我国近5年类NADC30 PRRSV毒株序列的重组及限制性片段长度多态性分析

NADC30-like PRRSV in China of 2016 to 2020:recombination analysis and restriction fragment length polymorphisms

为了解我国类NADC30猪繁殖与呼吸综合征病毒(PRRSV)毒株的流行和遗传变异情况,本研究利用生物信息学软件对本实验室2016-2020年获得的64条类NADC30毒株序列进行分析。Nsp2氨基酸序列比对结果表明,64株毒株均表现出与NADC30毒株一致的131个不连续的氨基酸缺失。其中,9株在Nsp2区域除该缺失特征外,另含有额外的氨基酸缺失。基于全基因组遗传演化分析结果表明,61株分布在Sublineage 1.8(类NADC30),3株分布在Lineage 8(HP-PRRSV)。而基于ORF5序列的遗传演化分析结果则更分散,56株分布在SubLineage 1.8,5株分布在Lineage 8,2株分布在Lineage 5(类VR2332),1株分布在Sublineage 1.5(类NADC34)。经RFP4和Simplot生物学软件进行重组分析结果表明,64株毒株中可能有44株为重组毒株,共表现出5种重组模式,其中以NADC30提供骨架,HP-PRRSV提供重组片段为主要模式。重组热点分布在非结构蛋白区域5′UTR~1500 nt(Nsp1)、5374~8177 nt(Nsp4~Nsp9)和结构蛋白区域12100~13279 nt(ORF2~ORF4)。RFLP结果表明,64条ORF5序列共存在12种RFLP模式,其中与美国高致病性变异株PRRSV RFLP1-4-4 Lineage1C相同的模式RFLP 1-4-4数量最多,共31株。综上所述,本研究为了解我国类NADC30毒株的遗传演化、重组分析和防控提供借鉴意义。

For the investigation into epidemic and genetic variation of NADC30-Like PRRSV strain,64 sequences of NADC30-like strain were analyzed by using bioinformatic software which had been identified and isolated in our laboratory from 2016-2020 in this study.The comparison between amino acid sequences of Nsp2 showed that the 131 amino acids noncontinuous deletion of all 64 strains were consistent with the deletion of NADC30 strain.Among them,9 strains also exhibited the additional deletion besides the above characteristic deletion in Nsp2 region.Results of genetic evolution analysis based on the whole genome showed that 61 strains belonged to Sublineage 1.8 and 3 strains belonged to Lineage8.Results of genetic evolution analysis of the ORF5 sequence were dispersed which showed that 56 strains belonged to Lineage 1.8,5 strains belonged to Sublineage 8,2 strains belonged to Lineage 5 and 1 strain belonged to Sublineage 1.5.Results of recombination analysis made by RFP4 and Simplot bioinformatics software indicated that 44 strains among 64 strains may be recombinant strain of the virus with five recombinant patterns,of which the main pattern was that NADC30acted as backbone and HP-PRRSV provided recombination fraction.Recombination hotspots located in the regions ofnonstructural proteins,5′UTR-1500nt(Nsp1)and 5374-8177nt(Nsp4-Nsp9)and of structural proteins,12100-13279nt(ORF2-ORF4).Results of RFLP showed that there were 12types of patterns in total of 64 ORF5 sequences among which RFLP 1-4-4 pattern was the most common pattern(the patterns of 31sequences were this type)which was the same as the pattern of American highly pathogenic variant PRRSV RFLP 1-4-4 Lineage1C.In summary,the results of this study can be used for reference of the investigation into genetic evolution,recombination analysis and prevention and control of NADC30-like in China.