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.

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.

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.

Prevalence and Countermeasures of Porcine Circovirus 3

The development history, etiology, epidemiology, clinical symptoms, pathological changes and diagnosis technology of porcine circovirus 3 were summarized, and countermeasures for prevention and control of the disease were put forward based on the prevalence status in China.

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.

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.

The Establishment of Immunochemistry Test Based on a Synthetic Peptide Antibody for the Detection of a Porcine Circovirus-Like Virus P1

Recently, a novel porcine circovirus-like virus P1 with a circular DNA genome of 0.648 kb was identified. P1 antigen was detected both in vitro and in vivo by synthetic peptide-derived polyclonal antibody-based immunochemistry. The designed peptides were synthesized by solid-phase technique, purified by high performance liquid chromatography, coupled to Keyhole limpet hemocyanin, and injected into rabbits to prepare polyclonal antibody. The emergence of positive cells revealed that synthetic peptide could elicit antibodies against P1 and viral protein could be synthesized. The polyclonal peptide antibodies described here was successfully applied to immunochemical staining and proved helpful in diagnosing P1.

Host immune response to infection with porcine circoviruses

Porcine circovirus type 2(PCV2),which serves as a major causative agent of PCV2-associated diseases and causes severe loss to the pig industry worldwide,can dysregulate the immune response and induce immunosuppression in PCV2-infected pigs.Similar to PCV2(porcine circovirus type 3(PCV3),a newly identified swine circovirus which might be closely associated with porcine dermatitis and nephropathy syndrome,reproductive disorder,and multisystemic inflammatoty responses,also interferes with host immune defense.Interaction between host immune system and PCVs is considered to be a crucial determinant of pathogenicity in pigs.Here,we sought to briefly discuss the current knowledge regarding the interaction of porcine circovirus type 2 and/or 3 with host immune cells and immune responses to better depict the viral immunomodulatory capacity,pathogenic mechanisms,and the future research direction in host immune responses to infection with PCV2 and PCV3.

Molecular detection of porcine circovirus type 3 in Shanxi Province,China

Porcine circovirus type 3(PCV3),which was first detected in the United States of America in 2015,is a potential threat to the swine industry.However,the prevalence of PCV3 in Shanxi Province,China,is unclear.In this research,the prevalence and genetic diversity of PCV3 were investigated in above area.Lung tissue samples(n=491)from 19 pig slaughterhouses across 11 cities throughout Shanxi Province were analyzed for PCV3 infection by PCR in 2019.The results showed that PCV3 positive rates in slaughterhouses and individuals were 100%(19/19)and 86.76%(426/491),respectively.PCV2 and PCV3 double-positive rates in slaughterhouses and individuals were 100%(19/19)and 59.27%(291/491),respectively.PCR positive samples were further sequenced and 8 PCV3 isolates were identified.The nucleotide homology of these isolates with other PCV3 isolates in NCBI database was 97.45-99.90%.A phylogenetic analysis,based on the complete genomic sequence and ORF2,divided these PCV3 strains into 2 major groups.Based on A24A/and R27/K amino acid mutations of capsid protein,the 8 identified PCV3 strains were separated to 2 clades.This was the first detailed investigation into the epidemiology of PCV3 in Shanxi Province.Our findings enabled us to assess the possibility of widespread transmission from this region.Thus,current findings establish a basis for further studies of genetic variations in PCV3 strains circulating in China.

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1型、2型、3型SYBR GreenⅠ实时荧光定量PCR鉴别方法的建立及应用

猪圆环病毒(PCV)包括猪圆环病毒1型(PCV1)、猪圆环病毒2型(PCV2)、猪圆环病毒3型(PCV3)。近些年来,PCV1、PCV2、PCV3基因型之间存在共感染,因此建立一种快捷、特异、敏感的PCV1、PCV2、PCV3的SYBR GreenΙ实时荧光定量PCR鉴别检测方法显得尤为必要。本试验通过特异性引物筛选,各项反应条件优化,建立了实时荧光定量PCR鉴别方法。结果表明:PCV1、PCV2、PCV3检测下限分别为40.3、25.2、22.4 copies/μL。与猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、猪伪狂犬病病毒(PRV)、猪细小病毒(PPV)均无交叉反应。批内及批间变异系数均小于1%。对98份PCV疑似阳性病料检测结果表明,PCV1、PCV2、PCV3感染率分别为10.2%、65.3%、53.1%,共感染率为7.1%。该方法具有良好的特异性、敏感性和稳定性,为PCV1、PCV2、PCV3的早期检测、定量检测及后期研究提供了技术手段。

伪狂犬病病毒和猪圆环病毒4型二重SYBR Green Ⅰ荧光定量PCR检测方法的建立

本研究设计、合成了分别靶向伪狂犬病病毒(PRV)gE基因和猪圆环病毒4型(PCV4)Cap基因保守区域的特异性引物,并在优化反应条件的基础上,建立了能够同时检测和鉴别PRV和PCV4的二重SYBR GreenⅠ荧光定量PCR方法。结果显示,该方法具有良好的线性关系,R2=0.999。在同一个反应体系中,能够特异地检测到PRV和PCV4的特定荧光信号,而其他病原体无信号;对PRV和PCV4的检测限分别为48.5 copies/μL和40.8 copies/μL。利用建立的二重荧光定量PCR方法对34份猪临床样品进行检测,结果显示,PRV检出率为44.12%(15/34),PCV4检出率52.94%(18/34),且二者混合感染的检出率为32.35%(11/34),比常规PCR方法更加灵敏。该方法是检测PRV和PCV4的一种快速、灵敏、特异的方法。