Isolation and characterization of porcine circovirus type-2 from sera of stillborn fetuses

In order to examine an association between porcine circovirus type-2 (PCV2) infection and reproductive failure in pigs, sera (n = 171) from stillborn fetuses were collected from 3 different farms with prolonged histories of reproductive problems. These sera were tested for the presence of antibodies to PCV2 using an immunoperoxidase monolayer assay. Of the 171 sera tested, 28 had PCV2 antibody titers of ≥ 1:16. When these 28 samples were tested by a polymerase chain reaction assay, 13 were found to contain PCV2 viral DNA. Of these 13 samples containing both PCV2 antibodies and viral DNA, 9 yielded PCV2 on virus isolation. Amino acid sequences comprising open reading frame 2 of PCV2 from 2 of these isolates were compared to PCV2 isolates from cases of post-weaning multi-systemic wasting syndrome (PMWS). The amino acid sequences of the 2 isolates from stillborn pigs were shown to be nearly identical to each other, as well as to other PCV2 isolates associated with reproductive failure. When compared with PMWS isolates, the isolates from the stillborn fetuses showed differences of at least 2 amino acids. These results confirm previous findings that transplacental infection of PCV2 occurs in the field and that stillbirths in pigs may be associated with PCV2 infections. At present, the significance of minor differences in amino acid sequences is not known.     

Serological Investigation and Genomic Characterization of PCV2 Isolates from Different Geographic Regions of Zhejiang Province in China

Sera collected from 46 swine farms in Zhejiang province were evaluated for the presence of antibodies to PCV2 using an indirect-fluorescent antibody procedure. In addition PCV2 isolated from superficial inguinal lymph node samples collected from 40-to 90-day-old pigs with clinical signs of post-weaning multisystemic wasting syndrome (PMWS) using the PK-15 cell line were sequenced and compared. Overall seroprevalence of PCV2 antibody averaged 58.34% for all samples. Breakdown of serology by groups was as follows: 59.38% for sows, 57.41% for post-weaning piglets, 44.83% for Landrace sows and 64.28% for Landrace piglets. The seroprevalence of Landrace sows was higher than that of Yorkshire and Duroc sows, but non-significant (p > 0.05). Serological analysis also showed that seroprevalence of PCV2 antibody was a negative correlation to that of PRRSV antibody. The complete genomes of five PCV2 isolates identified in the herds with PMWS consisted of 1767nt, containing the 11 potential ORFs. Genome of the virus isolates shared 93.8% to 99.8% identity with PCV2 reference strains from GenBank, 76.6% to 77.9% identity with PCV1. Phylogenetic analysis indicated that there were two subgenotypes within PCV2: subgenotype I (1767 nt) and subgenotype II (1768 nt).     

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。     

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。     

Use of a polymerase chain reaction assay and an ELISA to monitor porcine circovirus type 2 infection in pigs from farms with and without postweaning multisystemic wasting syndrome

OBJECTIVE: To determine whether correlations exist between viremia with porcine circovirus type 2 (PCV2) and serum antibody profiles and between detection of PCV2 in nasal cavities and viremia of pigs from farms with and without postweaning multisystemic wasting syndrome (PMWS). ANIMALS: 495 pigs, ranging from the late nursery stage to the early grower-finisher stage of production. PROCEDURE: Serum antibodies to PCV2 were studied with an ELISA that detects the ORF2 viral protein. Nasal swab specimens and serum samples were tested with a PCV2-specific PCR assay. RESULTS: PCV2 DNA and serum antibodies to PCV2 were detected in pigs from all farms, although in different proportions. Overall, PCV2 DNA was detected in greater percentages in serum samples and nasal swab specimens of pigs from farms with PMWS. Although viral DNA was detected in both serum samples and nasal swab specimens, PCV2 detection in nasal swab specimens was higher than in serum samples of pigs from all farms. Serum antibodies to PCV2 were detected in a greater percentage of pigs from farms with PMWS, compared with farms without PMWS. CONCLUSIONS AND CLINICAL RELEVANCE: A high prevalence of PCV2 infection was found in pigs from farms with and without PMWS. Besides the presence of PCV2, unknown additional factors may be necessary to induce the full expression of PMWS.     

Examination for a viral co-factor in postweaning multisystemic wasting syndrome (PMWS)

In order to test the hypothesis that a putative co-factor for the development of postweaning multisystemic wasting syndrome (PMWS) in pigs could be of viral origin, we performed extensive virological examinations on organ material from pigs diagnosed with PMWS originating from within a Danish PMWS-transmission study. Virus isolation attempts were carried out on a large panel of different cell types including primary pig kidney cells and lung macrophages, primary rabbit kidney cells and seven established cell lines (MARC-145, ST117, PK15, BHK21, HeLa, Vero, and MDCK). Although these represent cells with susceptibility to a wide range of known viruses, the results did not provide evidence for a specific virus other than PCV2 contributing to the development of PMWS. Furthermore, in order to test whether specific genotypes of PCV2 may trigger the switch from PCV2 infection to clinical disease, we compared complete DNA genome sequences of PCV2 derived from PMWS-positive as well as PMWS-negative pigs. On the basis of the DNA sequences, the PCV2 isolates were divided into two groups. Group 1 consisting of one isolate originating from a herd unaffected by PMWS, with group 2 consisting of nine isolates originating from four PMWS-affected herds, four PMWS-positive pigs plus one unaffected herd. The PCV2 genomes from the two groups showed 95.5% identity. Alignment analyses of the sequences encoding the replicase and capsid protein from group 1 and group 2 PCV2 isolates showed two amino acid differences encoded in the replicase protein, while 19 amino acid differences were predicted among the capsid protein sequences. The PCV2 DNA sequence analysis supports recent observations from studies in USA as well as Europe, which suggest that strain variations may influence the clinical outcome of PCV2 infection.     

猪圆环病毒2型感染对猪繁殖与呼吸综合征灭活疫苗体液免疫的影响

本试验采用ELISA方法对单独接种猪繁殖与呼吸综合征病毒(PRRSV)变异株灭活苗组(HP组,n=3)和PCV2感染且出现病毒血症后接种PRRSV变异株灭活苗组(PCV2/HP组,n=3)不同时相血清中的PRRSV抗体进行检测;并对HP和PCV2/HP组血清中PCV2特异的抗体和核酸分别进行ELISA和荧光定量PCR检测。结果表明,在首免后70 d HP组血清平均抗体效价极显著高于PCV2/HP组(P<0.01);首免后56、63和77 d HP组平均抗体效价明显高于PCV2/HP组。其中在首免后56和63 d HP组抗体阳性率均达67%(2/3),PCV2/HP组在相应时相抗体阳性率为0;在首免后70和77 d HP组抗体阳性率均达100%(3/3),PCV2/HP组在相应时相抗体阳性率仅为0和33%(1/3)。结果提示PCV2感染可在一定程度上抑制PRRSV变异株(JXA1)特异性的抗体反应。     

Multiply-primed rolling-circle amplification (MPRCA) of PCV2 genomes: Applications on detection, sequencing and virus isolation

Multiply-primed rolling-circle amplification (MPRCA) was used to amplify porcine circovirus type 2 (PCV2) genomes isolated from tissues of pigs with signs of post-weaning multisystemic wasting syndrome (PMWS). Two of the amplified PCV2 genomes were cloned in prokaryotic plasmids and sequenced. Both were nearly identical (1767 nt) except for one silent substitution in the region coding for the capsid protein (ORF2). In addition, they showed high nucleotide sequence similarity with PCV2 isolates from others countries (93–99%). To investigate whether the MPRCA amplified PCV2 genomes could be used to produce infectious virus, the cloned genomes were isolated from the plasmids, recircularized and used for transfection in PK-15 cells. This procedure led to the production of infectious virus to titres up to 10^5.55 TCID₅₀/mL. It was concluded that MPRCA is a useful tool to amplify PCV2 genomes aiming at sequencing and virus isolation strategies, where particularly useful is the fact that it allows straightforward construction of PCV2 infectious clones from amplified genomes. However, it was less sensitive than PCR for diagnostic purposes.     

猪瘟病毒-猪圆环病毒2型疫苗联合免疫效果的评价

为建立一种既有效又简便的猪瘟和猪圆环病毒病疫苗联合免疫方法,通过细胞感染试验和动物免疫试验,评价了猪圆环病毒2型(PCV2)灭活疫苗和猪瘟病毒(CSFV)冻干活疫苗联合免疫的可行性.通过细胞感染试验证明了PCV2灭活疫苗稀释CSFV冻干活疫苗不影响CSFV疫苗的病毒活性;将PCV2灭活疫苗稀释的CSFV冻干活疫苗免疫试...     

猪圆环病毒2型感染对猪瘟疫苗体液免疫应答的影响

采用ELISA方法对单独接种猪瘟疫苗组(CSFV组,n=3)、PCV2感染且出现病毒血症后接种猪瘟疫苗组(PCV2/CSFV组,n=3)及PCV2感染同时接种猪瘟疫苗组(CSFV/PCV2组,n=3)不同时相血清中的猪瘟抗体进行检测;并对PCV2感染对照组(PCV2组)及PCV2/CSFV和CSFV/PCV2组血清中PCV2特异的抗体和核酸分别进行ELISA和PCR检测.结果表明,在接种后52 d CSFV组血清中抗体的阻断值显著高于CSFV/PCV2组(P<0.05);接种后42 d和52 d CSFV组平均抗体效价明显高于PCV2/CSFV和CSFV/PCV2组,其中在52 d CSFV组抗体阳性率这100%(3/3)而PCV2/CSFV和CSFV/PCV2在相应时相抗体阳性率仅为67%(2/3).结果提示PCV2感染可在一定程度上抑制猪瘟疫苗特异性的抗体反应.     

猪圆环病毒2型天津及周边地区分离株的遗传演化分析

为了解天津及周边地区猪圆环病毒2型（porcine circovirus 2,PCV2）的流行毒株遗传变异情况,本研究应用基因克隆技术和生物信息学分析软件对24株PCV2分离株全基因及ORF2基因分别进行克隆、测序、同源性比对和遗传进化分析。全基因组序列分析结果显示,24株PCV2分离株全基因组序列有14株为1 767 bp,10株为1 768 bp。用DNAStar软件对全基因组序列同源性比对表明,24株PCV2分离株与NCBI上的PCV2参考株的全基因序列同源性为93.6%~99.8%,24株分离株之间的全基因组序列差异很小,同源性为94.0%~99.9%。同时,用DNAStar对PCV2分离株的ORF2基因序列同源性分析表明,其与NCBI上的PCV2 ORF2基因参考序列的核苷酸序列同源性为87.3%~99.3%,24株分离株ORF2基因核苷酸序列同源性为89.0%~100.0%,3株分离株（10084F4、R14040及R14086）的核苷酸序列同源性为100.0%。对全基因和ORF2基因的遗传进化分析表明,24株分离株的系统进化树被分为2个大分支,其中23株分离毒株属于PCV2b亚型,1株属于PCV2a亚型,未分离出PCV2c亚型。综上所述,天津及周边地区PCV2的流行模式仍以PCV 2b亚型为主,本研究为该地区PCV2的流行变异情况研究提供了一定的理论依据。     

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。     

Pigs naturally exposed to porcine circovirus type 2 (PCV2) generate antibody responses capable to neutralise PCV2 isolates of different genotypes and geographic origins

Porcine circovirus type 2 (PCV2) is the essential infectious agent for PCV2-systemic disease (PCV2-SD, formerly known as postweaning multisystemic wasting syndrome) and other pathological conditions. Recent studies indicated antigenic variability amongst different PCV2 isolates and suggested that single amino acid changes within the capsid protein determine differences in the level of neutralization by specific monoclonal antibodies. The objective of the present study was to examine the cross-reactivity of PCV2 antibodies induced in the context of a natural infection against different PCV2 isolates belonging to genotypes PCV2a and PCV2b. Sera taken from several farms from animals of varying health status (PCV2-SD and age-matched healthy pigs and a set of slaughter-aged animals) were assayed for neutralizing activity against four PCV2 isolates from both predominant genotypes (PCV2a and PCV2b) and of differing geographic origins (Europe and North-America). Results showed that most of studied pigs (79 out of 82) contained neutralizing antibodies (NA) able to neutralize all four studied viral strains. Overall, pigs had significantly higher NA titres against PCV2a than against PCV2b (P < 0.001). Accordingly, studied serums were able to better neutralize Burgos390L4 and Stoon-1010 strains (PCV2a) than L-33-Sp-10-54 and MO/S-06 strains (PCV2b) (P < 0.001). No differences between capabilities of seroneutralization of viruses from different geographic origin were observed. Present data suggests that sequence differences between PCV2 isolates translate to functional antigenic differences in viral neutralization in vivo.     

大庆市及周边地区非免疫猪群猪圆环病毒感染情况调查

为了解大庆市及周边地区猪群中PCV的感染情况,应用ELISA和PCR技术对采自大庆市5区4县及周边地区55个村屯/猪场未免疫PCV2疫苗猪血清样本649份,5家病猪场和6家屠宰场组织样本448套,进行血清学和分子生物学检查,并对检测结果进行了比较分析。结果显示,大庆市及周边地区未免疫PCV2疫苗猪血清样品PCV2抗体平均阳性率为54.70%,其中断奶仔猪样品阳性率为50.68%,育肥猪样品为70.00%,繁育母猪样品为54.70%。组织样品PCV1阳性率为4.46%,PCV2阳性率为22.32%。调查结果可为确定地区流行毒株序列提供原始材料和制定科学防控措施提供依据。     

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。     

Experimental infection of 3-week-old conventional colostrum-fed pigs with porcine circovirus type 2 and porcine parvovirus

This report describes an experimental infection with porcine circovirus type 2 (PCV2) in combination with porcine parvovirus (PPV) in 3-week-old conventional colostrum-fed pigs with maternal antibodies to both viruses. Two groups of four pigs each were inoculated with PCV2 and PPV. One of the groups received also a commercial inactivated vaccine against porcine pleuropneumonia to evaluate possible effects of the stimulation of the immune system of pigs on the infection. Another group of four pigs was kept as uninfected control. Clinical signs, rectal temperatures and body weights were recorded. Serum antibody titers to PCV2 and PPV were determined at weekly intervals. Pigs were killed 42 days after inoculation and tissue samples were examined for the presence of gross and microscopic lesions. Tissues were also analyzed for the presence of PCV2 and PPV DNA by PCR, and for the presence of PCV2 antigen by immunohistochemistry (IHC). All the pigs had serum antibodies to PCV2 and PPV at the beginning of the trial. None of them developed clinical symptoms or pathological lesions typical of post-weaning multisystemic wasting syndrome (PMWS), a disease associated to PCV2 infection. However, IHC and/or PCR analyses showed that clinically silent PCV2 infection developed in five of the eight inoculated pigs, regardless of the administration of the vaccine. In particular, PCV2 DNA and/or antigen were detected in most of the tissues examined in the two pigs with the lowest titer of maternal PCV2 antibodies at the beginning of the trial. PPV DNA was not detected in any of the samples examined. The five pigs with PCR and/or IHC evidence of PCV2 infection had a mean weight gain during the experiment lower than that of the inoculated PCR-negative pigs considered together and that of the control pigs. In conclusion, it would appear that passive immunity against PCV2 can play a role in preventing the development of PMWS, but is not able to prevent the establishing of clinically silent PCV2 infections. The dissemination and persistence of the virus in the tissues may depend on the level of PCV2 antibodies at the time of inoculation.     

Isolation and characterisation of circoviruses from pigs with wasting syndromes in Spain, Denmark and Northern Ireland

A porcine circovirus (PCV) was isolated from tissues of pigs with wasting syndromes from Spain, Denmark and N. Ireland. The antigenic profiles of these viruses were determined by indirect immunofluorescence assays using polyclonal antisera and monoclonal antibodies (mAbs) prepared against previously isolated PCVs. A rapid and convenient PCR-based test was developed and used for the genotyping of these PCV isolates. These PCV isolates were found to be antigenically and genomically similar to previously reported isolates of PCV from pigs with wasting disease (PCV2), but distinct from the isolate of PCV from continuous PK/15 cell cultures (PCV1).     

广东地区新生仔猪先天性震颤猪群中PCV3感染情况的调查

对广东省不同地区新生仔猪先天性震颤猪群中PCV3的流行病学情况进行调查。采集的病料样品,通过常规PCR进行PCV3检测与全基因扩增,分析其全基因的遗传进化关系。将获得的16株PCV3毒株核酸序列与其他环状病毒参考毒株对全基因组和Cap基因进行遗传变异分析,结果显示,新生仔猪的先天性震颤猪群中PCV3样本总阳性率高达58.2%。16株PCV3毒株之间全基因核苷酸序列同源性为99.5%~100%,与美洲代表毒株PCV3-US/MO2015全基因核苷酸序列同源性在98.8%~99.1%之间;16株PCV3毒株之间Cap基因的核苷酸序列同源性为99.7%~100%,与国内外参考毒株的核苷酸同源性为99.1%~100%。遗传进化分析显示,16株PCV3毒株都属于PCV3a分支。PCV3在我国广东省新生仔猪先天性震颤猪群中广泛存在。     

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.     

Distribution of antibodies against porcine circovirus type-2 (PCV2) in single site and multi-site farrow-to-finish farms in Brazil

A comparative serologic study was performed in seven single site (SS) farrow-to-finish farms and four multi-site (MS) farrow-to-finish farms, with or without post-weaning multisystemic wasting syndrome (PMWS). In each farm, 30 blood samples were collected for each category of the production cycle: sows, farrowing crate, nursery, grower pigs, and finishing pigs. Sera were evaluated for the presence of antibodies to porcine circovirus type-2 (PCV2) via immunoperoxidase monolayer assay. Serologic profiles for PCV2 were different between SS and MS farms. Seroconversion following the decline in maternal antibodies occurred at a later stage on SS farms (grower pigs) than MS farms (nursery pigs). MS farms tended to have lower antibody titers than SS farms in the categories of sow, piglet, and nursery, while higher antibody titers were found in grower pigs. Characterization of serologic profiles for different farms may provide important information for the adoption of vaccination programs.