新疆北疆部分地区PCV2的PCR检测及基因型分析

猪圆环病毒2型（PCV2）是猪源疾病中的主要致病原。为了解新疆北疆部分地区PCV2的感染情况及阳性毒株基因型。在石河子及北屯地区的屠宰场共采集了203份抗凝血液，采用PCR方法对血样进行PCV2检测，在分离的PCV2阳性毒株中，选取2株克隆、测序，并进行了同源性分析和遗传进化分析。结果在待检样品中，PCV2阳性感染率为7．9％（16／203）；两个阳性样品测得序列之间的同源性为100％，它们与PCV2a参照序列之间的同源性为86．4％--87．4％，与PCV2b参照序列之间的同源性为92．9％～96．4名；两个阳性样品的序列在构建的遗传进化树中与PCV2b参照序列同属于一个分支，亲缘关系较近。结果证实本研究中所获毒株属于PCV2b基因型，表明新疆北疆部分地区猪群中存在PCV2感染，其基因型为2b型。     

猪圆环病毒2型与猪TTV混合感染的流行病学调查

根据GenBank_h发表的猪圆环病毒2型（Porcinecircovirustype2,PCV2）基因组序列和TTV（Torquetenovirus）1、2型的UTR序列设计合成引物,建立了分别用于检测PCV2和TTV1、TTV2的PCR及巢式PCR方法。应用建立的PCR方法对送检的广东、福建和江西等7个省份258份血液和组织样品进行了PCV2、TTV1和TTV2的检测,确定猪群中PCV2与TTV1和／或TTV2混合感染情况。结果表明,94份样品表现为PCV2和TTV1的混合感染,占样品总数的36．4％;193份样品表现为PCV2和TTV2的混合感染,占74．8％;另外,还有一些样品为三重感染,占34．5％。由此可以看出,猪群中PCV2和／或TTV1和／或TTV2的混合感染很普遍。     

四川省PPV与PCV2的病原流行病学调查

从四川省21个规模化猪场采集样品273份,利用PCR方法检测并分析了猪细小病毒和猪圆环病毒的感染及混合感染情况,结果共检出PPV病原阳性样品47份（占17．22％）;PPV阳性猪场8个（占38．1％）;种猪的感染率较高,仔猪感染率相对较低;检出PCV2病原阳性样品143份（占52．38％）,PCV2阳性猪场18个（占85．7％）;PCV2感染随猪年龄的增长而升高;检出PPV和PCV2混合感染样品29份（占10．62％）;同时存在PPV和PCV2的猪场6个（占28．7％）,混合感染主要集中在母猪和保育仔猪阶段。仅有3个猪场未检出PPV和PCV2病原,占14．3％。该结果说明PPV与PCV2及其混合感染在四川省流行广泛,对养殖业构成了较严重的危害。     

病死猪圆环病毒2型检测及全基因序列分析

为准确检测猪圆环病毒2型（PCV2）并分析其基因序列情况，在序列比对分析的基础上，设计一对简并引物和一条探针，经体系优化建立了PCV2的荧光PCR方法。该方法可检出10拷贝的PCV2质粒DNA，不与猪瘟病毒等多种病毒发生交叉反应。对送检的2批次45份病死猪脏器进行检测，检出25份阳性样品，与套式PCR方法的复合率为95.6%；对3份强阳性样品使用一对全基因组扩增引物，扩增了PCV2型全基因片段；经克隆测序后分析，发现3个病毒基因分别属于2种基因亚型（PCV2b和PCV2d）。本研究对于PCV2的准确检测及其变异特征的了解具有参考意义。     

华东地区猪圆环病毒2型基因型鉴别及ORF2遗传变异分析

利用PCR方法对2014-2015年采集于华东地区部分猪场的病料进行猪圆环病毒2型(PCV2)检测,将所得阳性样品进行2a/2b鉴别分型。同时,选取15份阳性样品扩增ORF2完整序列,并利用DNAStar软件进行同源性分析和系统进化树分析。结果表明,335份病料中PCV2阳性样品146份,其中PCV2a型11份(7.53%)、PCV2b型127份(86.99%),共感染率5.48%,说明华东地区感染的PCV2以2b基因型为主,少数为2a基因型。同源性分析表明,试验所得15株ORF2序列同源性为89.9%~99.9%,与国内外参考毒株同源性为86.2%~99.7%,均存在较高的同源性。系统进化树表明,15株序列中有9株属于PCV2b型,4株属于PCV2d型,2株属于PCV2a型,未出现2c和2e基因型。本试验为进一步研究PCV2遗传变异趋况及新型疫苗的研制积累了有效材料。     

陕西省屠宰生猪PCV2检测及其遗传变异分析

为了解陕西省屠宰生猪猪圆环病毒2型(PCV2)的感染及其遗传变异情况,通过PCR方法,对2018—2019年采自陕西省咸阳和杨凌地区生猪屠宰企业的300份血样进行PCV2病原学检测,并扩增部分阳性样品的开放阅读框2(ORF2)基因,同时进行序列比对和遗传变异分析。试验结果显示,38份血样PCV2病原学检测为阳性,阳性率12.67%;选取的20份阳性血样,扩增ORF2基因并比对分析,结果11个属于PCV2b亚型(55%),9个属于PCV2d亚型(45%),说明PCV2b亚型和PCV2d亚型是陕西省屠宰生猪中PCV2的主要流行毒株。本研究结果有利于掌握陕西省屠宰生猪中PCV2毒株流行情况,从而为PCV2感染的针对性防治提供依据。     

中国部分地区猪圆环病毒2型的基因型分析

对中国11个省(市)部分猪场收集的812份病料,应用ORF2-PCR进行检测,发现有235份为PCV2阳性病料.利用鉴别PCR方法从235份阳性病料中,检出10份PCV2a基因型和133份PCV2b基因型,检出率分别为4.2%、56.6%.进一步对ORF2-PCR检测阳性的部分模板进行全基因组扩增,构建阳性重组质粒,对插入质粒的片段进行测序鉴定,利用DNAStar进行同源性分析,同GenBank参考毒株绘制系统发育树,从系统发育树中也可分出PCV2a、PCV2b 2种基因型.鉴别PCR和测序结果说明,我国部分地区流行的猪圆环病毒2型以PCV2b基因型为主,少数为PCV2a基因型,也有既非PCV2a又非PCV2b的基因型(PCV2c?).     

PCV3和PCV2双重PCR鉴别诊断方法的建立及应用

本研究根据GenBank中报道的PCV2和PCV3毒株全基因组序列,设计两对特异性引物,建立了一种多重PCR鉴别诊断方法,两种病毒的预期扩增片段分别大小分别为1 007 bp和505 bp。通过对扩增条件的优化,获得最佳的反应体系为25μL体系,最佳退火温度为56℃。该鉴别方法可以同时扩增PCV3和PCV2的特异性片段,而猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)、猪繁殖障碍与呼吸综合征病毒(PRRSV)、猪细小病毒(PPV)、猪乙型脑炎病毒(JEV)、轮状病毒(RV)、猪流行性腹泻病毒(PEDV)和猪德尔塔冠状病毒的基因组及阴性对照均未扩增出任何条带。该方法对PCV3和PCV2的最低检出量均为10 ng/μL。对采自湖北省内规模化猪场的260份淋巴结样品检测。结果显示,PCV3阳性率为16.9%(44/260),PCV2阳性率为46.5%(121/260),PCV3和PCV2混合感染率为4.6%(12/260),双重PCR检测与测序分析的符合率为100%。结果表明,本研究建立了一种简便、灵敏、稳定可靠且特异性强的双重PCR鉴别诊断方法,可为临床样品中PCV3和PCV2的快速鉴别诊断及流行病学研究提供技术支持。     

PCV2和PCV3双重纳米PCR方法的建立及初步应用

本研究旨在建立一种同时检测猪圆环病毒2型（PCV2）和猪圆环病毒3型（PCV3）的双重纳米PCR（nano-dPCR）方法,同时应用该方法对PCV2和PCV3进行检测。参考GenBank中登录的PCV2和PCV3型基因序列分别设计特异性引物,对nano-dPCR的反应条件进行优化;同时考察所建立的nano-dPCR检测方法的特异性和敏感性。结果显示,所建方法的特异性和敏感性良好,对PCV2和PCV3两种病毒的最低核酸检测量分别为93.2和91.6拷贝/μL,其敏感性比普通PCR高100倍。应用该方法对送检的265份临床样本进行检测,结果显示,PCV2和PCV3在猪群中存在普遍感染,PCV3和PCV2的阳性率分别为16.6%和14.7%,混合感染阳性率为6.8%。临床样品的检测结果表明,本试验建立的nano-dPCR方法为PCV2和PCV3早期感染进行快速、敏感鉴别诊断提供了新方法。     

多重POR检测CSFV和PRRSV与PCV2

根据GenBank上猪瘟病毒（CSFV）、猪繁殖与呼吸综合征（PRRSV）和猪圆环病毒2型（PCV2）的已发表序列,分别设计并合成了3对特异性扩增引物,建立CSFV、PRRSV和PCV2单项PCR检测方法,分别扩增出预期的466bp、202bp和706bp片段。在优化单项PCR反应条件基础上,建立了CSFV—PRRSV—PCV2多重PCR检测方法,为预防和控制上述三种病毒性传染病提供了一种快速、敏感、特异的检测方法。     

Infectivity of porcine circovirus type 2 DNA in semen from experimentally-infected boars

Porcine circovirus type 2 (PCV2) is an economically important pathogen. It has been demonstrated that PCV2 DNA can be detected in boar semen by PCR; however, the biological relevance of this is unknown. The objectives of this study were to determine if semen positive for PCV2 DNA is infectious (1) in a swine bioassay, or (2) when used for artificial insemination. For the first objective, 4-week-old pigs were inoculated intraperitoneally with PCV2 DNA-negative (bioassay-control; n = 3), PCV2a DNA-positive (bioassay-PCV2a; n = 3), or PCV2b DNA-positive (bioassay-PCV2b; n = 3) raw semen, or PCV2 live virus (bioassay-positive; n = 3), respectively. Pigs inoculated with PCV2 DNA-positive semen and PCV2 live virus became viremic and developed anti-PCV2 antibodies indicating that the PCV2 DNA present in semen was infectious. For the second objective, three Landrace gilts were inseminated with PCV2 DNA-negative semen (gilts-controls) from experimentally-infected boars, and six gilts were artificially inseminated with semen positive for PCV2a DNA (gilts-PCV2a; n = 3) or PCV2b DNA (gilts-PCV2b; n = 3). Serum samples collected from the gilts in all groups remained negative for anti-PCV2 antibodies for the duration of the experiment. In addition, fetal serum samples from all 105-day-gestation fetuses were negative for anti-PCV2 antibodies or PCV2 DNA. Under the conditions of this study, PCV2 DNA-positive semen was not infectious when used to artificially inseminate gilts; however, it was demonstrated to be infectious in a swine bioassay model and therefore is a potential means of PCV2 transmission amongst swine herds.     

猪圆环病毒2型分子流行病学及疫苗研究进展

猪圆环病毒2型(PCV2)在世界范围内广泛分布,主要以PCV2a和PCV2b两种基因型为主,PCV2b亚型为当前流行基因型。现有商品化疫苗主要针对PCV2a型病毒,也可对PCV2b型病毒产生部分交叉保护,但不能显著控制PCV2b型病毒在猪群中的传播。除此之外,由于已有的商品化疫苗以传统疫苗为主,存在种毒增殖难、疫苗生产周期长等缺点。因此,研发PCV2b新型疫苗可能成为未来PCV2疫苗研制的主要方向。对PCV2的分子流行病学及疫苗研究现状进行了综述,以期为PCV2疫苗的研究提供参考。     

猪圆环病毒2a/2b亚型病毒株毒力分析

本研究对367份猪内脏样本进行PCV2检测,对阳性样本进行2a/2b鉴别诊断,并采用ORF2测序方法对部分阳性样本进行测序。在发病猪群中PCV2b阳性率远高于健康猪群,而健康猪群中PCV2a的阳性率高于发病猪群,从而在临床表型方面论证了PCV2b毒力强于PCV2a。通过比较12株PCV2 ORF2推导氨基酸序列的关键氨基酸位点,9株PCV2b毒株在ORF2氨基酸76位为I,131位为T;而3株PCV2a毒株在ORF2氨基酸76位为L,131位为P,据比对结果可知,PCV2b毒株为强毒株,PCV2a毒株为弱毒株。研究结果表明,PCV2的2种亚型2a/2b无论是临床表型还是ORF2基因型,毒力表现都是有差异的,且PCV2b毒力要强于PCV2a。     

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.     

Effect of porcine circovirus type 2 (PCV2) vaccination on PCV2-viremic piglets after experimental PCV2 challenge

The objective of this study was to evaluate the effect of porcine circovirus type 2 (PCV2) vaccines on PCV2-viremic and -seropositive piglets born from naturally PCV2-infected sows against postnatal PCV2 challenge. The experimental design was aimed at mimicking commercial swine rearing conditions to evaluate the response of the PCV2 vaccine on PCV2-viremic and -seropositive piglets after experimental PCV2 challenge. PCV2a (or 2b)-viremic piglets received a PCV2 vaccine at 21 days of age followed by a PCV2b (or 2a) challenge at 49 days of age (28 days post vaccination). The PCV2 vaccines elicited a high level of humoral (as measured by immunoperoxidase monolayer assay and neutralizing antibody titers) and cellular (as measured by the frequency of PCV2-specific interferon-γ-secreting cells) immune response in the PCV2-viremic piglets after vaccination even in the presence of maternally derived antibodies (MDA). The initial infection of PCV2 in the pigs was not affected by PCV2 vaccination, however the challenging PCV2 was reduced by PCV2 vaccination on PCV2-viremic pigs. The results from this study demonstrate that the PCV2 vaccine used in this study is effective at reducing PCV2 viremia and lymphoid PCV2 DNA, even for PCV2-viremic pigs with passively acquired MDA at the time of vaccination.     

Evaluation of commercial polyclonal- and monoclonal-antibody-based immunohistochemical tests for 2 genotypes of Porcine circovirus type 2 and comparison with in-situ hybridization assays

The objective of the present study was to evaluate polyclonal- and monoclonal-antibody-based immunohistochemical (IHC) tests for the detection of 2 genotypes of Porcine circovirus type 2 (PCV2), a and b, in formalin-fixed, paraffin-embedded lymph-node tissue from pigs with experimental or natural postweaning multisystemic wasting syndrome and to compare the IHC results with those of in-situ hybridization (ISH) assays. The ISH assays proved more sensitive than the IHC tests for the detection of PCV2a and PCV2b. According to these findings, polyclonal-antibody-based IHC testing is the most practical routine diagnostic method for the detection of PCV2 regardless of genotype because IHC testing is less technically complex than ISH testing. However, ISH assays are useful to differentiate between PCV2a and PCV2b in surveillance programs for the monitoring of PCV2 in swine herds.     

PCV2b Cap蛋白重组杆状病毒的构建及鉴定

猪圆环病毒(PCV)能够引起多种综合征和疾病,给养猪业造成了较大的经济损失。PCV2毒株在2003年后以PCV2b取代PCV2a成为临床感染的猪群中最为流行的基因型。PCV基因组为单股负链环状DNA,其中ORF2编码病毒核衣壳蛋白Cap蛋白,Cap蛋白是主要的免疫原性蛋白。本研究利用Bac-to-Bac杆状病毒表达系统将PCV2b的Cap蛋白基因克隆到杆状病毒载体中,通过制备杆粒、转染sf9细胞,获得表达PCV2b Cap蛋白的重组杆状病毒毒株,从IFA、SDS-PAGE及Western-blotting分析结果可以看出,该重组杆状病毒毒株成功表达了PCV2b Cap蛋白,为制备PCV2b Cap蛋白亚单位疫苗打下基础。     

Postweaning multisystemic wasting syndrome produced in gnotobiotic pigs following exposure to various amounts of porcine circovirus type 2a or type 2b

In late 2005, a postweaning, high mortality syndrome spread rapidly through finishing barns in swine dense areas of the United States. Diagnostic investigations consistently detected porcine circovirus type 2 (PCV2) from diseased tissues. Subsequent genetic analysis revealed that the infectious agent was a PCV2 type termed "PCV2b". Prior to late 2004, only the PCV2a type, but not PCV2b, had been reported in North America. In this communication, we produce severe postweaning multisystemic wasting syndrome (PMWS) in gnotobiotic pigs using infectious PCV2a and PCV2b generated from DNA clones constructed from field isolates identified in the 2005 outbreak. Clinical signs exhibited by diseased pigs included anorexia, dyspnea and listlessness. Mortality was typically observed within 12h of onset of dyspnea. The most striking microscopic lesions in affected animals were severe hepatic necrosis and depletion of germinal centers in lymph nodes with associated abundant PCV2 viral antigen. Clinical signs and lesions observed in these studies were comparable to those reported in experiments with gnotobiotic pigs inoculated with a PCV2a isolate while concurrently receiving immune-stimulation or co-infection with porcine parvovirus or torque teno virus. The animals in these studies were confirmed to be free of detectable porcine parvovirus, porcine reproductive and respiratory syndrome virus, bovine viral diarrhea virus, swine hepatitis E virus, and aerobic and anaerobic bacteria. Seven out of 24 PCV2 inoculated pigs had a detectable congenital torque teno virus infection with no correlation to clinical disease. Thus, in these studies, both PCV2a and PCV2b isolates were singularly capable of inducing high mortality in the absence of any detectable infectious co-factor.     

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。