Interaction between host cell proteins and open reading frames of porcine circovirus type 2

박시원,박인병,강석진,배준범,전태훈 한국축산학회 2023 한국축산학회지 Vol.65 No.4

Postweaning multisystemic wasting syndrome (PMWS) is caused by a systemic inflammation after porcine circovirus type 2 (PCV2) infection. It was one of the most economically important pathogens affecting pig production worldwide before PCV2 vaccine was first introduced in 2006. After the development of a vaccine against PCV2a type, pig farms gradually restored enormous economic losses from PMWS. However, vaccine against PCV2a type could not be fully effective against several different PCV2 genotypes (PCV2b – PCV2h). In addition, PCV2a vaccine itself could generate antigenic drift of PCV2 capsid. Therefore, PCV2 infection still threats pig industry worldwide. PCV2 infection was initially found in local tissues including reproductive, respiratory, and digestive tracks. However, PCV2 infection often leads to a systemic inflammation which can cause severe immunosuppression by depleting peripheral lymphocytes in secondary lymphoid tissues. Subsequently, a secondary infection with other microorganisms can cause PMWS. Eleven putative open reading frames (ORFs) have been predicted to encode PCV2 genome. Among them, gene products of six ORFs from ORF1 to ORF6 have been identified and characterized to estimate its functional role during PCV2 infection. Acquiring knowledge about the specific interaction between each PCV2 ORF protein and host protein might be a key to develop preventive or therapeutic tools to control PCV2 infection. In this article, we reviewed current understanding of how each ORF of PCV2 manipulates host cell signaling related to immune suppression caused by PCV2.

바큘로바이러스를 이용한 돼지 2형 써코바이러스 유사입자 발현

김하현 ( Ha Hyun Kim ),박건용 ( Geon Yong Bak ),박준규 ( Jun Gyu Park ),김현정 ( Hyun Jeong Kim ),권형준 ( Hyoung Jun Kwon ),박수진 ( Su Jin Park ),정영주 ( Young Ju Jeong ),김채현 ( Chae Hyeon Kim ),( Myra Hosmillo ),( Mia 한국수의공중보건학회 2010 예방수의학회지 Vol.34 No.3

Porcine circovirus type 2 (PCV2) is associated with porcine circovirus diseases (PCVD), of which postweaning multisystemic wasting syndrome is considered to cause considerable economic losses in pig industry worldwide. As the virus-like particle (VLP) is a highly effective type of subunit vaccine and has unique advantages in terms of safety and immunogenicity, this study aimed to develop PCV2-like particles, which matched currently circulating Korean PCV2 and were applicable as vaccines. The ORF2 genes encoding PCV2 capsid protein were amplified from the PCV2 subgroup 1A/B Korean C275 isolate and the subgroup 2E C94 isolate by PCR assay with primer pair specific to PCV2 ORF2 gene, and were cloned into baculovirus transfer vector. Recombinant baculovirus was generated by cotransfection with the transfer vector and linear baculovirus DNA into the Sf9 cells, and then by plaque purification. Expression of PCV2 capsid protein was determined by the indirect immunofluorescence and Western blotting assays, and electron microscopy. By both immunological assays, PCV2 capsid antigen was detected in the Sf9 cells infected with the recombinant baculoviruses. The formation of empty virus particles, characteristic of VLP, was detected in the lysate of Sf9 cells infected with the recombinant baculoviruses by negative electron microscopy. From these results, VLPs of two genogroups of PCV2 were successfully expressed and generated in a baculovirus expression system. It is expected that the expressed VLPs of two genotypic groups can be used for control of PCV2 infection as good vaccine candidates.

Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

Lee, Jun Beom,Bae, Sung Min,Shin, Tae Young,Woo, Soo Dong Korean Society of Sericultural Science 2015 International Journal of Industrial Entomology Vol.30 No.2

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has type-specific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti-His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

이준범,배성민,신태영,우수동 한국잠사학회 2015 International Journal of Industrial Entomology Vol.30 No.2

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has typespecific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti- His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells

Comparison of three commercial one-dose porcine circovirus type 2 (PCV2) vaccines in a herd with concurrent circulation of PCV2b and mutant PCV2b

Jeong, J.,Park, C.,Choi, K.,Chae, C. Elsevier Scientific Pub. Co 2015 Veterinary microbiology Vol.177 No.1

Porcine circovirus associated disease (PCVAD) occurred in a farm where pigs had been routinely vaccinated with a commercial PCV2a vaccine. A mutant PCV2b (mPCV2b) was isolated from pigs with PCVAD, perhaps implying a perceived vaccine failure. The objective of this study was to determine and compare the efficacy of 3 one-dose PCV2a vaccines of varying antigen type and dose in the same pig farm with concurrent PCV2b and mPCV2b infection based on clinical (average daily weight gain; ADWG), virological (evidence of viremia), immunological (presence of PCV2-specific neutralizing antibody; NA and interferon-γ secreting cells; IFN-γ-SC), and pathological (lymphoid lesion and PCV2 antigen score within lesion) evaluation. Regardless of which commercial PCV2a vaccine was used, vaccinated animals improved ADWG, and reduced the amount of PCV2b and mPCV2b load in the blood compared to unvaccinated animals. The vaccination of piglets at 3 weeks of age effectively induced higher levels of PCV2b- and mPCV2b-specific NA and IFN-γ-SC compared to unvaccinated animals. A reduction in mPCV2b load in the blood coincided with the appearance of both mPCV2b-specific NA and IFN-γ-SC in the vaccinated animals. The microscopic lymphoid lesions and PCV2-antigen scores within the lymph nodes were significantly lower in vaccinated animals. The perceived vaccine failure could not be explained by incomplete protection of the commercial PCV2a vaccine against mPCV2b. The results of the present study demonstrated that currently available commercial PCV2a vaccines are protective against concurrent PCV2b and mPCV2b infection based on clinical, virological, immunological, and pathological evaluations under field conditions.

Expression of porcine circovirus type 2 capsid protein fused with partial polyhedrin using baculovirus

( Jun Beom Lee ),( Sung Min Bae ),( Tae Young Shin ),( Soo Dong Woo ) 한국잠사학회 2015 International Journal of Industrial Entomology Vol.30 No.2

Porcine circovirus type 2 (PCV2) is an important infectious swine virus causing postweaning multisystemic wasting syndrome (PMWS). PCV2 capsid protein, encoded by ORF2 has typespecific epitopes, is very immunogenic, and is associated with the induction of neutralizing antibodies. For the efficient production of capsid protein, recombinant Autographa californica nucleopolyhedroviruses were generated to express ORF2 fused with two forms of a partial polyhedrin. Recombinant capsid protein was produced successfully with the partial polyhedrin fusion form and the yield was high, as was shown by SDS-PAGE. Production of recombinant capsid proteins in insect cells was confirmed by Western blot analysis using anti- His monoclonal antibody, anti-ORF2 monoclonal antibody, and anti-PCV2 porcine serum. Fusion expression with amino acids 19 to 110 of the polyhedrin increased the production of recombinant capsid protein, but fusion with amino acids 32 to 85 did not. Additionally, PCV2 capsid protein is a glycoprotein; however, the glycosylation of recombinant protein was not observed. The results of an Enzyme-linked immunosorbent assay (ELISA) showed that recombinant capsid proteins could be utilized as antigens for fast, large-scale diagnosis of PCV2-infected pigs. Our results suggest that the fusion expression of partial polyhedrin is able to increase the production of recombinant PCV2 capsid protein in insect cells.

Classifying Host Susceptibility Using Porcine Circovirus Type 2 Viral Load and Antibody Titer

Kyu-Sang Lim(임규상),Eun-A Lee(이은아),Kyung-Tai Lee(이경태),Taehoon Chun(전태훈),Ki-Chang Hong(홍기창),Jun-Mo Kim(김준모) 한국생명과학회 2017 생명과학회지 Vol.27 No.3

양돈산업에 있어 돼지 써코바이러스 2형(PCV2)의 복합감염으로 인한 이유자돈의 질병 피해가 막대하다. PCV2 감염에 대한 숙주의 민감도는 상이한 것으로 알려져 있으며, 따라서 숙주의 민감도를 구분하는 것은, 이를 이용한 숙주의 저항성 향상 연구에 필수이다. 본 연구의 목적은 이유자돈군의 혈액 내에서 PCV2 바이러스에 대한 숙주의 민감도를 구분 짓고 구명하는데 있었다. 본 연구에서는 자연적으로 바이러스에 감염된 10주령의 이유자돈군으로 부터 혈청을 채취하여 PCV2 바이러스량과 항체가를 측정하고 혈구분석을 실시했다. 또한, 측정된 PCV2 바이러스량과 항체가를 기준으로 자돈군 내에서 저항성군과 민감성군을 선정하였고, 통계분석결과 저항성군에 비해 민감성군에서 백혈구 수가 현저히 줄어든 것을 확인하였다. 본 연구를 통해서 PCV2 감염에 대한 돼지의 민감도를 구분짓기 위한 PCV2 바이러스량과 항체가를 이용한 복합기준을 제시할 수 있었으며, 이유자돈군의 PCV2 관련 질병저항성 및 백혈구감소증을 확인할 수 있는 방법을 마련하였다. Porcine circovirus type 2 (PCV2) is a notorious and ubiquitous virus in the swine industry. The susceptibility of the host to PCV2 infection is considered to be one factor associated with the dynamics of PCV2. The objective of this study was to verify the criteria for host susceptibility to PCV2, using blood parameters of post-weaned pigs naturally infected with the virus. The PCV2 DNA viral load, antibody titer, and leukopenia characteristics were measured in the serum extracted from the pigs at the 10th week. We classified the pigs into high (>5.0), intermediate (3.0 to 5.0), and low (<3.0) groups on the basis of the PCV2 viral load (log copies/ml), or as positive (≤0.50) and negative (>0.50) groups on the basis of antibody titer (sample-to-negative corrected ratio). Moreover, using these two categorized parameters, we suggested the criteria for classification into the susceptible and resistant groups. Statistical analyses revealed that pigs in the susceptible group had a significantly higher viral load (p<0.001) and negative antibody titer (p<0.001), as well as significantly lower leukocyte counts (p=0.018) and lower amounts of several leukocyte components (p<0.05), than pigs in the resistant group. We concluded that the susceptible group could be considered to have PCV2-induced leukopenia. Therefore, we suggest that the combined classifications of viral loads and anti-PCV2 antibodies can be used to determine PCV2-induced leukopenia in the subclinical PCV2 infection of post-weaned pig populations.

Efficient Production of Porcine Circovirus Type 2 Capsid Protein using Baculovirus

이준범,배성민,김희정,이원우,허원일,최재방,우수동,신태영 한국잠사학회 2012 International Journal of Industrial Entomology Vol.24 No.1

Porcine circovirus type 2 (PCV2) is a single-stranded circular DNA virus associated with Postweaning multisystemic wasting syndrome (PMWS), which is considered to be an important infectious swine viral disease. PCV2 capsid protein encoded by ORF2 is a structural protein and expected as the high immunogenicity protein. In this study, we generated recombinant baculovirus containing ORF2 of PCV2 and analyzed the optimal conditions for the production of capsid protein in insect cell. Production and status of recombinant capsid protein in insect cell were confirmed by SDSPAGE and Western blot analysis using His tag antibody and anti-PCV2 serum. The yield of recombinant capsid protein was high like as shown visible on SDS-PAGE. Optimal multiplicity of infection (MOI) and infection time of recombinant virus were determined as 5 MOI and 4 days, respectively. ORF2 is known to have Nlinked glycosylation site, but we couldn’t detect the glycosylation of recombinant protein in insect cells.

Expression and evaluation of porcine circovirus type 2 capsid protein mediated by recombinant adeno-associated virus 8

Shuang Li,Bo Wang,Shun Jiang,Xiaohui Lan,Yongbo Qiao,Jiaojiao Nie,Yuhe Yin,Yuhua Shi,Wei Kong,Yaming Shan 대한수의학회 2021 Journal of Veterinary Science Vol.22 No.1

Background: Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide. Objectives: A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy. Methods: In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer. Results: The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2. Conclusions: The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.

Use of a multinomial logistic regression model to evaluate risk factors for porcine circovirus type 2 infection on pig farms in the Republic of Korea

Eu-Tteum Kim,Son-Il Pak 한국예방수의학회 2017 예방수의학회지 Vol.41 No.3

The current study identified risk factors associated with porcine circovirus type 2 (PCV2) infection on pig farms in the Republic of Korea using a multinomial logistic regression model to evaluate the PCV2 infection status of pigs at different growth stages. Compulsory disinfection of visitors (odds ratio [OR]: 0.019, 95% confidence interval [CI]: <0.001–0.378, p=0.0095), compulsory registration of visitors (OR: 0.002, 95% CI: <0.001–0.184, p=0.0070), regular blood testing (OR: 0.012, 95% CI: <0.001–0.157, p=0.0007), and running on-farm biosecurity learning programs for workers (OR: 0.156, 95% CI: 0.040 –0.604, p=0.0072 and OR: 0.201, 95% CI: 0.055–0.737, p=0.0155, respectively) were identified as factors which could reduce the risk of PCV2 infection. However, visitation by a regular veterinarian (OR: 32.733, 95% CI: 3.768–284.327, p=0.0016) was associated with PCV2 infection.