Construction of a replicative expression vector based on the porcine circovirus 2 replicon / 生物工程学报

The antigen gene expression level of a DNA vaccine is the key factor influencing the efficacy of the DNA vaccine. Accordingly, one of the ways to improve the antigen gene expression level of a DNA vaccine is to utilize a plasmid vector that is replicable in eukaryotic cells. A replicative DNA vaccine vector pCMVori was constructed based on the non-replicative pcDNA3.1 and the replicon of porcine circovirus 2 (PCV2) in this study. An EGFP gene was cloned into pCMVori and the control plasmid pcDNA3.1. The two recombinant vectors were transfected into PK-15 cell, and the plasmid DNA and RNA were extracted from the transfected cells. Real-time PCR was used to determine the plasmid replication efficiency of the two plasmids using plasmid before and after Bcl Ⅰ digestion as templates, and the transcription level of the Rep gene in PCV2 replicon was detected by RT-PCR. The average fluorescence intensity of cells transfected with the two plasmids was analyzed with software Image J, and the transcription level of EGFP was determined by means of real-time RT-PCR. The results showed that the replication efficiency of pCMVori in PK-15 cells incubated for 48 h was 136%, and the transcriptions of Rep and Rep' were verified by RT-PCR. The average fluorescence intensity of the cells transfected with pCMVori-EGFP was 39.14% higher than that of pcDNA3.1-EGFP, and the transcription level of EGFP in the former was also 40% higher than that in the latter. In conclusion, the DNA vaccine vector pCMVori constructed in this study can independently replicate in eukaryotic cells. As a result, the expression level of cloned target gene was elevated, providing a basis for developing the pCMVori-based DNA vaccine.

Analysis of genetic variation diversity of porcine circovirus-2 virus genome isolated from Shanxi area / 中国兽医学报

In order to study genetic variation diversity of porcine circovirus type 2 (PCV2) strains in Shanxi,the genomic sequences of nine PCV2 strains including SXQX,SXCZ,SXTY2,SXJC,SXJX,SXLL,SXPY,SXPG and SXXY recently isolated from some areas of Shanxi from 2013 to 2016,was cloned,sequenced and received by GenBank.The amplified PCV2 genomic sequences,ORF2 sequences and Cap protein amino acid of these nine strains were analysed and compared with those of published 28 PCV2 strains by DNAStar,drawing phylogenetic tree.The results showed that the genomic sequences of SXJX,SXJC and SXXY PCV2 strains were 1 768 bp,and the others were 1 767 bp,which accounted for 33％ and 67％,respectively.The homologies of nucleotide sequences of the nine strains were 94.7％-99.8％,the homologies of nucleotide sequences of the nine strains with the 28 isolates from different regions of the world PCV strain were 93.9％-99.9％,and the homologies of nucleotide sequences of the nine strains with the domestic vaccine strains were 95.1％-99.8％.The phylogenetic analysed that SXJX,SXJC and SXXY belonged to genotype PCV-2D,SXLL,SXPY and SXCZ belonged to genotype PCV-1C,and SXTY14,SXPG and SXQX belonged to genotype PCV-1A/1B.Thus it proved that the epidemic strain of PCV2 was mainly PCV-2b in Shanxi.The homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains were 90.0％-100.0％ and 87.1 ％-100.0％ respectively,the homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains with the 28 isolates from different regions of the world PCV strain were 87.6％-100.0％ and 84.1％-100.0％ respectively,and the homologies of ORF2 nucleotide sequences and Cap amino acid of the nine strains with the domestic vaccine strains were 91.0％-100.0％ and 89.3％-100.0％ respectively.The Cap amino acids of SXQX,SXJX,SXTY14,SXPG,SXJC and SXXY PCV2 were 233,ORF2 of SXQX,SXTY14 and SXPG located at 1 033-1 734 bp,ORF2 of SXXY,SXJX and SXJC located at 1 033-1 734 bp,and the Cap amino acids of SXCZ,SXLL and SXPY PCV2 were 234,ORF2 of them located at 1 030-1 734 bp,in addition,the positions of 1 030-1 734 bp were more three bases TCA than other ORF2 genome sequence of 1 767 bp,resulting in increasing a K (Lys) of amino acid sequencein at the 234 position.Also Cap protein of 9 PCV2 strains showed more amino acid variation in addition to the only high-ly conserved glycosylation sites (NYS) (pp.143-145 amino acid).It provided theoretical basis for the PCV2 immune prevention of research in Shanxi,and the data of basic theory of molecular pathogenesis of PCV2.

Assessing PCV2 antibodies in field pigs vaccinated with different porcine circovirus 2 vaccines using two commercial ELISA systems

Porcine circovirus type 2 (PCV2) is the primary causative agent for post-weaning, multisystemic, wasting syndrome. Consequently, serologic detection of and vaccination against PCV2 are important for the swine industry. Among several serological tests, the enzyme-linked immunosorbent assay (ELISA) is commonly used to measure anti-PCV2 antibody levels. In the present study, we used two commercial ELISA systems to comparatively evaluate anti-PCV2 antibodies in field pigs treated with three different PCV2 vaccines. Among a total of 517 serum samples, the results of the two ELISAs were fully concordant for 365 positive and 42 negative samples, indicating 78.7% agreement. In addition, the Pearson coefficient (0.636) indicated a moderate correlation between data from the two ELISAs. Results from the farms with pigs vaccinated with the three different PCV2 vaccines demonstrated that most of the vaccinated animals underwent seroconversion. However, the increase and duration of antibody titers varied depending on the vaccine, the presence of maternal antibodies, and the vaccination program. PCV2 serologic status and anti-PCV2 antibody levels of herds from this study could be utilized to determine the best timing for vaccination and assessing vaccination compliance.

The effects of sow viremia and maternal antibodies in porcine circovirus 2 on viral infection and weight of piglets / Efeito da viremia da porca e de anticorpos maternos para circovírus suíno 2 na infecção e no peso da leitegada

The aim of this study was to characterize the porcine circovirus 2 (PCV2) infections in farrowing sows and to evaluate an association with piglet viremia and weight. Twenty sows and 100 newborn piglets were studied. Colostrum and serum of the sows were obtained on the day of parturition. Milk samples were collected on day 20 postpartum. Blood samples were taken and the piglets were weighed on days 1, 20, 42, 63 and 84 postpartum. Colostrum, milk and serum were evaluated for PCV2 DNA load. Serum was evaluated for neutralizing antibodies. PCV2 DNA was found in 17/20 serum samples, 14/20 colostrum samples and 11/20 milk samples. On day 1 postpartum 29% of piglets were viremic. PCV2 viral load ranged from 3.02 to 6.75 log10 copies/mL considering all sampled days. There was no correlation between sow viremia, antibody levels or PCV2 load in colostrum and piglet viremia on day 1 postpartum. The PCV2 load in colostrum and milk was associated with viremia in piglets from weaning to 84 days postpartum. Piglets' PCV2 viremia and viral load could not be associated with weight throughout this study...

O objetivo deste estudo foi caracterizar o efeito do infecção pelo circovírus suíno 2 (PCV2) em porcas gestantes na viremia e no peso da leitegada. Vinte porcas e 100 leitões recém-nascidos foram acompanhados. Amostras de colostro e soro das porcas foram obtidas no dia do parto. Amostras de leite foram coletadas no dia pós-parto 20. Os leitões foram pesados e tiveram amostras de soro coletadas nos dias um, 20, 42, 63 e 84 pós-parto. Soro, colostro e leite foram testados para carga viral do PCV2. Soro foi avaliado para presença de anticorpos neutralizantes. O DNA do PCV2 foi encontrado em 14 de 20 amostras de colostro e em 11 de 20 amostras de leite. No dia pós-parto 1, 29% dos leitões foram virêmicos. A carga viral do PCV2 variou 3,02-6,75 log10 cópias / mL, considerando todos os dias amostrados. Não houve correlação entre viremia das porcas e os níveis de anticorpos no soro ou na carga de PCV2 no colostro e na viremia dos leitões com um dia de vida. A carga de PCV2 no colostro e no leite foi associada à viremia em leitões do desmame até 84 dias pós-parto. A carga viral do PCV2 em leitões não foi associada com o peso ao longo deste estudo...

Rapid Detection of Porcine Circovirus 2 Based on Double Molecular Beacons / 分析化学

A double-molecular beacons (DMB) based assay was developed for porcine circovirus 2(PCV2) detection. Two single-stranded DNA molecular beacons which could specifically hybridize with PCV2 genome DNA respectively in different sequence were designed according to the characteristics of the PCV2 genome sequences. The fluorescence signal was amplified 80 times by DMB, which was 2-4 times higher than that of single molecular beacon. Under the optimal conditions of 10 mmol/L MgCl2 , 20 mmol/L Tris-HCl (pH=8. 0), 40 ℃ and 30 min incubation time of DNA with DMB, the enlargement factor was increased linearly with DNA concentration over the range from 2 nmol/L to 200 nmol/L, with a detection limit of 1 nmol/L. The method was applied to detect PCV2 in genome of 18 swine fever samples and 8 PCV2 positive cases were found, which were confirmed by PCR method.

Enhancing mucosal immunity in mice by recombinant adenovirus expressing major epitopes of porcine circovirus-2 capsid protein delivered with cytosine-phosphate-guanosine oligodeoxynucleotides

A recombinant replication-defective adenovirus expressing the major epitopes of porcine circovirus-2 (PCV-2) capsid protein (rAd/Cap/518) was previously constructed and shown to induce mucosal immunity in mice following intranasal delivery. In the present study, immune responses induced by intranasal immunization with a combination of rAd/Cap/518 and cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) were evaluated in mice. The levels of PCV-2-specific IgG in serum and IgA in saliva, lung, and intestinal fluids were significantly higher in the group immunized with rAd/Cap/518 and CpG ODN than animals immunized with rAd/Cap/518 alone. The frequencies of IL-2-secreting CD4+ T cells and IFN-gamma-producing CD8+ T cells were significantly higher in the combined immunization group than mice immunized with rAd/Cap/518 alone. The frequencies of CD3+, CD3+CD4+CD8-, and CD3+CD4-CD8+ T cells in the combined immunization group were similar to that treated with CpG ODN alone, but significantly higher than mice that did not receive CpG ODN. PCV-2 load after challenge in the combined immunization group was significantly lower than that in the phosphate-buffered saline placebo group and approximately 7-fold lower in the group treated with CpG ODN alone. These results indicate that rAd/Cap/518 combined with CpG ODN can enhance systemic and local mucosal immunity in mice, and represent a promising synergetic mucosal vaccine against PCV-2.

Detection of Genetic characterization of Porcine circovirus 2 (PCV2) in Brazilian wildlife boars

A semi-intensive wildlife boars farm presented a clinical history of high mortality in 70 - 90 days-old pigs (> 50 %). Two 90 days-old animals with weight loss and wasting were necropsied and the samples tested for PCV2 by polymerase chain reaction (PCR). The genetic material of PCV2 was sequenced and classified into the PCV2a genotype together with PCV2 sequences obtained from samples of Poland, Brazil, Slovenia and Greece wild boars.

Detection of genetic characterization of Porcine circovirus 2 (PCV2) in Brazilian wildlife boars

A semi-intensive wildlife boars farm presented a clinical history of high mortality in 70 - 90 days-old pigs (> 50 %). Two 90 days-old animals with weight loss and wasting were necropsied and the samples tested for PCV2 by polymerase chain reaction (PCR). The genetic material of PCV2 was sequenced and classified into the PCV2a genotype together with PCV2 sequences obtained from samples of Poland, Brazil, Slovenia and Greece wild boars.

Obtaining of union expression virus-like particle of PPV VP2 and PCV2 ORF2 and its immunogenicity / 中国兽医学报

The amplified VP2 gene (PPV strain SC-1) and PCV20RF2 gene were inserted into the eukaryotic expression vector pPI-2.EGFP.Then the recombinant plasmid named pPI-2.EGFP.VP2.ORF2 was obtained.Mediated by liposome,the recombinant plasmid was transfected into Veto cells and expressed.Using immunofluorescence assay,the fluorescence of expression products were first detected at 20 h after transfection and peaked 36 h.Under electronmicroscope,virus-like particles (VLPs) can be observed in the transfected cells.To confirm the obtained VLPs to be recombinant particles,piglets were immunized using purified VLPs.The dynamic variation of blood T lymphocytes and serum antibody level of PPV and PCV2 were measured.The results showed that the ratio of CD3+,CD4+ T lymphocyte in peripheral blood lymphocyte of immunized piglets raised in a certain degree,the number of CD8+ T lymphocytes fell at 7-14 d after immunization,and then raised.Relatively high level of PP-V,PCV2 specific antibody could be detected.This indicated that the expression of recombinant plasmid pPI-2.EGFP.VP2.ORF2 was successful,the virus-like particles were formed and showed favourable immunogenicity.

Genetic Characterization of Porcine Circovirus Type 2 Detected from the Pigs in Commercial Swine Farms in Korea

Porcine circovirus type 2 (PCV-2) has been nowadays recognized as a major agent causing postweaning multisystemic wasting syndrome (PMWS) in pigs worldwide. PMWS most commonly affects the weaned piglets, being of increasing importance to the pig industry in Korea. Seven commercial farms affected with PMWS and 2 farms free from PMWS, located in the southern part of Gyeonggi province, were selected for this study. The peripheral mononuclear cells were tested for the presence of ORF2 gene by PCR, and 54 (68.4%) of 79 samples were positive. All of 9 herds tested included the positive cases. The positive rates by herds were 50 to 100% in the PMWS-affected herds and 40 to 62.5% in the PMWS-free herd. The nucleotide and amino acid sequences of ORF2 gene of 6 strains were characterized. Homologies among 6 strains revealed 92.1 to 100% in the nucleotide and 92.3 to 100% in the amino acid. The overall ranges of homologies for 25 strains comprised 2 Korean and 23 foreign strains were 91.1 to 100% in the nucleotide and 89.7 to 100% in the amino acid. Three regions of greater heterogeneity were found in immunorelevant epitopes of the capsid protein, and the sequences between 57 to 80 aa revealed higher mutation than other areas. In the phylogenetic tree analysis, KOR 71 strain was clustered together with Korean strains previously isolated in Korea. The remaining 5 strains were closely clustered with other European and Asian strains. The results will be valuable for improving our understanding of the molecular epidemiology of PCV-2 in Korea and development of preventive measures for PMWS.