scholarly journals The Monoclonal Antibody Recognized the Open Reading Frame Protein in Porcine Circovirus Type 2-Infected Peripheral Blood Mononuclear Cells

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 961
Author(s):  
Ling-Chu Hung
Keyword(s):  
Capsid Protein ◽  
Mononuclear Cells ◽  
P53 Protein ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Peripheral Blood Mononuclear ◽  
Reading Frame ◽  
Orf3 Protein ◽  
Infected Pbmcs

The purpose of this study in the context of the open reading frame 3 (ORF3) protein of porcine circovirus type 2 (PCV2) was especially its location and its relation to the capsid protein and the apoptosis protein in PCV2-infected porcine peripheral blood mononuclear cells (PBMCs). To detect the ORF3 protein, monoclonal antibodies (mAbs) were generated in this study. The mAb 7D3 binds to the ORF3 peptide (residues 35–66) and the native ORF3 protein in PCV2-infected PBMCs, as shown by immunofluorescence assay (IFA). The data show that 3–5% of PBMCs were positive for ORF3 protein or p53 protein. Further, 78–82% of PBMCs were positive for the capsid. This study confirmed the ORF3 protein not only colocalized with the capsid protein but also colocalized with the p53 protein in PBMCs. Immunoassays were conducted in this study to detect the capsid protein, the ORF3 protein, anti-capsid IgG, and anti-ORF3 IgG. The data show the correlation (r = 0.758) of the ORF3 protein and the capsid protein in the blood samples from the PCV2-infected herd. However, each anti-viral protein IgG had a different curve of the profile in the same herd after vaccination. Overall, this study provides a blueprint to explore the ORF3 protein in PCV2-infected PBMCs.

scholarly journals The Monoclonal Antibody Recognized Apoptosis-Associated Protein in PCV2-Infected Peripheral Blood Mononuclear Cells

2019 ◽  
Author(s):  
Ling-Chu Hung
Keyword(s):  
Binding Sites ◽  
Mononuclear Cells ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus ◽  
Antigen Binding ◽  
Peripheral Blood Mononuclear ◽  
Orf3 Protein ◽  
Induced Apoptosis

Porcine circovirus type 2 (PCV2) is a small non-enveloped DNA virus that causes swine immunosuppression by inducing apoptosis in lymphocytes. The ORF3 protein plays a major role in PCV2-induced apoptosis in porcine kidney cells, but there is little information regarding this protein in PCV2-infected lymphocytes. In this study, hybridoma screening and epitope mapping were determined by using an indirect ELISA. The mAb 7D3 against ORF3 peptide (residues 35–65) of PCV2 were generated in this study. In vivo situation, the mAb 7D3 recognized ORF3 protein existed in PCV2-infected apoptotic porcine PBMCs. It is noteworthy that thimerosal interfered with the binding of mAb 7D3 to epitope and it was diminished by adding cysteine. Additionally, thimerosal interacting with cysteine-containing peptide was demonstrated by the PTI assay. Furthermore, thimerosal specifically interacted with the antigen-binding sites of mAb 7D3. This study suggested that thimerosal blockade the occlusion of the antigen-binding sites of mAb 7D3 to bind ORF3 peptide (residues 35–65) via thimerosal interacting with cysteine residues which should be located within the antigen-binding sites of mAb 7D3. Overall, the mAb 7D3 has been characterized and it will be a valuables tool in future studies of ORF3 function and the wider mechanism of cell apoptosis caused by PCV2 infection. Similarly, these techniques will be useful for applications in detecting thimerosal too.

scholarly journals Antibody Recognition of Porcine Circovirus Type 2 Capsid Protein Epitopes after Vaccination, Infection, and Disease

2011 ◽  
Vol 18 (5) ◽  
pp. 749-757 ◽  
Author(s):  
Benjamin R. Trible ◽  
Maureen Kerrigan ◽  
Nicholas Crossland ◽  
Megan Potter ◽  
Kay Faaberg ◽  
...  
Keyword(s):  
Capsid Protein ◽  
Natural Infection ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Reading Frame ◽  
Antibody Recognition ◽  
Wasting Syndrome ◽  
Study Support

ABSTRACTOpen reading frame 2 (ORF2) of porcine circovirus type 2 (PCV2) codes for the 233-amino-acid capsid protein (CP). Baculovirus-based vaccines that express only ORF2 are protective against clinical disease following experimental challenge or natural infection. The goal of this study was to identify regions in CP preferentially recognized by sera from experimentally infected and vaccinated pigs and to compare these responses to those of pigs diagnosed with porcine circovirus-associated disease (PCVAD), including porcine multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The approach was to react porcine sera with CP polypeptide fragments followed by finer mapping studies using overlapping oligopeptides that covered amino acids 141 to 200. The results showed that vaccinated pigs preferentially recognized only the largest polypeptide fragment, CP(43-233). A subset of experimentally infected pigs and pigs with PDNS showed strong reactivity against a CP oligopeptide, 169-STIDYFQPNNKR-180. Alanine scanning identified Y-173, F-174, Q-175, and K-179 as important for antibody recognition. The results from this study support the notion of PCV2 modulation of immunity, including antibody responses that may represent a precursor for disease. The recognition of CP(169-180) and other polypeptides provides opportunities to devise diagnostic tests for monitoring the immunological effectiveness of vaccination.

scholarly journals Changes in CD4+, CD8+, CD4+ CD8+, and Immunoglobulin M-Positive Peripheral Blood Mononuclear Cells of Postweaning Multisystemic Wasting Syndrome-Affected Pigs and Age-Matched Uninfected Wasted and Healthy Pigs Correlate with Lesions and Porcine Circovirus Type 2 Load in Lymphoid Tissues

2002 ◽  
Vol 9 (2) ◽  
pp. 236-242 ◽  
Author(s):  
Laila Darwich ◽  
Joaquim Segalés ◽  
Mariano Domingo ◽  
Enric Mateu
Keyword(s):  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Porcine Circovirus Type ◽  
Postweaning Multisystemic Wasting Syndrome ◽  
Immunoglobulin M ◽  
Porcine Circovirus ◽  
Lymphoid Tissues ◽  
Peripheral Blood Mononuclear ◽  
Wasting Syndrome

ABSTRACT Forty-one 8- to 12-week-old wasted pigs were selected from several conventional farms with histories of postweaning multisystemic wasting syndrome (PMWS) and classified into two groups according to their porcine circovirus type 2 (PCV2) infection status, as determined by in situ hybridization (ISH). Twenty-four pigs tested positive for PCV2 (PCV2-positive group), while 17 pigs tested negative for PCV2 (PCV2-negative group). In addition, eight uninfected healthy pigs from an experimental farm were used as controls. Heparinized blood samples were taken to obtain peripheral blood mononuclear cells. The CD4+, CD8+, CD4+ CD8+ (double-positive [DP]), and immunoglobulin M-positive (IgM+) cell subsets were analyzed by flow cytometry with appropriate monoclonal antibodies. Histopathological studies were done to evaluate the apparent degrees of lymphocyte depletion in different lymphoid organs (superficial inguinal and mesenteric lymph nodes, Peyer's patches, tonsils, and spleen) and to determine the viral load of the PCV2 genome by using an ISH technique. Animals of the PCV2-positive group showed a significant downshift of the CD8+ and DP cell subsets compared to the other groups (P < 0.05). Moreover, in PCV2-positive pigs, the amount of PCV2 genome in lymphoid tissues was related to the degree of cell depletion in those tissues (P < 0.05) as well as to the relative decrease in IgM+ and CD8+ cells in peripheral blood. These data support the notion that PCV2-positive pigs might have an impaired immune response.

scholarly journals Regulator of G protein signalling 16 is a target for a porcine circovirus type 2 protein

2009 ◽  
Vol 90 (10) ◽  
pp. 2425-2436 ◽  
Author(s):  
Sirje Timmusk ◽  
Elodie Merlot ◽  
Tanja Lövgren ◽  
Lilian Järvekülg ◽  
Mikael Berg ◽  
...  
Keyword(s):  
G Protein ◽  
Porcine Circovirus Type ◽  
Porcine Circovirus Type 2 ◽  
Porcine Circovirus ◽  
Structural Protein ◽  
Reading Frame ◽  
Orf3 Protein ◽  
G Protein Signalling

Interaction studies have suggested that the non-structural protein encoded by open reading frame 3 (ORF3) of porcine circovirus type 2 (PCV2) binds specifically to a regulator of G protein signalling (RGS) related to human RGS16 (huRGS16). The full-length clone of RGS16 was generated from porcine cells and sequence analysis revealed a close relationship to huRGS16 and murine RGS16. In vitro pull-down experiments verified an interaction between porcine RGS16 (poRGS16) and ORF3 from PCV2. Using GST-linked ORF3 proteins from three different genogroups of PCV2 and from porcine circovirus type 1 (PCV1) in the pull-down experiments indicated that there were differences in their ability to bind poRGS16. Quantitative RT-PCR demonstrated that the expression of poRGS16 mRNA could be induced by a number of cell activators including mitogens (LPS and PHA), interferon inducers (ODN 2216 and poly I : C) and the neurotransmitter norepinephrine. Immunofluorescence labelling confirmed the induced expression of poRGS16 at the protein level and suggested that the PCV2 ORF3 protein co-localized with poRGS16 in LPS-activated porcine PBMC. Furthermore, poRGS16 appeared to participate in the translocation of the ORF3 protein into the cell nucleus, suggesting that the observed interaction may play an important role in the infection biology of porcine circovirus.

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