First Evidence of Porcine Circovirus Type 2 (PCV‐2) Infection of Pigs in the Czech Republic by Semi‐Nested PCR

Three oligonucleotide primers for semi‐nested polymerase chain reaction (PCR) were designed according to already published sequences of porcine circovirus types 1 (PCV‐1) and 2 (PCV‐2) isolates. These primers were used to detect PCV‐2 DNA. A positive amplification reaction was visualized from a DNA suspension containing as few as 10 copies of virus DNA. In total, 77 samples of inguinal lymph nodes and nasal swabs from pigs in the Czech Republic were used to detect the virus. Thirty‐seven of them were positive for PCV‐2 DNA. In order to confirm specificity of the PCR reaction, seven DNA fragments were sequenced. Czech PCV sequences were found to have a 92–97% homology with other known PCV‐2 strains and only 80–83% homology with PCV‐1 strains.     

Cloning and Sequence Analysis of Complete Genomes of Porcine Circovirus Type 2 (PCV-2) Jiangxi Strains

In order to understand the epidemiology and evolution of PCV-2 in Jiangxi province, a pair of primers was designed according to the PCV-2 gene sequence published in GenBank. After PCR amplification, we got the whole genome sequence of 9 strains PCV-2 isolates, and the nucleotide and protein sequences were analyzed, the genetic evolutionary tree was constructed. The results showed that the complete genome of 8 out of the 9 strains were 1 767 bp in length and one strain was 1 768 bp. By analyzing the whole genome sequences of the nucleotide,the homology of nucleotide sequences of the 9 strains was 94.7% to 99.9%.Compared with other whole genome sequences of PCV-2 in GenBank, the homology was 94.3% to 99.8%. The homology of nucleotide sequences of the ORF1 of the 9 strain was 96.9% to 100.0%. ORF2 and ORF3 encoding protein amino acid sequence had some locus mutation. Phylogenetic tree analysis showed that the 9 strains could be divided into 3 genotypes,5 strains belonged to PCV-2b, 3 strains belonged to PCV-2d, and 1 strain belonged to PCV-2a. This study was helpful to monitor and control of PCV-2 in Jiangxi province.     

猪圆环病毒2型江西株的全基因组克隆和序列分析

为了解江西地区猪圆环病毒2型（PCV-2）的流行和进化情况,根据GenBank上已发表的PCV-2全基因序列设计1对引物,PCR扩增后得到9条PCV-2全基因序列,并对其全基因序列核苷酸和蛋白序列进行分析,绘制遗传进化树。结果表明,江西地区流行的9株PCV-2中,基因组序列全长分为8株1 767 bp和1株1 768 bp,9株PCV-2的核苷酸同源性为94.7%~99.9%,与GenBank己发表的PCV-2分离株全基因组同源性介于94.3%~99.8%之间,而9株PCV-2的ORF1核苷酸序列同源性为96.9%~100.0%。ORF2和ORF3编码的蛋白氨基酸序列存在部分位点突变。遗传进化树显示为3种基因型：5株PCV-2b、3株PCV-2d、1株PCV-2a。本研究有助于江西地区PCV-2的监测和防制。     

两株猪圆环病毒2型ORF2基因的克隆和序列分析

根据GenBank中猪圆环病毒2型(PCV-2)ORF2基因序列,设计一对引物,从分离的PCV-2 GZ株、HN株的细胞培养物中扩增出ORF2基因(702 bp).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PCV-2的ORF2基因核苷酸序列同源性在90.6%～99.6%之间,推导的氨基酸序列同源性在88.9%～98.7%之间.     

PCR detection and characterization of type-2 porcine circovirus.

A polymerase chain reaction (PCR) assay was developed for detecting porcine circovirus (PCV). The assay readily detected type-2 PCV (PCV-2) and type-1 PCV (PCV-1). The PCR primers were designed based on DNA sequences conserved in all reported PCV genomes. Type 1 PCV and type 2 PCV both produced 438 bp amplification products, which were easily identified and differentiated from one another by restriction fragment length polymorphism (RFLP) analysis. Porcine circovirus was detected in 55% (931/1693) of randomly tested pigs with various clinical signs and lesions, most of which were difficult to differentiate from those associated with porcine reproductive and respiratory syndrome (PRRS). The PCR products from all positive clinical samples were identified by RFLP to be only PCV-2; DNA tested by PCR was extracted directly from one or more of lung, mesenteric or mediastinal lymph nodes, and tonsil. Type 2 PCV was also detected in 6% (2/34) of DNA extracted directly from semen of randomly chosen healthy boars. Positive PCR reactions from 554 diseased pigs were characterized by RFLP and categorized into 5 different profiles (A-E), of which 82.8% were PCV-2A (456/554), 3.0% were PCV-2B (17/554), 9.9% were PCV-2C (55/554), 1.1% were PCV-2D (6/554), and 3.2% were PCV-2E (18/554). The complete genomic nucleotide sequences of PCV-2A, B, C, D, and E were determined and found to have at least 95% homology compared with one another and with all other PCV-2 found in the GenBank database. All PCV-2 had less than 76% homology with PCV-1. This PCR assay will hopefully be useful to veterinary diagnostic laboratories for routine testing and surveillance of infection with PCV-2. The RFLP profiling system might be useful for preliminary characterization and identification of PCV isolates and might also benefit studies on the molecular epidemiology of PCV.     

猪细小病毒、伪狂犬病病毒和猪圆环病毒2型多重PCR检测方法的建立及应用

根据猪细小病毒(PPV)、伪狂犬病病毒(PRV)和猪圆环病毒2型(PCV-2)的基因序列,分别选取各自的保守区段设计引物,通过反应条件的优化,建立了检测PPV、PRV和PCV-2的多重PCR方法.用建立的方法对采自陕西省部分猪场的286份病料及血样进行检测,从对临床健康猪全血样品中PPV、PRV和PCV-2的检测结果看...     

猪圆环病毒2型PCR检测方法的建立及其在临床检测中的应用

选择猪圆环病毒2型(PCV-2)基因保守区设计1对引物P1和P2,扩增536 bp的片段,该方法可以特异地检测出PCV-2的DNA,而对猪细小病毒、猪瘟病毒、猪伪狂犬病毒及未接PCV-2的PK-15细胞均呈阴性;该法能检测出29 ng/L的病毒DNA。对扩增片段进行测序,结果表明扩增片段属于PCV-2。应用该方法对2008年度上海及周边地区送检的91份临床样本进行了PCV-2的检测,结果表明,阳性样本为52份,阳性率为57.14%。研究结果表明,建立的PCR方法检测PCV-2具有较好的敏感性和特异性,可用于PCV-2感染疑似病例的诊断及其分子流行病学调查。     

Molecular diversity of Brazilian strains of porcine circovirus type 2 (PCV-2)

Porcine circovirus 2 (PCV-2) is associated with a broad range of syndromes. In this study, eight pig tissue samples from two Brazilian states were analyzed using six PCR primer pairs amplifying a 1705-bp fragment of the PCV-2 genome. The NJ distance-based method was used for the phylogenetic analysis with the eight field strains herein, 15 GenBank sequences and using PCV-1 as an out-group. This yielded two major clusters (A and B) for this viral species, with the Brazilian strains segregating with European and Asian sequences. Nucleotide identity was 99.7 to 100% among the sequences. This information can be used in further studies of pathogenesis related to PCV-2 in Brazil.     

The emergence of porcine circovirus 2b genotype (PCV-2b) in swine in Canada

Since late 2004, the swine industry in the province of Quebec has experienced a significant increase in death rate related to postweaning multisystemic wasting syndrome (PMWS). To explain this phenomenon, 2 hypotheses were formulated: 1) the presence of a 2nd pathogen could be exacerbating the porcine circovirus 2 (PCV-2) infection, or 2) a new and more virulent PCV-2 strain could be infecting swine. In 2005, 13 PMWS cases were submitted to the Quebec provincial diagnostic laboratory and PCV-2 was the only virus that could be found consistently by PCR in all 13 samples. The PCR detection results obtained for other viruses revealed the following: 61.5% were positive for porcine reproductive and respiratory syndrome virus, 30.8% for swine influenza virus, 15.4% for porcine parvovirus, 69.2% for swine torque teno virus (swTTV), 38.5% for swine hepatitis E virus (swHEV) and 84.6% for Mycoplasma hyorhinis; transmissible gastroenteritis virus and porcine respiratory coronavirus (TGEV/PRCV) was not detected. Sequences of the entire genome revealed that these PCV-2 strains belonged to a genotype (named PCV-2b) that has never been reported in Canada. Further sequence analyses on 83 other Canadian PCV-2 positive cases submitted to the provincial diagnostic laboratory during years 2005 and 2006 showed that 79.5% of the viral sequences obtained clustered in the PCV-2b genotype. The appearance of the PCV-2b genotype in Canada may explain the death rate increase related to PMWS, but this relationship has to be confirmed.     

猪圆环病毒2型不对称PCR检测方法的建立

为研究制备单链DNA的方法,根据GenBam已发表的PCV-2全基因组序列,设计合成2条特异性引物和1条通用引物,通过pGM-T-PCV-2重组质粒的构建、引物浓度优化、单链PCR产物的杂交测序鉴定,以及敏感性和特异性试验,对PCV-2不对称PCR方法进行了研究.结果显示,上、下游引物用量比为20:1～40:1时,可明...     

猪Ⅱ型圆环病毒ORF2基因的克隆及其序列分析

根据 GenBank 中猪圆环病毒Ⅱ型(PCV- 2)ORF2基因序列,设计一对引物,应用PCR从疑似断奶仔猪多系统消耗综合征(PMWS)的死亡仔猪组织病料中扩增出 ORF2 基因(702 bp)。将此基因片段克隆入 pMD -18 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基因与德国分离株AF201897核苷酸序列同源性为99.5%与其它PCV- 2 的 ORF2 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。     

FRRSV和PCV-2双重PCR检测方法的建立及应用

根据猪繁殖与呼吸综合征病毒(PRRSV)美洲型标准株(ATCCVR-2332)的ORF7保守序列和猪圆环病毒2型(PCV-2)(AF381175)的ORF2基因保守序列,设计合成了两对特异性引物。用这两对引物,通过优化的PCR条件,对PRRSV阳性毒株反转录后的cDNA模板和PCV-2毒株的DNA模板进行双重PCR扩增,同时得到两条与试验设计相符的432bp(PRRSV)和630bp(PCV-2)特异性条带,建立了同时检测PRRSV和PCV-2的双重PCR方法。并用此方法对在安徽省不同地区所采集的72头份病猪的淋巴结、肺、肝、脾、肾等组织进行检测,证明建立的PCR方法可用于临床诊断。     

检测PRRSV—Ⅰ、PRRSV—Ⅱ和PCV-2多重PCR方法的建立及初步应用

猪繁殖与呼吸综合征病毒（Porcine Reproductive and Respiratory Syndrome Virns,PRRSV）欧洲株（PRRSV-Ⅰ）、北关株（PRRSV-Ⅱ）常与猪圆环病毒2型（Porcine Circorirnstype 2,PCV-2）呈混合感染,严重影响养猪业的经济效益。本文根据GenBank上已发表的PRRSV北美型（PRRSV-Ⅰ）、PRRSV欧洲型（PRRSV-Ⅱ）、圆环病毒2型（PCV-2）的基因组全序列,分别设计了3对能特异性扩增PRRSV-Ⅰ、PRRSV-Ⅱ和PCV-2的引物,建立并优化了可同时检测PRRSV-Ⅰ、PRRSV-Ⅱ和PCV-2三种病毒的多重PCR方法,通过对临床病料的检测,证实了此方法具有高特异性、高灵敏度、高效率、低成本的特点,适合大量样本的分析与鉴定。利用该方法能在24h内对样品进行检测并获得结果,因此本方法的建立对这3种病毒的早期快速诊断有十分重要的意义。     

Ring tests to evaluate the performance of Porcine circovirus-2 (PCV-2) polymerase chain reaction (PCR) assays used in North American diagnostic laboratories

Two laboratory studies involving 11 laboratories were undertaken to assess the performance of North American Porcine circovirus-2 (PCV-2) polymerase chain reaction (PCR) assays. Laboratories received identical submissions containing randomly coded positive and negative control samples, and serially diluted PCV-2-spiked samples. In study 1 and 2, respectively, spiked samples contained measured amounts of PCV-2 virus or DNA. All but 1 assay detected DNA in the most concentrated spiked sample. There were no statistical differences in the proportion of positive or negative samples reported by quantitative (n = 7) versus non-quantitative (n = 6) assays. Across both studies, the false positive rate was 17% (4 out of 23), and 17% (2 out of 12) of assays cross-reacted with PCV-1. The most sensitive assay detected PCV-2 DNA levels about 100 000 times lower the least sensitive assay. This study demonstrated that the PCR assays available in North American diagnostic labs vary considerably in their detection limits and quantification.     

3株新城疫山东分离株HN基因全序列测定和分析

用设计的特异性引物,通过RT-PCR法对1999和2004年间分离自山东省的具有一定代表性的3株鸡新城疫病毒(ShD-2-04,ShD-3-99,ShD-5-04)进行HN基因的扩增和克隆,并对其进行核苷酸序列测定和分析。结果表明:3个毒株HN基因开放性阅读框架(ORF)均为1716bp,编码571个氨基酸,属强毒的C群;3株山东分离毒的核苷酸同源性为98.2%~98.8%,氨基酸同源性为98.4%~98.8%,其中ShD-2-04和ShD-5-04核苷酸及氨基酸同源性均为最高,达到99.8%和99.8%;将3株山东分离毒株与国内外标准强毒株及弱毒株的HN基因核苷酸及氨基酸同源性分别进行比较:3毒株与国内外标准毒株HN的核苷酸同源性为82.7%~87.2%,氨基酸同源性为87.6%~90.0%,表明已发生一定的变异。     

两株猪圆环病毒2型的分离及基因组序列分析

为比较猪圆环病毒2型毒株的遗传变异特性,从2009年河南郑州和山东东营的猪场疑似猪断奶后多系统衰弱综合征(PMMS)病料中分离到2株病毒,经PCR检测初步鉴定为猪圆环病毒2型,并对病毒全基因组进行扩增,用DNA Star对序列进行比较分析.结果表明,分离到的河南郑州和山东东营PCV-2毒株全基因组长度均为1 767 b...     

猪瘟病毒与猪圆环病毒2型混合感染的检测

对广西南宁市、贵港市、崇左市共5个规模化猪场送检病猪的组织器官样品(脾脏和淋巴结),应用已建立的检测猪瘟病毒(CSFV)的RT-PCR技术和检测圆环病毒2型(PCV-2)的PCR技术,快速准确地扩增出了CSFV和PCV-2特异的目的基因片段,从而证实为猪瘟病毒和猪圆环病毒2型混合感染。     

陕西省部分地区猪圆环病毒2型全基因组的遗传变异分析

为了解陕西省猪圆环病毒2型(PCV2)的遗传变异情况,根据GenBank登录的PCV2全基因组序列,设计1对引物,从陕西省部分地区规模化猪场疑似PCV2感染的病猪采集病料9份,应用PCR扩增PCV2的全基因,其中6份为阳性,并对扩增的6个PCV2全基因组序列进行测序和序列分析。基因测序表明,PCV2基因组全长为1 767bp;对6株病毒序列进行同源性比较,6个毒株全基因之间核苷酸同源性为98.3%~100%,与GenBank上已发表的国内外毒株全基因组比较,同源性为96.3%~97.8%;与近几年陕西株比较发现基因变异程度不稳定,与2013年分离株(KX352154.1)同源性最高,2015年分离株(KX352159.1)次之,反而与2014年分离株(KX068219.1)最低;对6个毒株的ORF1和ORF2基因进行同源性比较,6个毒株的ORF1和ORF2基因的核苷酸同源性分别为98.1%~100%和98.2%~100%,与GenBank上已发表的国内外参考株ORF1和ORF2基因进行同源性比较,同源性分别为97.6%~99.8%和91.5~96.0%。陕西省流行的PCV2基因组较为保守,变异不大,同源性很高。     

猪圆环病毒3型PCR检测方法的建立与Cap基因序列分析

建立一种检测PCV3流行毒株感染的PCR方法,并分析Cap基因序列变异情况,为PCV3流行病学调查提供技术支持.根据GenBank中公开的PCV3全基因序列,分别设计了质粒构建引物和检测引物,通过构建阳性质粒、灵敏度试验、特异性试验、临床样品检测试验以及测序分析,建立PCR检测方法.结果显示:建立的PCR方法检测的极限...     

Evaluation of a nested polymerase chain reaction assay to differentiate between two genotypes of Porcine circovirus-2.

Porcine circovirus-2 (PCV-2) is associated with several diseases in pigs, including postweaning multisystemic wasting syndrome (PMWS). A new genotype of PCV-2 was isolated from swine farms with and without clinical PMWS in North America. The new genotype was differentiated in a separate cluster by phylogenetic analyses and is now named PCV-2b compared with PCV-2a for the previously known genotype. The purpose of this study was to develop and evaluate a nested polymerase chain reaction (nPCR) assay to detect and differentiate between PCV-2a and PCV-2b. Genotype-specific primer sets were designed by using sequence data published for different PCV-2 strains. Specificity and sensitivity of the nPCR were examined by using PCV-2 isolates with known genotype. Nested PCR was found to be highly specific and sensitive for detecting and differentiating between the PCV-2 genotypes compared with the conventional 1-step PCR assay. Nested PCR was applied to detect PCV-2 and to identify the genotype in serum samples from swine farms with and without a clinical history of PMWS. Of 60 serum samples collected from 4 farms during clinical PMWS outbreaks, PCV-2a and PCV-2b were detected in 6 and 49 samples, respectively. Six of the 10 samples from one of the 4 farms had both PCV-2a and PCV-2b. Of 20 serum samples from 2 farms without PMWS, 11 were positive for PCV-2a only. These results suggest that the differential nPCR can be used to detect PCV-2 and to differentiate the 2 genotypes from field samples.