广西北部湾地区猪圆环病毒2型检测及其ORF2遗传变异分析

为了解猪圆环病毒2型(Porcine circovirus type 2,PCV2)在广西北部湾地区的流行情况和其ORF2基因的遗传变异规律,本研究采用PCR方法对2016年采自广西北部湾地区规模猪场和农村散养户的306份疑似PCV2感染猪的血液和组织样品进行了PCV2的病原学检测,同时对分离的4株PCV2 ORF2基因进行PCR扩增、克隆和测序,并与Gen Bank中登录的34株国内外PCV2参考株ORF2序列进行比对和遗传进化分析。结果表明:PCV2检测阳性样品为129份,阳性率为42.16%。随机选取的4份阳性病料PCV2 ORF2基因测序结果显示,4个毒株遗传关系上均属于Group 1(PCV2b),其中BBW-1和BBW-2分离株属于1A分支且分别与Fd3株(AY321984)和NL_PMWS_3株(AY484415)同源性较高,BBW-3和BBW-4分离株属于1B分支且BBW-4分离株与Hu Zhou0301株(AY536756)和NL_Control_1株(AY484407)同源性较高,其ORF2基因核苷酸和氨基酸同源性分别为98.9%~99.5%和93.9%~95.4%,说明该地区PCV2的优势基因型是PCV2b。研究结果不仅有利于掌握广西北部湾地区PCV2流行毒株同源性情况,而且将为该地区(porcine circovirus associated disease,PCVAD)的防治和新型疫苗的研制提供依据。     

猪细环病毒1b型ORF3基因克隆及序列分析

为明确福建省猪细环病毒（porcine torque teno sus virus,PTTSuV）1b型（PTTSuV-1b）ORF3基因的遗传进化特征,本研究根据GenBank中登录的PTTSuV-1b基因组特征设计特异性引物,对福建省某猪场患有仔猪断奶后多系统衰竭综合征(PMWS)的猪血清进行PTTSuV-1b分段扩增,并分别对PCR扩增产物进行胶回收后克隆测序,将测序结果经BLAST分析后进行序列拼接。试验结果表明,所扩增的目的片段编码有完整的PTTSuV-1b ORF3蛋白,全长为600 bp,编码有199个氨基酸。将获得的PTTSuV-1b型福建株与GenBank中PTTSuV-1b型的ORF3基因进行比对分析,其与FJ/China/2010/TTV2/2株核苷酸同源性最高,为99.7%,与西班牙PTTSuV-1b分离株TTV2_G43核苷酸同源性为97.3%,与SC株核苷酸同源性稍低,但也达94.0%;而与猪细环病毒K2型德国家猪分离株472142株核苷酸同源性仅为60.7%,与猪细环病毒1a型西班牙分离株PTTV1_1914株核苷酸同源性仅为46.8%。从遗传进化关系上看,PTTSuV-1b ORF3基因在遗传进化上呈2个大的遗传进化分支（分支Ⅰ和分支Ⅱ）,本研究分离株处于分支Ⅰ。     

广西地区猪圆环病毒2型和猪圆环病毒3型基因型监测及遗传进化分析

为了明确猪圆环病毒2型(Porcine circovirus type 2,PCV-2)和猪圆环病毒3型(Porcine circovirus type 3,PCV-3)在广西地区的分子流行病学情况,试验通过PCR方法对2017年来自广西不同地区的80份猪肺脏样品进行PCV-2和PCV-3核酸检测,并对PCV-2和PCV-3的阳性样品进行ORF2基因的扩增和测序,同时利用生物信息学软件DNAStar和MEGA 6.06对PCV-2和PCV-3的ORF2基因进行同源性分析和构建遗传进化树。结果表明:2017年广西地区PCV-2和PCV-3的总感染率分别为43.8%(35/80)和33.8%(27/80),PCV-2和PCV-3的共感染率为16.3%(13/80)。试验获得的19个PCV-2 ORF2基因序列之间的核苷酸同源性为93.6%～99.9%,对应氨基酸同源性为93.1%～100%;19个PCV-2 ORF2基因序列与国内外参考毒株ORF2基因序列的核苷酸同源性为82.8%～99.9%,对应氨基酸同源性为83.3%～99.6%。试验获得的2个PCV-3 ORF2基因序列之间的核苷酸同源性为99.8%,对应氨基酸同源性为100%;2个PCV-3 ORF2基因序列与国内外参考毒株ORF2基因序列的核苷酸同源性为97.7%～99.8%,对应氨基酸同源性为96.2%～100%。获得的19个PCV-2 ORF-2基因序列中,12个为PCV-2b亚型,7个为PCV-2d亚型;获得的2个PCV-3 ORF2基因序列为PCV-3a亚型,与丹麦和中国湖南分离得到的PCV-3亲缘关系较近。说明PCV-2和PCV-3在广西地区猪群中感染率很高,PCV-2感染主要以PCV-2b亚型为主,PCV-3之间高度保守,分布无地域性规律。     

新疆首次检测到猪圆环病毒3型及其全基因组进化分析

为了解新疆某规模化猪场猪圆环病毒3型(Porcine circovirus type 3,PCV-3)的流行现状、分子生物学特征和遗传演化规律,试验应用巢式PCR技术对新疆某规模化猪场的45份血液样本进行PCV-3基因检测和测序;利用TempliPhi 100 amplification Kit对检测到的PCV-3阳性样本进行全基因组扩增,将扩增得到的基因组片段克隆到Trans-T1载体中,经测序、拼接获得PCV-3基因组全长序列;利用MEGA 6. 0. 6和MegAlign软件对其全基因组进行遗传进化分析和同源性比较。结果表明:在新疆地区首次检测到PCV-3,且该猪场PCV-3的阳性率为20%(9/45),并获得PCV-3全基因组序列,长度为2 000 bp,命名为PCV-3/CN/Xinjiang-11/2018;与美国株PCV-3-US-SD 2016处于同一进化分支,属于3b亚群;与国内PCV-3/Pig/CN/Hebei20170320的同源性最高(99. 4%),与PCV-3-CN/Fujian-820-2016和PCV-3-CN/Fujian-318-2017的同源性最低(97. 8%),与国外美国株PCV-3-US-SD 2016的同源性最高(99. 1%)。说明新疆地区猪场已经存在PCV-3,应该引起养猪业的重视。     

猪Ⅱ型圆环病毒广东分离株的全基因组克隆及序列分析

根据GenBank中猪Ⅱ型圆环病毒(PCV-2)基因序列,设计两对特异性引物.将PCV-2广东分离株(GD1株)用PK15细胞系培养,从细胞培养物中提取病毒总DNA并以之为模板,用PCR方法分段扩增病毒全基因组,分别克隆到pMD18-T载体,对阳性质粒进行序列测定.用DNAstar对序列进行拼接,得到PCV-2 GD1株基因组全序列,全长为1767个碱基,包涵2个开放阅读杠(ORF1和ORF2),分别编码与复制相关的Rep蛋白和结构蛋白Cap.通过BLAST对所测GD1株核苷核酸序列早国内外已登录GenBank中的PCV-2病毒核苷酸序更进行同源性比较,与PCV-2参考毒株的核苷酸同源性介于94.4%～99.7%之间,与德国株PCV-2(AF201897)的同源性最高,为99.7%;与猪I型圆环病毒(PCV-1)参考株(AY184287)的同源性仅为70%.     

猪圆环病毒2型ORF2主要抗原区基因的克隆与分析

根据GenBank中猪2型圆环病毒（PCV-2）北京AY176626株ORF2序列设计1特异性引物,从疑似断奶仔猪多系统衰竭综合征（PMWS）的死亡仔猪组织病料和经PK15盲传的培养物中扩增出了ORF2表位基因,将其克隆到pMD18-T载体上,筛选获得了重组质粒pMD18-T-ORF2。对此基因片段进行测序分析结果表明,与美国AF264038同源性为92%,与北京AY176626的同源性为98%,与广州AY651850的同源性为98%。     

猪圆环病毒2型浙江株ORF2基因的克隆及序列分析

为了研究猪圆环病毒2型(PCV-2)ORF2基因的分子特征,试验采用PCR方法从疑似圆环病毒感染的猪组织病料中扩增ORF2基因(702 bp),将纯化后的PCR产物克隆入pMD18-T载体,筛选鉴定重组质粒T-ORF2,并进行序列测定。结果表明:克隆的PCV-2 ORF2与加拿大分离株(AF118097)、美国分离株(AY099495)的同源性最高,核苷酸序列和推导的氨基酸序列同源性分别达98.7%和97.9%,与其他PCV-2毒株同源性分别为97.0%~98.6%和94.0%~97.9%。     

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。     

猪圆环病毒2型滨州分离株全基因组的克隆与序列分析

为了解猪圆环病毒2型(PCV-2)滨州地方分离株的起源及遗传变异,从滨州不同区域疑似病猪病料中克隆了3株PCV-2的全基因组序列,并进行序列测定及分析。核苷酸同源性比较显示,PCV-2分离株BZ-1、BZ-2及BZ-3与GenBank上登录的14株PCV-2同源性为95.6%~99.9%;核苷酸遗传进化树分析显示,BZ-1株与BZ-3株处在同一分支,而BZ-2株处在另一分支。     

上海市猪圆环病毒2型全基因组的克隆与序列分析

参照国外发表的猪圆环病毒2型(Porcine circovirus type 2,PCV-2)全基因组序列,设计合成了1对特异性引物,从上海市临床病料中提取PCV-2基因组DNA,进行PCR全基因扩增。回收PCR产物,将其插入pMD18-T载体,并对筛选出的阳性质粒进行测序。应用DNAStar序列分析软件,对所测PCV-2序列与GenBank中登录的国内外PCV-2毒株进行同源性比较。结果显示,PCV-2上海株与国内外毒株的核苷酸同源性高达95.0%～100%。进化树分析结果显示,其中9株PCV-2病毒属于PCV-2b亚型,3株PCV-2病毒属于PCV-2a亚型,且9株PCV-2b亚型毒株部分核苷酸位点显示其属于强毒力毒株。研究表明,上海市PCV-2感染以PCV-2b亚型为主,且氨基酸位点表明其具有较高的毒力。     

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.     

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.     

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...     

Porcine circovirus type 2 infection before and during an outbreak of postweaning multisystemic wasting syndrome on a pig farm in the UK

The presence of porcine circovirus type 2 (PCV-2) and other pathogens before and during an outbreak of postweaning multisystemic wasting syndrome (PWMS) in pigs is evaluated in this study. At the time of the outbreak on a large commercial pig farm in the UK, serum samples and data were collected in two independent on-going research projects, one in weaned pigs and the other in sows. Serum samples of growing pigs and sows were PCV-2-antibody and PCR positive before and during the PMWS outbreak. Upon sequencing, PCV-2 isolates collected before the outbreak were identified as PCV-2a, and isolates collected during the outbreak were identified as PCV-2b, suggesting a shift of PCV-2 genotypes present on the farm. Pigs in the weaner study were from sows originating from different breeders and an association of sow origin and PCV-2 serostatus in offspring was found. Further, pigs had higher odds to be PCV-2 antigen positive if the sow was PCV-2 antibody positive around farrowing, the sow was of higher parity, and were less likely to test antigen positive if the sow was sourced from a particular breeder. The findings of this study highlight the potential role of the immune status of the sow on the occurrence of PMWS.     

Porcine circovirus-2 DNA concentration distinguishes wasting from nonwasting pigs and is correlated with lesion distribution, severity, and nucleocapsid staining intensity.

The emergence of severe porcine circoviral disease in North America is associated with Porcine circovirus-2 genotype b (PCV-2b), which has led to speculation that PCV-2b is more virulent than PCV-2a. The objectives of this study were to 1) correlate the PCV-2 DNA concentration and lesions in wasting (WST) and age-matched healthy (HLTH) pigs from 2 clinically affected farms, and unaffected (UNFCT) pigs from a farm with no prior clinical or diagnostic history of PCVD; and 2) to determine the initial estimates of sensitivity and specificity of PCV-2 quantitative polymerase chain reaction (qPCR). PCV-2b was confirmed in all 3 farms. Compared with HLTH pigs, WST pigs demonstrated significantly more prevalent thymic atrophy, failure of normal pulmonary collapse, and ascites (P < 0.017 for all). The HLTH and UNFCT pigs had significantly more pronounced lymphoid germinal centers and proliferative paracortical T-dependent zones, compared with WST pigs (P < 0.017). Across all tissues, PCV-2 DNA concentrations were significantly higher in WST compared with HLTH and UNFCT pigs (P < 0.017 for all). The PCV-2 DNA concentrations were strongly correlated with PCV-2 nucleocapsid staining intensity in lymph node, spleen, Peyer's patches, lung, liver, and kidney (0.60 < or = r < or = 0.84). In the current study, the PCV-2 DNA log10 cutoff concentrations best able to distinguish WST from HLTH and UNFCT pigs were between 7.0 and 8.0 per gram for tissues, and between 4.0 and 5.0 per milliliter for sera. The presence of PCV-2b in UNFCT pigs is evidence that PCV-2b by itself is not sufficient to induce severe disease.     

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.     

华南地区猪圆环病毒和猪繁殖与呼吸综合征病毒检测分析

为了解华南地区猪圆环病毒及猪繁殖与呼吸综合征病毒的最新流行情况,采集了华南地区11个规模猪场各饲养阶段猪血清807份,用套式PCR(nPCR)检测猪圆环病毒1型(PCV-1)和猪圆环病毒2型(PCV-2);采集了若干猪场2010年1月至2011年8月期间有咳嗽、喘气、消瘦及疑似PDNS等临床症状的猪血清312份,以及无临床症状猪血清104份,以nPCR检测PCV-2,以一步法反转录PCR(RT-PCR)检测猪繁殖与呼吸综合征病毒。结果发现,所调查的11个规模猪场中只有5个检出PCV-1,所有猪场均检出PCV-2。PCV-2阳性率为30.61%,而PCV-1阳性率仅为4.21%;经产母猪和7周龄以上保育猪PCV阳性率最高;有临床症状的猪血清PCV-2阳性率为58.65%,PRRSV阳性率为37.82%,PCV-2阳性猪群中有39.89%的猪同时感染PRRSV;有症状猪群7月～9月的PCV-2感染率最高,而1月～3月最低;无临床症状猪血清PCV-2阳性率为27.9%,PRRSV阳性率为0.96%,PCV-2与PRRSV无混合感染。证明PCV尤其是PCV-2在华南地区仍广泛传播并流行,而且PCV-2与PRRSV混合感染致病情况较多。PCV-2的感染率与季节有一定的相关性,种猪带毒情况严重。     

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.     

广东省猪圆环病毒2型感染的血清学调查

为了解广东省猪群中猪圆环病毒2型(PCV-2)的感染情况,应用酶联免疫吸附试验(ELISA)对广东部分地区的764份不同年龄段的猪血清进行PCV-2抗体检测,对PCV-2与猪猪繁殖与呼吸综合征病毒(PRRSV)、伪狂犬病病毒(PRV)混合感染情况做了进一步调查.结果表明,PCV-2抗体检测平均阳性率为61.91％(47...     

利用宏基因组学鉴定藏猪腹泻粪便病毒种群

为进一步了解藏猪腹泻粪便中病毒种群，从青藏高原地区16个藏猪场采集腹泻粪便样本146份，将样本混合成一个pool样，提取RNA反转录成cDNA，建库后利用TruSeq Illumina测序，对测序的序列进行整理分析及相关病毒的分子特征进行研究。数据分析结果表明：藏猪腹泻粪便样本病毒种群包括11个病毒科的19种病毒，主要以线性和环状的小DNA病毒为主，如猪粪相关环状病毒7型、猪腺病毒和猪博卡病毒（PBoV）等；与腹泻相关的病原有猪流行性腹泻病毒（PEDV）、猪圆环病毒2型（PCV-2）和牛病毒性腹泻病毒1型（BVDV-1）3种；与腹泻相关的新发病原有porcine bufavirus、rabovirus和pasivirus 3种。利用SOAP软件组装出猪细小病毒6型（PPV-6）、PCV-2、PBoV-2完整或接近全长的基因组和戊型肝炎病毒（HEV）完整的ORF2基因序列，系统发育结果显示PPV-6和PCV-2与参考毒株有较近的遗传进化关系，PBoV-2与参考毒株有较远的遗传进化关系，单独聚为一簇，可能是一个全新的基因型。为进一步调查PCV-2在藏猪腹泻粪便中的检出率，对采集146份样本进行检测，检出率为10.96%（16/146，95% CI：6.4%~17.2%），且与四川地区PCV-2流行株有较近的亲缘关系。本研究发现藏猪腹泻粪便中病毒种类复杂多样，且可能存在与其他动物和人交叉感染情况，具有重要的公共卫生学意义，也为藏猪腹泻病防控提供一定的理论依据。