猪繁殖与呼吸综合征病毒RFLP 1-4-4 L1C株的分离鉴定及其遗传演化分析  

Isolation,identification and epidemiological analysis of RFLP 1-4-4 L1C strain of porcine reproductive and respiratory syndrome virus

作　　者：田笑笑 黄昕怡 夏大松 王海伟[1] 田志军[1] 蔡雪辉[1] 安同庆[1] TIAN Xiao-xiao;HUANG Xin-yi;XIA Da-song;WANG Hai-wei;TIAN Zhi-jun;CAI Xue-hui;AN Tong-qing(State Key Laboratory of Veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2022年第6期583-590,597,共9页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(32072851、31941019);兽医生物技术国家重点实验室开放课题(SKLVBF202110)。

摘　　要：猪繁殖与呼吸综合征病毒(PRRSV)RFLP 1-4-4 lineage1C(L1C)变异株自2020年在美国出现以来,给养猪业造成了严重的经济损失。我国目前尚无RFLP 1-4-4 L1C变异株的报道。为了解我国PRRSV RFLP 1-4-4株的流行情况,本研究对我国15个省219份疑似PRRSV感染的临床样品进行了PRRSV ORF5基因的RT-PCR检测与测序。结果显示,41份(18.72%)为PRRSV阳性样品,其中有1份来自黑龙江省样品中的PRRSV ORF5基因为1-4-4模式。将该样品经PAM分离病毒并经PCR和间接免疫荧光试验鉴定,结果显示分离到一株RFLP模式为1-4-4的PRRSV,命名为HLJ-80。采用PCR对分离病毒的全基因组分段扩增并测序拼接后显示其基因组全长15013 nt,利用MegAlign软件分析分离病毒与PRRSV 6株代表株的全基因组序列、ORF基因序列的同源性及其NSP2的氨基酸序列;利用MEGA6.0软件分析分离株ORF5基因序列并绘制其系统发生树(ML法);采用RDP4分析分离株全基因组序列的重组事件。全基因组序列的同源性分析结果显示,HLJ-80株与NADC30株的同源性最高,为93.3%,与其他代表株的同源性均低于90%。除ORF2基因外,其余基因序列均与NADC30株同源性最高(90.6%~99.3%)。ORF5基因的遗传进化树显示,HLJ-80株属于RFLP 1-4-4 L1C株。NSP2氨基酸序列分析结果显示,与VR-2332株相比,HLJ-80株NSP2存在131个氨基酸(111+1+19)的不连续缺失,缺失位置与NADC30株一致。上述结果表明,HLJ-80株与NADC30株的亲缘关系较近。重组分析结果显示,HLJ-80株有可能与PRRSV 2014-81株、SD53-1603株高度同源的病毒株重组而来。将本研究中的41株分离株ORF5基因序列,及GenBank中1996年~2021年我国3125条共计3166条北美洲型PRRSV ORF5基因序列,按照PRRSV RFLP 1-4-4模式和L1C分支谱系分类,并分析我国RFLP 1-4-4 L1C株在时间和地理上的分布;将2016年~2021年GenBank中7个国家全部北美洲型PRRSV ORF5基因序列(共计1918条),按照PRRSV RFLP分类,分析2016年以来我�Porcine reproductive and respiratory syndrome virus(PRRSV)restriction fragment length polymorphism(RFLP)1-4-4 lineage1C(L1C)variant has caused serious economic losses to the pig industry since its emergence in the United States in2020.RFLP 1-4-4 L1C variant has not been reported in China.In order to understand the prevalence of PRRSV RFLP 1-4-4 strain in China,219 clinical samples of suspected PRRSV infection from 15 provinces in China were detected by RT-PCR and sequenced.The results showed that 41 samples(18.72%)were positive for PRRSV,among which one sample from Heilongjiang province was PRRSV with 1-4-4 RFLP pattern,and this virus strain was isolated by PAM and named HLJ-80.The whole genome of the isolated virus was amplified by PCR,sequenced,and showed the full length of 15013nt.MegAlign software was used to analyze the homology of neucleotide sequence and amino acid sequence of NSP2 of the isolated virus and 6 representative PRRSV strains.MEGA6.0 software was used to map the ORF5 nulceotide sequences of PRRSV isolates and different branches(ML method).RDP4 was used to analyze the recombination events of the whole genome sequence of the isolate.The homology analysis result showed that HLJ-80 strain had the highest homology with NADC30 strain(93.3%),and the homology with other representative strains was less than 90%.Except for ORF2 gene,the other gene sequences had the highest homology with NADC30 strain(90.6%-99.3%).The genetic evolution tree of ORF5 gene showed that HLJ-80 strain belonged to RFLP 1-4-4 L1C strain.Compared with VR-2332 strain,HLJ-80 strain NSP2 protein had 131 amino acid discontinuous deletion(111+1+19),which was consistent with NADC30 strain.Together,these results indicated that HLJ-80 strain had close genetic to NADC30 strain.Recombination analysis results suggested that HLJ-80 strain might be a recombined strain from PRRSV 2014-81 strain and SD53-1603 strain.Fourty-one ORF5 gene sequences obtained in this study and 3125 North American PRRSV ORF5 gene sequences identified in China from199

关 键 词：猪繁殖与呼吸综合征病毒 限制性酶切片段长度多态性 分支谱系 RFLP 1-4-4 L1C 

分 类 号：S852.65[农业科学—基础兽医学]