猪圆环病毒2型山东株全基因组的克隆与序列分析

根据GenBank已发表的猪圆环病毒2型（PCV-2）全基因组序列,设计2对特异性引物,对山东省不同地区规模化猪场采集的疑似断奶仔猪多系统消耗综合征（PMWS）组织病料进行了PCV-2全基因组克隆和序列分析。结果表明,PCV-2核苷酸序列较稳定,不同地区8个PCV-2毒株全基因组序列均由1 767 bp组成,彼此间核苷酸序列同源性达97.3%～99.8%,亲缘关系密切;与GenBank已发表的PCV-2参考毒株的同源性介于95.6%～99.8%。而ORF2的核苷酸序列同源性只有91.6%～99.9%。     

Genetic typing of porcine circovirus type 2 (PCV-2) isolates from Slovakia

Of 120 clinical specimens obtained from pigs bred on 28 PMWS-affected farms in Slovakia, porcine circovirus type 2 (PCV-2) was detected by single PCR in 77 samples. A short 224 bp fragment of ORF2 was used for preliminary grouping of isolates by phylogenetic analysis. Nucleotide sequences of the entire ORF2 region provided more precise genetic typing and segregation of preselected isolates (n = 10) into two known genotypes, PCV-2a (n = 1) and PCV-2b (n = 9). Complete genome sequencing of three selected isolates allowed their definitive grouping into genotype PCV-2b, cluster 1A or genotype PCV-2a, cluster 2D. No correlation between the mutations and the geographic origin of isolates was observed. Results confirmed that many PCV-2 isolates are genetically very stable since similar viruses circulate in Central and Western Europe.     

Quantification of porcine circovirus type 2 (PCV-2) within- and between-pen transmission in pigs

PCV-2 within- and between-pen transmission was quantified by estimating the daily transmission rate beta and the basic reproduction ratio (R(0)) using a stochastic SEIR (Susceptible, Exposed, Infectious, Removed) model fitted on experimental data. Within-pen transmission was quantified by using four groups of eight SPF (specific pathogen-free) pigs (four infected and four susceptible pigs having direct contact). Between-pen transmission was studied in two groups of 16 SPF pigs (eight infected and eight susceptible pigs having indirect contact (10 cm distance)). Pigs were monitored twice a week (blood samples) and were tested for PCV-2 antibodies (ELISA test) and viral genome load in sera (real-time PCR). Transmission parameters beta(within) and beta(between) were estimated using a maximum likelihood method and the duration of infectiousness, to compute R(0), was estimated with a parametric survival model. Different assumptions were made to determine the end of infectiousness (seroconversion, seroconversion and decline in viral genome load, permanent infectiousness). R(0[within]) (8.9 (5.1-15.4)) was greater when the end of infectiousness was assumed to be related to both seroconversion and a decline of PCV-2 genome load in sera (average duration of infectiousness = 32 days) compared with only seroconversion as the indicator of recovery (R(0[within]) = 5.5 (3.3-9.0)). Whatever the assumption, between-pen R(0) (0.58 (0.23-1.47)) was always significantly lower than within-pen R(0). Only beta(within) was sensitive to the assumption on end of infectiousness and decreased with increasing duration of infectiousness. These results showed that PCV-2 transmission is influenced by contact structure that appears worth being taken into account in an epidemic model.     

Genotypic shift of porcine circovirus type 2 from PCV-2a to PCV-2b in Spain from 1985 to 2008

Changes in porcine circovirus type 2 (PCV-2) genotypes were evaluated before, during and after outbreaks of post-weaning multisystemic wasting syndrome (PMWS) in (1) a retrospective study using pig sera collected in Spain from 1985 to 2008 and (2) a longitudinal study using pig sera collected from two farms in Spain over periods of 7 and 14 years. In both studies, there was a rapid genotypic shift from PCV-2a to PCV-2b that was related to the peak of PMWS epizootics in Spain and the appearance of PMWS on the two farms studied longitudinally.     

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.     

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.     

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.     

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型广东分离株的遗传演化分析

为了解广东地区猪圆环病毒2型(PCV2)毒株的遗传变异情况,从广东不同地区的规模化猪场采集疑似断奶仔猪多系统衰竭综合征(PMWS)病猪的淋巴结、肺脏和脾脏。对PCR鉴定为阳性的病料在PK-15细胞上增殖培养6代,采用PCR和相关分子生物学软件对12个分离株进行全基因组克隆和测序分析。结果显示,分离株基因组全长为1 767 nt或1 768 nt,核苷酸序列同源性为94.6%~99.9%。遗传演化分析表明,12个分离株分布于3个大群,其中5株属于PCV2b,4株属于PCV2d,3株属于PCV2e,提示广东地区PCV2流行毒株已发生变化。     

猪圆环病毒2型江苏分离株的遗传进化分析

采用PCR方法扩增了15个猪圆环病毒2型（PCV 2）江苏分离株的基因组DNA,以这些毒株的ORF2核苷酸序列进行遗传进化分析。经序列比较发现,所有分离株均属于2b基因群,其中7株为1A/1B亚群、8株为1C亚群;毒株间核苷酸同源性为93.6%~100%,所编码的Cap蛋白氨基酸同源性为92.3%~100%。PCV 2江苏分离株Cap蛋白的主要变异区域为53~90、121~151和190~210位氨基酸;R59、R89、S90、S121、T134、S169、A190、E210为1A/1B亚群分离株的特征氨基酸,而F8、I53、N68、L89、T90、T121、N134、R169、D210、I215、K234则是1C亚群分离株的特征氨基酸。     

Epidemiology and horizontal transmission of porcine circovirus type 2 (PCV2)

Porcine circovirus type 2 (PCV2) is a small, non-enveloped, circular, single-stranded DNA virus of economic importance in the swine industry worldwide. Based on the sequence analyses of PCV2 strains, isolates can be divided into five subtypes (PCV2a-e). PCV2 is an ubiquitous virus based on serological and viremia data from countries worldwide. In addition, PCV2 DNA was discovered in archived samples prior to the first recognition of clinical disease. Recently, a worldwide shift in PCV2 subtype from PCV2a to PCV2b occurred. PCV2 DNA can be detected in fecal, nasal, oral and tonsillar swabs as well as in urine and feces from both naturally and experimentally infected pigs. PCV2 DNA can be detected early in the infectious process and persists for extended periods of time. The effectiveness of disinfectants for reducing PCV2 in vitro is variable and PCV2 is very stable in the pig environment. Limited data exist on the horizontal transmission of PCV2. Direct transmission of PCV2 between experimentally or naturally infected animals and na?ve animals has been documented and the incorporation of clinical or subclinically infected animals into a population represents a risk to the herd. Indirect transmission through the oral, aerosol or vaccine routes is likely a lesser risk for the transmission of PCV2 in most swine populations but may be worth evaluating in high heath herds. The objective of this review was to discuss data on the epidemiology and horizontal transmission of PCV2.     

Genetic diversity of porcine circovirus type 2 from pigs in Republic of Korea

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS). In this study, 38 PCV2 cases obtained from different pig farms with different health conditions in Republic of Korea were sequenced and analyzed. Phylogenetic analysis showed that our cases had a greater variation and the existence of two PCV2 groups with at least four subgroups (1A, 1C, 2D, and 2E). Most cases obtained from PMWS-affected herds were in group 1, whereas cases with no clinical signs compatible with PMWS (wasted non-PMWS) were included within group 2. Moreover, four cases from the wasted non-PMWS belonged to PCV2-group 1. Therefore, our results suggest that PCV2-group 1 is more related to PMWS than group 2.     

猪圆环病毒2型山东分离株全基因组的克隆与序列分析

根据猪圆环病毒2型(PCV-2)全基因组序列,设计一对特异性引物,从疑似断奶仔猪多系统衰竭综合征(PMWS)病料中扩增出PCV-2基因组DNA,将基因片段克隆于pMD18-T载体,筛选获得含有相应片段的阳性重组质粒pMD18-T-PCV-2。对筛选出的阳性质粒进行测序。结果表明,PCV山东株的全基因组为1 767 bp,与国内外毒株核苷酸同源性高达99.6%。     

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型福建分离株ORF2基因遗传变异分析

为了解福建部分地区感染猪圆环病毒2型(PCV2)的基因型及遗传变异情况,采集了9份来自福建4个地区临床疑似PMWS仔猪肺、肾、淋巴结组织,病毒核酸提取后用PCV2ORF2特异引物进行PCR扩增、凝胶电泳,鉴定5份阳性样品,阳性率55.6%,回收、纯化阳性条带后送生物公司测序。Base by Base软件分析发现5份阳性样品ORF2的变异程度很小,主要是1~7个碱基点突变,有1处2个碱基连续突变,没有插入或缺失突变,同源性为98.91%~99.84%。进化树分析显示5份阳性样品都属于Group 1群(PCV2b型)中的1A/1B亚型,表明来自不同地区的样品之间变异较小。结合GenBank中已收录的所有福建18株PCV2ORF2序列进行分析,发现PCV2b基因型占95.6%,其中1A/1B亚型占72.7%,1C亚型占27.3%,结果表明PCV2b基因型中的1A/1B亚型在福建地区广泛流行并占主导地位。研究结果对本地区选择相匹配的疫苗进行有效防控具有重要意义。     

江苏部分地区猪圆环病毒2型的分子流行病学检测

根据猪圆环病毒2型（PCV2）基因序列,设计1对特异性PCV2的鉴定引物,利用PCR方法对65份疑似PCV2感染的病料进行检测。PCR检测为阳性的病料经处理后,接种无PCV污染的Dulac细胞进行间接免疫荧光检测。对各分离毒株的全基因组进行PCR扩增、测序和基因型分析。结果表明：共分离鉴定21株PCV2,均属2b基因型,其中有16株为1C群,5株为1A/1B群。研究显示PCV2b 1C群已在江苏成为流行毒株。     

A meta-analysis on experimental infections with porcine circovirus type 2 (PCV2)

A meta-analysis was performed with the aim to identify factors with a relevant influence on the expression of clinical postweaning multisystemic wasting syndrome (PMWS) under experimental conditions. Data from 44 studies were included in the analysis. Several variables were studied: number of pigs in the experiment, intake of colostrum, serological status against porcine circovirus type 2 (PCV2), strain of PCV2 used for inoculation, the route and dose of inoculation, and use of potential triggering factors (such as co-infections, vaccinations, or immunomodulator products). Multiple correspondence analysis and log-linear regression methods were used to establish the relationships between the studied variables and the number of PCV2 infected pigs that developed PMWS. Based on the results of the meta-analysis, the most successful animal experiment aimed to develop PMWS should include: (1) colostrum-deprived pigs, (2) age of inoculation below 3 weeks, (3) high doses of PCV2 inoculum, (4) PCV2 strain from genotype 1, and (5) co-infection with another swine pathogen as a triggering factor.     

Porcine circovirus type 2 (PCV-2) coinfections in US field cases of postweaning multisystemic wasting syndrome (PMWS).

The prevalence of different pathogens detected in combination with porcine circovirus type 2 (PCV-2) was studied retrospectively in field cases of postweaning multisystemic wasting syndrome (PMWS) diagnosed at the Iowa State University Veterinary Diagnostic Laboratory, Ames, Iowa, between January 2000, and September 2001. The presence of PCV-2 antigen in lymphoid tissues and/or lung, demonstrated by immunohistochemistry, together with moderate to severe lymphoid depletion and/or granulomatous lymphadenitis, was used as the criteria for the diagnosis of PMWS. A total of 484 cases fulfilled these criteria. Most of the cases (294/369) of PMWS occurred in pigs between the ages of 8 and 18 weeks, with a peak at 10 weeks of age. Porcine reproductive and respiratory syndrome virus was detected in 51.9% of the cases, Mycoplasma hyopneumoniae in 35.5%, bacterial septicemia in 14.0%, bacterial pneumonia in 7.6%, swine influenza virus in 5.4%, and PCV-2 alone in 1.9%. In cases with bacterial septicemia the most frequently isolated pathogen was Streptococcus suis. In cases with bacterial pneumonia, Pasteurella multocida was the most prevalent.     

猪圆环病毒2型细胞培养适应毒株的培育和鉴定

从临床表现为仔猪断奶后多系统衰竭综合征（PMWS）淋巴组织病料，经聚合酶链式反应（PCR）证实为猪圆环病毒2型（PCV2）感染，采用无污染的猪肾细胞系（PK15）分离培养，并连续传代培育成一株细胞培养适应毒，命名为PCV2／LG株。分离毒株经细胞培养，于第25代后毒价显著升高，于第35代毒价可达10^5.6TCID 50/mL。采用免疫过氧化物酶单层细胞染色法（IPMA）、免疫电镜技术、分子克隆及核酸序列分析等鉴定表明，分离株感染细胞后病毒抗原主要分布在细胞核及细胞质中；病毒感染的阳性细胞呈散在分布，阳性细胞数可达50％以上；免疫电镜观察到与PCV2特异抗体结合形成的病毒免疫复合物呈实心小颗粒样粒子团，病毒粒子直径约为17nm；病毒抗原基因组由1768个核苷酸组成，与GenBank登录的8个PCV2基因组序列同源性达96．2％以上。用2mL的病毒细胞培养物（10^5.6TCID 50/mL）接种30日龄PCV2抗体阴性仔猪3头，可引起典型PMWS临床症状。本研究为进一步开展该病毒的致病性、疫苗免疫、诊断及分子生物学等研究奠定了基础。