Complete Genome Sequence of Recombinant Porcine Circovirus Type 2 Strain SD-3

We report here the genome sequence of a recombinant porcine circovirus type 2 strain SD-3, isolated from a commercial swine farm with an outbreak of postweaning multisystemic wasting syndrome (PMWS) in pigs in Shandong Province of China. The complete circular genome of this isolate is 1,767 nucleotides in length. This recombinant isolate has the ORF1 regions from PCV2a viruses and ORF2 regions from PCV2b. The findings will help us to understand the molecular evolution of porcine circovirus type 2 and the relationship between porcine circovirus type 2 and disease.     

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。     

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.     

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。     

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.     

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.

     

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.     

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).     

猪Ⅱ型圆环病毒四川分离株鉴定及其ORF2基因序列分析

猪圆环病毒(Porcine circovirus,PCV),属于圆环病毒科(Circoviridae),圆环病毒属(Circovirus),血清型为PCV-1和PCV-2[1]。PCV-1首先由Tis-cher[2]于1974年从PK-15猪肾传代细胞系中分离获得,PCV-2首先由Clark[3]报道了是断奶仔猪多系统衰竭综合征(PMWS)的主要病原,随即相继报道了PCV-2与PDNSS(猪皮炎与肾炎综合症)、NP(增生性坏死性肿炎)、PRDC(猪呼吸道综合征)、繁殖障碍、先天性颤抖、肠炎等疾病亦有重要关联[4,5];它常与猪呼吸与繁殖综合征病毒(PRRSV)或猪细小病毒(PPV)并发感染或继发细菌感染[6]。我国自从2000年郎…     

猪圆环病毒2型的PCR检测方法应用

本研究登录Genbank对猪圆环病毒2型基因的序列进行分析,用Primer 5.0设计了ORF2基因的扩增引物,试图选择一种比较合理的PCR方法检查PCV2感染的病原,以期这种PCR方法可以有效分析猪综合征障碍病毒(PRRSV)、猪细小病毒(PPV)、猪瘟病毒的扩增(HCV)、猪伪狂犬病毒(PRV)等比较常见的病原。研究结果表明,在PCV2进行扩增阳性样品检测中,发现一条异常447 bp的DNA条带,对产物测序结果进行扩增分析,证明其是PCV ORF2基因序列。敏感性检验分析表明检测样本DNA浓度时达到了9.8×10^-4ng/μL。PCR实验具有良好的稳定性和重复性。根据对66例临床病猪的分析表明,在PCV2的检测中阳性感染率是27.26%,这可能受到HCV、PRRSV、PRV、PPV等多种病毒的感染,其中混合感染的比例达到了72.23%。     

In Vitro and In Vivo Isolation of a Novel Rearranged Porcine Circovirus Type 2

Here, we present the first report of a novel rearranged porcine circovirus type 2 (PCV2) strain named BIV, isolated from both in vitro and in vivo sources. The complete circular genome of BIV is 896 nucleotides in length. The data will help us to update current knowledge of the replication of PCV2 viruses in cell culture and of their molecular evolution, as well as their diagnosis.     

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.     

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。     

Cytotoxicity of ORF3 Proteins from a Nonpathogenic and a Pathogenic Porcine Circovirus

Porcine circovirus type 2 (PCV2) infection is associated with significant and serious swine diseases worldwide, while PCV1 appears to be a nonpathogenic virus. Previous studies demonstrated that the ORF3 protein of PCV2 (PCV2ORF3) was involved in PCV2 pathogenesis via its proapoptotic capability (J. Liu, I. Chen, Q. Du, H. Chua, and J. Kwang, J. Virol. 80:5065-5073, 2006). If PCV2ORF3-induced apoptosis is a determinant of virulence, PCV1ORF3 is hypothesized to lack this ability. The properties of PCV1 and PCV2 ORF3, expressed as fusion proteins to an enhanced green fluorescent protein (eGFP), were characterized with regard to their ability to cause cellular morphological changes, detachment, death, and apoptosis. PCV1ORF3 significantly induced more apoptotic cell death and was toxic to more different cell types than PCV2ORF3 was. PCV1ORF3-associated cell death was caspase dependent. PCV1ORF3 also induced poly(ADP-ribose) polymerase 1 (PARP) cleavage; however, whether PARP was involved in cell death requires further studies. Truncation of PCV1 and elongation of PCV2 ORF3 proteins revealed that the first 104 amino acids contain a domain capable of inducing cell death, whereas the C terminus of PCV1ORF3 contains a domain possibly responsible for enhancing cell death. These results suggest that the pathogenicity of PCV2 for pigs is either not determined or not solely determined by the ORF3 protein.Lymphocyte depletion and the presence of porcine circovirus type 2 (PCV2) genome and antigens (4, 6, 21) are hallmarks of PCV-associated disease (PCVAD), a wasting and immunosuppressive ailment of postweaned pigs. Despite two decades of research, little is known about the molecular pathogenesis of PCVAD.PCV2 is the smallest known autonomous vertebrate virus containing a 1.7-kb single-stranded, ambisense DNA genome (32). The virus has two major open reading frames (ORFs) that encode the replication proteins (Rep and Rep′) involved in the initiation of virus replication (17) and a capsid protein (Cap), forming the capsid of the virion (32). A third ORF encodes an ORF3 protein that has been characterized as an inducer of apoptosis (14). Abrogation of ORF3 expression attenuated PCV2 pathogenesis in BALB/c mice (13) and specific-pathogen-free piglets (9). This led to the hypothesis that ORF3 is involved in PCV2 pathogenesis by inducing apoptosis in infected lymphocytes, leading to lymphocyte depletion and ultimately immunosuppression (13, 25). The closely related, yet nonpathogenic porcine circovirus type 1 (PCV1) also has a third open reading frame, but the properties of the ORF3 protein of PCV1 (PCV1ORF3) have not been characterized. Analysis of over 250 PCV2 variants and 30 PCV1 variants shows a consistent single-nucleotide substitution in the ORF3 coding sequence of PCV2 (PCV2ORF3), resulting in a stop codon and a protein that is half the size of PCV1ORF3 (PCV2ORF3 is made up of 104 amino acids [aa] compared to PCV1ORF3, which is made up of 206 aa). A comparison between PCV1 and PCV2 ORF3 translated regions reveals only 60% amino acid sequence identity (5), making ORF3 the most variable protein among the three identified major proteins of PCV. If ORF3 is a determinant of virulence of PCV2 via its apoptotic capability, PCV1ORF3 is hypothesized to lack the ability to induce apoptotic cell death. This report demonstrates the differences in cytotoxic properties between PCV1ORF3 and PCV2ORF3. Interestingly, PCV1ORF3 appeared to be more cytotoxic than PCV2ORF3, activating a caspase-dependent apoptotic pathway and potentially a caspase-independent, poly(ADP-ribose) polymerase 1 (PARP) cleavage pathway. Further analysis of truncated PCV1ORF3 and elongated PCV2ORF3 showed that different ORF3 proteins had similar patterns of cytotoxicity, although full-length PCV1ORF3 was the most potent inducer of cell death.     

Complete Genome Sequence of Porcine Circovirus 2b Strain Shandong

Shandong is a porcine circovirus 2b (PCV2b) strain that was isolated and purified from tissue samples from pigs with postweaning multisystemic wasting syndrome (PMWS) in the Shandong Province of China. Here, we report the complete genome sequence of strain Shandong, which may aid in understanding the molecular characteristics of this strain.     

Immunogenicity and pathogenicity of chimeric infectious DNA clones of pathogenic porcine circovirus type 2 (PCV2) and nonpathogenic PCV1 in weanling pigs

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS), whereas the ubiquitous porcine circovirus type 1 (PCV1) is nonpathogenic for pigs. We report here the construction and characterization of two chimeric infectious DNA clones of PCV1 and PCV2. The chimeric PCV1-2 clone contains the PCV2 capsid gene cloned in the backbone of the nonpathogenic PCV1 genome. A reciprocal chimeric PCV2-1 DNA clone was also constructed by replacing the PCV2 capsid gene with that of PCV1 in the backbone of the PCV2 genome. The PCV1, PCV2, and chimeric PCV1-2 and PCV2-1 DNA clones were all shown to be infectious in PK-15 cells, and their growth characteristics in vitro were determined and compared. To evaluate the immunogenicity and pathogenicity of the chimeric infectious DNA clones, 40 specific-pathogen-free (SPF) pigs were randomly assigned into five groups of eight pigs each. Group 1 pigs received phosphate-buffered saline as the negative control. Group 2 pigs were each injected in the superficial inguinal lymph nodes with 200 micro g of the PCV1 infectious DNA clone. Group 3 pigs were each similarly injected with 200 micro g of the PCV2 infectious DNA clone, group 4 pigs were each injected with 200 micro g of the chimeric PCV1-2 infectious DNA clone, and group 5 pigs were each injected with 200 micro g of the reciprocal chimeric PCV2-1 infectious DNA clone. As expected, seroconversion to antibodies to the PCV2 capsid antigen was detected in group 3 and group 4 pigs. Group 2 and 5 pigs all seroconverted to PCV1 antibody. Gross and microscopic lesions in various tissues of animals inoculated with the PCV2 infectious DNA clone were significantly more severe than those found in pigs inoculated with PCV1, chimeric PCV1-2, and reciprocal chimeric PCV2-1 infectious DNA clones. These data indicated that the chimeric PCV1-2 virus with the immunogenic ORF2 capsid gene of pathogenic PCV2 cloned into the nonpathogenic PCV1 genomic backbone induces a specific antibody response to the pathogenic PCV2 capsid antigen but is attenuated in pigs. Future studies are warranted to evaluate the usefulness of the chimeric PCV1-2 infectious DNA clone as a genetically engineered live-attenuated vaccine against PCV2 infection and PMWS.