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1.
Int J Biol Macromol ; 278(Pt 3): 134955, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39173309

RESUMO

As one genotype of porcine circovirus (PCV) identified in 2016, PCV3 has brought huge hidden dangers to the global swine industry together with PCV2. Virus-like particles (VLPs) of capsid protein (Cap) of PCV2 serve as an alternative nano-antigen delivery strategy to efficiently induce antiviral immune response against PCV2 and/or other covalently displayed swine pathogens. However, the current understanding is limited on the capability of PCV3 as a nano-vaccine vehicle. Here we systematically compared the characteristics and the immunogenic efficacy of PCV3 Cap (Cap3) and PCV2 Cap (Cap2) in a VLP form. Cap3 VLPs presented higher internalization efficiency into cells and cytokines production compared to those of Cap2. Meanwhile, cross-reactive immunity between Cap3 VLPs and Cap2 VLPs was detected. Furthermore, to evaluate the function of Cap3 VLPs and Cap2 VLPs as vaccine vehicles carrying foreign proteins, the non-structural protein 6 of porcine reproductive and respiratory syndrome virus (PRRSV) was fused to C-terminus of Cap. Cap3-based chimeric particles induced a higher level of nsp6-specific immune response and PRRSV inhibition. Collectively, these self-assembling, Cap-based VLPs offer a compelling platform for enhancing the effectiveness of subunit vaccinations against newly emerging diseases and hold great promise for the development of Cap3-based chimeric subunit vaccines.


Assuntos
Proteínas do Capsídeo , Circovirus , Vacinas de Partículas Semelhantes a Vírus , Vacinas Virais , Animais , Circovirus/imunologia , Circovirus/genética , Suínos , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas Virais/imunologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Anticorpos Antivirais/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/imunologia
2.
Front Immunol ; 15: 1438371, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39081314

RESUMO

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.


Assuntos
Infecções por Circoviridae , Circovirus , Herpesvirus Suídeo 1 , Animais , Circovirus/imunologia , Circovirus/genética , Camundongos , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Vacinas Virais/imunologia , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/genética , Suínos , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/sangue , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Vacinas Sintéticas/imunologia , Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Feminino , Camundongos Endogâmicos BALB C
3.
Vet J ; 307: 106199, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39038778

RESUMO

Porcine circoviruses (PCVs) contain four types: PCV1, PCV2, PCV3, and PCV4, all of which can infect pigs. Among them, PCV1 is non-pathogenic, and PCV2 can cause porcine circovirus diseases (PCVD) or porcine circovirus-associated diseases (PCVAD). Although the pathogenicity of PCV3 and PCV4 is still controversial, increasing evidence shows that PCV3 and PCV4 can cause PCV-related disease. However, mixed infection of PCV2, PCV3, and PCV4 with other pathogens often occurs in large-scale pig breeding, bringing severe economic losses to the global pig industry. In this study, the soluble recombinant proteins of PCV2, PCV3, and PCV4 Cap were expressed by the prokaryotic expression system and biotinylated to combine with the Streptavidin magnetic beads, followed by immunogenicity evaluation of the recombinant proteins. Furthermore, we also assessed the efficacy and immunogenicity of trivalent recombinant proteins conjugated with different adjuvants in mice. The results showed that the highly effective anti-PCV serum was successfully prepared, and the recombinant proteins conjugated with different adjuvants produced various degrees of humoral and cellular immunity in mice. Three recombinant proteins are effective immunogens, and the trivalent proteins coupled with the aluminum adjuvant or GM-CSF-CpG for two-dose immunization can stimulate prominent humoral and cellular immunity against PCVs in vivo. The soluble recombinant proteins are the most promising candidate for developing a trivalent vaccine against PCVs (PCV2, PCV3, and PCV4) infection simultaneously.


Assuntos
Proteínas do Capsídeo , Infecções por Circoviridae , Circovirus , Circovirus/imunologia , Circovirus/genética , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Infecções por Circoviridae/imunologia , Suínos , Camundongos , Doenças dos Suínos/virologia , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/imunologia , Proteínas Recombinantes/imunologia , Feminino , Vacinas Virais/imunologia , Camundongos Endogâmicos BALB C , Anticorpos Antivirais/sangue
4.
Int Immunopharmacol ; 139: 112701, 2024 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-39024747

RESUMO

Current evidence suggests that porcine circovirus type 2 (PCV2) infection induces immunosuppression in piglets. Sophora subprostrate polysaccharide (SSP) exhibits various pharmacological activities, including immunoregulatory, anti-inflammatory, antiviral, and antioxidant properties. However, the acts of lncRNAs in regulating the therapeutic effects of SSP on PCV2-infected RAW264.7 cells remains poorly understood. This study aimed to investigate the molecular mechanisms by which lncRNAs regulate PCV2-induced immunosuppression during SSP treatment. Our findings revealed that 1699 mRNAs, 373 lncRNAs, and 129 miRNAs were differentially expressed in PCV2-infected RAW264.7 cells. Additionally, 359 mRNAs, 271 lncRNAs, and 79 miRNAs exhibited differential expression in SSP-treated PCV2-infected RAW264.7 cells. GO and KEGG analyses indicated that the candidate genes were enriched in the TNF/NF-κB signaling pathway. Furthermore, based on GO and KEGG pathway analysis, a ceRNA network involving chemokine (C-X-C motif) ligand 2 (CXCL2), miR-217-x, and MSTRG.5823.1 was constructed. We demonstrated that lncRNA MSTRG.5823.1 localized to the cytoplasm. Moreover, we found that silencing or overexpressing lncRNA MSTRG.5823.1 significantly modulated PCV2-induced immunosuppression by regulating the activation of the TNF/NF-κB signaling pathway. Specifically, lncRNA MSTRG.5823.1 overexpression increased the expression of TNF/NF-κB signaling pathway-related genes and proteins in PCV2-infected RAW264.7 cells. Conversely, silencing lncRNA MSTRG.5823.1 decreased their expression. Rescue assays further revealed that the suppressive effects of miR-217-x overexpression on TNF/NF-κB signaling pathway-related genes and proteins could be reversed by MSTRG.5823.1 overexpression. These findings highlight the critical role of lncRNA MSTRG.5823.1 in PCV2 infection progression and suggest a new strategy for the prevention and treatment of PCV2 infection.


Assuntos
Infecções por Circoviridae , Circovirus , NF-kappa B , Polissacarídeos , RNA Longo não Codificante , Transdução de Sinais , Sophora , Animais , Camundongos , Circovirus/imunologia , NF-kappa B/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos , Infecções por Circoviridae/imunologia , Polissacarídeos/farmacologia , Suínos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Tolerância Imunológica/efeitos dos fármacos
5.
Sci Rep ; 14(1): 13815, 2024 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-38877168

RESUMO

This study was aimed to investigate the frequency of PiCV recombination, the kinetics of PiCV viremia and shedding and the correlation between viral replication and host immune response in young pigeons subclinically infected with various PiCV variants and kept under conditions mimicking the OLR system. Fifteen racing pigeons originating from five breeding facilities were housed together for six weeks. Blood and cloacal swab samples were collected from birds every seven days to recover complete PiCV genomes and determine PiCV genetic diversity and recombination dynamics, as well as to assess virus shedding rate, level of viremia, expression of selected genes and level of anti-PiCV antibodies. Three hundred and eighty-eight complete PiCV genomes were obtained and thirteen genotypes were distinguished. Twenty-five recombination events were detected. Recombinants emerged during the first three weeks of the experiment which was consistent with the peak level of viremia and viral shedding. A further decrease in viremia and shedding partially corresponded with IFN-γ and MX1 gene expression and antibody dynamics. Considering the role of OLR pigeon rearing system in spreading infectious agents and allowing their recombination, it would be reasonable to reflect on the relevance of pigeon racing from both an animal welfare and epidemiological perspective.


Assuntos
Doenças das Aves , Infecções por Circoviridae , Circovirus , Columbidae , Eliminação de Partículas Virais , Animais , Columbidae/virologia , Circovirus/genética , Circovirus/imunologia , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Infecções por Circoviridae/epidemiologia , Infecções por Circoviridae/imunologia , Doenças das Aves/virologia , Doenças das Aves/epidemiologia , Doenças das Aves/imunologia , Viremia/epidemiologia , Viremia/virologia , Viremia/imunologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Genoma Viral , Recombinação Genética , Genótipo , Replicação Viral , Filogenia
6.
Vet Microbiol ; 295: 110151, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38870752

RESUMO

Porcine circovirus type 2 (PCV2) stands as a predominant etiological agent in porcine circovirus-associated diseases. To manage the spread of the disease, it is necessary to develop a next-generation vaccine expressing PCV2 antigens that target the prevailing genotype such as PCV2d. A bacterial-mediated vaccine delivery by live-attenuated Salmonella has attracted interest for its low-cost production and highly effective vaccine delivery. Thus, in this study, we utilized the advantages of the Salmonella-mediated vaccine delivery by cloning PCV2d cap and rep into a eukaryotic expression plasmid pJHL204 and electroporation into an engineered live-attenuated Salmonella Typhimurium JOL2500 (Δlon, ΔcpxR, ΔsifA, Δasd). The eukaryotic antigen expression by JOL2995 (p204:cap) and JOL2996 (p204:rep) was confirmed in vitro and in vivo which showed efficient antigen delivery. Furthermore, vaccination of mice model with the vaccine candidates elicited humoral and cell-mediated immune responses as depicted by high levels of PCV2-specific antibodies, CD4+ and CD8+ T cells, and neutralizing antibodies, especially by JOL2995 (p204:cap) which correlated with the significant decrease in the viral load in PCV2d-challenged mice. Interestingly, JOL2996 (p204:rep) may not have elicited high levels of neutralizing antibodies and protective efficacy, but it elicited considerably higher cell-mediated immune responses. This study demonstrated Salmonella-mediated vaccine delivery system coupled with the eukaryotic expression vector can efficiently deliver and express the target PCV2d antigens for strong induction of immune response and protective efficacy in mice model, further supporting the potential application of the Salmonella-mediated vaccine delivery system as an effective novel approach in vaccine strategies for PCV2d.


Assuntos
Infecções por Circoviridae , Circovirus , Vetores Genéticos , Salmonella typhimurium , Vacinas Virais , Animais , Circovirus/imunologia , Circovirus/genética , Camundongos , Salmonella typhimurium/imunologia , Salmonella typhimurium/genética , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/imunologia , Suínos , Antígenos Virais/imunologia , Antígenos Virais/genética , Camundongos Endogâmicos BALB C , Anticorpos Antivirais/sangue , Feminino , Anticorpos Neutralizantes/sangue , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia
7.
Microbiol Spectr ; 12(8): e0087024, 2024 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-38916319

RESUMO

Since Porcine Circovirus 3 (PCV3) was first identified in 2016, our understanding of the humoral response is still relatively scarce. Current knowledge of the PCV3 humoral response is primarily based on field studies identifying the seroprevalence of PCV3 Cap-induced antibodies. Studies on the humoral response following experimental PCV3 infection have conflicting results where one study reports the development of the Cap IgG response 7 days postinfection with no concurrent Cap IgM response, while a second study shows a Cap IgM response at the same time point with no detection of Cap IgG. The dynamics of the PCV3 Cap and Rep IgG following maternal antibody transfer and experimental infection have not been well characterized. Additionally, the cross-reactivity of convalescent serum from PCV2 and PCV3 experimentally infected animals to serologic methods of the alternate PCV has limited evaluation. Here, we show that maternally derived antibodies were detectable in piglet serum 7-9 weeks postfarrowing for the Cap IgG and 5-weeks-post farrowing for the Rep IgG using Cap- and Rep-specific enzyme linked immunosorbent assays (ELISA) and immunofluorescent assays (IFA) methods. Following experimental inoculation, Cap IgG was detected at 2-weeks-post inoculation and Rep IgG detection was delayed until 4-weeks-post inoculation. Furthermore, convalescent serum from either PCV2 or PCV3 methods displayed no cross-reactivity by serological methods against the other PCV. The information gained in this study highlights the development of both the Cap- and Rep-specific antibodies following experimental infection and through the transfer of maternal antibodies. The increased understanding of the dynamics of maternal antibody transfer and development of the humoral response following infection gained in the present study may aid in the establishment of husbandry practices and potential application of prophylactics to control PCV3 clinical disease. IMPORTANCE: Research on Porcine Circovirus 3 (PCV3) immunology is vital for understanding and controlling this virus. Previous studies primarily relied on field observations, but they have shown conflicting results about the immunological response against PCV3. This study helps fill those gaps by looking at how antibodies develop in pigs, especially those maternal-derived, and their impact in neonatal pigs preventing PCV3-associated disease in piglets. In addition, we look at the dynamics of antibodies in experimental infections mimicking infection in pigs in the grower-phase condition. Understanding this process can help to develop better strategies to prevent PCV3 infection. Also, this research found that PCV2 and PCV3 do not cross-react, which is crucial for serological test development and results interpretation. Overall, this work is essential for improving swine health and farming practices in the face of PCV3 infections.


Assuntos
Anticorpos Antivirais , Infecções por Circoviridae , Circovirus , Imunidade Humoral , Imunidade Materno-Adquirida , Imunoglobulina G , Doenças dos Suínos , Animais , Circovirus/imunologia , Suínos , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Doenças dos Suínos/virologia , Doenças dos Suínos/imunologia , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Feminino , Imunoglobulina M/sangue , Imunoglobulina M/imunologia , Ensaio de Imunoadsorção Enzimática , Reações Cruzadas/imunologia
8.
mSphere ; 9(7): e0022524, 2024 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-38926905

RESUMO

Porcine circovirus type 4 (PCV4), a recently identified circovirus, is prevalent in numerous provinces in China, as well as in South Korea, Thailand, and Europe. PCV4 virus rescued from an infectious clone showed pathogenicity, suggesting the economic impact of PCV4. However, there remains a lack of understanding regarding the immunogenicity and epitopes of PCV4. This study generated a monoclonal antibody (MAb) 1D8 by immunizing mice with PCV4 virus-like particles (VLPs). Subsequently, the epitope recognized by the MAb 1D8 was identified by truncated protein expression and alanine scanning mutagenesis analysis. Results showed that the 225PKQG228 located at the C-terminus of the PCV4 Cap protein is the minimal motif binding to the MAb. Homology modeling analysis and immunoelectron microscopy revealed that the epitope extends beyond the outer surface of the PCV4 VLP. Moreover, the epitope is highly conserved among PCV4 strains and does not react with other PCVs. Together, the MAb 1D8 recognized epitope shows potential for detecting PCV4. These findings significantly contribute to the design of antigens for PCV4 detection and control strategies. IMPORTANCE: Porcine circovirus type 4 (PCV4) is a novel circovirus. Although PCV4 has been identified in several countries, including China, Korea, Thailand, and Spain, no vaccine is available. Given the potential pathogenic effects of PCV4 on pigs, PCV4 could threaten the global pig farming industry, highlighting the urgency for further investigation. Thus, epitopes of PCV4 remain to be determined. Our finding of a conserved epitope significantly advances vaccine development and pathogen detection.


Assuntos
Anticorpos Monoclonais , Anticorpos Antivirais , Proteínas do Capsídeo , Circovirus , Epitopos de Linfócito B , Circovirus/imunologia , Circovirus/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/química , Animais , Anticorpos Monoclonais/imunologia , Camundongos , Suínos , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito B/genética , Anticorpos Antivirais/imunologia , Camundongos Endogâmicos BALB C , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Doenças dos Suínos/virologia , Doenças dos Suínos/imunologia , Feminino
9.
Vet Microbiol ; 293: 110088, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38640639

RESUMO

Orf virus (ORFV), a member of the genus Parapoxvirus, possesses an excellent immune activation capability, which makes it a promising immunomodulation agent. In this study, we evaluated ORFV as a novel adjuvant to enhance the immune response of mice to a subunit vaccine using porcine circovirus type 2 (PCV2) capsid (Cap) protein as a model. Our results showed that both inactivated and live attenuated ORFV activated mouse bone marrow-derived dendritic cells and increased expression of immune-related cytokines interleukin (IL)-1ß, IL-6, and TNF-α. Enhanced humoral and cellular immune responses were induced in mice immunized with PCV2 Cap protein combined with inactivated or live attenuated ORFV adjuvant compared with the aluminum adjuvant. Increased secretion of Th1 and Th2 cytokines by splenic lymphocytes in immunized mice further indicated that the ORFV adjuvant promoted a mixed Th1/Th2 immune response. Moreover, addition of the ORFV adjuvant to the PCV2 subunit vaccine significantly reduced the viral load in the spleen and lungs of PCV2-challenged mice and prevented pathological changes in lungs. This study demonstrates that ORFV enhances the immunogenicity of a PCV2 subunit vaccine by improving the adaptive immune response, suggesting the potential application of ORFV as a novel adjuvant.


Assuntos
Adjuvantes Imunológicos , Infecções por Circoviridae , Circovirus , Citocinas , Vírus do Orf , Vacinas de Subunidades Antigênicas , Vacinas Virais , Animais , Circovirus/imunologia , Camundongos , Vacinas de Subunidades Antigênicas/imunologia , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas Virais/imunologia , Vacinas Virais/administração & dosagem , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/veterinária , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Adjuvantes Imunológicos/administração & dosagem , Citocinas/imunologia , Vírus do Orf/imunologia , Proteínas do Capsídeo/imunologia , Feminino , Imunidade Celular , Células Dendríticas/imunologia , Carga Viral , Anticorpos Antivirais/sangue , Imunidade Humoral , Suínos , Adjuvantes de Vacinas , Camundongos Endogâmicos BALB C , Células Th1/imunologia
10.
Viruses ; 16(4)2024 04 22.
Artigo em Inglês | MEDLINE | ID: mdl-38675986

RESUMO

Porcine circovirus type 2 (PCV2) infection can cause immunosuppressive diseases in pigs. Vascular endothelial cells (VECs), as the target cells for PCV2, play an important role in the immune response and inflammatory regulation. Endothelial IL-8, which is produced by porcine hip artery endothelial cells (PIECs) infected with PCV2, can inhibit the maturation of monocyte-derived dendritic cells (MoDCs). Here, we established a co-culture system of MoDCs and different groups of PIECs to further investigate the PCV2-induced endothelial IL-8 signaling pathway that drives the inhibition of MoDC maturation. The differentially expressed genes related to MoDC maturation were mainly enriched in the NF-κB and JAK2-STAT3 signaling pathways. Both the NF-κB related factor RELA and JAK2-STAT3 signaling pathway related factors (IL2RA, JAK, STAT2, STAT5, IL23A, IL7, etc.) decreased significantly in the IL-8 up-regulated group, and increased significantly in the down-regulated group. The expression of NF-κB p65 in the IL-8 up-regulated group was reduced significantly, and the expression of IκBα was increased significantly. Nuclear translocation of NF-κB p65 was inhibited, while the nuclear translocation of p-STAT3 was increased in MoDCs in the PCV2-induced endothelial IL-8 group. The results of treatment with NF-κB signaling pathway inhibitors showed that the maturation of MoDCs was inhibited and the expression of IL-12 and GM-CSF at mRNA level were lower. Inhibition of the JAK2-STAT3 signaling pathway had no significant effect on maturation, and the expression of IL-12 and GM-CSF at mRNA level produced no significant change. In summary, the NF-κB signaling pathway is the main signaling pathway of MoDC maturation, and is inhibited by the PCV2-induced up-regulation of endothelial-derived IL-8.


Assuntos
Circovirus , Interleucina-8 , Transdução de Sinais , Doenças dos Suínos , Animais , Diferenciação Celular , Células Cultivadas , Infecções por Circoviridae/virologia , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Circovirus/imunologia , Técnicas de Cocultura , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Células Endoteliais/virologia , Células Endoteliais/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , NF-kappa B/metabolismo , Suínos , Doenças dos Suínos/virologia , Doenças dos Suínos/imunologia , Doenças dos Suínos/metabolismo
11.
Microb Pathog ; 190: 106630, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38556102

RESUMO

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.


Assuntos
Anticorpos Antivirais , Proteínas do Capsídeo , Circovirus , Escherichia coli , Proteínas Recombinantes , Vacinas de Partículas Semelhantes a Vírus , Animais , Circovirus/imunologia , Circovirus/genética , Suínos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/genética , Proteínas do Capsídeo/imunologia , Proteínas do Capsídeo/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Camundongos , Anticorpos Antivirais/imunologia , Anticorpos Antivirais/sangue , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/genética , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Vacinas Virais/genética , Desenvolvimento de Vacinas , Antígenos Virais/imunologia , Antígenos Virais/genética , Imunoglobulina G/sangue , Análise Custo-Benefício , Feminino , Interferon gama/metabolismo , Imunogenicidade da Vacina
12.
J Virol ; 96(13): e0014322, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35658531

RESUMO

Differentiation of infected from vaccinated hosts (DIVH) is a critical step in virus eradication programs. DIVH-compatible vaccines, however, take years to develop, and are therefore unavailable for fighting the sudden outbreaks that typically drive pandemics. Here, we establish a protocol for the swift and efficient development of DIVH assays, and show that this approach is compatible with any type of vaccines. Using porcine circovirus 2 (PCV2) as the experimental model, the first step is to use Immunoglobin G (IgG) sero-dynamics (IsD) curves to aid epitope discovery (IsDAED): PCV2 Cap peptides were categorized into three types: null interaction, nonspecific interaction (NSI), and specific interaction (SI). We subsequently compared IsDAED approach and traditional approach, and demonstrated identifying SI peptides and excluding NSI peptides supports efficient diagnostic kit development, specifically using a protein-peptide hybrid microarray (PPHM). IsDAED directed the design of a DIVH protocol for three types of PCV2 vaccines (while using a single PPHM). Finally, the DIVH protocol successfully differentiated infected pigs from vaccinated pigs at five farms. This IsDAED approach is almost certainly extendable to other viruses and host species. IMPORTANCE Sudden outbreaks of pandemics caused by virus, such as SARS-CoV-2, has been determined as a public health emergency of international concern. However, the development of a DIVH-compatible vaccine is time-consuming and full of uncertainty, which is unsuitable for an emergent situation like the ongoing COVID-19 pandemic. Along with the development and public health implementation of new vaccines to prevent human diseases, e.g., human papillomavirus vaccines for cervical cancer; enterovirus 71 vaccines for hand, foot, and mouth disease; and most recently SARS-CoV-2, there is an increasing demand for DIVH. Here, we use the IsDAED approach to confirm SI peptides and to exclude NSI peptides, finally to direct the design of a DIVH protocol. It is plausible that our IsDAED approach is applicable for other infectious disease.


Assuntos
Anticorpos Antivirais , Infecções por Circoviridae , Epitopos , Imunoglobulina G , Vacinas Virais , Animais , Anticorpos Antivirais/sangue , COVID-19 , Infecções por Circoviridae/imunologia , Circovirus , Modelos Animais de Doenças , Epitopos/análise , Epitopos/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Peptídeos , SARS-CoV-2 , Suínos , Doenças dos Suínos/imunologia , Vacinas Virais/imunologia
13.
Viruses ; 14(2)2022 01 20.
Artigo em Inglês | MEDLINE | ID: mdl-35215787

RESUMO

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus (PCVs) are two major viruses that affect pigs. Coinfections between PRRSV and PCV2 are frequently reported in most outbreaks, with clinical presentations involving dyspnea, fever, reduced feed intake, weight loss, and death in fattening pigs. The NADC30-like PRRSV and PCV2d are the main circulating virus strains found in China. This study determines the impact of NADC30-like PRRSV and PCV2d mono-infection and coinfection on the immune system, organ pathology, and viral shedding in five-week-old post-weaned pigs. Pigs were randomly divided into six groups: PBS, PRRSV, PCV2, PRRSV-PCV2 coinfection (co), and PRRSV-PCV2 or PCV2-PRRSV sequential infections. Fever, dyspnea, decreased feed intake, weight loss, and pig deaths occurred in groups infected with PRRSV, Co-PRRSV-PCV2, and PRRSV-PCV2. The viral load was higher in Co-PRRSV-PCV2, PRRSV-PCV2, and PCV2-PRRSV than those mono-infected with PRRSV or PCV2. Additionally, cytokines (IFN-γ, TNF-α, IL-4, and IL-10) produced by pigs under Co-PRRSV-PCV2 and PRRSV-PCV2 groups were more intense than the other groups. Necropsy findings showed hemorrhage, emphysema, and pulmonary adhesions in the lungs of pigs infected with PRRSV. Smaller alveoli and widened lung interstitium were found in the Co-PRRSV-PCV2 and PRRSV-PCV2 groups. In conclusion, PRRSV and PCV2 coinfection and sequential infection significantly increased viral pathogenicity and cytokine responses, resulting in severe clinical signs, lung pathology, and death.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Circovirus/patogenicidade , Coinfecção/virologia , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/fisiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/patogenicidade , Animais , China , Infecções por Circoviridae/genética , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/virologia , Circovirus/genética , Coinfecção/genética , Coinfecção/imunologia , Coinfecção/mortalidade , Feminino , Interleucina-10/genética , Interleucina-10/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Pulmão/imunologia , Pulmão/virologia , Masculino , Síndrome Respiratória e Reprodutiva Suína/genética , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/mortalidade , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Suínos , Virulência
14.
Viruses ; 13(9)2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34578257

RESUMO

Porcine circovirus type 2 (PCV2), the causative agent of a wasting disease in weanling piglets, has periodically evolved into several new subtypes since its discovery, indicating that the efficacy of current vaccines can be improved. Although a DNA virus, the mutation rates of PCV2 resemble RNA viruses. The hypothesis that recoding of selected serine and leucine codons in the PCV2b capsid gene could result in stop codons due to mutations occurring during viral replication and thus result in rapid attenuation was tested. Vaccination of weanling pigs with the suicidal vaccine constructs elicited strong virus-neutralizing antibody responses. Vaccination prevented lesions, body-weight loss, and viral replication on challenge with a heterologous PCV2d strain. The suicidal PCV2 vaccine construct was not detectable in the sera of vaccinated pigs at 14 days post-vaccination, indicating that the attenuated vaccine was very safe. Exposure of the modified virus to immune selection pressure with sub-neutralizing levels of antibodies resulted in 5 of the 22 target codons mutating to a stop signal. Thus, the described approach for the rapid attenuation of PCV2 was both effective and safe. It can be readily adapted to newly emerging viruses with high mutation rates to meet the current need for improved platforms for rapid-response vaccines.


Assuntos
Anticorpos Antivirais/sangue , Circovirus/genética , Circovirus/fisiologia , Vacinas Virais/imunologia , Replicação Viral/genética , Animais , Anticorpos Neutralizantes/imunologia , Proteínas do Capsídeo/genética , Infecções por Circoviridae/imunologia , Circovirus/classificação , DNA Viral/sangue , Imunidade Celular , Suínos , Doenças dos Suínos/virologia , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Virais/administração & dosagem , Replicação Viral/imunologia
15.
PLoS Pathog ; 17(9): e1009940, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34543359

RESUMO

Viruses use diverse strategies to impair the antiviral immunity of host in order to promote infection and pathogenesis. Herein, we found that PCV2 infection promotes the infection of DNA viruses through inhibiting IFN-ß induction in vivo and in vitro. In the early phase of infection, PCV2 promotes the phosphorylation of cGAS at S278 via activation of PI3K/Akt signaling, which directly silences the catalytic activity of cGAS. Subsequently, phosphorylation of cGAS at S278 can facilitate the K48-linked poly-ubiquitination of cGAS at K389, which can been served as a signal for recognizing by the ubiquitin-binding domain of histone deacetylase 6 (HDAC6), to promote the translocation of K48-ubiquitinated-cGAS from cytosol to autolysosome depending on the deacetylase activity of HDAC6, thereby eventually resulting in a markedly increased cGAS degradation in PCV2 infection-induced autophagic cells relative to Earle's Balanced Salt Solution (EBSS)-induced autophagic cells (a typical starving autophagy). Importantly, we found that PCV2 Cap and its binding protein gC1qR act as predominant regulators to promote porcine cGAS phosphorylation and HDAC6 activation through mediating PI3K/AKT signaling and PKCδ signaling activation. Based on this finding, gC1qR-binding activity deficient PCV2 mutant (PCV2RmA) indeed shows a weakened inhibitory effect on IFN-ß induction and a weaker boost effect for other DNA viruses infection compared to wild-type PCV2. Collectively, our findings illuminate a systematic regulation mechanism by which porcine circovirus counteracts the cGAS-STING signaling pathway to inhibit the type I interferon induction and promote DNA virus infection, and identify gC1qR as an important regulator for the immunosuppression induced by PCV2.


Assuntos
Infecções por Circoviridae/metabolismo , Circovirus/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Interferon Tipo I/metabolismo , Nucleotidiltransferases/metabolismo , Animais , Infecções por Circoviridae/imunologia , Circovirus/imunologia , Infecções por Vírus de DNA/imunologia , Infecções por Vírus de DNA/metabolismo , Células HEK293 , Humanos , Interferon Tipo I/imunologia , Nucleotidiltransferases/imunologia , Suínos , Doenças dos Suínos/virologia
16.
Front Immunol ; 12: 688294, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34394082

RESUMO

Polarization of macrophages to different functional states is important for mounting responses against pathogen infections. Macrophages are the major target cells of porcine circovirus type 2 (PCV2), which is the primary causative agent of porcine circovirus-associated disease (PCVAD) leading to immense economic losses in the global swine industry. Clinically, PCV2 is often found to increase risk of other pathogenic infections yet the underlying mechanisms remain to be elusive. Here we found that PCV2 infection skewed macrophages toward a M1 status through reprogramming expression of a subset of M1-associated genes and M2-associated genes. Mechanistically, induction of M1-associated genes by PCV2 infection is dependent on activation of nuclear factor kappa B (NF-κB) and c-jun N-terminal kinase (JNK) signaling pathways whereas suppression of M2-associated genes by PCV2 is via inhibiting expression of jumonji domain containing-3 (JMJD3), a histone 3 Lys27 (H3K27) demethylase that regulates M2 activation of macrophages. Finally, we identified that PCV2 capsid protein (Cap) directly inhibits JMJD3 transcription to restrain expression of interferon regulatory factor (IRF4) that controls M2 macrophage polarization. Consequently, sustained infection of PCV2 facilitates bacterial infection in vitro. In summary, these findings showed that PCV2 infection functionally modulated M1 macrophage polarization via targeting canonical signals and epigenetic histone modification, which contributes to bacterial coinfection and virial pathogenesis.


Assuntos
Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/patogenicidade , Infecções por Circoviridae/virologia , Circovirus/patogenicidade , Coinfecção , Macrófagos/microbiologia , Macrófagos/virologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/patogenicidade , Infecções por Actinobacillus/imunologia , Infecções por Actinobacillus/metabolismo , Actinobacillus pleuropneumoniae/imunologia , Animais , Células Cultivadas , Montagem e Desmontagem da Cromatina , Infecções por Circoviridae/imunologia , Circovirus/imunologia , Modelos Animais de Doenças , Epigênese Genética , Interações Hospedeiro-Patógeno , Fatores Reguladores de Interferon/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , NF-kappa B/metabolismo , Fenótipo , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Transdução de Sinais
17.
Dis Markers ; 2021: 9434944, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34257749

RESUMO

The clinical outcome of porcine circovirus 3 (PCV3) infection is still controversial. Herein, a novel PCV3 isolate (PCV3-China/DB-1/2017) with the molecular characterization of 24A and 27K in the Cap protein was used to inoculate three-week-old cesarean-derived, colostrum-deprived piglets. The nine PCV3 DB-1 inoculated piglets exhibited no obvious clinical symptoms or macroscopic lesions. PCV3 displayed a broad histotropism, including the heart, liver, spleen, lung, kidney, brain, lymph nodes, and tonsil, and the lungs and lymph nodes contained a higher quantity of viral genomes compared to that of the other organs. From 7 days after PCV3 DB-1 inoculation, the piglets showed obvious IgG antibody responses against PCV3 rCap-VLPs. The cumulative results demonstrated that PCV3 trend to low pathogenicity.


Assuntos
Infecções por Circoviridae/veterinária , Infecções por Circoviridae/virologia , Circovirus/patogenicidade , Doenças dos Suínos/virologia , Animais , Anticorpos Antivirais/metabolismo , Infecções Assintomáticas , Biomarcadores/metabolismo , China , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/patologia , Circovirus/genética , Circovirus/imunologia , Circovirus/isolamento & purificação , Genoma Viral , Distribuição Aleatória , Sus scrofa , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/patologia , Carga Viral , Proteínas Virais/genética , Proteínas Virais/imunologia
18.
Dev Comp Immunol ; 122: 104112, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33971216

RESUMO

Chicken Infectious Anaemia (CIA) Virus (CAV) inhibits the function of multiple immune compartments. Mortality due to clinical infection is controlled in broilers by passive immunization derived from vaccinated breeders. Therefore, serological tests are often used in chicks to determine maternally-derived antibodies (MDA). We used a vaccine overdose-induced model of CIA. The model replicated the most common features of the disease. This model was used to determine the role of MDA in the protection of chicks. Hatchlings were tested for anti-CAV titers by ELISA and were sorted into groups based on antibody levels. SPF chicks were used as a no-antibody control. Lower specific antibody levels seemed to facilitate viral entry into the thymus, but viral levels, CD4+ and CD8+ counts, thymus architecture, and haematocrit were preserved by MDA, regardless of its levels. Levels of MDA are not correlated with protection from CIA, but are important for the progression CAV infection.


Assuntos
Anticorpos Antivirais/sangue , Vírus da Anemia da Galinha/imunologia , Galinhas/imunologia , Infecções por Circoviridae/imunologia , Imunidade Materno-Adquirida/imunologia , Vacinas Virais/imunologia , Animais , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Circoviridae/veterinária , Ensaio de Imunoadsorção Enzimática , Feminino , Hematócrito , Imunização Passiva , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Gravidez , Timo/virologia , Vacinação/veterinária , Vacinas Atenuadas/imunologia
19.
Viruses ; 13(4)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916308

RESUMO

Mink refractory diarrhea is a seasonal disease that occurs in many mink farms in China. Mink circovirus (MiCV) has been recognized as the causative agent of the disease. The aim of the study was to develop a subunit vaccine against mink refractory diarrhea. A recombinant baculovirus strain expressing the capsid protein was constructed using the baculovirus expression vector system (BEVS). A subunit vaccine was developed based on the capsid protein with appropriate adjuvant. Then, a field trial was carried out in two districts in order to evaluate the efficiency of the subunit vaccine. The field trial indicated that in total, only 1.8% of the minks developed typical diarrhea in the vaccinated group compared with 74.5% in the control group. The vaccination could significantly reduce the infection rate of MiCV among the mink herds and could restrain the virus' shedding from feces. Furthermore, the vaccinated group had a higher average litter size in the following year compared to the control group. Collectively, the results indicated that the subunit vaccine based on the capsid protein can provide reliable protection against MiCV infection.


Assuntos
Anticorpos Antivirais/sangue , Baculoviridae/genética , Proteínas do Capsídeo/genética , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Diarreia/prevenção & controle , Vison/virologia , Vacinas Virais/imunologia , Animais , Capsídeo/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/imunologia , China , Infecções por Circoviridae/imunologia , Circovirus/genética , Diarreia/virologia , Feminino , Masculino , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Virais/administração & dosagem
20.
J Nanobiotechnology ; 19(1): 34, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526021

RESUMO

BACKGROUND: The widespread popularity of porcine circovirus type 2(PCV2) has seriously affected the healthy development of the pig industry and caused huge economic losses worldwide. A rapid and reliable method is required for epidemiological investigation and evaluating the effect of immunization. However, the current methods for PCV2 antibody detection are time-consuming or very expensive and rarely meet the requirements for clinical application. we have constructed the platform for expressing the nanobody(Nb)­horseradish peroxidase(HRP) fusion protein as an ultrasensitive probe to detect antibodies against the Newcastle disease virus(NDV), previously. In the present work, an Nb-HRP fusion protein-based competitive ELISA(cELISA) for rapid and simple detection antibodies against PCV2 was developed using this platform to detect anti-PCV2 antibodies in clinical porcine serum. RESULTS: Using phage display technology, 19 anti-PCV2-Cap protein nanobodies were screened from a PCV2-Cap protein immunized Bactrian camel. With the platform, the PCV2-Nb15­HRP fusion protein was then produced and used as a sensitive reagent for developing a cELISA to detect anti­PCV2 antibodies. The cut­off value of the cELISA is 20.72 %. Three hundreds and sixty porcine serum samples were tested by both newly developed cELISA and commercial kits. The sensitivity and specificity were 99.68 % and 95.92 %, respectively. The coincidence rate of the two methods was 99.17 %. When detecting 620 clinical porcine serum samples, a good consistent (kappa value = 0.954) was found between the results of the cELISA and those of commercial kits. CONCLUSIONS: In brief, the newly developed cELISA based PCV2-Nb15­HRP fusion protein is a rapid, low-cost, reliable and useful nanobody-based tool for the serological evaluation of current PCV2 vaccine efficacy and the indirect diagnosis of PCV2 infection.


Assuntos
Anticorpos Antivirais/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Doenças dos Suínos/imunologia , Animais , Anticorpos Antivirais/sangue , Camelus/imunologia , Infecções por Circoviridae/sangue , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/imunologia , Circovirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/economia , Peroxidase do Rábano Silvestre/imunologia , Imunização , Masculino , Proteínas Recombinantes de Fusão/imunologia , Sensibilidade e Especificidade , Anticorpos de Domínio Único/imunologia , Suínos/sangue , Suínos/imunologia , Suínos/virologia , Doenças dos Suínos/sangue , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/virologia , Fatores de Tempo
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