地区PCV2的分子流行病学特征，分析PCV2的遗传演化规律。[方法] 本研究利用PCR方法对384份断奶仔猪多系统衰竭综合征疑似发病猪样本进行检测，并对随机选取的42份阳性样品进行PCV2全基因组的测定和分析。[结果] 华东地区普遍存在PCV2的感染，阳性率为41.15%。序列扩增获得42株PCV2全长基因组，同源性和系统进化树分析表明，基于PCV2 ORF2和全长核苷酸序列构建的系统进化树结构是基本一致的。从病料中测得的42株PCV2与11株参考毒株序列的ORF2基因的核苷酸和氨基酸序列同源性分别为87.0%-100.0%和82.5%-100.0%。遗传进化分析显示，53株PCV2毒株可分为4个基因型，即PCV2a、PCV2b、PCV2c和PCV2d。本文获得的42株序列ORF2基因的核苷酸和氨基酸序列同源性分别为89.6%-100.0%和86.8%-100%，可分为3个基因型，即PCV2a、PCV2b和PCV2d，其中69.05%（29/42）属于PCV2d基因型，为优势基因型；PCV2b未在安徽和浙江出现。Cap蛋白的氨基酸序列分析表明，42株PCV2毒株总体多样性较低，存在4个主要的高变区，且不同基因型具有代表性突变位点。本研究获得的42株Cap序列在抗体识别的关键位点存在一定程度变异，这是否导致PCV2抗原结构、致病性以及疫苗效果的改变，还有待深入分析。[结论] 2010-2015年间，PCV2在华东地区猪群中存在较高的感染率，遗传演化相对稳定，基因型无明显地域差异，且PCV2d为优势基因型。42株PCV2 Cap序列有明显差异，但同一基因型具有代表性突变位点。本研究为探讨华东地区PCV2的遗传进化和致病机理提供了参考依据。

关键词:猪圆环病毒2型;ORF2基因;分子流行病学;遗传变异

Abstract:

[Objective] Porcine circovirus type 2 (PCV2) is one of the major etiological agents of porcine circovirus-associated diseases (PCVAD) and could cause severe economic loss to the swine industry worldwide. However, the mechanism of pathogenesis, epidemiology and molecular genetic evolution are not fully understood. Samples obtained from East China between 2010 and 2015 were detected for PCV2. In addition, 42 of 158 PCV2 positive samples were used for the whole genome amplification and sequencing to explore the genetic relationship, molecule epidemiology and genetic evolution of PCV2. [Methods] All of 384 field samples from the clinical diseased pigs between 2010 and 2015 in Jiangsu, Anhui and Zhejiang provinces of China, were screened by specific PCR to detect PCV2. The whole genome of PCV2 positive samples selected randomly was sequenced and analyzed. [Results] PCV2 is distributed widely among swine populations in East China and 158 clinical samples were positive for PCV2, indicating that the prevalence of PCV2 infection in pigs was about 41.15%. The phylogenetic tree constructed by the neighbor-joining method based on the whole genome or ORF2 gene was highly similar. The nucleotide homology and amino acid homology between the ORF2 gene of 42 isolates and 11 reference strains was between 87% to 100% and 82.5% to 100%, respectively. Phylogenetic analysis of the ORF2 genome showed that 53 strains were presented in 4 sub-genotypes (PCV2a, PCV2b, PCV2c and PCV2d). Based on comparing the nucleotides and amino acid sequences of ORF2, 42 isolates obtained in this study shared 89.6% to 100% and 86.8% to 100% identity, respectively. Genetic evolution analysis indicated that 42 isolates could be divided into three genotypes as PCV2a, PCV2b and PCV2d (69.05%), and PCV2b did not appear in Anhui and Zhejiang. Among the three genotypes, PCV2d was the predominant genotype circulating in the swine population of three provinces. Comparisons of the amino acid sequence of Cap protein revealed that 42 isolates in this study have a low variation, four major hypervariable genome regions and special substitutions among the different PCV2 genotypes. There were some aa changes in the reported antibody epitope regions of the Cap protein. However, whether those mutations could result in antigenic variations or change the pathogenicity of PCV2 and the vaccine effect should be explored further. [Conclusion] The results demonstrated that genetic evolution of PCV2 was relatively stable and PCV2b genotype was dominant in the porcine population in East China from 2010 to 2015. According to amino acid alignment data of Cap protein, different substitutions were observed in different genotypes and some representative substitutions exist in each genotype. This study provides a reference for investigating the genetic relationship, genetic evolution and pathogenesis of PCV2 in East China.

Key words:porcine circovirus type 2;ORF2 gene;molecular epidemiology;genetic variation