2017年~2023年河南省猪圆环病毒2型的流行调查及遗传变异分析

Epidemiological investigation and genetic variation analysis of porcine circovirus type 2 in Henan Province from 2017 to 2023

为了解河南省猪圆环病毒2型(PCV2)的流行及变异情况,本研究采集该地区规模化猪场2017年1月~2023年6月939份表现为繁殖障碍和呼吸道症状的病猪血液或组织样品,采用PCR方法进行PCV2检测。结果显示,PCV2总阳性率为31.42%(295/939);2017年~2022年PCV2阳性率逐年降低,分别为58.65%(61/104)、49.48%(48/97)、28.57%(36/126)、16.15%(31/192)、11.52%(19/165)和7.87%(7/89),而2023年迅速上升,达到56.02%(93/166)。利用PCR扩增29份PCV2阳性样品的全基因组序列并测序,采用Meg Align分析PCV2流行株全基因组序列与Gen Bank中4株PCV2参考株全基因组序列的同源性;采用MEGA 11软件利用NJ法构建PCV2流行株ORF2基因与Gen Bank中24株PCV2参考株ORF2基因的系统发育树;采用Meg Align分析PCV2流行株Cap蛋白氨基酸序列的变异特征;采用RDP4和Sim Plot分析PCV2流行株全基因组的重组特征。全基因组同源性分析结果显示,本研究鉴定的PCV2全基因组序列之间的同源性为95.1%~100%,与PCV2参考株的同源性为93.7%~98.6%,其中与疫苗株PCV2a LG(HM038034)、PCV2b DBN-SX07-2(HM641752)和PCV2d SH(AY686763)的同源性分别为94.8%~96.2%、95.3%~98.6%和96.6%~97.8%,与来自丹麦的代表株PCV2c DK1980PMWSfree株(EU148503)的同源性为93.7%~95.1%。此外,两株2023年的PCV2流行株HN230522和HN231217与参考株的同源性仅为94.5%~97.9%。进化树结果显示,有19株PCV2流行株与PCV2d基因型参考株聚为一个分支,10株与PCV2b基因型参考株聚为一个分支。其中今年出现的PCV2流行株HN230522和HN231217株虽然属于PCV2d基因型,但处于一个相对独立的分支。Cap蛋白氨基酸序列分析结果显示,与疫苗株PCV2a LG(HM038034)、PCV2b DBN-SX07-2(HM641752)和PCV2d SH株(AY686763)相比,部分PCV2流行株在构象表位区(aa47~aa85和aa165~aa200)存在R^(48)H、A^(59)K/R、G^(85)D、P^(151)T、N^(178)S、R^(180)K和G^(197)S的突变,在基因型特异性结构域(aa190~aa191/aa206/aa210)存在K^(206)I的突变,且HN230522株在核定位信号区(NLS)(aa1~aa41)存在特有的V^(30)L突变,HN231217株在基因型特异性结构域(aa89~aa91)存在特有的^(89)RTV^(91)残基。重组分析结果显示,有高达65.52%(19/29)的PCV2流行株存在疑似的重组片段,且重组片段全部位于ORF1中。上述结果表明,今年以来河南省猪场PCV2阳性率快速上升,且流行株出现了较大变异,应加强对PCV2流行动态和遗传变异的监测。本研究为河南省PCV2分子流行病学、疫苗研究提供了参考依据。

In order to study the prevalence and variation of porcine circovirus type 2 (PCV2) in Henan Province,939 pig blood or tissue samples with clinical symptoms of reproductive disorders and respiratory diseases were collected from large-scale pig farms from January 2017 to June 2023,and PCV2 detection was performed by PCR.The results showed that the overall positive rate of PCV2 was31.42%(295/939).And the PCV2 positive rate decreased year by year from 2017 to 2022,which were 58.65%(61/104),49.48%(48/97),28.57%(36/126),16.15%(31/192),11.52%(19/165) and 7.87%(7/89),respectively.However,it increased rapidly in 2023 reaching56.02%(93/166).To further analyze the genetic evolution characteristics of the PCV2 epidemic strains,the whole genome of 29 PCV2positive samples was amplified by PCR and sequenced.Meg Align software was used to analyze the whole genome nucleotide homology between the PCV2 epidemic strains and four representative PCV2 strains in Gen Bank.Meg Align was also used to analyze variation characteristics of the amino acid sequence of the Cap protein of PCV2 epidemic strains.A phylogenetic tree of ORF2 gene of PCV2epidemic strains and 24 PCV2 representative strains in Gen Bank was constructed by MEGA 11 software using NJ method.RDP4 and Sim Plot software were used to analyze the recombination characteristics of the whole genome of PCV2 strains.The results showed that the nucleotide homology was 95.1%-100%between the PCV2 whole genomes identified in this study.In addition,the homology with four PCV2 representative strains was 93.7%-98.6%,among which the homology between the PCV2 strains and vaccine strains PCV2a LG (HM038034),PCV2b DBN-SX07-2 (HM641752) and PCV2d SH (AY686763) was 94.8%-96.2%,95.3%-98.6% and 96.6%-97.8%,respectively,and that between the PCV2 strains and PCV2c DK1980PMWSfree (EU148503) stain from Denmark was 93.7%-95.1%.Besides,two new PCV2 strains from 2023,HN230522 and HN231217,were highly variable and the homology with the four PCV2 reference strains was only 94.5%-97.9%.Phylogenetic tree results showed that 19 PCV2 epidemic strains were clustered into one branch with the PCV2d genotype reference strains,and 10 strains were clustered into one branch with the PCV2b genotype reference strains.Among them,the PCV2 epidemic strains HN230522 and HN231217 belonged to the PCV2d genotype,but were in a relatively independent branch.The amino acid sequence analysis of Cap protein showed that compared with the three PCV2 vaccine strains,PCV2a LG (HM038034),PCV2b DBN-SX07-2 (HM641752) or PCV2d SH (AY686763),some PCV2 epidemic strains had amino acid mutations R^(48)H,A^(59)K/R,G^(85)D,P^(151)T,N^(178)S,R^(180)K and G^(197)S in the conformational epitopes (aa47-aa85 and aa165-aa200),and there was a mutation of K^(206)I in the genotype-specific domain (aa190-aa191/aa206/aa210).Moreover,the HN230522 strain had a unique V^(30)L mutation in the nuclear localization signal region (NLS)(aa1-aa41) and the HN231217 strain had a unique^(89)RTV^(91)residue in the genotype-specific domain (aa89-aa91).Recombinant analysis results revealed that up to 65.52%(19/29) of the PCV2 epidemic strains might have suspected recombinant fragments,and all the recombinant fragments were located in ORF1.These results indicate that the positive rate of PCV2 has increased rapidly in Henan Province since 2023,and the PCV2 epidemic strains have large mutations,which may escape the vaccine-induced immune protection.Thus,the monitoring of the epidemic dynamics and genetic variation of PCV2should be strengthened.This study would help in understanding the molecular epidemiology,vaccine research and effective prevention and control of PCV2 infection in Henan Province.