2021年~2022年山东部分地区猪繁殖与呼吸综合征病毒的遗传进化分析

Genetic evolution analysis of PRRSV in some areas of Shandong Province from 2021 to 2022

为了解山东部分地区PRRSV的分子流行及遗传进化情况,本研究对2021年~2022年采自山东部分地区的214份疑似PRRSV感染猪的临床样品通过RT-PCR检测,并经PCR扩增PRRSV阳性样品的ORF5基因和Nsp2基因,采用MegAlign和MEGA X软件对这两个基因进行同源性、遗传进化及其编码的关键氨基酸位点突变及分析。结果显示,214份临床样品中检出51份PRRSV阳性样品,检出率为23.83%。表明山东部分地区PRRSV的感染较为严重。经PCR扩增并测序得到26条ORF5基因序列和20条Nsp2基因序列。26条ORF5基因序列之间的同源性为98.7%~77.8%,与6株PRRSV参考株(经典株VR2332、Ch-1a、疫苗株JXA1、QYYZ及流行株NADC30、NADC34)相应基因序列的同源性在80.1%~98.7%,其中210412等17株PRRSV ORF5基因序列与NADC30株ORF5基因序列的同源性为80.1%~91.9%;进化树结果显示,26条ORF5基因分布于4个谱系中,分别为Lineage1 (220406等22株PRRSV ORF5基因)、Lineage3 (220714和211111株)、Sub Lineage8.7 (HP-PRRSV-Like)(210806株)、Sub Lineage5.1(VR2332-Like)(210629株)谱系;20条Nsp2基因主要分布于3个谱系中,其中220110等13株PRRSV Nsp2基因位于Sub Lineage1.8 (NADC30-Like)谱系,210629株位于Lineage5谱系,与VR2332株遗传距离较近;220714等6株PRRSV Nsp2基因位于Lineage8谱系。上述结果表明,山东部分地区流行的主要为PRRSV NADC30株,属于PRRSV-2,且流行的PRRSV的谱系较复杂,主要以Lineage1、Lineage8及Sub Lineage1.8谱系为主。关键氨基酸位点变异分析结果显示,仅Sub Lineage1.8谱系中的211221和220109株GP5氨基酸序列表现为强毒株特征R^(13)及R^(151);210806株与RespPRRS MLV均为A^(137);除210806株在非中和表位(aa180~aa197)处的变化较为活跃外,其余GP5氨基酸序列与参考株GP5氨基酸序列的变异特征基本一致;在T细胞表位(aa117~aa131)中,Sub Lineage1.8谱系中的多数流行株(包括本研究中的211110等12株)均出现L^(120)S、L^(120)F或I^(125)F、I^(125)L变异。210629株Nsp2氨基酸序列与以VR2332株为代表的Lineage5谱系的相应氨基酸序列高度一致,仅存在少数氨基酸的变异;220114等5株PRRSV Nsp2氨基酸序列在aa481、aa532~aa560处分别缺失1+29个氨基酸,与Sub Lineage8.7谱系缺失特征一致,尤其是210806株除在aa532~aa560缺失29个氨基酸外,在aa476~aa512还缺失37个氨基酸;220110等13株PRRSV Nsp2氨基酸序列在aa323~aa433、aa483和aa504~aa522处分别缺失111+1+19个氨基酸,与Sub Lineage1.8谱系缺失特征一致。上述结果表明,各谱系PRRSV GP5氨基酸序列之间存在明显差异,同一谱系PRRSV GP5氨基酸序列与参考株相应氨基酸序列有相似性,但也存在一定差异,其中以Sub Lineage1.8 GP5氨基酸的变异最为活跃。20条Nsp2氨基酸序列与参考株Nsp2氨基酸序列具有一定相似性,但也存在部分的缺失和变异。也表明,目前山东部分地区PRRSV的流行情况较为复杂,仍以NADC30-Like PRRSV为优势流行株,对PRRS的防控形式仍很严峻。本研究为了解山东部分地区PRRSV流行变异情况及PRRS的防控提供参考依据。

To understand the molecular epidemiology and genetic evolution of PRRSV in some areas of Shandong Province,214 clinical samples suspected to be infected with PRRSV collected there in 2021-2022 were detected by RT-PCR,and the ORF5 and Nsp2 genes of PRRSV were amplified in positive samples by PCR.The resulting sequences were analyzed for the homology,genetic evolution and key amino acid site mutation with the MegAlign and MEGA X sofiwares.The results showed that 51 of 214 clinical samples were detected as PRRSV positive samples and the positive detection rate was 23.83%.It shows that there is PRRSV infection in some areas of Shandong.Twenty-six ORF5 gene sequences and 20 Nsp2 gene sequences were obtained by PCR amplification and sequencing.The homology is 98.7%-77.8%among the 26 ORF5 gene sequences and it is 80.1%-98.7%comparing with the corresponding gene sequences of six PRRSV reference strains(classic strain VR2332,Ch-la,vaccine strain JXA1,QYYZ and epidemic strains NADC30 and NADC34)among which 17 PRRSV ORF5 gene sequences like 210412 strain share 80.1%-91.9%of homology with those of NADC30 PRRSV reference strains.The phylogenetic tree analysis showed that 26 ORF5 genes were distributed in four lineages,namely Lineagel(22 PRRSV ORF5 genes such as 220406 strain),Lineage3(220714 and 21111 strain),Sublineare8.7(HP-PRRSV-Like)(210806 strain)and Sublineare5.1(VR 2332-Like)(210629 strain).Twenty Nsp2 genes are mainly distributed in three lineages,among which 13 PRRSV Nsp2 genes,such as 220110 strain,are located in SubLineagel.8(NADC30-Like)pedigree;210629 strain is located in Lineage5 pedigree,which is close to VR2332.Six PRRSV Nsp2 genes,such as 220714 strain,are located in Lineage8 pedigree.The above results show that PRRSV NADC30 strain belongs to PRRSV-2,and the pedigree of PRRSV is complex,mainly including Lineagel,Lineage8 and SubLineagel.8.The results of mutation analysis of key amino acid sites showed that only 211221 and 220109 strains of PRRSV GP5 protein in SubLineage1.8(NADC30-Like)pedigree showed the characteristics of R^(13)and R^(151);210806 strain is A^(137)just like RespPRRS MLV;all the amino acid sequence of GP5 protein is basically the same as that of reference strains in non-neutralizing epitopes(aa180-aa197)except for 210806 strain,which is more active.Among the T cell epitopes(aal17-aal31),most of the epidemic strains in SubLineagel.8 pedigree(including 12 strains such as 211110 in this study)have L^(120)S,L^(120)F or I^(125)F,I^(125)L mutations.The amino acid sequence of Nsp2 in 210629 strain is highly consistent with the corresponding that of Lineage5 pedigree represented by VR2332,but with a few amino acid variations.The amino acid sequences of Nsp2 in five PRRSV,such as 220114 strain,were deleted by 1+29 amino acids at a481,aa532-aa560,respectively,which was consistent with the deletion characteristics of SubLineage8.7(HP-PRRSVLike).Especially,there were 37 amino acids deletion at aa476-aa512 in strain 210806,as well as 29 amino acids deletion at aa532-aa560.The amino acid sequences of Nsp2 in 13 PRRSV,such as 220110 strain,were deleted by 111+1+19 amino acids at aa323-aa433,aa483 and aa504-aa522,respectively,which was consistent with the deletion characteristics of SubLineagel.8 pedigree.The above results showed that there were obvious differences among the amino acid sequences of PRRSV GP5 protein in different lineages,and these sequences in the same lineages were similar to those of reference strains,but there were also some differences,among which the amino acid mutation of Sublineagel.8 ORF5 was the most active.The amino acid sequences of 20 Nsp2 are similar to those of the reference strain,but there are also some deletions and variations.The above results show that the epidemic situation of PRRSV in some areas of Shandong is complicated,and NADC30-Like PRRSV is still the dominant epidemic strain,and the situation of prevention and control of PRRS is still very severe.This study provides a reference for understanding the epidemic variation of PRRSV in some areas of Shandong Province and the prevention and control of PRRS.