Simultaneous detection and genotyping of porcine reproductive and respiratory syndrome virus (PRRSV) by real-time RT-PCR and amplicon melting curve analysis using SYBR Green

The feasibility of using a SYBR Green-based real-time RT-PCR assay (SYBR Green ReTi RT-PCR) followed by melting curve analysis (MCA) for detecting and genotyping porcine reproductive and respiratory syndrome virus (PRRSV) was assessed. The SYBR Green ReTi RT-PCR and a previously reported two-step, non-nested RT-PCR assays were simultaneously tested on selected European (EU) and North American (US) PRRSV strains and isolates collected from diverse clinical, temporal, and geographical origins. The validation experiments showed that the optimised SYBR Green ReTi RT-PCR can sensitively and specifically detect PRRSV, consistently detecting as little as 0.03TCID(50)/sample of each virus genotype, with no type-bias and no amplification signal for other swine pathogens. After MCA, two well-differentiated melting temperature (T(m)) profiles for each virus genotype were obtained, as sequencing confirmed it. High repeatability was obtained for the T(m) values, with intra-run coefficients of variation (CoVs) of 0.25 and 0.32 and inter-run CoVs of 0.42 and 0.52 for EU and US genotypes, respectively. The sensitivity of the SYBR Green ReTi RT-PCR (100%) was higher than that of the RT-PCR (95.7%) when testing field isolates. This greater sensitivity of the SYBR Green ReTi RT-PCR was further confirmed by the detection of a higher proportion of PRRSV-positive diagnostic specimens (29.7%) than by the RT-PCR (28.5%). The SYBR Green ReTi RT-PCR test detected infection as early as 2 dpi in the sera of experimentally infected pigs regardless of virus genotype, and discriminated negative (non-inoculated), EU- and US-infected pigs. In conclusion, the reported SYBR Green ReTi RT-PCR assay coupled with MCA can detect and type PRRSV and may be useful as an alternative diagnostic assay in diverse PRRSV epidemiological circumstances.     

两株高致病性PRRSV湖北分离株ORF5与Nsp2基因序列分析

2013年从湖北两个发病猪场采集疑似猪繁殖与呼吸综合征病料,接种Marc-145细胞,可致明显细胞病变,各分离到1株猪繁殖与呼吸综合征病毒(HBHS株和HBXN株)。采用RTPCR方法,对ORF5基因序列和Nsp2基因部分序列进行扩增并测序,并用DNA MAN软件将测序结果与国内外发表的13株参考毒株进行比对分析。结果显示,2株分离株Nsp2基因与国内高致病性JXA1、GD2007、GD2008毒株的氨基酸同源性很高,介于98.5%~99.5%;且该基因与国内其他变异株有完全一致的缺失特征;ORF5基因与国内高致病性毒株的氨基酸同源性为98.1%~99.5%,且系统进化树遗传距离很近,同处一个基因群中,而与CH-1a等经典毒株较远。这两株分离株均属于PRRSV美洲型变异株,此结论为该病的防治及疫苗的设计奠定了基础。     

2013年-2014年江西地区PRRSV分离株NSP2及ORF5基因的遗传变异分析

为研究江西地区猪繁殖与呼吸综合征病毒(PRRSV) ORF5基因的变异情况及NSP2基因的结构特征,采用RT-PCR方法扩增了12份江西地区猪场疑似患PRRS的猪肺脏样品中的ORF5全序列和NSP2部分序列,应用DNAStar和Mega 6.0等软件对所得序列进行同源性比对及遗传变异分析。12株PRRSV ORF5核苷酸同源性为83.7%~99.8%,氨基酸同源性为82.1%~99.5%;与参考毒株JXA1、VR-2332和LV的核苷酸同源性分别为84.9%~99.7%、85.2%~91.0%和62.4%~64.8%。对阳性病料进行了NSP2基因部分序列的扩增,测序结果显示12株PRRSV均属于美洲型毒株,12株PRRSV的NSP2部分序列均存在30个氨基酸的不连续缺失,与高致病性PRRSV有相同的缺失特征。12株PRRSV的ORF5遗传进化树分析显示,10株与高致病性PRRSV处在同一进化分支,进一步说明高致病性PRRSV已成为江西地区的优势流行毒株。     

猪繁殖与呼吸综合征病毒7个分离株全基因序列测定及遗传进化分析

为了解猪繁殖与呼吸综合征病毒(PRRSV)的变异情况,本研究利用RT-PCR方法对2006年～2009年在我国部分地区分离鉴定的7株PRRSV分别进行9段基因片段扩增,测序分析表明,获得的病毒全基因序列与PRRSV JXA1变异株高度同源。并与GenBank中登录的其他70株PRRSV全基因序列进行遗传分析,根据构建的遗传进化树分析表明,中国大陆PRRSV分离株包含美洲型和欧洲型,美洲型可分为3个亚群,亚群1主要为以JXA1株为代表的病毒株,本研究中的7个分离株均为亚群1,其GP5主要中和抗原表位高度变异;亚群2主要为以CH-1a株为代表的病毒株;亚群3主要为以VR-2332株为代表的病毒株。欧洲型病毒株BJEU06-1、NMEU09-1分属于不同亚型。本实验为深入研究该病毒的遗传与变异及其分子流行病学研究奠定基础。     

2005年-2010年我国部分地区PRRSV流行毒株的遗传变异分析

为了掌握高致病性猪繁殖与呼吸综合征病毒(PRRSV)的变异情况,揭示该病的发生规律,根据GenBank登录的PRRSV基因序列设计引物,采用RT-PCR法对2005年-2010年间送检的282份病料进行了PRRSV核酸检测,对其中9份阳性样品进行了ORF5～7基因片段扩增和测序,所得序列与GenBank下栽的PRRSV...     

Molecular characterization of recent Korean porcine reproductive and respiratory syndrome (PRRS) viruses and comparison to other Asian PRRS viruses

Twenty-eight PRRS viruses (PRRSVs) isolated from various pig farms in Korea between 2002 and 2003 were sequenced for open-reading frame (ORF) 5 and/or full-length genome and compared with numerous PRRSVs reported from North America, Europe and Asia. All Korean isolates examined were genetically of the North American genotype. The ORF5 sequence of one isolate was identical to Ingelvac PRRS MLV vaccine virus. ORF5 nucleotide sequence divergence of the remaining 27 Korean PRRSVs from VR-2332, the prototype of the North American PRRSV and parental strain of the MLV vaccine virus, ranged from 1.3% to 12.9%, which corresponded to 2.0% to 14.9% divergence at the amino acid level, raising a concern on the efficacy of the MLV vaccine. Phylogenetic analyses of ORF5 and/or full-length sequences revealed that the Korean PRRSVs formed a clade distinct from PRRSVs reported from other Asian countries (China, Taiwan, Japan, and Thailand). Our study demonstrated that PRRSVs of the North American genotype were introduced to the Korean swine population some time ago and have evolved independently from PRRSV in other Asian countries, suggesting that geographic separation might influence the molecular evolution of PRRSV. This should be taken into consideration when a national PRRS prevention and control policy for international trade is established.     

2018年广东部分地区猪繁殖与呼吸综合征病毒流行毒株ORF5基因变异分析

为了解广东省猪繁殖与呼吸综合征病毒(PRRSV)流行毒株ORF5基因遗传变异情况,采用RT-PCR对2018年采自广东部分地区疑似患有PRRS的猪肺组织样品进行PRRSV ORF5基因扩增以及克隆测序,并进行生物信息学分析。结果表明,成功扩增出18株PRRSV流行毒株的ORF5基因片段。ORF5基因序列分析表明,18株PRRSV流行毒株ORF5基因核苷酸同源性为83.7%~99.8%,PRRSV流行毒株与参考毒株的同源性为62.1%~99.8%。基于ORF5基因的遗传进化树分析表明,18株PRRSV流行株均为美洲型毒株。其中,10株与以JXA1为代表的高致病性毒株亲缘较近,2株与新型高致病性毒株FZ16A相似;1株与以NT1为代表的疫苗返强毒株亲缘较近,1株与以R98为代表的疫苗毒株亲缘性较近,4株与广东新报道的GM2和QYYZ毒株亲缘性较近。DNA推导氨基酸序列分析表明,18株流行株的氨基酸序列与国内已报道的代表株相比发生不同程度的变异,GP5抗原表位上存在着差异。研究结果揭示了广东地区PRRSV有新型强毒株、重组毒株以及疫苗返强毒株的流行,提示养殖者谨慎、合理使用疫苗,防止疫苗毒株返强和毒株重组,为该地区防控PRRS提供参考。     

猪繁殖与呼吸综合征病毒的分离与鉴定

从广东地区发病猪场的病料中,分离到1株致Marc-145细胞病变的病毒ShB6。扩增猪繁殖与呼吸综合征病毒(PRRSV)主要结构蛋白基因ORF2-ORF7并进行序列分析,结果表明,该分离株与国内PRRSV分离株HB-1(sh)/2002的同源性为96.9%;与ATCC VR-2332株的同源性为91%;而与Lelystad株的同源性仅为59.8%;用美洲型PRRSV单抗进行免疫组化染色,结果显示在细胞病变处呈现明显的阳性着色(为棕黄色)。综合可见,所分离的病毒为美洲型PRRSV。     

Molecular evolution of porcine reproductive and respiratory syndrome virus isolates from central China

To investigate the genetic diversity of prevailing porcine reproductive and respiratory syndrome virus (PRRSV) in Henan Province of China, 61 ORF5 gene sequences, originating from Henan Province during 2003–2010, were subjected to amino acid variation and phylogenetic analysis. The analyzed PRRSV ORF5 sequences carried evidence of one unique recombination event. Phylogenetic analysis revealed that all Henan isolates belonged to type 2 genotype and were divided into two subgroups. The dominant isolates had shifted from subgroup 1 to subgroup 2 during 2003–2010. Amino acid variation analysis of the glycoprotein 5 revealed that Henan PRRSV strains tended to accumulate more substitutions within the N-terminus and hypervariable region. Selective pressure analysis revealed evidence that some ORF5 sites have likely evolved in response to immune pressure.     

山东PRRSV流行株ORF5、ORF6、ORF7基因序列的分子特征

采用RT-PCR技术对2001～2007年分离自山东地区10株(ShanDong-3、SD-JN、SD-ZQ、SD1、SD2、SD3、SD4、SD5、SD6和SD7)猪繁殖与呼吸综合征病毒(PRRSV)进行ORF5、ORF6和ORF7基因的扩增、克隆和测序,与已知序列的毒株的相应片段进行同源性分析比较,并对其分子特征进行分析.结果表明:该10株病毒仍属北美洲型,其中2001～2002年分离的SD1株、SD2株和2006年分离的SD6株核苷酸序列之间的ORF5、ORF6、ORF7同源性分别为99.2%,99.8%,100%,均与北美洲原型代表株(VR-2332株)和疫苗毒MLVRespPRRS Repro USA遗传距离较近,同属一大分支;2006～2007年分离的PRRSV ShanDong-3、SD-JN、SD-ZQ 、SD3,SD4,SD5, SD7分离株ORF5、ORF6、ORF7同源性分别为97.8%～100%,99.4%～99.8%,99.2%～99.7%,均与国内96年Ch-1a和2002年HB-1株及2006年分离鉴定的国内高致病性分离株(JXA1、Shanghai、HEB1)同属一个大的分支.首次证实目前山东省同时存在高致病性PRRSV和传统PRRSV,并且有由传统PRRSV向高致病性PRRSV演化的趋势.     

2014年-2016年广西地区猪繁殖与呼吸综合征病毒Nsp2和ORF5基因的遗传多样性分析

为了解广西地区猪繁殖与呼吸综合征病毒(PRRSV)流行毒株的遗传进化情况,对2014年-2016年来自广西各地的部分PRRSV阳性病料进行Nsp2和ORF5基因的扩增和测序分析.结果获得34个Nsp2基因序列和45个ORF5基因序列,均属于美洲型毒株.Nsp2基因间核苷酸序列的同源性为91.8％~100％,与PRRSV美洲型毒株VR-2332、CH-1a、JXA1及NADC30株核苷酸序列的同源性分别为81.3％~84.3％、88.9％~92.1％、94.3％~99.3％和73.5％~75.1％,而与PRRSV欧洲型毒株LV株核苷酸序列的同源性为51.5％~53.2％.ORF5基因间核苷酸序列的同源性为82.8％~100％,与PRRSV美洲型毒株VR-2332、CH-1a、JXA1及NADC30株核苷酸序列的同源性分别为83.7％~99.5％、85％~95％、83.8％~99.7％和83.2％~86.4％,而与PRRSV欧洲型毒株LV株核苷酸序列的同源性为62.4％~64.5％.基于Nsp2和ORF5基因推导的氨基酸序列绘制的遗传进化树中,广西地区的毒株主要分布在以JXA1为代表的Ⅳ亚群.表明当前广西PRRSV流行毒株以JXA1株为代表的高致病性美洲型毒株为主,各毒株Nsp2和ORF5基因序列存在一定的差异,尚未发现欧洲型毒株和美洲型NADC30类毒株.     

Genetic and immunobiological diversities of porcine reproductive and respiratory syndrome genotype I strains

Genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV) has been based on ORF5/GP5 and ORF7/N protein variations. Complete viral genome studies are limited and focused on a single or a few set of strains. Moreover, there is a general tendency to extrapolate results obtained from a single isolate to the overall PRRSV population. In the present study, six genotype-I isolates of PRRSV were sequenced from ORF1a to ORF7. Phylogenetic comparisons and the variability degree of known linear B-epitopes were done considering other available full-length genotype-I sequences. Cytokine induction of all strains was also evaluated in different cellular systems. Non structural protein 2 (nsp2) was the most variable part of the virus with 2 out of 6 strains harboring a 74 aa deletion. Deletions were also found in ORF3 and ORF4. Phylogenetic analyses showed that isolates could be grouped differently depending on the ORF examined and the highest similarity with the full genome cluster was found for the nsp9. Interestingly, most of predicted linear B-epitopes in the literature, particularly in nsp2 and GP4 regions, were found deleted or varied in some of our isolates. Moreover, 4 strains, those with deletions in nsp2, induced TNF-α and 3 induced IL-10. These results underline the high genetic diversity of PRRSV mainly in nsp1, nsp2 and ORFs 3 and 4. This variability also affects most of the known linear B-epitopes of the virus. Accordingly, different PRRSV strains might have substantially different immunobiological properties. These data can contribute to the understanding of PRRSV complexity.     

杂交野猪猪繁殖与呼吸综合征病毒分离及其ORF6、ORF7基因的序列分析

试验旨在研究杂交野猪猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndrome virus, PRRSV) 辽宁分离株的遗传变异情况及分子生物学特征.用Marc-145细胞从辽宁某杂交野猪场疑似猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome, PRRS)病猪血液中分离到1株病毒,该分离毒株经Marc-145细胞6次传代后出现稳定的细胞病变,采用RT-PCR方法对分离病毒进行ORF6和ORF7基因的扩增、克隆和测序,并与已知序列毒株的相应片段进行同源性比对.结果表明,分离毒株的ORF6、ORF7基因与国内外美洲型毒株的核苷酸同源性分别为96.0%～100.0%、94.5%～99.4%;氨基酸同源性分别为89.6%～100.0%、87.3%～98.7%;与欧洲型代表毒株LV的ORF6、ORF7基因差异较大,核苷酸同源性分别为70.4%、70.1%,氨基酸同源性分别为48.8%、49.7%.推测辽宁杂交野猪体内分离毒株在基因型上属于美洲型毒株.     

Genetic variation and phylogenetic analyses of the ORF5 gene of acute porcine reproductive and respiratory syndrome virus isolates

Swine herds in the US have experienced recent outbreaks of a severe form of porcine reproductive and respiratory syndrome (designated acute or atypical PRRS) characterized by abortion and high mortality in pregnant sows. Most of the affected herds had been vaccinated with modified live-vaccines (MLVs) against PRRS. To explore the possible mechanism of the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. The complete ORF5 gene of eight acute PRRSV isolates from herds experiencing acute PRRS outbreaks in Iowa and North Carolina was amplified and sequenced. Sequence analyses revealed that these acute PRRSV isolates shared 88-95% nucleotide and 88-96% amino acid sequence identities to each other, 87-97% nucleotide and 84-96% amino acid sequence identities with other North American PRRSV isolates and the MLVs. Most of the amino acid substitutions locate in the putative signal sequence and two short hypervariable regions at the amino terminus. The ORF5 gene sequence of the acute PRRSV isolate 98-37120-2 from a non-vaccinated swine herd in Iowa is very closely related to that of the RespPRRS MLV, with 97% nucleotide and 96% amino acid sequence identities. Phylogenetic analysis revealed that all eight acute PRRSV isolates are clustered within the North American genotype. Several minor branches that are not associated with geographic origins were also identified within the North American genotype. One acute PRRSV isolate (98-37120-2) is clustered with the RespPRRS MLV and several Danish isolates that were confirmed to be derived from the RespPRRS MLV. The ORF5 gene sequences of other seven acute isolates are more related to those of several earlier PRRSV isolates and the PrimePac MLV than to that of the RespPRRS MLV. Our results showed that the acute PRRSV isolates analyzed in this study differed from each other in ORF5 genes, although they all clustered within the North American genotype. The data from this study do not fully support the hypothesis that the emergence of acute PRRS is due to reversion of MLVs to a pathogenic phenotype, as only one of the eight acute isolates was shown to be very closely related to the RespPRRS MLV.     

两株高致病性PRRSV ORF6和ORF7基因克隆及序列分析

应用RT PCR方法从实验室分离的两株高致病性PRRSV SX、ZQ株中扩增出ORF6和ORF7,将其分别克隆、测序。用DNAStar 软件分析所测序列,并与VR 2332株、LV株、周边国家及国内分离株进行核苷酸和推导氨基酸同源性比较,并绘制系统进化树,结果ORF6、ORF7核苷酸与北美洲型的同源性为91.1％～100％,与欧洲型的同源性为66.2％～70.7％,推导氨基酸与北美洲型的同源性为91.2％～100％,与欧洲型的同源性为62.3％～82.3％。证明新分离到的PRRSV SX株、ZQ株仍属北美洲型。SX株ORF6、ORF7核苷酸与国内新分离到的高致病性PRRSV JXA1株同源性分别为99.8%、100%;ZQ株ORF6、ORF7核苷酸与国内新分离到的高致病性PRRSV JXA1株同源性分别为99.6%、99.7%。     

Sequence analysis of the membrane protein gene and nucleocapsid gene of porcine reproductive and respiratory syndrome virus isolated from a swine herd in Hungary

Porcine reproductive and respiratory syndrome virus (PRRSV) was isolated from blood samples taken at a pig farm in Hungary from pigs showing clinical signs of the disease. The virus (ABV 32) was identified as belonging to the European genotype by using type-specific monoclonal antibodies. This was confirmed by comparing the sequence of the membrane protein gene (ORF 6) and the nucleocapsid gene (ORF 7) with the American VR2332 and the European LV genotype reference strain, respectively. Analysis of the amino acid sequence of the ORF 6 and ORF 7 of ABV 32 revealed five amino acid changes in both ORFs when compared with LV, of which two changes in ORF 7 were only found in the Spanish isolates. Additionally, the ORF 7 sequence was compared with corresponding sequences of a total of 21 other European strains. Phylogenetic analysis using the PHYLIP package confirmed the close relationship between the Hungarian and the Spanish isolates. Of all the isolates analysed, ABV 32 and LV were the least related.     

猪繁殖与呼吸综合征病毒HS株结构蛋白基因的克隆与序列分析

根据GenBank中猪繁殖与呼吸综合征病毒(PRRSV)美洲株(VR-2332)基因序列,设计合成了ORF2a、ORF3、ORF4、ORF5、ORF6和ORF7基因的引物.利用RT-PCR扩增出PRRSV HS株各基因的cDNA片段,将扩增的各cDNA片段克隆入pMD18-T载体并测序.应用DNA Man软件,将测序结果与国内外已发表野毒株和疫苗株(VR-2332、Resp MLV、16244B、HN1、BJ-4、CH1-a、HB-1、HB-2、LV)的相应基因进行序列比较,并绘制系统进化树.结果表明,PRRSV HS株与美洲型的相应基因核苷酸同源性为83.6%～99.7%,与LV株的相应基因核苷酸同源性为38.9%～49%;推导的氨基酸与美洲型相应基因的同源性为86.6%～99.6%,与LV株的同源性为54.2%～78.2%.系统进化树表明,PRRSV HS株属于美洲型,与HN1、VR-2332、RespMLV、16244B、BJ-4亲缘关系较近.