Genetic diversity of porcine circovirus type 2 (PCV2) in the Romanian wild boar population

In this study, we have analyzed 23 PCV2 ORF2 sequences recovered from wild boar population in Romania. The PCV2 sequences were originated from different geographical regions in Romania, and collected between 2008 and 2009 during the classical swine fever virus (CSFV) surveillance campaign. Complete open reading frame 2 (ORF2) nucleotide sequences were obtained and compared with sequences mainly from European and Asian isolates. The Romanian sequences were identified as belonging to previously described clusters 2a and 2b, with high degree of heterogeneity (PCV2 ORF2 nucleotide homology ranged between 90.1% and 100%). Interestingly, for cluster 2a, the majority of the sequences (8 from a total number of 9) clustered mainly with the Asian isolates (especially China, but also India and South Korea), with three exceptions from Europe previously reported in Germany, Belgium and The Netherlands.     

猪圆环病毒2型云南株分离鉴定及ORF2序列分析

本研究根据GenBank中已经发表的猪圆环病毒2型基因序列,设计了2对PCV2特异性引物,从疑似断奶仔猪多系统衰竭综合征(postweaning multisystemic wasting syndrome,PMWS)感染病例中检测出3株云南省PCV2流行株,通过ve-ro细胞分离病毒,并用透射电子显微镜观察到了约17nm的病毒样粒子的存在。经同源性比较分析,3个分离株之间核苷酸同源性为99.5%～99.8%,与甘肃省PCV2分离株核苷酸同源性最低(90.7%),浙江省分离株核苷酸同源性最高(99.7%),与南美洲分离株核苷酸同源性最低(92.0%),瑞典分离株核苷酸同源性最高(99.4%),这可能与云南省猪种引进有关。     

2008～2011年中国部分地区猪圆环病毒2型的分子流行病学调查

为了解2008~2011年中国部分地区猪圆环病毒2型(Porcine circovirus type 2,PCV2)分子流行病学变化趋势,本实验室共采集福建省、江西省、广东省、安徽省、浙江省、河南省、河北省、广西壮族自治区、内蒙古自治区、上海市、江苏省和山西省共12个省(市、区)的健康猪群和发病猪群共452份样品,对其进行病原学检测,并通过扩增、克隆和测序共获得31株PCV2 ORF2基因编码序列。结果显示,452份样品中,有354份样品检测为PCV2阳性,感染率高达78.3%。对31株ORF2基因序列的分析和比对结果表明,31株PCV2均为PCV2b基因型,其中有21株归类于PCV1A/1B基因亚群,而10株为PCV1C亚群;对31株PCV2 ORF2编码氨基酸序列比对分析表明PCV2基因亚型具有其特异的氨基酸变异位点,这对于临床上区别PCV2亚型具有一定的指导意义。从基因水平上来说,自2008年以来中国以PCV 1A/1B亚群为主要流行致病株,但值得我们关注的是,PCV 1C亚群毒株从无到有并有逐渐增多的趋势,将来可能在PCV2的流行毒株中占据主要地位。     

猪圆环病毒Ⅱ型广东分离株全基因组的克隆和序列分析

分离了9株猪圆环病毒Ⅱ型（PCV2）广东地方分离株，并进行了全基因组序列测定；对这9株PCV2广东分离毒株的ORF1和ORF2基因的序列分析表明ORF2的变异程度要比ORF1的变异程度大；对PCV2衣壳蛋白的氨基酸序列同源性比较发现了PCV2毒株间存在1个氨基酸变异程度较大的区域和2个氨基酸变异程度较小的区域，其中前两个区域与两个主要的免疫反应区域相对应；将这9个毒株的全基因组序列与GenBank上收录的18个PCV2毒株的全基因组序列基因进化树分析表明，这9个PCV2广东分离株彼此之间及与国内PCV2分离株、欧洲株之间更接近，而与韩国、中国台湾、日本和美洲毒株之间则稍远，因而在选PCV2疫苗免疫时，建议尽量使用国内生产的疫苗或自制组织灭活疫苗进行免疫。     

猪圆环病毒2型GD株ORF2基因的序列分析

根据GenBank中猪圆环病毒2型（PCV2）0RF2基因序列，设计一对引物，应用PCR从本室鉴定分离的PCV2 GD株的细胞培养物中扩增出ORF2基因（702bp）。将此基因片段克隆入pMD18-T载体，筛选获得重组质粒pMD—ORF2并对其测序，结果表明所克隆的ORF2基因与其他PCV2的0RF2基因核苷酸序列同源性在92．1％～99．9％之间，推导的氨基酸序列同源性在90．2％～99．5％之间。     

Study of the genetic variability of porcine circovirus type 2 detected in Serbia and Slovenia

Recent variants of porcine circovirus type 2 (PCV2) were obtained from tissues of domestic pigs with porcine circovirus associated disease and from randomly selected wild boar samples from Serbia and Slovenia. A 450-base-pair nucleotide sequence was obtained by PCR from the ORF2. The derived nucleotide and amino acid sequences were aligned and compared to the corresponding region of closely related PCV2 sequences determined in previous years and retrieved from the GenBank. The 30 Serbian and 17 Slovenian PCV2 sequences clustered into three previously determined genotypes (PCV2a: 7), (PCV2b: 38) and (PCV2d: 2). Three major variable regions, concerning 29 amino acid position substitutions within the ORF2, were observed, which further supports the segregation of the detected strains into three separate genotypes. This study indicates that PCV2b is the predominant genotype in Serbia and Slovenia and the detected PCV2 strains are closely related to those previously described in Europe and in other parts of the world.     

Seroprevalence of porcine circovirus type 2 in swine populations in Canada and Costa Rica

Porcine circovirus (PCV) was recently divided into 2 antigenically distinct types that differ (65% amino acid identity) in the protein encoded by open reading frame 2 (ORF2). Porcine circovirus 1 is apparently non-pathogenic and, in contrast, PCV2 is associated with porcine multisystemic wasting syndrome (PMWS). Our objective was to determine the extent of exposure of normal pigs in Canada and Costa Rica to PCV2. Recombinant DNA techniques were used to produce an antigen from ORF2 of PCV2 that was suitable for the detection of antibody in swine sera. The presence of PCV2 nucleotide sequences was detected using polymerase chain reaction (PCR) techniques. Using these tests, specific antibody and nucleotide sequences were demonstrated in sera from a cohort of pigs during a PMWS outbreak. Antibody was detected in normal, healthy hogs slaughtered in Canada (82.4% of 386) and in Costa Rica (14.6% of 322). This is the first report indicating the presence of PCV2 in Latin America. More than 50% of these sera also contained PCV2 nucleotide sequence. Although these hogs were healthy when slaughtered, they were infected with PCV2 and may have previously been ill. The widespread occurrence of PCV2 in swine suggests that this virus is adapted to replication in porcine tissue.     

吉林省猪圆环病毒3型的分子流行病学调查及遗传演化分析

为了解猪圆环病毒3型(porcine circovirus type 3,PCV3)在吉林省的流行情况和分子生物学特性,本研究通过PCR方法对吉林省2015-2017年的484份血清样品进行PCV3检测,将PCV3检测阳性的样品进行ORF2基因扩增和测序,并利用生物信息学软件DNAStar和Mega 6.06对ORF2基因的分子生物学特性进行分析。结果显示,吉林省2015-2017年PCV3样品总感染率和猪场感染率分别为28.1%(136/484)和65.8%(25/38),且呈逐年上升趋势。同源性分析结果表明,本研究获得的4株PCV3 ORF2基因的核苷酸和氨基酸序列同源性分别为98.3%～98.9%和97.7%～99.5%,4株PCV3 ORF2基因与国内外参考毒株ORF2基因的核苷酸和氨基酸序列同源性分别为97.7%～99.7%和96.7%～100%。遗传进化分析表明,PCV3存在2个亚群:PCV3a和PCV3b。本试验分离的PCV3毒株分别位于2个亚群上,1株属于PCV3a亚群,3株属于PCV3b亚群。PCV3毒株Cap蛋白第24(A、V)和27位(R、K)氨基酸的不同可能与PCV3毒株的进化相关。本试验结果表明,PCV3在吉林省猪群和猪场中存在很高的感染率,PCV3毒株之间高度保守,本研究结果为PCV3的分子特性研究提供了参考依据。     

我国部分地区猪圆环病毒2型分离株的遗传变异分析

为了解我国猪圆环病毒2型(PCv2)流行毒株遗传变异情况,本研究对本实验室分离到的19株PCV2分离株通过病毒全基因组克隆和测序分析,将其分为2个大基因群和3个亚群,其基因组分别为1 766 nt、1 767 nt和l 768 nt,各占15.8%、73.7%和10.5%.以1 767 nt毒株为基准,其基因组第39或1 039位有1个碱基缺失后突变成1 766 nt毒株;第1 040位有1个碱基插入后突变成1 768 nt毒株.对19株病毒ORF2编码的Cap蛋白分析发现,有4株病毒编码基因发生了突变,出现了705 nt和708 nt两种突变型,使ORF2编码的Cap蛋白C末端分别有1和2个氨基酸增加.同时,本实验选用Acc I和Fba I内切酶对19株病毒基因组PCR产物进行了限制性片段长度多态性(PCR-RFLP)分析,其结果可作为流行毒株分型鉴别依据.     

北京市猪场猪圆环病毒3型感染状况调查及其ORF2基因遗传进化分析

为了解猪圆环病毒3型(PCV3)在北京市不同区县猪场中的流行情况,研究建立PCR检测方法,对来自北京市8个区县56个养殖场的1177份临床样品进行检测,并对获得的部分ORF2全基因序列进行遗传进化分析。结果显示,PCV3总体阳性率为1.0%(12/1177),猪场阳性率为8.9%(5/56)。对PCV3阳性样本进行ORF2基因测序及同源性比对,共测得12株PCV3 ORF2全长基因序列,其中包括6株不同的ORF2全长基因序列。结果显示,该6株序列之间的核苷酸相似性为97.7%～99.5%,推导氨基酸序列的相似性为97.2%～100%;与参考毒株之间的核苷酸同源性为96.0%～99.5%,推导氨基酸同源性为92.1%～100.0%;进化树显示北京毒株属于PCV3b基因型。试验表明,PCV3在北京多个猪场呈现一定的流行趋势,流行毒株以PCV3b基因型为主。     

Genomic analysis of PCV2 isolates from Danish archives and a current PMWS case-control study supports a shift in genotypes with time

Porcine circovirus type 2 (PCV2) is the primary cause of Postweaning Multisystemic Wasting Syndrome (PMWS) in pigs. PCV2, however, is found in both PMWS-affected herds and non-affected herds. The objective of this study was to clarify if PCV2 genome nucleotide sequences isolated from pigs from PMWS-affected herds and non-affected herds cluster phylogenetically in two separate groups. All isolates (45) belonged to PCV2 group 1 and shared a nucleotide sequence identity of 99.4-100% indicating a very homogeneous PCV2 population in Denmark. Phylogenetic analysis of the PCV2 isolates revealed no distinctive clustering of case- and control-herds suggesting that there is no link between PCV2 sequences and herd disease status. The appearance of only PCV2 group 1 isolates in this study (isolates from 2003/2004) led us to determine if PCV2 nucleotide sequences had changed in Denmark over time. Interestingly, all PCV2 isolates from before the first outbreak of PMWS (2001) belonged either to a new PCV2 group identified for the first time in this study and named group 3 (isolates from 1980, 1987 and 1990) or PCV2 group 2 (isolates from 1993 and 1996). The shift from PCV2 group 2 to 1 was confirmed on a more global scale by placing all full genome PCV2 sequences submitted to GenBank from 1997 to 2006 in either of the groups by phylogenetic analysis. The analysis showed that the shift happened in 2003 or even earlier. This may indicate that PCV2 group 1 is a more adapted form of PCV2 and possibly could be more pathogenic.     

2013~2017年江西省猪圆环病毒2型分子流行病学调查

为掌握江西地区猪圆环病毒2型（PCV2）的分子流行病学及其遗传变异情况,本研究运用实验室已建立的PCR方法对2013~2017年采集自江西省南昌市、宜春市、新余市、赣州市、吉安市、九江市、上饶市、抚州市、景德镇市、鹰潭市等10个地区的1 082份疑似PCV2感染猪病料组织进行病原学检测,并通过PCR扩增、克隆和基因测序对PCV2的全基因组序列进行分析。研究表明,1 082份病料中有610份为PCV2阳性,总阳性率为56.4%,其中2013~2017年阳性率分别为52.7%、48.8%、58.5%、71.0%和67.4%。共获得89株PCV2全基因组序列,其中有83株为PCV2b基因型,其中53株归类于PCV2b-1C基因亚群,30株归为PCV2b-1A/1B基因亚群;6株为PCV2a型。测序毒株与参考毒株ORF2基因核苷酸、氨基酸序列同源性分别为88.9%~100.0%和86.0%~100.0%;氨基酸序列分析表明,Cap蛋白存在20个氨基酸突变位点。本研究表明,江西地区猪群中PCV2的主导基因型为PCV2b,其中又以PCV2b-1C基因亚群占据主导地位,同时也存在少量PCV2a基因型。     

广东省4株PCV2型分离株全基因组进化分析

【目的】 了解目前广东省猪圆环病毒2型（Porcine circovirus type 2,PCV2）分离株的基因型和进化特征,为广东省PCV2防控及疫苗株的筛选提供参考依据。【方法】 运用PCR方法将4份鉴定为PCV2阳性样品进行PCV2全基因组序列扩增、测序及遗传进化分析;利用MegAlign软件对4株PCV2广东分离株ORF2氨基酸序列与国内外参考毒株进行关键位点氨基酸变异分析;应用DNAStar中Protean软件的Jameson-Wolf方法对4株PCV2广东分离株ORF2基因编码的Cap蛋白与4株疫苗株进行抗原指数预测分析。【结果】 测序结果表明,4株PCV2广东分离株序列长度均为1 767 bp。遗传进化树结果表明,4株分离株均属于PCV2d基因型,且核苷酸相似性在97.7%~99.1%之间,与国内外54株参考毒株相似性在91.7%~99.8%之间,其中与PhuTho/G40312/2018株（Viet Nam,登录号：LC602996）、QZ1410株（江苏,登录号：MG732832）、GXBB1501211株（广西,登录号：MH756609）亲缘关系最为接近。关键氨基酸位点变异分析表明,在Cap蛋白上有8个氨基酸特异性突变位点,分别是Y3C、F8Y、T56S、R116K、V123I、K164E、R169G及T216A。抗原指数分析发现,广东省分离株的Cap蛋白抗原指数与4株疫苗株相比差异较大,主要集中在第7－12、47－53、80－90、160－170和205－213位氨基酸这5个区域。【结论】 从广东省部分地区分离的4株PCV2均为2d亚型,其Cap蛋白氨基酸序列存在多个突变位点,抗原指数与疫苗株差异较大,提示广东省PCV2的流行趋势逐渐以2d亚型为主。     

4株猪圆环病毒2型凉山州分离株全基因组进化分析

为了解猪圆环病毒2型(PCV2)凉山州分离株的基因型,本研究采用PCR方法对采自凉山州3个发病猪场的4个PCV2阳性样品进行全基因组序列的扩增、克隆、测序分析,并绘制遗传进化树.结果显示:4个PCV2凉山州分离株全基因组序列长度均为1767 bp,核苷酸同源性为99.4％～99.8％,与国内外101株PCV2参考毒株核...     

猪圆环病毒2型广东分离株ORF2基因的克隆和序列分析

为了解广东部分地区猪圆环病毒2型(porcine circovirus type 2,PCV2)流行毒株的基因型和遗传变异情况,本研究根据GenBank中PCV2的核苷酸序列设计针对ORF2基因的1对特异性引物,从分离于广东省的8株PCV2分离株BL、HD、Li、GD01、GD02、HD01、HD02和HD03株的细胞培养物中扩增ORF2基因,扩增片段克隆到pMD18-T上,获得重组质粒,并对其进行测序。序列分析结果表明,8株PCV2分离株ORF2基因与其他PCV2 ORF2基因核苷酸序列同源性为89.7%~100%,氨基酸序列同源性为87.2%~99.6%,进化分析结果表明,其中7株分离株处于同一分支,属于PCV2b-1A/1B,HD01株属于PCV2b-1C。     

凉山州腹泻仔猪猪圆环病毒2型感染情况调查

为了解凉山州腹泻仔猪猪圆环病毒2型(PCV2)的感染情况,采用PCR方法对采自四川省凉山州西昌市和喜德县7个猪场85份腹泻仔猪粪便、病变组织等样品进行PCV2检测,并将扩增到的PCV2 ORF2基因片段进行测序和序列分析。结果显示,PCV2阳性样品有22份,阳性率为25.9%,阳性猪场占71.4%(5/7)。22个ORF2基因片段长度均为459 bp,核苷酸同源性为92.8%～100%,氨基酸同源性为93.5%～100%。构建的系统进化树显示22个测序序列分别处于2个分支:PCV2b和PCV2d,但未形成明显的地理分支。结果表明凉山州猪群中PCV2感染较为普遍,且以PCV2d亚型为主,并存在一定程度的遗传变异。     

Genome Sequence Analysis of Porcine Circovirus Type 2 Strains from Shanxi Province

In order to study genetic variation of porcine circovirus type 2 (PCV2) strains in Shanxi province, the genomic sequences of four PCV2 strains which were SXXZ1, SXXZ2, SXTY and SXJZ were isolated recently from some areas of Shanxi province, they were amplified, cloned and sequenced. The amplified PCV2 genomic sequences of these four strains were analyzed and compared with that of 34 published PCV2 stains by DNAStar and drawing phylogenetic tree. The results showed that the genomic sequences of SXJZ PCV2 strain was 1 768 bp, and the others were 1 767 bp, which accounted for 25% and 75%, respectively. The homologies of nucleotide sequences of the four strains were 95.9% to 100.0%, the homologies of nucleotide sequences of the four strains with the 34 isolates from different regions of the world PCV strain were 94.5% to 99.9%, and the homologies of nucleotide sequences of the four strains with the domestic vaccine strains were 95.6% to 99.8%. The phylogenetic tree analysis showed that SXXZ1, SXXZ2 and SXTY belonged to PCV-2b genotype, and SXJZ belonged to PCV-2e genotype. The evolutionary relationship among SXXZ1, SXXZ2 and the QY strain of Guangdong were closer, we also found a recent evolutionary relationship between SXTY and AUT5 of Austria, and a recent evolutionary relationship between SXJZ and FJ of Fujian. Besides the evolutionary relationship among SXXZ1, SXXZ2, SXTY and the DBN-SX07-2 of PCV2 vaccine of Shanxi province, a recent evolutionary relationship between SXJZ and LG of PCV2 vaccine were found. Thus PCV2 strains were popular based on PCV-2b in Shanxi province, also PCV-2e was isolated and existed. It laid a foundation for the molecular epidemiology, genetic variation, prevention and control of PCV2 in Shanxi province.     

猪圆环病毒2型山西株全基因组序列分析

为了解山西省猪圆环病毒2型（PCV2）的遗传变异情况,本试验采用PCR方法对山西省分离的4株猪圆环病毒流行株（SXXZ1株、SXXZ2株、SXTY株和SXJZ株）的全基因组进行了扩增、克隆和测序,并将其全基因组序列与国内外34株主要流行毒株进行核苷酸同源性与系统进化树分析。结果显示,4株PCV2山西流行株全基因组核酸序列全长SXJZ株为1 768 bp,其余均为1 767 bp,分别占25%和75%。4株毒株核苷酸同源性为95.9%~100.0%,与国内外34株参考株同源性为94.5%~99.9%,与国产疫苗株同源性为95.6%~99.8%;PCV2全基因组序列进化分析表明,本研究分离的SXXZ1株、SXXZ2株和SXTY株属于PCV-2b基因型,SXJZ株属于PCV-2e基因型,其中SXXZ1株和SXXZ2株与广东QY株的进化关系最近,SXTY与奥地利AUT5株的进化关系最近,SXJZ与福建FJ株的进化关系最近;而SXXZ1株、SXXZ2株和SXTY株与山西PCV2疫苗DBN-SX07-2株的进化关系较近,SXJZ株与国内PCV2疫苗LG株的进化关系较近。从而证实在山西省流行的PCV2毒株以PCV-2b为主,同时还分离出了PCV-2e亚型,表明山西省存在PCV-2e亚型毒株。本试验结果为山西省PCV2的分子流行病学、遗传变异及防控奠定了基础。     

猪圆环病毒2型广西株的分离和全基因组序列分析

从广西表现为断奶仔猪多系统衰竭综合征的猪群中分离到1株猪圆环病毒2型(PCV-2),命名为GXB株.对GXB株的全基因组进行PCR扩增,扩增产物克隆至PMD18-T载体.测序结果表明,全基因组为1 767 bp,与GenBank上已知的8株PCV-2参考株序列的同源性在95.0%～99.6%之间.序列分析表明,GXB株基因组包含11个读码框(ORF),其中ORF1和ORF2是最主要的读码框,分别编码314个和233个氨基酸,与其他PCV-2毒株的ORF1、ORF2氨基酸的同源性分别为97.8%～100%、91.5%～98.7%.对GXB株ORF2编码的Cap蛋白基因进行功能分析,表明含有1个潜在的糖基化位点,3个明显的亲水区,有较强的抗原性和亲水性,为作为主要的免疫原性蛋白基因提供了依据.     

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。