猪圆环病毒2型分离株优势基因型分析与检测

为探讨猪圆环病毒2型(PCV2)流行株基因型的变化,本研究对1999年～2009年NCBI中登录的556个PCV2分离株全基因序列进行分析。结果表明,1999年～2002年所登录的PCV2的优势流行毒株为基因A型(PCV2A);从2003年开始基因B型(PCV2B)逐渐为优势流行毒株,在PCV2分离株中基因B型所占比例,2004年为95.4%,2007年～2009年均超过76.0%。2003年～2009年国内的PCV2分离株有287个,其中250个为PCV2B,37个为PCV2A,表明PCV2B是国内猪群中的主要优势毒株。通过建立PCV2A和PCV2B PCR检测方法,对70份临床病料的检测显示,PCV2检出率为34.29%(24/70),基因型均为PCV2B,无PCV2A的检出,表明PCV2B为检测猪群中的主要优势毒株。     

Characterization of porcine circovirus type 2 in Taiwan

In an effort to understand the genetic diversity of porcine circovirus type 2 (PCV2) and the prevalence of PCV2 infection in Taiwanese herds, we have sequenced the complete genomes from PCV2-infected specimens and individually measured the antibody titer against PCV2 from pigs reared in Taiwan between the years 2000 and 2002. A total of 623 specimens originating from pigs displaying varied clinical signs were screened with the polymerase chain reaction (PCR). Results showed that 309 pigs (49.6%) tested positive for PCV2. Eight of the positive specimens were used for the amplification of the complete viral genome. Sequence comparison of the complete genomes indicated that the 8 Taiwanese PCV2 isolates shared 95-99% similarity. Phylogenetic analysis of all 40 PCV2 isolates from North America, Europe, Asia and Taiwan revealed that those isolates were grouped together in one large group containing two minor subgroups. The Taiwanese PCV2 isolates were classified into the two minor subgroups. The prevalence of serum antibodies to PCV2 in pigs was investigated, and results showed that approximately 83.5% of the pigs in Taiwan were seropositive. Finishing pigs possess the highest titers of antibodies, while 9-week-old pigs contained the lowest titers for specific antibodies. Our results suggest that PCV2 infections have become common in Taiwanese pig farms.     

猪圆环病毒2型的分离与鉴定

利用PCR方法,从疑似断奶猪多系统衰竭综合征(PMWS)的自然病例的淋巴结和脾脏中扩增出了预期长度的猪圆环病毒2型(Porcine Circovirus type 2,PCV2)的DNA片段,并用限制性内切酶对PCR产物进行了酶切鉴定。将经PCR扩增和酶切鉴定为PCV2阳性的组织样品匀浆液接种到无PCV污染的PK15细胞中传代,经间接免疫荧光检查,在接毒细胞内观察到了特异性免疫荧光;用接毒细胞制备超薄切片,在电镜下观察到了直径大约为17nm的圆形病毒样粒子和大量不同形态的胞浆内包涵体。由此表明分离出的病毒为猪圆环病毒2型。     

猪圆环病毒2型毒株的分离鉴定

猪圆环病毒2型(PCV2)可引起断奶后仔猪多系统衰竭综合征(PMWS)、猪皮炎肾病综合征(PDNS)、繁殖障碍等相关疾病[1],其中以PMWS最为严重.该病主要侵害6～12周龄仔猪,引起渐进性消瘦、呼吸困难、腹泻和黄疸等,给世界养猪业造成了巨大的经济损失.     

猪圆环病毒2型标记毒株的构建与iDNA疫苗的初步研究

为实现携带分子标记的猪圆环病毒2型标记毒株的构建,对其进行感染性DNA(iDNA)疫苗的初步研究,即构建的感染性克隆质粒既能发挥DNA疫苗的作用,同时又能在动物机体内拯救出活病毒,发挥病毒活疫苗的作用,本研究以PCV2Cap蛋白末端具有赖氨酸延伸特征的GZ-RH1株为材料,构建该毒株的感染性克隆质粒pcDNA3.1(+)-PCV2,并在病毒基因组中引入1个SalⅠ酶切位点作为鉴别拯救病毒的分子标记。将该感染性克隆质粒转染PK-15传代细胞进行病毒拯救后,通过IPMA、PCR-RFLP以及全基因组测序等试验对拯救病毒进行检测和鉴定,并初步测定了拯救病毒在体外细胞培养的增殖能力。结果表明,利用构建的感染性克隆质粒拯救获得的PCV2标记毒株,经盲传10代后毒价可到105.3 TCID50/mL,分子标记能够稳定存在,该感染性克隆质粒可以应用到PCV2iDNA疫苗的初步研究中。昆明小鼠接毒试验结果表明,构建的感染性克隆质粒在小鼠体内也具有感染性,能够拯救出PCV2标记毒株,且可以诱导产生抗PCV2的抗体,与体外拯救的标记毒株免疫小鼠具有类似的体内生物学特性,表明PCV2iDNA新型疫苗构建策略是可行的。本研究为进一步研究PCV2iDNA疫苗奠定了基础。     

Detection and genetic characterization of porcine circovirus type 2 (PCV2) in pigs from Croatia

Porcine circovirus type 2 (PCV2) from the Circoviridae family has recently been associated with two serious diseases of swine, post-weaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). During 2002, several outbreaks of clinical disease in pigs with weights ranging from 10 to 70 kg occurred on four farms in different locations in Croatia. The signs were consistent with PMWS and PDNS. Apart from progressive weight loss, pneumonia and/or diarrhoea, multifocal erythematous skin lesions and dermal necrosis were also observed. The PCR results obtained from PCV2 specific oligonucleotide primers confirmed a PCV2 infection. In addition, archive samples that were classical swine fever virus positive and derived from domestic pigs during an outbreak in 1997 were included in this study and one out of the three isolates was found to be positive for PCV2. For a better epizootiological understanding, genetic typing of representative isolates was carried out and compared with available isolates reported in the GenBank databases.     

猪圆环病毒2型感染的检测与流行病学调查

根据GenBank上发表的PCV2 ORF2和PRRSV ORF7序列设计合成引物,建立分别用于检测PCV2和PRRSV的PCR和RT-PCR方法,并对2003年9月至2005年5月采自189个可疑发病猪场的389份病料进行了PCV2和PRRSV的检测。结果显示,来自33个猪场的35份样品为PCV2阳性,猪场PCV2阳性率为17.1%;PCV2与PRRSV混合感染率为5.1%,PCV2阳性猪场中PRRSV感染率为57.6%。流行病学统计结果表明,安徽省猪场PCV2阳性率最高;安徽省和广西省猪场PCV2和PRRSV的混合感染最严重;春夏季节PCV2阳性率明显高于秋冬;消瘦、被毛粗乱是PCV2感染的主要特征;淋巴结水肿或出血,肾脏肿大、出血或呈灰白色以及脾脏病变是PCV2感染的主要病理变化;仔猪7～8周龄发病率最高。     

猪圆环病毒2型SH毒株免疫原性

用D-氨基葡萄糖处理PK-15细胞,将PCV2-SH分离株增殖传代至第50代,测定病毒滴度TCID50均为10-6.25/mL.将第20代和第50代病毒液分别灭活乳化制成灭活疫苗,进行猪体免疫保护试验.结果显示,这2个不同代次的病毒制成的疫苗都能刺激机体产生较高水平的ELISA抗体,攻毒后2个免疫组的猪均没有明显的临床症状,相对日增重相似,同时病理变化和病毒血症程度明显减轻.结果表明,PCV2-SH株具有稳定的免疫原性,并可以提供有效的免疫保护作用.     

Replication and transmission of porcine circovirus type 2 in mice

Little information is known about infection, replication and transmission of porcine circovirus type 2 (PCV2) in species other than swine. Two sets of animal experiments were carried out to investigate the susceptibility of mice to PCV2 and to study their possible role in maintaining and transmitting the virus. In the first experiment 14 mice were inoculated with PCV2 by the intraperitoneal route with 5 x 10(2) TCID50 of the PCV2-ROM strain (Cadar et al., 2007). In a second experiment 24 mice were divided into two groups (A and B); mice in Group A (n = 18) were inoculated orally with 1 x 10(5) TCID50 PCV2-ROM and mice in Group B (n = 6) were left uninoculated until day 12 post inoculation (p.i.), when they were mixed with Group A. The animals were sacrificed at intervals for postmortem investigation and virus genome detection by polymerase chain reaction (PCR). The PCR results indicated that PCV2 could replicate in mice infected intraperitoneally or by the oral route, and that the virus can be transmitted directly from mouse to mouse.     

Efficacy of bivalent vaccines of porcine circovirus type 2 and Mycoplasma hyopneumoniae in specific pathogen-free pigs challenged with porcine circovirus type 2d

BackgroundPorcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MHP) are economically significant pathogens in the pig industry. The use of combined vaccines against PCV2 and MHP is one of the most effective ways of protecting pigs from both diseases, and it has become a part of general management.ObjectivesThis study evaluated the efficacy of two new bivalent vaccines of PCV2 and MHP (Myco-X and Myco-XD) in SPF pigs. Myco-X and Myco-XD are a combined vaccine of MHP with PCV2b and PCV2d, respectively.MethodsSixteen pigs were divided into four groups: Myco-X-vaccinated challenged, Myco-XD-vaccinated challenged, unvaccinated challenged, and unvaccinated unchallenged. Two milliliters of Myco-X were administered intramuscularly, and 0.5 mL of Myco-XD was injected intradermally at 3 wk of age. The pigs were challenged with virulent PCV2d via the intramuscular and intranasal route 4 wk post-vaccination.ResultsAll vaccinated pigs showed effective reduction of the clinical signs, the PCV2d load in the blood and nasal swab samples, as well as lung and lymphoid tissue lesions in the challenge test. Compared to unvaccinated challenged animals, the vaccinated challenged animals showed significantly higher (p < 0.05) levels of anti-PCV2 IgG, PCV2d-specific interferon-γ (IFN-γ), and anti-MHP IgG.ConclusionsBased on clinical, microbiological, serological, and pathological assessments, this study confirmed that both combined vaccines could protect pigs against PCV2 infection caused by PCV2d. On the other hand, further research on the efficacy evaluation of these new vaccines against the MHP challenge and PCV2d/MHP co-challenge is needed.     

Association of porcine circovirus type 2 with vascular lesions in porcine pneumonia

The aim of this study was to evaluate the vasculature in porcine circovirus type 2-infected (PCV2-infected) lungs and to identify the PCV2 subtypes involved in porcine pneumonia. Pulmonary samples from 140 pigs, 2 weeks to 7 months of age, from 36 Hungarian commercial herds with clinical signs of respiratory disease were examined for the presence of respiratory pathogens, with bacterial culture, pathologic evaluation, and immunohistochemistry for PCV2, porcine reproductive respiratory syndrome virus, and swine influenza virus. PCV2 was the most commonly identified pathogen (49 cases) among the 74 of 140 cases (53%) with respiratory pathogens. PCV2 was detected immunohistochemically in the wall of 13% to 100% of pulmonary vessels (mean, 89%) in 38 of 49 cases (78%). Detection of PCV2 antigen was positively correlated with the presence of vascular lesions (P < .001, odds ratio [OR]: 159.54). Other pathogens capable of vascular injury in swine were found in 29 of 49 of the PCV2-positive cases (59%). The probability of detecting vascular lesions in PCV2-infected lung was higher than in infection with porcine reproductive respiratory syndrome virus (P < .002, OR: 14.63), Pasteurella multocida infection (P < .001, OR: 5.75), or Streptococcus spp. infection (not significant, OR: 1.45). Sequence analysis of open reading frame 2 amplicons was possible in 6 PCV2-positive cases, from which 5 cases proved to be PCV2b subtype and 1 case, PCV2a subtype. In conclusion, PCV2 antigen was commonly colocalized with pulmonary vascular lesions in pneumonia in Hungarian swine, and PCV2b was the dominant subtype.     

猪圆环病毒2型诊断技术研究进展

猪圆环病毒2型(PCV2)是新发现的迄今为止最小的动物病毒,是猪断奶后多系统衰竭综合征(PMWS)的病原.PMWS给世界养猪业造成了巨大的经济损失.本文综述了猪圆环病毒2型的诊断技术,包括抗原和抗体的检测技术.     

猪圆环病毒2型遗传标记毒株的构建及生物学特性鉴定

以临床分离的猪圆环病毒2型（PCV2）为材料，利用PCR法扩增病毒基因组，将2个基因组顺式连接插入到质粒载体中构建感染性分子克隆。通过引物设计替换碱基，在病毒基因组内插入Sal Ⅰ限制性内切酶位点作为遗传标记，经重组质粒转染细胞获得带有遗传标记的新毒株。采用免疫过氧化物酶单层细胞试验、免疫电镜、核酸序列分析表明，在病毒感染细胞中检出病毒特异抗原，其抗原性、病毒形态学及基因序列与亲本毒株一致。鉴于新病毒基因组内插入一个SalⅠ酶切住点，用PCR与限制性片段长度多态性分析法可与野生型病毒相鉴别。新毒株经细胞连续传60代，体外培养增殖性能稳定，毒价可达10^6.6CID50/mL。取病毒培养物经静脉和滴鼻途径接种35日龄抗体阴性仔猪4头，接种后表现出体温升高、进行性消瘦、被毛粗糙及体表淋巴结肿胀症状，迫杀后多种脏器中均能检测到病毒抗原与核酸。研究表明，利用感染性分子克隆手段构建带有遗传标记的PCV2新毒株，为进一步开展该病毒的致病性、疫苗免疫、分子诊断等研究奠定了基础。     

广西猪圆环病毒2型感染的流行病学调查

结合流行病学、临床症状、病理变化，采用PCR技术，对2004年1月至2005年5月采自广西14个市97个疑似猪圆环病毒2型（PCV2）感染发病猪场的197份组织病料（脾、肺、淋巴结）进行了PCV2检测；同时，对鉴定为PCV2阳性的组织病料和猪场进行了猪生殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪流感病毒（SIV）和猪伪狂犬病病毒（PRV）的检测，另外，对6个地（市）11个生猪屠宰场采集的外观健康屠宰猪的295份组织样品（脾、肺、淋巴结）进行了PCV2检测。结果显示，在197份组织样品中检出PCV2阳性病料108份，平均阳性率为54．82％（108／197），阳性猪场62个，平均阳性率为63．92％（62／97）。PCV2与PRRSV、CSFV、SIV、PRV混合感染的组织病料总阳性率为42．13％（83／197），混合感染的猪场总阳性率为57．73%（56／97）。从21头外观健康屠宰猪的组织样品中检测到PCV2，阳性率为7．10％。由此可见，PCV2感染在广西猪群中已普遍存在，混合感染和健康带毒现象使病情更加复杂。     

First report of porcine circovirus type 2 infections in Cuba

To obtain information about the porcine circovirus type 2 (PCV2) infection status of pigs in Cuba and the probable association of PCV2 with other porcine viruses, tissue samples collected from ill pigs were evaluated using polymerase chain reaction (PCR). The PCR analysis showed that 67.7% of the samples (23/34) from seven swine herds of six different geographic regions were detected to be positive for PCV2. Ten of the 23 PCV2 positive samples (43.5%) shown a concurrent infection with porcine parvovirus (PPV) and 17 of 23 PCV2 positive samples (73.9%) exhibited a concomitant infection with classical swine fever virus (CSFV). This study is the first report of PCV2 infecting pigs with different clinical conditions in Cuban swine herds and provides evidence of PCV2 co-infection with PPV and CSFV in the field.     

某猪场猪2型圆环病毒感染的诊断

猪2型圆环病毒(PCV2)是引起断奶仔猪多系统衰竭综合征(PMWS)的主要病原,此外,PCV2还可能与猪皮炎肾病综合征、猪呼吸系统综合征、仔猪先天性震颤等疾病有关.PMWS临床症状主要表现为进行性消瘦,生长发育受阻,皮肤苍白或有黄疸,体表淋巴结肿大,腹泻,贫血和呼吸道症状等.     

应用套式PCR方法检测猪圆环病毒2型

根据Genbank中发表的猪圆环病毒2型(PCV2)全基因序列,设计2对PCV2特异性引物,建立套式PCR检测方法。外测引物p1、p2扩增片段长度为647bp(92-738),内测引物p3、p4扩增长度为219bp(319-537)。其中用外部引物可扩增DNA含量到10-5mg/mL,而套式PCR则比普通PCR灵敏性还要提高103倍。用该方法对山东、安徽和河北10省的899份临床发病猪的肺脏和淋巴结样品进行检测,结果有329份样品检测出阳性,平均阳性检测率达36.6%。由此可见,PCV2感染在全国范围内的发病猪群中普遍存在。     

猪圆环病毒2型检测与免疫技术研究进展

对年近来国内外对猪圆环病毒2型(PCV2)的检测技术进展和疫苗研究进展作了简要回顾。PCV2实验室检验技术有间接免疫荧光(IFA)、间接酶联免疫吸附试验(ELISA)、免疫过氧化物酶单层细胞试验(IPMA)、单克隆抗体以及多种聚合酶链式反应(PCR)技术等。在疫苗研究方面,国外已研发多种PCV2灭活疫苗,国内亦有人正在研发PCV2灭活苗(SH株)。在PCV2基因工程疫苗研究方面主要有重组活载体疫苗和核酸疫苗两类。     

2009年我国猪群中猪圆环病毒2型感染情况调查

为了解我国猪群中猪圆环病毒2型（Porcine circovirustype2,PCV2）的感染情况,分析感染与猪年龄的关系,本试验于2009年在我国28个省市的71个中小规模场、22个屠宰场和62家散养户采集了2905份猪血清样品,用PCR法对PCV2进行检测。结果显示,检出阳性血清429份,阳性率为14．8％。在被调查的场／P中,86．4％屠宰场、52．1％中小规模场和58．1％散养户为PCV2感染阳性,其中48．6％的PCV2阳性中小规模场的群阳性率低于10％。分析中小规模场PCV2感染情况和猪年龄之间的关系发现,在5个年龄组中,2～4周龄组未检出PCV2感染,5周龄以上各组均检出PCV2感染,其中11～14周龄组和15～26周龄组阳性率较高,高于5～7周龄组和8～10周龄组。     

猪圆环病毒2型致病机制研究进展

猪圆环病毒2型是引起断奶仔猪多系统衰竭综合征(PMW S)的主要病原。PMW S主要以进行性消瘦和多系统病理损伤为特征,近些年来流行很广,给世界养猪业带来巨大损失。本文对该病毒致病机制的研究进展作一综述。