畜牧兽医学报  2020, Vol. 51 Issue (12): 3181-3186. DOI: 10.11843/j.issn.0366-6964.2020.12.027    PDF    
1株重组猪繁殖与呼吸综合征病毒的基因组特征
谭祥梅1, 吴瑕1, 陈鹏飞1, 赵雄伟1, 周书亭1,2, 虞凌雪1, 童武1, 高飞1, 姜一峰1, 于海1, 童光志1,3, 周艳君1,3     
1. 中国农业科学院上海兽医研究所, 上海 200241;
2. 上海交通大学农业与生物学院, 上海 200240;
3. 江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009
摘要:猪繁殖与呼吸综合征病毒(PRRSV)不断发生变异,其防控难度也逐渐增加。为了及时监测PRRSV在自然感染背景下的遗传进化特点,本研究从上海某发病猪场的临床样品中分离获得1株PRRSV毒株,命名为SHpd1/2018株,并对其生物学特性和基因组序列进行分析。结果表明,该毒株基因组全长为15 018 bp(不含poly A),不能被针对HP-PRRSV强毒HuN4株的Nsp2的单抗所识别,但其增殖特性与强毒HuN4株相似。序列分析表明,SHpd1/2018株与HP-PRRSV强毒株的相似性最高(与HuN4相似性为94.3%)。重组分析结果显示,SHpd1/2018株是以HP-PRRSV-like为主要亲本毒株,NADC30-like为次要亲本毒株的重组病毒,两个重组断点nt 2002和nt 3205均位于Nsp2基因的高变区。研究证实SHpd1/2018株是1株发生变异重组的PRRSV分离株,该毒株是上海某猪场保育猪发病的主要原因。
关键词猪繁殖与呼吸综合征病毒    SHpd1/2018    生物学特性    基因重组分析    
Genome Characterization of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus
TAN Xiangmei1, WU Xia1, CHEN Pengfei1, ZHAO Xiongwei1, ZHOU Shuting1,2, YU Lingxue1, TONG Wu1, GAO Fei1, JIANG Yifeng1, YU Hai1, TONG Guangzhi1,3, ZHOU Yanjun1,3     
1. Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China;
2. School of Agriculture and Biology, Shanghai Jiaotong University, Shanghai 200240, China;
3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou 225009, China
Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) mutates continuously, and it is increasingly difficult to control it. To monitor the genetic evolution characteristics of PRRSV timely under the condition of natural infection, a novel PRRSV variant named SHpd1/2018 was isolated from PRRSV positive clinical samples. The results showed that the SHpd1/2018, with a total genome length of 15 018 bp (excluding poly A), showed similar proliferation characteristics to HP-PRRSV strain HuN4. However, it could not be recognized by the monoclonal antibody against HuN4-Nsp2. The sequence analysis indicated that SHpd1/2018 had the greatest homology with HP-PRRSV-like strains, reaching 94.3% with HuN4 strain. The results of recombination analysis showed that SHpd1/2018 was recombined from HP-PRRSV-like strain (main parent strain) and NADC30 strain (secondary parent strain), and both the two recombination breakpoints nt2002 and nt3205 are located in the hypervariable regions of the Nsp2 gene. In short, we confirmed that the isolated strain SHpd1/2018 is a recombinant PRRSV, which may be the main cause of the disease in the pig farm in Shanghai.
Key words: porcine reproductive and respiratory syndrome virus    SHpd1/2018    phylogenetic analysis    recombination analysis    

猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome, PRRS)是严重危害全球养猪业的病毒性传染病之一,给养猪业造成巨大经济损失[1-2]。2006年,高致病性猪繁殖与呼吸综合征病毒(HP-PRRSV)在我国首次出现,并引起大规模的疫情暴发[3],此后,HP-PRRSV-like毒株成为在我国猪群中广泛流行的优势毒株。近几年,在我国又出现了发生重组变异的PRRSV分离株[4-5],其致病性虽然比HP-PRRSV毒株低,但其在猪群中造成的持续性感染对猪的生长性能影响严重。PRRSV的变异和重组对现有疫苗的防控提出了新的挑战,有必要及时监测PRRSV遗传进化和流行情况。本研究从上海某发病猪场的PRRSV阳性样品中分离获得了1株基因组发生高度变异和重组的PRRSV,分析证实该毒株是由HP-PRRSV-like毒株和NADC30-like毒株经重组而来的新的变异毒株,研究结果可为指导临床有效预防PRRS流行提供科学依据。

1 材料与方法 1.1 细胞、病毒及抗体

Marc-145细胞、高致病性PRRSV HuN4株和疫苗毒HuN4-F112株均由中国农业科学院上海兽医研究所猪传染病研究室保存[3, 6];9份临床样品采自上海某猪场中发病的保育猪群,该猪场此前一直采用经典弱毒疫苗进行免疫预防;抗PRRSV N和Nsp2蛋白单抗由本研究室制备并保存[7-8];FITC标记的羊抗鼠IgG荧光二抗均购自Thermo Scientific公司。

1.2 样品检测及病毒生物学特性分析

利用PCR方法(Nsp2检测引物见表 1)检测样品,将PRRSV阳性样品接种于Marc-145细胞进行病毒分离和传代,对第5代培养物进行毒株的鉴定,通过噬斑形态观察和增殖曲线绘制分析其生物学特性。

表 1 PRRSV检测以及全基因组扩增引物 Table 1 Primers for PRRSV detection and full genome sequencing
1.3 病毒全长基因扩增和序列分析

提取第5代病毒基因组RNA,设计扩增PRRSV全基因组的引物(表 1),将全基因分为8个片段(A、B、C、D、E、F、G和H)分别进行RT-PCR扩增和克隆测序,并利用DNAstar软件的Seqman对各基因片段进行全长基因拼接。同时,利用MEGA7.0软件分析分离毒株的遗传进化关系,利用RDP4软件和Simplot软件对分离毒株进行基因重组分析。

2 结果 2.1 SHpd1/2018分离株生物学特性分析

对采集的9份临床样品进行PCR检测,结果显示,有7份样品仅呈PRRSV阳性。分离毒株在Marc-145细胞上引起的CPE形态与HuN4株相似,将其命名为SHpd1/2018株。利用抗PRRSV N和Nsp2蛋白特异抗体对其进行IFA检测,结果显示,SHpd1/2018分离株可被针对PRRSV N蛋白的特异性单抗所识别,但是却不被针对Nsp2蛋白的单抗所识别(图 1A),其在Marc-145细胞形成的CPE、噬斑形态和增殖特点与HP-PRRSV HuN4株相似(图 1BC)。推测SHpd1/2018分离株Nsp2基因与HP-PRRSV-like毒株和CH-1a-like毒株不同,可能存在变异。

A. SHpd1/2018分离株的IFA鉴定;B. SHpd1/2018分离株在Marc-145细胞中的多步生长曲线;C. SHpd1/2018分离株的噬斑形态分析 A. IFA identification of SHpd1/2018; B. Multi-step growth curve of SHpd1/2018 in Marc-145 cells; C. Plaque morphology of SHpd1/2018 strain 图 1 SHpd1/2018分离株的IFA鉴定及生物学特性分析 Fig. 1 IFA identification of SHpd1/2018 and its biological characteristics
2.2 SHpd1/2018分离株遗传进化分析

扩增SHpd1/2018分离株全基因组并拼接,显示其全长基因为15 018 bp(不含polyA)。序列分析结果显示,SHpd1/2018株与经典毒株VR2332、CH-1R以及HP-PRRSV毒株JXA1、HuN4的核苷酸相似性分别为86.5%、90.9%和94.3%,与HP-PPRSV弱毒疫苗株JXA1-P80和HuN4-F112的相似性为93.8%,与NADC30株的相似性为86.7%,表明SHpd1/2018株与HP-PRRSV强毒株的亲缘关系较近。其Nsp2基因与NADC30毒株有相同的氨基酸缺失模式“111+1+19”(图 2A);进化树分析结果显示,SHpd1/2018与HP-PRRSV like株的全长基因和ORF5基因同属于谱系8(图 2BC),而Nsp2基因则与NADC30 like毒株同属于谱系1(图 2D),结果表明,SHpd1/2018分离株可能存在基因重组现象。

A. SHpd1/2018分离株Nsp2氨基酸缺失区域比对分析; B. SHpd1/2018分离株全基因进化树分析;C. SHpd1/2018分离株ORF5进化树分析;D. SHpd1/2018分离株Nsp2进化树分析 A. Comparison analysis of amino acid deletion regions in Nsp2 of SHpd1/2018; B. Phylogenetic analysis of SHpd1/2018 complete sequence; C. Phylogenetic analysis of SHpd1/2018 ORF5 gene; D. Phylogenetic analysis of SHpd1/2018 Nsp2 gene 图 2 SHpd1/2018分离株的基因序列分析 Fig. 2 Genome sequence analysis of SHpd1/2018
2.3 SHpd1/2018分离株重组分析

通过RDP4软件进行重组分析,结果显示,经过RDP、GENECONV、BootScan、MaxChi、Chimaera、SiScan和3Seq 7种方法分析,证实SHpd1/2018毒株是以谱系8的HP-PRRSV like强毒株为主要亲本毒,以NADC30-like毒株为次要亲本毒重组而来,其重组断点为nt 2002和nt 3205(P < 0.001)(图 3A)。利用Simplot软件对分析结果进行可视化,结果显示,两个重组断点将其分隔为3个基因区域,A(nt 1~nt 2001)、B(nt 2002~nt 3205)和C(nt 3206~nt 15018),对不同基因区域进行进化树分析,结果显示,A和C基因区域位于HP-PRRSV类毒株分支,而B区域则位于NADC30类毒株的分支(图 3B),由此证实SHpd1/2018株是由HP-PRRSV-like强毒株和NADC30-like毒株重组而来的变异病毒。

A. SHpd1/2018分离株全长基因重组分析; B. SHpd1/2018分离株不同基因区域进化树分析 A. Recombination analysis of SHpd1/2018 full-length genome; B. Phylogenetic analysis of SHpd1/2018 in different regions 图 3 PRRSV SHpd1/2018分离株的重组分析 Fig. 3 Whole genome recombination analysis of PRRSV SHpd1/2018 strain
3 讨论

PRRSV是一种极易发生突变的RNA病毒,由于它的RNA依赖的RNA聚合酶缺乏校对能力,导致PRRSV在复制过程容易出现突变和错配而产生大量变异毒株,PRRSV的持续变异不仅增加了其遗传多样性,而且其致病性也发生了不同的改变[9-10]。近年来, 随着临床针对PRRSV预防策略的无序变化,一些发生新变异和重组的PRRSV不断被发现,这些变异毒株的出现可能成为该病广泛流行的潜在威胁,因此,针对新变异的PRRSV监测不容忽视。在本研究中获得的SHpd1/2018分离株,基于全长基因组和ORF5基因的进化树分析表明其属于谱系8,而基于Nsp2基因分析表明, 其属于谱系1。经过基因重组分析证实,SHpd1/2018分离株主要是以HP-PRRSV-like强毒株为主要亲本毒,作为病毒骨架,以NDAC30-like毒株为次要亲本毒发生重组而来的变异毒株。其基因骨架序列与HP-PRRSV强毒株的亲缘关系最高,其次是HP-PRRSV弱毒疫苗毒株。除此而外,该毒株还存在大量新的氨基酸突变,其中,Nsp2编码区中发生的突变最多,与HP-PRRSV类毒株和经典毒株相比,Nsp2核苷酸/氨基酸相似性均低于80%,与NADC30毒株的核苷酸/氨基酸相似性也低于90%,推测这些新发生突变的氨基酸可能是PRRSV为了调节其对宿主适应性的一种自我保护的进化策略。结合该毒株在临床上显示出对保育猪具有较强的致病性,提示有必要持续监测临床上PRRSV流行毒株的遗传演化趋势,并有针对性地制定科学的PRRSV防控策略。

4 结论

获得1株PRRSV毒株——SHpd1/2018株,系以HP-PRRSV-like为主要亲本毒株,NADC30-like为次要亲本毒株的重组病毒。

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