伪狂犬病病毒贵州分离株与疫苗株遗传进化和抗原表位分析

Phylogenetic Evolution and Epitope Analysis of PseudorabiesVirus Guizhou Isolates and Vaccine Strains

【目的】了解贵州省猪群伪狂犬病病毒(Pseudorabies virus,PRV)毒株与疫苗株的分子特征,探究贵州省PRV变异株的遗传演化情况并对比其与疫苗株抗原差异性。【方法】通过分子克隆和生物信息学技术对本实验室在2015-2021年间贵州省分离到的5株PRV毒株的gB、gC、gD基因和商品化疫苗株(C株和HB2000株)进行克隆测序,利用本实验室序列库中的2株PRV贵州株及GenBank上登录的疫苗毒株序列,进行系统遗传进化和B细胞线性抗原表位差异性分析。【结果】结果显示,7株贵州株与疫苗株gB、gC、gD全基因组的核苷酸序列相似性分别为98.1%~100%、94.7%~100%和98.8%~100%,7株贵州株与疫苗株gB、gC、gD全基因组的氨基酸序列相似性分别为96.4%~100%、90.9%~100%和97.0%~100%;氨基酸替换分析结果显示,7株PRV贵州株和疫苗毒株间gB、gC、gD基因存在56、68和23处氨基酸位点差异,其中gC基因氨基酸位点差异最大;GZQX2017株缺失gE基因,在遗传进化树中GZQX2017株与Bartha-K61、Becker和NIA3等国外株位于同一大进化分支,属于PRVⅠ型;其余6株贵州株gE基因在第48和496位各插入1个天冬氨酸(D),符合流行变异株的基因特征,与国内流行PRV变异株位于另一大进化分支,属于PRVⅡ型;以GZQX2017株gB、gC、gD全基因作为目标基因,以Bartha-K61株作为主要亲本、Fa株作为次要亲本,gB、gC全基因均能检测到重组信号,gD全基因重组信号不明显;相比Bartha-K61株,gB、gC、gD蛋白抗原表位数量和位点存在差异。【结论】7株PRV贵州株中,GZQX2017株与疫苗Bartha-K61株、C株属于PRVⅠ型,GZQX2017株可能是由Bartha-K61株为主要亲本进行重组产生的gE基因自然缺失株,后续有望将其作为候选疫苗株进行进一步研究;其余6株贵州株与Fa株、HB2000株等属于PRVⅡ型,具有流行变异毒株的流行特征。本研究结果对了解中国贵州省流行PRV毒株分子特征具有十分重要的意义。

【Objective】This study was aimed to understand the molecular characteristics of Pseudorabies virus(PRV)strains and vaccine strains in Guizhou province,the genetic evolution of PRV variants in Guizhou province and the antigenic differences between PRV strains and vaccine strains were investigated.【Method】gB,gC,gD genes of 5 PRV strains isolated from Guizhou province from 2015 to 2021 and commercial vaccine strains(C strains and HB2000 strains)were cloned and sequenced by molecular cloning technology and bioinformatics technology.Using the sequences of two PRV Guizhou strains in the sequence library of our laboratory and the vaccine strains registered on GenBank,systematic genetic evolution and differential analysis of B cell linear antigen epitopes were conducted.【Result】The results showed that the genome-wide nucleotide similarities of gB,gC and gD genes of the 7 Guizhou strains and vaccine strains were 98.1%to 100%,94.7%to 100%and 98.8%to 100%,respectively.The genome-wide amino acid similarities of gB,gC and gD genes of the 7 Guizhou strains and vaccine strains were 96.4%to 100%,90.9%to 100%and 97.0%to 100%,respectively.Amino acid replacement analysis showed that there were 56,68 and 23 amino acid sites differences in gB,gC and gD genes between the 7 PRV Guizhou strains and vaccine strains,among which the amino acid sites of gC gene were the most different.GZQX2017 strain lacked gE gene.In the genetic evolution tree,GZQX2017 strain and Bartha-K61,Becker,NIA3 and other foreign strains were located in the same large evolutionary branch,belonging to PRV typeⅠ.The remaining six Guizhou strains inserted an aspartic acid(D)at the 48th and 496th positions respectively in gE gene,which was in line with the genetic characteristics of the epidemic variant,and located in another major evolutionary branch with the domestic epidemic PRV variant,belonging to PRV typeⅡ.When the whole genome of GZQX2017 strain gB,gC and gD genes were used as target genes,Bartha-K61 strain was used as the main parent and Fa strain was used as the secondary parent,both gB and gC genes could detect the recombinant signal,but the recombinant signal of gD gene was not obvious.Compared with Bartha-K61 strain,there were differences in the number and sites of gB,gC and gD protein epitopes.【Conclusion】Among the 7 PRV strains in Guizhou,GZQX2017 belonged to PRV typeⅠwith vaccine Bartha-K61 and C strains.GZQX2017 might be the natural deletion of gE gene produced by recombination with Bartha-K61 as the main parent,which was expected to be used as a candidate vaccine strain for further study.The other 6 strains from Guizhou,Fa strain and HB2000 strain belonged to PRV typeⅡ,which had the epidemic characteristics of epidemic mutant strains.This study was of great significance for understanding the molecular characteristics of PRV strains prevalent in Guizhou province,China.