Preparation and Identification of Specific Monoclonal Antibody against Porcine Circovirus Type 2

BALB/c mice were immunized using synthetic tandem polypeptide of Cap protein epitope of porcine circovirus type 2 （PCV2） as the antigen. By using lym-phocyte hybridoma technique, a hybridoma cellline stably secreting monoclonal an-tibody against PCV2-rCap protein was successful y obtained and named as 670#. The ascites titer of the obtained monoclonal antibody was 1∶100 000. Western blot results showed that the monoclonal antibody could react with prokaryotical y ex-pressed PET32a-ORF2 recombinant protein, eukaryotical y expressed ORF1-ORF2 tandem protein and PCV2 whole virus celllysate. Indirect EILSA demonstrated that the monoclonal antibody could bind with ORF1-ORF2 tandem protein. Indirect im-munofluorescence assay （IFA） indicated that the monoclonal antibody could identify native PCV2 virus. The preparation of this monoclonal antibody provided technical tools for epitope analysis and molecular diagnosis of PCV2 virus.

Genetic Variation of Porcine Circovirus Type 2 Isolate 201105ZJ

[Objective] This study aimed to investigate the genetic variation of porcine circovirus type 2 （PCV2） in China. [Method] The strain was isolated from infected samples by cel passage and preliminarily identified by PCR and IFA. Ful-length genome of the isolated strain was obtained by specific amplification for homology and phylogenetic analysis. [Result] A PCV2 strain was successful y isolated and named 201105ZJ, which could proliferate in PK15 cel lines. Specific fragments could be amplified by specific PCR assay. According to results of IFA assay, specif-ic immunofluorescence was observed; the TCID50 was low （102.67）; the ful-length genome sequence of the isolated strain was 1 768 bp, sharing 94.1%-96.8% ho-mology with 13 reference strains; to be specific, the isolated strain exhibited the highest homology of 96.8% with AF055392PCV2a; the isolated strain 201105ZJ and reference strain AF055392 belonged to genotype PCV2a, exhibiting a distant genetic relationship with genotype PCV2c. [Conclusion] Characteristics of genetic variation of PCV2 isolate 201105ZJ provided theoretical basis for vaccine development, investi-gation of PCV2 pathogenesis, and prevention and control of porcine circovirus-as-sociated diseases （PCVAD） in East China.

A survey on porcine circovirus type 2 infection and phylogenetic analysis of its ORF2 gene in Hangzhou,Zhejiang Province,China

Porcine circovirus type 2 (PCV2) is closely related to the postweaning multisystemic wasting syndrome (PMWS). In this study, the pig serum and tissue samples collected from different regions of Hangzhou District in Zhejiang Province of China between 2003 and 2005 were analyzed by enzyme-linked immunosorbent assay (ELISA) for PCV2 antibody and by polymerase chain reaction (PCR) for ORF2 gene. The results show that out of 1250 randomly collected serum samples, 500 sera (40%) were seropositive for PCV2. PCR results demonstrate that Hangzhou PCV2 with more than 50% Chinese PCV2 strains and French PCV2 formed Cluster A. Only one PCV2 from Hangzhou belonged to Cluster B with some other Chinese PCV2 and Netherlands’s isolates. Cluster C consisted of PCV2 isolates from China, US, Canada, UK and Germany. The results indicate that the PCV2 infection was widespread in Hangzhou.

Development of a Loop-Mediated Isothermal Amplification Assay for Porcine Circovirus Type 2

In this study, the loop-mediated isothermal amplification (LAMP) method was used to develop a rapid and simple detection system for porcine circovirus type 2 (PCV2). According to the PCV2 sequences published in GenBank, multiple LAMP primers were designed targeting conserved sequences of PCV2. Using the DNA extracted from PCV2 isolates HUN-09 and SD-09 as the template, LAMP reactions in a PCV2 LAMP system was performed, the amplification products were detected by adding SYBR Green I and could be observed directly by the naked eye. The results showed highly-efficient and specific amplification in 30 min at 63°C with a LAMP real-time turbidimeter. Furthermore, PCV2 DNA templates, with a detection limit of 5.5×10-5 ng of nucleic acid, indicated that this assay was highly sensitive. The results obtained with the naked eye after SYBR Green I staining were consistent with those detected by the real-time turbidimeter, showing the potential simplicity of interpretation of the assay results. The LAMP assay appeared to have greater accuracy than PCR and virus isolation for the analysis of 18 clinical samples. In addition it offers higher specificity and sensitivity, shorter reaction times and simpler procedures than the currently available methods of PCV2 detection. It is therefore a promising tool for the effective and efficient detection of PCV2.

Mouse models of porcine circovirus 2 infection

PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases(PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.

Genetic Diversity of Intragenomic Rearrangement of Porcine Circovirus Type 2 in vitro and in vivo

We characterized the genome sequences of defective-interfering(DI) particle DNA of porcine circovirus type 2(PCV2) by sequencing and bioinformatics analyses. DI particles were both generated by serial passage of PCV2 in PK15 cells and obtained from sera of pigs with postweaning multisystemic wasting syndrome(PMWS). These subviral isolates ranged from 358 nt to 1 125 nt genomes. Investigating the complexity and diversity of PCV2 DI in vivo and in vitro can help elucidating the evolutionary history of PCV2.

引起典型PDNS的PCV-2病原分子特征分析

为了了解引起贵州省贵定县某猪场育肥猪群出现的典型猪皮炎肾病综合征(PDNS)的PCV-2的分子特征,试验首先对采自该猪场患病猪血清进行PCR扩增和克隆测序,然后采用多种分子生物学软件及网站对PCV-2的基因型、基因结构、推导蛋白等进行研究。结果表明:PCR扩增得到大小为1767 bp的条带,与GenBank中CZ246毒株序列(登录号为MZ995482)的相似性最高,将获得的毒株命名为PCV-2 GZGD2022。PCV-2 GZGD2022株为PCV-2d基因型,与PCV-2d参考株NLControl4(登录号为AY484410)相比,含10个开放阅读框(ORF),缺失ORF8,其ORF5终止密码子提前,大小仅为21 bp。该毒株在基因组第1042位有1个碱基T的缺失,但导致Cap蛋白C末端延伸的是脯氨酸(P)而非赖氨酸(K)。与疫苗株(LG株、DBN-SX07-2株、ZJ株、SH株)相比,在Cap蛋白的抗原表位区有7个特异性突变点(F53I、R59K、A68N、T134N、A190T、A68N、T134N)。上述疫苗株之间磷酸化位点的差异主要集中在Cap蛋白上,在88 aa处多了1个丝氨酸磷酸化位点,在47 aa、134 aa、149 aa处少了1个苏氨酸磷酸化位点,在55 aa和218 aa处少了1个酪氨酸磷酸化位点。Rep蛋白有3个N糖基化位点(23NPS25、256NQT258和286NAT288),Cap蛋白有1个N糖基化位点(143NYS145)。Rep、Cap蛋白为混合型蛋白。此外,Rep、Cap蛋白有一些特殊功能结构域,如前者的PH-LIKE、NACHT等结构域和后者的乙酰化修饰位点(1MTYPR6)与核定位信号(NLS)。说明引起贵州省贵定县某猪场猪群出现典型PDNS的致病株为PCV-2d基因型,该毒株缺失ORF8,编码蛋白的氨基酸、抗原表位和磷酸化位点的改变可能是导致其毒力较强、引起典型PDNS的原因。

Epidemiology Survey of Porcine Parvovirus and Porcine Circovirus Type 2 in Sichuan Province

[ Objective] The study aimed to analyze the prevalence of Porcine Parvovirus （PPV） and Porcine Circovirus Type 2 （ PCV2 ） and the mixed infection in Sichuan Province to lay the foundation for further predicting the tendency of the plague and formulating appropriate prevention and control strategies. [ Method] Two hundred and seventy -three samples were collected from 21 large pig farms in Sichuan province, and epidemiology of PPV and PCV2 were investigated by PCR detecting. [ Result] The positive rate of PPV was 17.22% （47/273）, and positive rate of pig farms was 38.1% （8/21）, meanwhile it also displayed the feature that infection rate of boar was higher than that of piglets; The positive rate of PCV2 was 52.38% （143/273）, and positive rate of pig farms was 85.7% （18/21）, and it showed the trend that the infection rate of PCV2 was rising with the growth of the age. The co-infection rate of PPV and PCV2 was 10.62% （29/273）, and co-infection rate of pig farms was 28.7% （6/21）, which mainly concentrated in the sow and nursery piglets. Only 14.3% （3/21） pig farms was epidemiologically negative of PPV and PCV2. [ Conclusion] PPV and PCV2 and co-infection was widely popular in Sichuan province, and it did more serious harm to the pig-raising industry.

Recent Advances in Epidemiology of Porcine Circovirus Type 2 and Diagnosis of Porcine Circovirus-Associated Diseases

Porcine cimovirus （PCV） is the smallest animal virus so far and has two serotypes. PCV1 is nonpathogenic, but PCV2 is pathogenic and causes post-weaning multisystemic wasting syndrome （ PMWS）. Factors to induce PMWS include immunity and infection status of sows, infec- tion time, mixed infection, PCV2 variants, physical status of gilts, and feeding management. For final diagnosis, histopathological changes and ex- istence of PCV2 in lymphoid tissues are professional standards, because fluorescence quantitative RT-PCR is not enough specific or sensitive. The commemial PCV2 vaccines can reduce occurrence of PMWS and PCV-related diseases. This paper reviews recent advances in epidemiology of PCV2 as well as diagnosis and control of PMWS.

An Immunochromatographic Strip Test for Rapid Detection of Antibodies against Porcine Circovirus Type 2

Porcine cimovirus type 2 (PCV2) infection causes huge economic losses, but no serological method is available for batch detection of field samples. The aim of the study was to develop a method for large-batch detection of PCV2 infection. Colloidal gold-labeled staphylococcal protein A (SPA) was sprayed on glass fibers to prepare a conjugate pad. The recombinant ORF2 protein of PCV2 was blotted on the test line of the nitrocellulose (NC) membrane, and pig IgG was streaked on the control line of the NC membrane. Then, the immunochromatographic strip was used for detection of antibodies against PCV2. The results show that the strip test was simple and the results could be determined within 10 min with naked eyes. The test strip was highly specific for pig serum against PCV2 and no cross-reaction with pig serum against other pathogens was observed. The test strip had close similarity with ELISA. Storage at RT for 6 months did not affect the specificity and sensitivity obviously. A total of 324 clinical pig sera were detected by both ELISA and the developed test strip, and the coincidence was 98.77%. Therefore, the developed immunochromatographic strip is specific, sensitive, stable, fast and simple, and it is suitable for on-site detection of antibodies against PCV2.

Phylogenetic Relationship Analysis of the Complete Genomes of Porcine Circovirus Type 2( PCV2) Strains Isolated from Hainan Province

[ Objective] This study aimed to investigate the molecular characteristics of porcine circovirus type 2 （PCV2） strains isolated from Hainan Province. [ Method] The complete genome of PCV2 was amplified from PMWS-suspected samples by PCR for sequence analysis. [ Result] A total of eight PCV2 strains were isolated and identified. All the eight isolates belonged to genotype PCV2b, among which seven isolates belonged to subgenotype PCV2b-1 C, and one isolate be- longed to subgenotype PCV2b-IA/1B. ORF2 gene of PCV2 isolates from Hainan Province was 705 bp in length, encoding 234 amino acids. Antigenic epitopes of Cap protein exhibited certain changes. Nucleotide sequences and deduced amino acid sequences of ORF2 gene shared 95.3% -99.7% and 93.6% - 100% simi- larities among eight PCV2 isolates from Hainan Province, respectively. Moreover, nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 91.0% -99.9% and 91.0% -99.6% similarities with other PCV2 strain isolated from China （AY682994, AF381175, JX945577, JX682407, AY180397 ）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.0% - 97.0% and 88.0% -97.9% similarities with PCV2 isolates from other countries （ NC_005148, JQ994268, KJ187306, AF201307, AF454546, AY＂/Sd020）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 98.2% -100% and 94.9% -100% similarities with vaccine strain SH, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.6% -91.7% and 89.7% - 91.0% similarities with vaccine strain LG, respectively. [ Conclusion] This study provided theoretical hasis for the prevention and control of PCV2 and selection of vaccine strains in Hainan Province.

Establishment and Application of a Real-time Fluorescent Quantitative PCR Method for Detection of Porcine Circovirus Type 2

[ Objective ] To establish a real-time fluorescent quantitative polymerase chain reaction （PCR） method with SYBR Green I for the detection of porcine circovirus type 2 （PCV2）. [Methods] Specific primers were designed to amplify the conserved gene segments of PCV2 with a size of 177 bp by PCR. The ampli- fied gene was cloned into the vector of pMD 18-T and transformed into DHSct to screen positive clones. After being extracted and purified, the recombinant plasraids pMD 18-T-177 were taken as the standard DNA templates to establish the fluorescence quantitative PCR method for the detection of PCV2, and the PCR re- action conditions were optimized. [ Results] Ct value of the established PCR method showed a good linear relationship with the standard DNA templates within a viral load of 3.21 × 100 -4.16 × 108 copies/μL , the correlation coefficient was O. 998 8 and the slope was - 3.286. The method did not show any cress-reactions with the genomes of PRRSV, PCV1, CSFV, PRV, PPV and Escherichia coli. Sensitivity of this method was proved to be 3.21 × 10 copies/μL, which was 1 000 times higher as conventional PCR method. Variation coefficients of the repeated trims among same batch or different batches were both less than 3.00%. Positive rate of clinical samples detected by the established PCR method was 58.94%, which was significantly higher than the detection rate by conventional PCR. [ Conclusions ] A reM-time fluorescent quantitative PCR method with SYBR Green I for the detection of PCV2 was established, which was better for conducting the quan- titative analysis and the early diagnosis of PCV2 infection.

Isolation and Identification of Porcine Circovirus Type 2 in Taizhou

In September 2011, an infectious disease suspected to be postweaning multisystemic wasting syndrome （PMWS） broke out in some pig farm in Taizhou. The inguinal lymph node, liver and lung tissues were collected and grinded into tissue suspension. The suspension was subjected to PCR detection, and the positive product was sequenced. The suspension of positive samples was filtered with 0.22 μm filter membrane, and the filtrate was inoculated onto PK15 cells. After five generations of blind passages, the cell viral liquid was collected and extracted for DNA, which was subjected to PCR detection and indirect immunofluorescence. The results showed that the isolate was porcine circovirus type 2 （PCV2） and designated as TAIZ110926. The target sequence was committed to NCBI with a serial number： KF039888.

Expression of Porcine Circovirus Type 2 ORF2 Gene in Myocardial Cells of Detached Cherry Valley Duck

The objective of this study was to construct the recombinant eukaryotic expression plasmid of ORF2 gene harboring enhanced green fluorescent protein （EGFP） report gene. ORF2 gene of porcine circovirus type 2 cloned by PCR was ligated to the expression vector pEGFP-N1, which contains enhanced green fluorescent protein （EGFP） report gene, the recombinant eukaryotic expression plasmid pEGFP-N1-ORF2 was constructed successfully and was transfected into pre- pared duck myocardial cells （DMCs） by lipofectin. According to the result, the fluorescence expression was directly detected with fluorescence microscope, and the expression of ORF2 were analyzed by RT-PCR and indirect immunofluorescence assay （IFA） respectively. About 48 h after transfection, green fluorescent can be observed on transfected cells ： T-PCR and IFA were positive. This indicated that ORF2 gene of PCV2 was expressed efficiently in transfected duck myocardial cells.

Epidemiological Investigation and Genetic Characterization of Type 2 PCV2 （Type 2 Porcine Circovirus） in Mexican Commercial Herds

PCV2 （Porcine circovirus type 2） is considered as the essential infectious agent of PMWS （post weaning multisystemic wasting syndrome） in pigs. Serological studies have shown that the virus is ubiquitous. Currently, there are many reports about the epidemiology and genetic characteristics of this virus around the world, but in Mexico it has not been studied. More than 3,500 samples of serum, rectal swabs, tissues and semen of 34 Mexican porcine farms from 10 important producer regions were analyzed by PCR. Results show that 97% of the farms were positive. Both genotypes, PCV2a and PCV2b were detected. It also found that the most prevalent genotype in Mexico is PCV2b. Regarding to amino acid sequence; three major heterogenic regions were present in the positions 59-91,123-136 and 185-210.

Research on Propagation Characteristics of DifferentIsolates of Porcine Circovirus Type 2 in vivo

[Objective]The aim of this study is to explore the dynamic changes of Porcine circovirus type 2 (PCV2) and the propagation differences between different PCV2 strains.[Method]A rapid, sensitive and SYBR Green I-based real-time quantitative PCR assay was developed to detect PCV2 in mice. The Balb/c mice were inoculated with 200 μL of 1×10^6 TCID 50 /mL different strains of PCV2;the serum and tissues of mice were collected at different time. SYBR Green I fluorescent quantitative PCR was used to determine the viral titer in the serum and tissues of mice.[Result]The results indicated that the SYBR Green I PCR could speci fically detect PCV2 with high specificity. All strains were detected in the serum 14 d after infection, and 2007HA strain had the highest level of 1.21×10^8 copies/mL. The titers of all strains decreased 21 d after infection and then increased 28 d after infection. In addition, 2010NJ strain had the highest titer in serum 28 d after infection. The two PCV2b isolates, 2010NJ and 2009ZJ, had the highest titer in lungs and spleens. [Conclusion]All results showed that different PCV2 isolates have different proliferation ef ficiencies in Balb/c mice, even if they belong to the same subtype. In addition, the proliferation rate of 2009ZJ in visceral organs was significantly higher than that in serum. However, this phenomenon is not obvious for other strains. These results laid a foundation for the analysis of proliferation characteristics and pathogenicity of different PCV2 strains in vivo.

Inducible Expression on Multi-epitope of Porcine Circovirus Type 2 and Its Immunological Competence

In order to obtain induction expressed porcine circovirus type 2（PCV2）multi-epitope with good immunogenicity in vitro.Major epitopes of PCV2 were screened in the test,epitopes were composed sequentially,the c DNA sequences were artificially synthesized.Bam HⅠand SalⅠwere directionally cloned into prokaryotic expression vector PEGX-4T-1 multiple cloning site,then BL21 competent cells were transformed,positive clones were screened,IPTG inducible expression was conducted.Expression on target gene was analyzed by SDS-PAGE,fusion protein polypeptide was extracted and purified,immunocompetence of the expressed multi-epitope protein was identified by Westernblot.BALB/c mouse was immuned by fusion protein polypeptide,the antibody was determined by ELISA,immunogenicity was evaluated.Results showed that expression recombinant plasmid pEGX-4T-1-ep contained with seven PCV2 antigen epitopes had been constructed successfully.SDS-PAGE analysis showed that fusion protein polypeptide was expressed effectively in Escherichia coli（E.coli）,and the molecular weight was about 35ku,which existed in the form of solubility.Results of Westernblot showed that the extraction and purification of fusion protein polypeptide and PCV2 positive serum had good reactogenicity.Results of ELISA showed that the purified fusion protein polypeptide could stimulate the body to produce PCV2 specific antibody which had good immunogenicity.Results indicated that the constructed PCV2 multi-epitope had good expression characteristics in vitro,and the expression protein had good immunogenicity.The study provided a basic for the study on PCV2 epitope screening,functional identification and multi-epitope vaccine.

cGAS蛋白促进PCV-2茎环结构诱导cGAS-STING信号通路天然免疫反应研究

为了探索猪圆环病毒2型(PCV-2)DNA茎环结构是否可以激活环鸟苷酸腺苷酸合成酶(cGAS),以及是否诱导cGAS-STING信号通路天然免疫反应,试验将含有PCV-2茎环结构的单链DNA(SL-DNA)转染至PK15细胞中,通过Western-blot检测cGAS蛋白是否在PK15细胞中表达;将SL-DNA转染至过表达及敲除cGAS蛋白的PK15细胞中,通过实时荧光定量PCR检测细胞中cGAS、干扰素刺激基因(STING)、TANK结合激酶1(TBK1)、干扰素调节因子3(IRF3)、干扰素-β(IFN-β)、白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)基因的表达水平。结果表明:SL-DNA转染后,PK15细胞中cGAS蛋白表达量上升;过表达cGAS蛋白的PK15细胞与PK15细胞相比,SL-DNA转染后cGAS、STING、TBK1、IRF3、IFN-β、IL-1β、IL-6、TNF-α基因的相对表达量均极显著升高(P<0.01);敲除cGAS蛋白的PK15细胞与PK15细胞相比,SL-DNA转染后cGAS、STING、TBK1、IRF3、IFN-β、IL-1β、IL-6、TNF-α基因的相对表达量均极显著降低(P<0.01)。说明cGAS蛋白可以识别PCV-2的茎环结构,并且促进其诱导的cGAS-STING信号通路天然免疫反应。

Porcine Circovirus 2:Immunopathogenesis and Recent Developments in Vaccines

Porcine circovirus 2 (PCV2) is currently considered an important etiologic agent of swine and its infection has potentially serious economic impact on the swine industry worldwide. This virus is frequently associated with postweaning multisystemic wasting syndrome (PMWS), and also with other clinical conditions such as porcine dermatitis and nephropathy syndrome (PDNS), late-term abortions, reproductive failure in sows, proliferative and necrotizing pneumonia and congenital tremors. The term porcine circovirus-associated disease (PCVAD) is currently used to refer to any of these diseases when they are associated with PCV2 infection. The PCV2 was recognized as a pathogen in 1997, and many questions regarding its biology and pathogenesis remain unanswered. Currently, some studies have shown the production of new vaccine candidates and field efficacy testing of commercial vaccines. This review discusses some major points concerned with immunopathogenesis and vaccines for PCV2 infection.

猪圆环病毒2型疫苗及其效力评价方法比较研究

为探索对不同猪圆环病毒2型(Porcine circovirus type2,PCV2)疫苗产品进行统一质量评价的可行性,对国内该类疫苗产品及其现有效力评价方法进行了归纳和比较研究。结果发现,目前猪圆环病毒2型疫苗类产品种类有25个,且疫苗毒株数量多达10余个,效力检验方法包括传统的免疫攻毒法、血清学方法和多种不同的替代方法,但尚没有统一的效力评价体系。本研究可为进一步完善PCV2疫苗效力评价方法、统一评价疫苗效力、提高产品质量提供参考。