两株田间重组猪繁殖与呼吸综合征病毒的分离鉴定、进化分析及其致病性研究

Isolation,Identification and Evolution Analysis of Two Recombinant PRRSV Strains in Fields and Their Pathogenicity to Piglets

为了解2型猪繁殖与呼吸综合征病毒（PRRSV2）田间毒株重组特征,本研究对两株分离物（HENXX-8、HENJY-2）进行全基因测序、进化分析及毒力测定,并对其全基因序列进行重组分析。结果显示,两个毒株均属于PRRSV 2,两者与其代表株VR-2332的核苷酸相似性分别为85.6%、85.7%;与高致病性PRRSV（HPPRRSV）毒株JXA1、TJ和WUH4株分别为85.7%-85.8%、85.4%-85.5%;与经典毒株CH-1a、HB-2（sh）分别为84.7%-85.7%、84.5%-85.7%;而与所有NADC30类毒株均在90.0%以上。全基因、ORF5及Nsp2序列进化分析结果均显示两个分离物与国内报道的类NADC30毒株遗传距离较近,同处于一个分支。全基因组重组分析结果表明,两个分离毒株存在明显重组现象,且重组模式均以类NADC30毒株为骨架病毒,与HP-PRRSV毒株发生重组。但重组对象不同：HENJY-2是由类NADC30毒株与TJ株发生重组;而HENXX-8则是由类NADC30毒株与WUH4、TJ株发生3毒株间重组。由于重组部位序列缺乏特异性分子标志,因此无法确定与类NADC30发生重组的是HP-PRRSV还是其减毒的疫苗毒株。两株分离物的重组部位与早期分离毒株HENANHEB、JL580等有所不同,均未涉及到ORF5基因,而是集中在Nsp1-Nsp2以及ORF2a-ORF3区域,主要发生在病毒基因组的靠5#端（30-7 000bp）和3#端（11 000-13 000bp）处。对部分重要基因的核苷酸相似性分析结果显示,两个分离株的ORF2a、ORF3基因与HP-PRRSV毒株相似性最高,表明重组病毒的这两个基因可能来自HPPRRSV毒株。致病性试验结果表明,参考毒株HENXC-4的毒力略高于分离毒株HENXX-8,主要表现在体温升高、日均增重降低和肺部病变上。但两个分离物均未引起发病猪死亡。以上结果表明,目前PRRSV2在田间的基因重组事件存在随机性,提示不同毒株在田间的存在会加剧PRRSV重组事件的发生和流行、毒株类型更加复杂,从而增加了临床防控难度。因此,慎重使用活疫苗并持续监测活疫苗毒株在田间的变异动态,对更好防控本病具有一定的临床指导意义。

In order to understand the recombination characteristics of field porcine reproductive and respiratory syndrome virus（PRRSV）,the whole genome of PRRSV isolates（HENXX-8 and HENJY-2）was sequenced and analyzed for genetic evolution,virulence and recombination.The result showed that HENXX-8 and HENJY-2 belonged to the North American genotype（species PRRSV2）,and shared 85.6% and85.7% nucleotide identity with the reference strain VR-2332,respectively.Compared to the Chinese strains,HENXX-8 and HENJY-2 shared 85.7%-85.8% and 85.4%-85.5% nucleotide identities with the highly pathogenic PRRSV（HP-PRRSV）strains JXA1,TJ and WUH4,and 84.7%-85.7% and84.5%-85.7% with the Chinese classical PRRSV strains CH-1 aand HB-2（sh）,respectively,while90.0% with all reference NADC30-like strains.Complete genomic sequence alignment and phylogenetic analysis revealed that HENXX-8 and HENJY-2 are most closely related to the NADC30-like strains recently reported in China and they were in the same subgroup.Recombination analysis indicated that the two isolates had a similar recombination model with NADC30-like strain as skeleton virus recombined with ORF2 a and ORF3 genes of HP-PRRSV.The major difference between two recombinants is that HENJY-8 was recombined by two strains NADC30-like and TJ,while HENXX-8 was recombined by three strains NADC30-like,WHU4 and TJ.Since the sequence at the recombination site lacking specific molecular markers,so it was difficult to distinguish whether ORF2 aor ORF3 genes in two recombinants were from HP-PRRSV or its attenuated live vaccine strain.The recombination sites of the two isolates were different from those of the earlier isolates HENAN-HEB and JL580,etc,and were focused in the Nsp1-Nsp2 and ORF2 a-ORF3 regions mainly occurred in the 5′（30-7,000 bp）and 3′（11,000-13,000 bp）of the viral genome.Based on the nucleotide similarity analysis of some important genes ORF2 aand ORF3 of PRRSV,the two isolates had the highest similarity with HP-PRRSV,which indicated that the recombinant gene might be from the HP-PRRSV strain.The pathogenicity of the two isolates was slightly different and the virulence of HENXC-4 was slightly higher than that of HENXX-8.The pathogenicity mainly represented in increased body temperature,lower average daily weight gain and lung lesions.But neither isolate caused the death of diseased pigs.The above result indicated that the recombination of PRRSV in the field herds was random and that the presence of different strains in the field would benefit the occurrence and prevalence of PRRSV recombination events.Thus the strains would be more complex,resulting in increasing difficulty for disease control.Therefore,using live vaccines carefully,and continuously monitoring the evolution of live vaccine strain in the field may benefit the control of PRRSV.