猪2型圆环病毒ORF1与ORF2基因和伪狂犬病毒基因的重组与表达的研究

根据GenBank发布的猪2型圆环病毒(PCV2 )序列(AY0 35 82 0 ) ,设计两对特异性引物,采用PCR方法,分别扩增了猪2型圆环病毒ORF1和ORF2基因。将ORF1和ORF2基因的PCR产物回收并酶切后,依次插入到伪狂犬病毒gE gI双缺失通用转移载体pIECMV中,构建了猪2型圆环病毒_伪狂犬病毒重组中间转移质粒pIEORF1-ORF2。采用脂质体介导法,将重组中间转移质粒pIEORF1_ORF2与伪狂犬病毒TK^- gE^- LacZ⁺ 基因组共转染IBRS_2细胞,待发生细胞病变后收集病毒液进行空斑纯化,利用检测PCV2ORF1基因和ORF2基因的PCR方法筛选重组病毒TK^- gE^- gI^- ORF1-ORF2⁺ ,用Southernblotting鉴定重组病毒,并用Westernblotting检测ORF1_ORF2融合蛋白的表达情况,在此基础上也测定了重组病毒在不同细胞上的增殖滴度。结果表明,外源基因ORF1和ORF2已成功插入到TK^- gE^- LacZ⁺ 亲本株的基因组中,并获得了表达,表达的蛋白可与PCV2阳性血清发生反应。同时发现ORF1和ORF2基因的插入不影响重组病毒的增殖特性,其毒力与亲本株相当。     

Complete genome sequence of a novel field strain of rearranged porcine circovirus type 2 in southern china

We report here the complete genomic sequence of a novel porcine circovirus type 2 (PCV2) strain, which is supposed to be the result of natural genetic recombination between the ORF1 gene of genotype PCV2b-1B and the ORF2 gene of PCV2b-1C. Further analyses revealed that this novel PCV2 strain arose from recombination between PCV2a and PCV2b strains within the ORF2 gene. To our knowledge, this is the first report of both inter- and intragenotype PCV2 gene rearrangement in the field, and it will help in understanding the epidemiology and molecular characteristics of porcine circovirus type 2(PCV2) in southern China.     

Complete Genome Sequence of Porcine Circovirus 2b Strain CC1

A porcine circovirus 2 (PCV2) strain, designated CC1, was isolated and purified from tissue samples from pigs with wasting syndromes in China. We report the complete genome sequence of PCV2b strain CC1 with a deletion of C at position 1053 resulting in elongation of open reading frame 2 (ORF2) and formation of ORF5. There were 11 ORFs in the genome.     

嵌合猪圆环病毒PCV1-2的构建及其感染性初步鉴定

猪Ⅱ型圆环病毒(PCV2)是当前严重危害养猪业的重要病原之一。目前,世界上还没有有效疫苗用于该病毒的免疫预防。该研究利用PCR方法,将PCV2的ORF2基因替换猪Ⅰ型圆环病毒(PCV1)的ORF2基因,构建了以PCV1基因组为骨架的嵌合病毒(PCV1-2)分子克隆(pSK2PCV1-2)。将该分子克隆转染PK-15细胞并连续盲传5代,用RT-PCR方法可以在转染后盲传的细胞中检测到PCV1的ORF1 mRNA和PCV2的ORF2 mRNA,但检测不到PCV1的ORF2 mRNA和PCV2的ORF1 mRNA。间接免疫荧光检测显示在盲传第5代的细胞中有PCV2 ORF2蛋白的表达,表达蛋白主要分布于细胞核。该研究初步证实构建的PCV1-2分子克隆转染细胞后可以形成具有感染性的嵌合病毒,从而为更深入研究嵌合病毒生物学特性奠定了基础。     

猪2型圆环病毒原核表达产物的免疫原性测定

目的检测猪2型圆环病毒(PCV2)全基因组原核表达产物的免疫原性。方法根据PCV2JXL株序列(GenBank登录号AY491310),设计合成引物,利用多聚酶链式反应(PCR)从含有PCV2接种猪肾细胞PK15中扩增了Cap蛋白的氨基端片段(737-421nt),克隆入上游带有谷光苷肽-S-转移酶的原核表达载体pGEX-6p-1中,获得重组质粒pGEX-PCV737-421。PCV2Cap蛋白羧基端(426-37nt)和Rep蛋白已在过去的研究中分别融合表达。利用IPTG对3种重组大肠杆菌BL21进行诱导,进行聚丙烯酰胺凝胶电泳分析(SDS-PAGE)以及对PCV2阳性猪多抗血清的蛋白免疫印迹试验,在此基础上,将3种SDS-PAGE分离粗纯的重组表达产物碾碎后分别免疫BALB/c小鼠,用获得的抗鼠血清与PCV2病毒感染PK15细胞做间接免疫荧光抗体试验(IFA)。结果PCV2Cap蛋白的氨基端片段能在大肠杆菌中表达,且Cap蛋白羧基端和Rep蛋白的原核表达产物都能在免疫印迹试验中被PCV2阳性猪血清检测出特异性条带,重组蛋白免疫鼠血清可在IFA试验中观察到特异性的荧光分布,即检测到培养细胞中的病毒抗原。结论PCV2全基因组都可以用原核系统高效表达,且表达产物具有免疫原性。     

GST pull-down结合质谱筛选猪圆环病毒2型ORF4潜在互作蛋白

猪圆环病毒2型编码的ORF4蛋白是近年来发现的新蛋白。迄今为止,人们对ORF4所参与的细胞生物学过程知之甚少。本研究首先构建了带双标签的真核表达载体pCMV-N-Flag-GST,再将ORF4基因插入该载体中,形成pCMV-N-Flag-GST-ORF4。将质粒转染293T细胞表达ORF4后,通过GSTPull-down试验捕获细胞内潜在与ORF4互作的蛋白库。经SDS-PAGE分离及银染后,对所得的特异性条带进行质谱鉴定,筛选出5个与ORF4潜在互作的蛋白,包括丝氨酸/苏氨酸蛋白磷酸酶6催化亚基、α心肌蛋白、β肌动蛋白、SEC-14样蛋白5和肌球蛋白myosin 9。上述研究结果为深入揭示ORF4在病毒感染细胞过程中发挥的作用提供新的思路与方向。     

家养野猪源猪圆环病毒2型安徽株的分离鉴定及其全基因组序列分析

目的对安徽省临诊疑似PMW S家养野猪病例进行猪圆环病毒2型(PCV2)分离鉴定,并对分离株的全基因组进行克隆与序列分析。方法应用PK-15细胞进行PCV2的分离与增殖,根据PCR、IFA、电镜技术进行PCV2分离株的鉴定,克隆分离株的全基因组,并对序列进行分析。结果获得1株来自安徽家养野猪源PCV2分离株,命名为YZ0901。该毒株全基因组长为1 767 bp,属于PCV2b基因型。与GenBank已发表的国内外参考毒株的同源性介于93.9%~99.2%,与安徽分离毒株彼此之间的同源性介于93.4%~99.5%。结论安徽省家养野猪中存在PCV2感染,分离毒株与家猪源病毒差异不大,PCV2核苷酸序列比较稳定,其进化不存在明显的地域相关性,家养野猪源PCV2的基因型与当地PCV2流行株的基因型密切相关。     

Protective efficacy of a DNA vaccine encoding capsid protein of porcine circovirus‐like virus P1 against porcine circovirus 2 in mice

The capsid protein is the major immunogenic protein of porcine circovirus 2 (PCV2). The nucleotide sequence of porcine circovirus‐like virus P1 shares high homology with open reading frame (ORF) 2 of PCV2, and ORF1 of P1 encodes its structural protein. Mice were vaccinated twice intramuscularly with a plasmid expressing the P1 ORF1 protein (pcDNA3.1(+)‐ORF1) at 2‐week intervals. All animals vaccinated with pcDNA3.1(+)‐ORF1 developed higher specific anti‐P1 antibody levels, and had less PCV2 viremia and milder histopathological changes than PCV2‐challenged mice in the control group. Our results show that the P1 DNA vaccine elicited immune responses against PCV2 infection in a mouse model.

     

In vitro intragenomic rearrangement of porcine circovirus type 2

We report here for the first time the genome sequence of a rearranged porcine circovirus type 2 (PCV2) strain, CH-IVT1, isolated from PCV2-infected PK-15 cells. The complete circular genome of the CH-IVT1 is 605 nucleotides (nt) in length. The finding will help us to understand the molecular evolution of PCV2 and the relationship between PCV2 and PCV-associated diseases.     

2010–2015年华东地区猪圆环病毒2型的分子流行病学分析

【目的】猪圆环病毒2型(PCV2)是严重危害世界养猪业的重要传染病,即猪圆环病毒相关疾病(PCVAD)的重要病原,其致病机制及流行规律尚未阐明。本研究对2010–2015年间采集自中国华东地区的病料进行PCV2检测,以探讨中国华东地区PCV2的分子流行病学特征,分析PCV2的遗传演化规律。【方法】本研究利用PCR方法对384份断奶仔猪多系统衰竭综合征疑似发病猪样本进行检测,并对随机选取的42份阳性样品进行PCV2全基因组的测定和分析。【结果】华东地区普遍存在PCV2的感染,阳性率为41.15%。序列扩增获得42株PCV2全长基因组,同源性和系统进化树分析表明,基于PCV2 ORF2和全长核苷酸序列构建的系统进化树结构是基本一致的。从病料中测得的42株PCV2与11株参考毒株序列的ORF2基因的核苷酸和氨基酸序列同源性分别为87.0%–100.0%和82.5%–100.0%。遗传进化分析显示,53株PCV2毒株可分为4个基因型,即PCV2a、PCV2b、PCV2c和PCV2d。本文获得的42株序列ORF2基因的核苷酸和氨基酸序列同源性分别为89.6%–100.0%和86.8%–100%,...     

Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines.     

Enhancement of the immunogenicity of a porcine circovirus type 2 DNA vaccine by a recombinant plasmid coexpressing capsid protein and porcine interleukin‐6 in mice

The development of effective vaccines against porcine circovirus type 2 (PCV2) has been accepted as an important strategy in the prophylaxis of post‐weaning multisystemic wasting syndrome; a DNA vaccine expressing the major immunogenic capsid (Cap) protein of PCV2 is considered to be a promising candidate. However, DNA vaccines usually induce weak immune responses. In this study, it was found that the efficacy of a DNA vaccine expressing Cap protein was improved by simultaneous expression of porcine IL‐6. A plasmid (pIRES‐ORF2/IL6) separately expressing both Cap protein and porcine IL‐6 was constructed and compared with another plasmid (pIRES‐ORF2) expressing Cap protein for its potential to induce PCV2‐specific immune responses. Mice were vaccinated i.m. twice at 3 week intervals and the induced humoral and cellular responses evaluated. All animals vaccinated with pIRES‐ORF2/IL6 and pIRES‐ORF2 developed specific anti‐PCV2 antibodies (according to enzyme‐linked immunosorbent assay) and a T lymphocyte proliferation response. The percentages of CD3⁺, CD3⁺CD8⁺, and CD3⁺CD4⁺ subgroups of peripheral blood T‐lymphocytes were significantly higher in mice immunized with pIRES‐ORF2/IL6 than in those that had received pIRES‐ORF2. After challenge with the virulent PCV2 Wuzhi isolate, mice vaccinated with pIRES‐ORF2/IL6 had significantly less viral replication than those vaccinated with pIRES‐ORF2, suggesting that the protective immunity induced by pIRES‐ORF2/IL6 is superior to that induced by pIRES‐ORF2.     

Complete genome sequence of a highly prevalent porcine circovirus 2 isolated from piglet stool samples in China

Porcine circovirus type 2 (PCV2) is the etiologic agent of porcine circovirus-associated disease. Here, we first report the complete genome sequence of PCV2 strain JSTZ, which was isolated from piglet stool samples and is highly prevalent in China. It will help in understanding the epidemiology and molecular characteristics of PCV2.     

Complete Genome Sequence of Porcine Circovirus 2d Strain GDYX

Porcine circovirus type 2 (PCV2) is the etiologic agent of porcine circovirus-associated disease, and it is mainly divided into five genotypes. Here, we report the complete genome sequence of PCV2 strain GDYX, which belongs to PCV2d and has a unique amino acid variation at position 169 (S to G).     

Complete Genome Sequence of a Novel Porcine Circovirus Type 2b Variant Present in Cases of Vaccine Failures in the United States

Two genomes of a new porcine circovirus type 2 (PCV2) strain associated with cases of perceived failure of PCV2 vaccination were sequenced and analyzed. Based on the genome, this is the first report of this mutant of PCV2b in the United States. The genomic knowledge of this mutant PCV2b will improve understanding of the epidemiology of PCV and potentially inform the development of new and more effective vaccines for PCV2.     

安徽省猪圆环病毒2型毒株的分离与鉴定

目的从病原学角度了解安徽省猪群中PCV2的感染情况。方法PCR检测临床疑似PCV2感染的病料,阳性病料接种于PK15细胞中进行PCV2的分离和增殖,再经PCR检测、间接免疫荧光试验、电镜观察,以及ORF2基因序列分析等鉴定。结果9份临床病料中均扩增出630bp的PCV2特异性DNA片段,分离鉴定获得5株PCV2,分离毒株之间及其与27株国内外参考毒株之间的ORF2基因序列同源性在87.3%～99.8%。结论首次从病原学角度证实安徽省猪群中PCV2感染的普遍存在,分离毒株与国内外参考毒株的同源性均较高。     

A novel porcine circovirus type 2a strain, 10JS-2, with eleven-nucleotide insertions in the origin of genome replication

Here, we report a novel porcine circovirus type 2a (PCV2a) strain with 11 nucleotides (nt) inserted in the origin of genome replication (Ori). This is the first report of a PCV2a strain with nucleotide insertion in Ori. Our study will help further epidemiological studies and extend our knowledge of evolutionary characteristics of PCV2.     

猪圆环病毒2型与猪细小病毒共感染对猪肺泡巨噬细胞吞噬功能及其干扰素表达水平的影响

【目的】分析猪圆环病毒2型(PCV2)与猪细小病毒(PPV)共感染对猪肺泡巨噬细胞(PAM)吞噬功能及其干扰素表达水平的影响,为进一步阐明猪断奶后多系统衰减综合征的发病机制提供实验依据。【方法】将48头5周龄健康仔猪随机分为PCV2组、PPV组、PCV2/PPV组和对照组,每组12头。PCV2和PPV组经口鼻途径分别接种PCV2或PPV,PCV2/PPV组同时接种PCV2和PPV,对照组接种细胞营养液。感染后3、7、14和35d(dpi)从每组随机选择3头剖杀,检测PAM的存活率、吞噬活性及其α1型干扰素(IFN-α1)、γ干扰素(IFN-γ)mRNA的表达水平。【结果】PCV2组和PCV2/PPV组PAM的存活率于3、7、14dpi均低于对照组,35dpi均与对照组PAM的存活率接近,PCV2与PCV2/PPV两组之间差异不显著(P>0.05);3个病毒感染组PAM的吞噬功能均显著下降(P<0.01),其中PCV2/PPV组的吞噬功能低于PCV2组(P<0.05);PCV2/PPV组PAM中IFN-α1和IFN-γ mRNA的水平持续低于PCV2、PPV组和对照组的水平(P<0.01)。【结论】PCV2与PPV共感染没有引起PAM的活力进一步下降,但导致其吞噬功能及IFN-α1和IFN-γmRNA的表达水平持续降低。