基于全基因组分析2017-2021年福建省猪繁殖与呼吸综合征病毒基因组特征

Genomic Characteristics of Porcine Reproductive and Respiratory Syndrome Virus in Fujian Province from 2017 to 2021 based on Whole Genome

通过对福建地区规模化猪场猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)进行病毒分离及全基因组序列分析,从而了解PRRSV流行情况及基因组特征,为猪场防控PRRS提供理论基础。2017—2021年从福建地区规模化猪场采集疑似感染PRRSV的组织病料和血清,进行病毒分离鉴定,采用RT-PCR方法对PRRSV分离株进行全基因组序列扩增,应用DNAstar 7.0软件进行序列分析,使用MEGA 7.0软件邻近法进行遗传演化分析,并利用生物软件SimPlot 3.51和RDP4.10对PRRSV基因组序列进行重组分析。成功分离得到32株PRRSV-2,其全基因组大小为14938~15439 bp(不包括ployA),32株PRRSV之间基因组核苷酸相似性为82.4%~99.4%,与PRRSV-2代表毒株(VR-2332、JXA1、NADC30、QYYZ)和PRRSV-1代表毒株(LV)之间核苷酸相似性分别为81.4%~99%和59.8%~60.6%。分别基于PRRSV全基因组与ORF5基因构建的遗传进化树对32株PRRSV进行分型,结果显示福建省呈现多种谱系毒株并存,两种分型结果符合率为75%(24/32)。PRRSV基因组中Nsp2和ORF5的变异程度最大,Nsp2蛋白存在不同程度的氨基酸(1~155aa)缺失,GP5蛋白主要中和表位发生广泛的变异。32株PRRSV存在较高的重组概率(27/32)且重组模式复杂多样,重组模式有9种:L1+L8、L1+L3、L8+L5、L8+L3、L1(Sublineage1.8+Sublineage1.5)+L8、L1+L5+L8、L1+L3+L8、L1+L3+L5、L1+L3+L5+L8,其中19株以NADC30-like PRRSV为主要亲本,重组方式主要为L1+L8和L1+L8+L3,重组热点主要分布在Nsp1、Nsp2、Nsp9和ORF3基因。综上,福建省主要流行毒株为以NADC30-like PRRSV为主要亲本的重组毒株,对PRRSV进行分型应当以全基因组结果为准。

To investigate the prevalence and genomic characterization of porcine reproductive and respiratory syndrome virus(PRRSV)in Fujian province and provid a theoretical basis for the prevention and control of PRRS,the complete genome of 32 isolates from Fujian province from 2017 to 2021 were analyzed.Tissues and serum were collected from suspected of being infected with PRRSV in Fujian during 2017-2021 were used for virus isolation and full-length genomics sequences amplification by RT-PCR.Genome analyses were performed using the DNASTAR 7.0 package.Phylogenetic trees were constructed by the MEGA 7.0 software using the neighbor-joining method,the genomic recombination of PRRSV were analyzed by using RDP 4.10 and Simplet SimPlot 3.51 softwares.Thirty-two PRRSV-2 strains were successfully isolated.The full length of 32 isolates were determined to be 14938-15439 bp,excluding the poly(A)tail and shared 81.4%-99%identity with PRRSV-2 representative strains and only 59.8%-60.6%with LV.Genotyped of 32 PRRSVs were tested by genetic evolutionary tree constructed by the whole genome and ORF5 gene,and the results showed 75%agreement.Genomic sequence analysis showed Nsp2 and ORF5 are the most variable protein in PRRSV genome,Nsp2 protein has different degrees of amino acid deletion(1-155 aa)and GP5 had high variability in the primary neutralizing epitope(PNE).Recombination analyses revealed a high recombination probability(27/32)and complex recombination patterns were found in the 32 PRRSV strains,including:L1+L8,L1+L3,L8+L5,L8+L3,L1(Sublineage1.8+Sublineage1.5)+L8,L1+L5+L8,L1+L3+L8,L1+L3+L5,L1+L3+L5+L8.Additionally,PRRSV genomic recombination hotspots mainly distributed in Nsp1,Nsp2,Nsp9 and ORF3 genes.In summary,the epidemiological and evolutionary characteristics of PRRSV indicating the circulating strains in Fujian Province are recombinant strains with NADC30-like PRRSV as the main parent.Genotypes of PRRSV should be based on the results of the full-length genome.