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1.
J Appl Microbiol ; 128(1): 74-87, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31574195

RESUMO

AIMS: Porcine circovirus type 2 (PCV2) can cause postweaning, multisystemic wasting syndrome in pigs, which leads to enormous losses in the swine industry worldwide. Here, a genetically engineered Lactococcus strain expressing the main protective antigen of PCV2, the Cap protein, was developed to act against PCV2 infection as an oral vaccine. METHODS AND RESULTS: Expression of the Cap protein was confirmed via western blot, ELISA and fluorescence microscopy. Over 90% of the recombinant pAMJ399-Cap/MG1363 survived a simulated gastrointestinal transit. It also survived the murine intestinal tract for at least 11 days. Then, the safety and immunogenicity of pAMJ399-Cap/MG1363 in orally immunized mice was evaluated. The levels of the sIgA, IgG and cytokines (IL-4 and IFN-γ) obtained from the mice immunized with pAMJ399-Cap/MG1363 were significantly higher than those in the control groups. CONCLUSIONS: pAMJ399-Cap/MG1363 can survive in the gastrointestinal transit and effectively induce mucosal, cellular and humoral immune response against PCV2 infection via oral administration. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the potential of the genetically engineered Lactococcus lactis as a candidate for an oral vaccine against PCV2.


Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/imunologia , Lactococcus lactis/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Proteínas do Capsídeo/genética , Citocinas/sangue , Imunogenicidade da Vacina , Lactococcus lactis/genética , Camundongos , Vacinação , Vacinas Virais/administração & dosagem
2.
Int J Nanomedicine ; 14: 7533-7548, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571862

RESUMO

Background: The influenza A virus (IAV) is known for its high variability and poses a huge threat to the health of humans and animals. Pigs play a central role in the cross-species reassortment of IAV. Ectodomain of matrix protein 2 (M2e) is the most conserved protective antigen in IAV and can be used to develop nanovaccines through nanoparticles displaying to increase its immunogenicity. However, the high immunogenicity of nanoparticles can cause the risk of off-target immune response, and excess unwanted antibodies may interfere with the protective efficacy of M2e-specific antibodies. Therefore, it is necessary to select reasonable nanoparticles to make full use of antibodies against nanoparticles while increasing the level of M2e-specific antibodies. Porcine circovirus type 2 (PCV2) is the most susceptible virus in pigs and can promote IAV infection. It is meaningful to develop a vaccine that can simultaneously control swine influenza virus (SIV) and PCV2. Methods: In the present study, M2e of different copy numbers were inserted into the capsid (Cap) protein of PCV2 and expressed in Escherichia coli to form self-assembled chimeric virus-like particles (VLPs) nanovaccine. BALB/c mice and pigs were immunized with these nanovaccines to explore optimal anti-IAV and anti-PCV2 immunity. Results: Cap is capable of carrying at least 81 amino acid residues (three copies of M2e) at its C-terminal without impairing VLPs formation. Cap-3M2e VLPs induced the highest levels of M2e-specific immune responses, conferring protection against lethal challenge of IAVs from different species and induced specific immune responses consistent with PCV2 commercial vaccines in mice. In addition, Cap-3M2e VLPs induced high levels of M2e-specific antibodies and PCV2-specific neutralizing antibodies in pigs. Conclusion: Cap-3M2e VLP is an economical and promising bivalent nanovaccine, which provides dual protection against IAV and PCV2.


Assuntos
Circovirus/imunologia , Vírus da Influenza A/imunologia , Nanopartículas/uso terapêutico , Infecções por Orthomyxoviridae/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos/imunologia , Especificidade de Anticorpos/imunologia , Aves/virologia , Proteínas do Capsídeo/química , Proliferação de Células , Citocinas/metabolismo , Cães , Feminino , Humanos , Imunidade Humoral , Influenza Aviária/imunologia , Influenza Aviária/prevenção & controle , Influenza Aviária/virologia , Influenza Humana/imunologia , Influenza Humana/prevenção & controle , Influenza Humana/virologia , Linfócitos/citologia , Células Madin Darby de Rim Canino , Camundongos Endogâmicos BALB C , Testes de Neutralização , Proteínas Recombinantes/isolamento & purificação , Suínos , Vírion/imunologia , Vírion/ultraestrutura
3.
BMC Vet Res ; 15(1): 342, 2019 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-31619295

RESUMO

BACKGROUND: The objective of this study was to assess the efficacy of a trivalent vaccine mixture and compare it to the respective monovalent vaccines against Mycoplasma hyopneumoniae, porcine circovirus type 2 (PCV2), and porcine reproductive and respiratory syndrome virus (PRRSV). RESULTS: Pigs that were triple challenged with M. hyopneumoniae, PCV2, and PRRSV following vaccination with the trivalent vaccine mixture exhibited a significantly better growth performance when compared to unvaccinated and challenged pigs. A statistical difference was not found when comparing pig populations which were vaccinated with the trivalent vaccine followed by a triple challenge and pigs vaccinated with monovalent M hyopneumoniae vaccine followed by mycoplasmal single challenge in the following areas: M. hyopneumoniae nasal shedding, the number of M. hyopneumoniae-specific interferon-γ secreting cells (IFN-γ-SC), and mycoplasmal lung lesion scores. Pigs vaccinated with the trivalent vaccine mixture followed by a triple challenge resulted in a similar reduction of PCV2 viremia, an increase in the number of PCV2-specific IFN-γ-SC and reduction in interstitial lung lesion scores when compared to pigs vaccinated with a PCV-2 vaccine and challenged with PCV2 only. Lastly, there was a significant difference in the reduction of PRRSV viremia, an increase in PRRSV-specific IFN-γ-SC and a reduction of interstitial lung lesion scores between pigs vaccinated with the trivalent vaccine mixture followed by a triple challenge and pigs vaccinated with a monovalent PRRSV vaccine followed by PRRSV challenge only. CONCLUSION: The trivalent vaccine mixture was efficacious against a triple challenge of M. hyopneumoniae, PCV2, and PRRSV. The trivalent vaccine mixture, however, did not result in equal protection when compared against each respective monovalent vaccine, with the largest vaccine occurring within PRRSV.


Assuntos
Circovirus/imunologia , Mycoplasma hyopneumoniae/imunologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Doenças dos Suínos/prevenção & controle , Vacinação/veterinária , Animais , Vacinas Bacterianas/imunologia , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Feminino , Masculino , Pneumonia Suína Micoplasmática/imunologia , Pneumonia Suína Micoplasmática/prevenção & controle , Síndrome Respiratória e Reprodutiva Suína/imunologia , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Sus scrofa , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/microbiologia , Doenças dos Suínos/virologia , Vacinas Combinadas/administração & dosagem , Vacinas Combinadas/imunologia , Vacinas Virais/imunologia
4.
Int J Mol Sci ; 20(18)2019 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-31505747

RESUMO

Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are the main pathogens for mycoplasmal pneumonia of swine (MPS) and post-weaning multisystemic wasting syndrome (PMWS), respectively. Infection by these pathogens often happens together and causes great economic losses. In this study, a kind of recombinant baculovirus that can display P97R1P46P42 chimeric protein of Mhp and the capsid (Cap) protein of PCV2 was developed, and the protein location was identified. Another recombinant baculovirus was constructed without tag proteins (EGFP, mCherry) and was used to evaluate the immune effect in experiments with BALB/c mice and domestic piglets. Antigen proteins P97R1P46P42 and Cap were expressed successfully; both were anchored on the plasma membrane of cells and the viral envelope. It should be emphasized that in piglet immunization, the recombinant baculovirus vaccine achieved similar immunological effects as the mixed commercial vaccine. Both the piglet and mouse experiments showed that the recombinant baculovirus was able to induce humoral and cellular responses effectively. The results of this study indicate that this recombinant baculovirus is a potential candidate for the further development of more effective combined genetic engineering vaccines against MPS and PMWS. This experiment also provides ideas for vaccine development for other concomitant diseases using the baculovirus expression system.


Assuntos
Vacinas Bacterianas , Circovirus , Engenharia Genética , Mycoplasma hyopneumoniae , Vacinas Virais , Animais , Vacinas Bacterianas/genética , Vacinas Bacterianas/imunologia , Circovirus/genética , Circovirus/imunologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Mycoplasma hyopneumoniae/genética , Mycoplasma hyopneumoniae/imunologia , Células Sf9 , Spodoptera , Vacinas Virais/genética , Vacinas Virais/imunologia , Vacinas Virais/farmacologia
5.
J Vet Sci ; 20(4): e35, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31364320

RESUMO

The major immunogenic protein capsid (Cap) of porcine circovirus type 2 (PCV2) is critical to induce neutralizing antibodies and protective immune response against PCV2 infection. This study was conducted to investigate the immune response of recombinant adenovirus expressing PCV2b Cap and C-terminal domain of Yersinia pseudotuberculosis invasin (Cap-InvC) fusion protein in pigs. The recombinant adenovirus rAd-Cap-InvC, rAd-Cap and rAd were generated and used to immunize pigs. The phosphate-buffered saline was used as negative control. The specific antibodies levels in rAd-Cap-InvC and ZJ/C-strain vaccine groups were higher than that of rAd-Cap group (p < 0.05), and the neutralization antibody titer in rAd-Cap-InvC group was significantly higher than those of other groups during 21-42 days post-immunization (DPI). Moreover, lymphocyte proliferative level, interferon-γ and interleukin-13 levels in rAd-Cap-InvC group were increased compared to rAd-Cap group (p < 0.05). After virulent challenge, viruses were not detected from the blood samples in rAd-Cap-InvC and ZJ/C-strain vaccine groups after 49 DPI. And the respiratory symptom, rectal temperature, lung lesion and lymph node lesion were minimal and similar in the ZJ/C-strain and rAd-Cap-InVC groups. In conclusion, our results demonstrated that rAd-Cap-InvC was more efficiently to stimulate the production of antibody and protect pigs from PCV2 infection. We inferred that InvC is a good candidate gene for further development and application of PCV2 genetic engineering vaccine.


Assuntos
Vacinas contra Adenovirus/administração & dosagem , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Imunização/veterinária , Doenças dos Suínos/prevenção & controle , Adesinas Bacterianas/genética , Adesinas Bacterianas/imunologia , Animais , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Feminino , Proteínas Recombinantes/imunologia , Sus scrofa , Suínos , Doenças dos Suínos/virologia , Vacinas Sintéticas/administração & dosagem , Yersinia pseudotuberculosis/genética
6.
Vet Microbiol ; 235: 86-92, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31282383

RESUMO

Although PCV2 infections generally cause mild disease in pigs, concurrent co-infections with other pathogens can damage the immune system and cause more severe diseases, collectively termed porcine circovirus associated diseases (PCVAD). Involvement of porcine parvovirus (PPV, a common cause of reproductive failure in naïve dams) in PCVAD caused by PCV2, has been reported. As this co-infection can be difficult to eliminate, there is a critical need to develop an effective vaccine to protect against PPV or synergistic effects of PCV2 and PPV under field conditions. In this study, we designed chimeric PCV2 virus-like particles (cVLPs) displaying a B-cell epitope derived from PPV1 structural protein around the surface of the 2-fold axes of PCV2 VLPs, based on 3D-structure analysis of the PCV2 capsid. The cVLPs were successfully prepared, verified by transmission electron microscopy and chromatography, with robust antibody titers against PCV2 and PPV1 produced in mice and guinea pigs. In addition, in guinea pigs challenged with 106 TCID50 PCV2, cVLPs conferred more effective immune protection (based on viral load) than a commercial PCV2 vaccine. Finally, antibody responses and immune protection against PPV were also evaluated. In guinea pigs vaccinated with cVLPs, although PPV antibodies detected by a hemagglutination inhibition (HI) assay appeared later after vaccination in the PCV2 cVLPs group than in the commercial PPV vaccine group, there were fewer PPV genomic DNA copies in the PCV2 cVLPs group than in a PBS group. In conclusion, guinea pigs vaccinated with cVLPs developed effective protective immunity against PCV2 challenge, with some protective immunity against PPV. This study provided valuable research data to pursue molecular design of chimeric epitopes PCV2 VLPs.


Assuntos
Infecções por Circoviridae/veterinária , Coinfecção/veterinária , Epitopos de Linfócito B/imunologia , Imunidade Humoral , Infecções por Parvoviridae/veterinária , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Linfócitos B/imunologia , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Circovirus/imunologia , Coinfecção/virologia , Feminino , Cobaias , Camundongos , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/prevenção & controle , Parvovirus Suíno/imunologia , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinas Atenuadas/imunologia , Vacinas de Partículas Semelhantes a Vírus/imunologia
7.
J Appl Microbiol ; 127(3): 658-669, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31183947

RESUMO

AIMS: Purification of porcine circovirus type 2 (PCV2) using Gram-positive enhancer matrix (GEM) surface display technology and immunogenicity evaluation of the purified antigen. METHODS AND RESULTS: A recombinant bifunctional protein containing a protein anchor domain and a 'virus anchor' domain was designed as a protein linker (PL) between PCV2 and GEM particles. By incubating with PL and GEM particles sequentially, PCV2 could be purified and enriched through a simple centrifugation process with GEM surface display technology. Our data showed that one unit (2·5 × 109 particles) of GEM particles with 80 µg PL could purify 100 ml of PCV2-containing culture supernatant (viral titre: 106·5 TCID50 per ml-1 ) with a recovery rate up to 99·6%. The impurity removal efficiency of this method, calculated according to decreased total protein content during purification, was approximately 98%. Furthermore, in vivo experimentation showed that piglets immunized with purified PCV2 could elicit strong immune responses to prevent against PCV2 infection. CONCLUSION: Porcine circovirus type 2 could be efficiently purified and enriched with GEM display technology via a crucial PL, and the purified PCV2 could elicit effective immune responses against PCV2 infection. SIGNIFICANCE AND IMPACT OF THE STUDY: The GEM-based purification method established here is cost-efficient and high-throughput, and may represent a promising large-scale purification method for PCV2 vaccine production.


Assuntos
Circovirus/imunologia , Vacinas Virais/imunologia , Vacinas Virais/isolamento & purificação , Animais , Técnicas de Visualização da Superfície Celular , Infecções por Circoviridae/prevenção & controle , Proteínas Recombinantes , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia
8.
PLoS One ; 14(6): e0219175, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31251772

RESUMO

Infections with immunosuppressive pigeon circovirus (PiCV) pose the most severe health problem to the global pigeon breeding. The vaccination with immunogenic PiCV recombinant capsid protein (PiCV rCP) is a potential tool for disease control. Because of the high prevalence of PiCV asymptomatic infections, the subclinically infected pigeons will be vaccinated in practice. The aim of this study was to answer a question if vaccination of asymptomatic, infected with PiCV pigeons induces a similar immune response to PiCV rCP as in uninfected birds. One hundred and twenty 6-week-old carrier pigeons were divided into 4 groups (2 groups of naturally infected and uninfected with PiCV individuals). Birds from groups V and V1 were vaccinated twice with PiCV rCP mixed with an adjuvant, whereas pigeons from groups C and C1 were immunized with an adjuvant only. The expression of genes encoding IFN-γ, CD4, and CD8 T lymphocyte receptors; the number of anti-PiCV rCP IgY-secreting B cells (SBC) and anti-PiCV rCP IgY were evaluated 2, 21, 39 and 46 days post vaccination (dpv). Study results showed that the expression of CD8 and IFN-γ genes was higher in both groups of infected pigeons than in the uninfected birds, irrespective of vaccination. In the uninfected birds, the expression of these genes was insignificantly higher in the vaccinated pigeons. The anti-PiCV rCP IgY-SBC were detected on 2 and 23 dpv and seroconversion was noted on 23 and 39 dpv in V and V1 groups, respectively. In the light of the results obtained, it could be concluded that pigeon circovirus recombinant capsid protein elicits the immune response in both naturally infected and uninfected pigeons, but its rate varies depending on PiCV infectious status. The infection with PiCV masks the potential cellular immune response to the vaccination with PiCV rCP and leads to the suppression of humoral immunity.


Assuntos
Doenças das Aves/tratamento farmacológico , Proteínas do Capsídeo/administração & dosagem , Infecções por Circoviridae/veterinária , Circovirus/metabolismo , Vacinas Virais/administração & dosagem , Animais , Anticorpos Antivirais/metabolismo , Doenças das Aves/imunologia , Antígenos CD8/genética , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/tratamento farmacológico , Infecções por Circoviridae/imunologia , Circovirus/imunologia , Columbidae , Interferon gama/genética , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/imunologia , Resultado do Tratamento , Regulação para Cima , Vacinação/veterinária , Vacinas Virais/imunologia
9.
Virol J ; 16(1): 57, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31046793

RESUMO

BACKGROUND: Porcine circovirus type 2 (PCV2) is an economically important viral pathogen for swine industry worldwide. However, current PCV2 vaccines provide incomplete protection against the PCV2d, which has recently emerged as the predominant pathogenic form of PCV2. METHODS: To develop a novel DNA vaccine with high efficacy against PCV2d virus, we fused the ORF2 of PCV2d to three copies of the minimum-binding domain of the complement C3 cascade terminal component, C3d-P28. Expression of ORF2 alone (pVO) or fused C3d-P28 (pVOC3) were verified by immunofluorescent assay. Vaccine efficacy was tested by measured the DNA copy and T and B cell immune response. RESULTS: Vaccination with pVOC3 reduced the levels of PCV2 genomic DNA after pigs were infected with either PCV2b or PCV2d genotypes, produced potent antibodies against PCV2, and stimulated PCV2-specific interferon-γ secreting cells. CONCLUSION: Results suggested pVOC3 would be a safe and effective DNA vaccine to confer cross-protection against both PCV2b and PCV2d genotypes in pigs.


Assuntos
Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/veterinária , Circovirus/genética , Complemento C3d/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas de DNA/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Proteínas do Capsídeo/genética , Infecções por Circoviridae/prevenção & controle , Circovirus/imunologia , Complemento C3d/genética , Proteção Cruzada , DNA Viral/genética , Genoma Viral , Genótipo , Masculino , Suínos , Doenças dos Suínos/virologia , Vacinação , Vacinas de DNA/genética , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologia , Vacinas Virais/genética
10.
Transbound Emerg Dis ; 66(4): 1454-1461, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059197

RESUMO

PCV2 is globally spread pathogen involved in a number of diseases (PCVD). Commonly used vaccines against PCV2 are proved to be highly efficacious. The role of recently discovered PCV3 for pig health and interference with PCV2 remains unknown. The study performed on serum samples from seven farms vaccinated against PCV2 and four non-vaccinated showed very low prevalence of PCV2 viremia in the former (3 out of 106 positive serum pools) and high prevalence of PCV2 viremia in the latter (35 out of 60 positive pools). Mean log10 PCV2 genome equivalents were lower in vaccinated farms (4.8 ± 0.6 log10  copies/ml) than in non-vaccinated farms (6.3 ± 1.3 log10  copies/ml). PCV3 was detected in 31 out of 106 and 12 out of 60 serum pools from vaccinated and non-vaccinated farms, respectively. Mean log10 PCV3 genome equivalents were significantly (p < 0.05) lower in vaccinated farms (3.9 ± 0.8 log10  copies/ml) than in non-vaccinated farms (4.4 ± 0.6 log10  copies/ml). Concurrent PCV2 and PCV3 infection was rare and found only in 1 out of 529 and 4 out of 292 individual serum samples from vaccinated and non-vaccinated farms, respectively. Our results showed lack of impact of PCV3 circulation on PCV2 vaccine efficacy. On the other hand, intensive PCV2 circulation and high viremia detected in non-vaccinated farms did not seem to increase the level of PCV3 infection.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Viremia/veterinária , Animais , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Feminino , Reação em Cadeia da Polimerase/veterinária , Suínos , Doenças dos Suínos/virologia , Viremia/prevenção & controle , Viremia/virologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-30961815

RESUMO

Primary sclerosing cholangitis (PSC) is a chronic, cholestatic liver disease of unknown cause. In the study, we found that duck circovirus (DuCV) induces PSC in natural and reproductive cases. PSC in DuCV naturally infected ducks was investigated by PCR and histopathology. A model of PSC was developed in one-day old duck by infection of DuCV. Effects on serum levels of liver enzymes and histology were evaluated, and DuCV tropism for bile duct in liver was analyzed by immuohistochemistry. Pathology observation of natural or reproductive DuCV infected ducks showed that the lesion of liver were characterized by cholangiocytic injuries and progressive fibrous obliteration of the biliary tree associated with lymphocytes infiltration. ALT, AST, ALP, GGT, ALB, TBIL and TP were significantly increased in serum of DuCV infected ducks. DuCV showed higher tropism for epithelial cells of bile duct than other cells in PSC.


Assuntos
Ductos Biliares/virologia , Doenças das Aves/fisiopatologia , Colangite Esclerosante/fisiopatologia , Colangite Esclerosante/virologia , Circovirus/fisiologia , Tropismo Viral/fisiologia , Animais , Ductos Biliares/citologia , Doenças das Aves/virologia , Colangite Esclerosante/imunologia , Circovirus/imunologia , Patos , Humanos , Fígado/patologia , Fígado/virologia , Linfócitos/imunologia
12.
Vet Microbiol ; 231: 87-92, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30955830

RESUMO

The objective of this study was to compare the efficacy of a commercial porcine circovirus type 2a (PCV2a) subunit vaccine against experimental PCV2a, PCV2b, and PCV2d challenge. A total of 105 pigs were randomly divided into 7 groups (15 pigs per group). At 21 days old the pigs were intramuscularly administered the PCV2a vaccine as a 1.0 mL dose. Four weeks following vaccination, pigs were challenged with either Korean PCV2a, PCV2b, or PCV2d. All vaccinated pigs showed a significant (P < 0.05) reduction of clinical signs, PCV2 viremia, lymphoid lesions, and lymphoid PCV2 antigen levels compared to unvaccinated control pigs. Vaccination resulted also in significantly higher (P < 0.05) titers of neutralizing antibody against PCV2, and an increase in the frequency of PCV2-specific interferon-γ secreting cells (IFN-γ-SC). The vaccine showed similar protection among the vaccinated groups regardless of the genotype of the challenge. Interestingly, vaccinated pigs had higher levels of neutralizing antibody titers against PCV2a compared to PCV2b or PCV2d while the number of PCV2a-, PCV2b-, and PCV2d-specific IFN-γ-SC were similar. Taken together, the results presented here demonstrate that a PCV2a vaccine can be effective against experimental PCV2a, PCV2b, and PCV2d challenge.


Assuntos
Anticorpos Antivirais/sangue , Infecções por Circoviridae/veterinária , Doenças dos Suínos/prevenção & controle , Vacinas Virais/uso terapêutico , Animais , Anticorpos Neutralizantes/sangue , Infecções por Circoviridae/imunologia , Infecções por Circoviridae/prevenção & controle , Circovirus/genética , Circovirus/imunologia , DNA Viral/sangue , Fazendas , Genótipo , Injeções Intramusculares , Interferon gama/imunologia , Gado , Distribuição Aleatória , Suínos , Doenças dos Suínos/imunologia , Vacinação , Vacinas de Subunidades/imunologia , Vacinas de Subunidades/uso terapêutico , Vacinas Virais/imunologia
13.
BMC Vet Res ; 15(1): 79, 2019 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-30841883

RESUMO

BACKGROUND: Porcine circovirus type 3 (PCV3), recently widely isolated from pigs with various clinical conditions, is likely globally epidemic. However, development of serological diagnosis for PCV3 in pigs is ongoing. Our objectives were to: 1) establish an indirect ELISA, using PCV3 capsid protein (Cap) prepared by Baculovirus Expression Vector System (BEVS) as a high-quality coating antigen for detection of PCV3-associated antibodies in serum samples; and 2) use this ELISA to conduct a serological survey for PCV3 in various regions of Hunan province, China. RESULTS: The PCV3 positive rate to the ELISA assay (total of 190 serum samples) was higher in sows with reproductive failure compared to healthy sows (34/85, 40.0% versus 30/105, 28.6%), with similar results using qPCR assays. Further, in an additional 1038 serum samples collected from January 2016 to May 2018 in various regions of Hunan province and tested with this established ELISA, 20 to 84% were positive for PCV3 (according to region of sera collection), with high PCV3 seroprevalence (> 50%) in herds in Changde, Hengyang and Yueyang. Moreover, among serum samples from herds in Shaoyang and Changde, PCV3 seroprevalence was higher in sows than in other classes of pigs (i.e., suckling piglets, nursery pigs, gilts, growing-finishing pigs and boars). CONCLUSIONS: We developed a full-length PCV3 Cap-based ELISA using a eukaryotic expression system with excellent potential to elucidate PCV3 epidemiology. Based on this assay, PCV3 has been circulating in Hunan province. PCV3 prevalence was lower in healthy sows than in those with reproductive failure. Further studies are warranted to identify the PCV3 responsible for high seroprevalence in sows and determine pathogenesis of PCV3 in sows with reproductive failure.


Assuntos
Infecções por Circoviridae/veterinária , Circovirus/isolamento & purificação , Ensaio de Imunoadsorção Enzimática/veterinária , Doenças dos Suínos/virologia , Animais , Baculoviridae , Proteínas do Capsídeo , China/epidemiologia , Infecções por Circoviridae/diagnóstico , Infecções por Circoviridae/epidemiologia , Circovirus/imunologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Genitália Feminina/patologia , Genitália Feminina/virologia , Imunoglobulina G/imunologia , Masculino , Estudos Soroepidemiológicos , Suínos/sangue , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/epidemiologia
14.
PLoS Pathog ; 15(3): e1007562, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30822338

RESUMO

Postweaning multisystemic wasting disease (PMWS) in piglets caused by porcine circovirus type 2 (PCV2) is one of the major threats to most pig farms worldwide. Among all the PCV types, PCV2 is the dominant genotype causing PMWS and associated diseases. Considerable efforts were made to study the virus-like-particle (VLP) assembly and the specific PCV2-associated epitope(s) in order to establish the solid foundation for engineered PCV2 vaccine development. Although the N-terminal fragment including Nuclear Localization Signal (NLS) sequence seems important for recombinant PCV2 capsid protein expression and VLP assembly, the detailed structural and functional information regarding this important fragment are largely unknown. In this study, we report crystal structure of PCV2 VLP assembled from N-terminal NLS truncated PCV2 capsid protein at 2.8 Å resolution and cryo-EM structure of PCV2 VLP assembled from full-length PCV2 capsid protein at 4.1Å resolution. Our in vitro PCV2 VLP assembly results show that NLS-truncated PCV2 capsid protein only forms instable VLPs which were easily disassembled in solution, whereas full-length PCV2 capsid protein forms stable VLPs due to interaction between 15PRSHLGQILRRRP27 (α-helix) and 33RHRYRWRRKN42 (NLS-B) in a repeated manner. In addition, our results also showed that N-terminal truncation of PCV2 capsid protein up to 27 residues still forms PCV2 particles in solution with similar size and immunogenicity, while N-terminal truncation of PCV2 capsid protein with more than 30 residues is not able to form stable PCV2 particles in solution, demonstrating the importance of interaction between the α-helix at N-terminal and NLS-B in PCV2 VLP formation. Moreover, we also report the cryo-EM structure of PCV2 VLP in complex with 3H11-Fab, a PCV2 type-specific neutralizing antibody, at 15 Å resolution. MAb-3H11 specifically recognizes one exposed epitope located on the VLP surface EF-loop (residues 128-143), which is further confirmed by PCV1-PCV2 epitope swapping assay. Hence, our results have revealed the structural roles of N-terminal fragment of PCV2 capsid protein in PCV2 particle assembly and pinpointed one PCV2 type-specific neutralizing epitope for the first time, which could provide clear clue for next generation PCV2 vaccine and diagnostic kits development.


Assuntos
Proteínas do Capsídeo/imunologia , Circovirus/metabolismo , Circovirus/ultraestrutura , Animais , Anticorpos Antivirais/imunologia , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , Circovirus/imunologia , Epitopos , Sinais de Localização Nuclear , Síndrome Definhante Multissistêmico de Suínos Desmamados/metabolismo , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes , Suínos , Vacinas de Partículas Semelhantes a Vírus/imunologia , Vacinas de Partículas Semelhantes a Vírus/metabolismo , Vacinas Virais/biossíntese , Vacinas Virais/imunologia
15.
Appl Microbiol Biotechnol ; 103(8): 3453-3464, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30863876

RESUMO

This study described the production, characterization, and application of monoclonal antibodies (mAbs) against porcine circovirus type 2 (PCV2). Twelve stable hybridomas were produced by immunization with purified PCV2a/LG strain and characterized by immunoperoxidase monolayer assay (IPMA), Western blotting, and neutralization assays. All mAbs could react with the PCV2 Cap protein and neutralize PCV2a/LG strain. One of them, mAb 3A5, reacted to all PCV2 strains from PCV2a, PCV2b, and PCV2d and it could be applied to detect PCV2 antigen and antibodies. It was shown that the mAb 3A5 could be used to locate PCV2 antigen in PK15 cells and the inguinal lymph nodes of PCV2b/YJ stain-infected piglets. Furthermore, this mAb could immunoprecipitate the Cap protein in PCV2-infected PK15 cells. Meanwhile, a capture ELISA based on mAb 3A5 was developed and used to specifically test PCV2 antigen from cultures; a linear relationship was observed between the optical density at 405 nm of the ELISA and viral titers (200-12,800 TCID50/mL), with a correlation coefficient of 0.9999. Finally, a competitive ELISA based on mAb 3A5 was developed to specifically detect antibodies in PCV2-infected and immunized pigs, and its sensitivity was higher than that of the blocking ELISA. This study suggested that the mAb 3A5 could be used in several convenient and efficient methods for PCV2 clinical and pathological studies, as well as surveillance in pigs and seroconversion monitoring in the vaccinated pigs.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Doenças dos Suínos/diagnóstico , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Proteínas do Capsídeo/imunologia , Linhagem Celular , Infecções por Circoviridae/diagnóstico , Circovirus/genética , Genótipo , Imunoensaio , Suínos
16.
Vet Microbiol ; 228: 69-76, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30593382

RESUMO

The porcine circovirus (PCV) is one of the most economically important infection diseases of pigs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) or Flic as an immune adjuvant has been shown to enhance the immunogenicity of vaccines in previous study. However, the composite biological adjuvants stimulate more effective immunological response. In this study, the porcine GM-CSF (pGM-CSF) and FliC protein were expressed by pSUMO in E.coli Rosetta (DE3) and purified by Ni-NTA Sepharose, respectively. The immunogenicity of PCV vaccine with pGM-CSF and FliC was firstly evaluated to identify the immunoenhancement in mice. The results indicated that mice immunized with vaccine + pGM-CSF + FliC enhanced immune responses ability significantly and quickly. Then, the immune response level of PCV vaccine with pGM-CSF and FliC was assessed in piglets. The results indicated that pigs immunized with vaccine + pGM-CSF + FliC showed significantly higher PCV antibody level than those immunized with vaccine and single adjuvant or vaccine alone. Furthermore, pigs in the vaccine + pGM-CSF + FliC group elicited stronger CD4+ and CD8 + T cells proliferative responses than those in all other groups and showed the effectively up-regulated transcriptional level of IL-1, IL-8 and IL-17 stimulating the immune system. We demonstrated that GM-CSF and FliC as composite biological adjuvants of the vaccine showed a stronger immune response and it is promising application for vaccines to against PCV.


Assuntos
Adjuvantes Imunológicos , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/virologia , Feminino , Imunidade Humoral , Imunização/veterinária , Camundongos , Camundongos Endogâmicos BALB C , Suínos , Doenças dos Suínos/virologia
17.
Vet Microbiol ; 227: 34-40, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30473349

RESUMO

PCV2 is a single-stranded DNA virus that we previously found to induce IFN-ß production via RIG-I and MDA-5. cGAS is known to be the most important DNA sensor for the recognition of cytosolic DNA; however, it remains unclear whether the interferon production induced by PCV2 is associated with cGAS. In the present study, PCV2 infection was found to increase the level of cGAS and STING expression, promote the release of cyclic dinucleotide cGAMP, and induce STING dimerization and translocation into the nucleus of PK-15 cells. These findings indicate that PCV2 infection activates both cGAS and STING. Furthermore, the knockdown of cGAS and STING decreased both the mRNA expression and promoter activity of IFN-ß, demonstrating that the cGAS/STING signaling pathway contributes to the production of IFN-ß. In addition, a knockdown of cGAS and STING also decreased the PCV2 viral load and infectivity. Therefore, PCV2 infection activates the cGAS/STING signaling pathway to induce IFN-ß production and the knockdown of cGAS and STING decreases viral replication in PK-15 cells. These results provide further insight into the relationship between PCV2 and host innate immunity.


Assuntos
Circovirus/imunologia , Interferon beta/imunologia , Proteínas de Membrana/imunologia , Nucleotidiltransferases/imunologia , Transdução de Sinais/imunologia , Replicação Viral/imunologia , Animais , Linhagem Celular , Infecções por Circoviridae/virologia , Circovirus/genética , Circovirus/fisiologia , Imunidade Inata , Interferon beta/biossíntese , Interferon beta/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nucleotídeos Cíclicos/genética , Nucleotídeos Cíclicos/imunologia , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Transdução de Sinais/genética , Suínos
18.
Viruses ; 10(11)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30384424

RESUMO

Pigeon circovirus (PiCV) is the most frequently diagnosed virus in pigeons and is thought to be one of the causative factors of a complex disease called the young pigeon disease syndrome (YPDS). The development of a vaccine against this virus could be a strategy for YPDS control. Since laboratory culture of PiCV is impossible, its recombinant capsid protein (rCP) can be considered as a potential antigen candidate in sub-unit vaccines. The aim of this basic research was to evaluate the immune response of pigeons to PiCV rCP. Sixty six-week-old carrier pigeons were divided into two groups (experimental immunized with PiCV rCP mixed with an adjuvant, and control immunized with an adjuvant only), and immunized twice in a 21-day interval. On the day of immunization and on two, 23, 39, and 46 days post first immunization (dpv), samples of blood, spleen, and bursa of Fabricius were collected from six birds from each group to examine anti-PiCV rCP IgY, anti-PiCV rCP IgY-secreting B cells (SBC), IFN-γ gene expression, and percentage of T CD3⁺, CD4⁺, CD8⁺, and B IgM⁺ lymphocytes. The results indicated a correct immune response to PiCV rCP both in humoral and cell-mediated immunity, which was manifested by seroconversion since 23 dpv, by a significantly higher anti-PiCV rCP IgY-SBC number on two and 23 dpv, and significantly higher IFN-γ gene expression since two dpv. There were no significant differences or trends noted between particular T and B lymphocyte subpopulations. To conclude, PiCV rCP may be deemed immunogenic and could be considered as an antigen candidate in sub-unit vaccines against PiCV infections in pigeons.


Assuntos
Doenças das Aves/imunologia , Doenças das Aves/virologia , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/veterinária , Circovirus/imunologia , Columbidae/imunologia , Columbidae/virologia , Interações Hospedeiro-Patógeno/imunologia , Animais , Ensaio de Imunoadsorção Enzimática , ELISPOT/métodos , Citometria de Fluxo , Imunidade , Imunoglobulina M/imunologia , Imunoglobulinas/imunologia , Interferon gama/genética , Proteínas Recombinantes , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo
19.
Appl Microbiol Biotechnol ; 102(24): 10541-10550, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30338355

RESUMO

Mixed infection of porcine circovirus type 2 (PCV2) and foot-and-mouth disease virus (FMDV) is devastating to swine populations. To develop an effective vaccine that can protect the pigs from the infection of PCV2 and FMDV, we used the neutralizing B cell epitope region (aa 135-160) of FMDV to replace the regions aa 123-151 and aa 169-194 of the PCV2b Cap protein to generate a recombinant protein designated as Capfb. The Capfb protein was expressed in Escherichia coli system and the purified Capfb protein assembled into virus-like particles (VLPs) through dialysis. The ability of the Capfb protein to induce effective immune response against FMDV and PCV2b was tested in mice and guinea pigs. The results showed that the Capfb-VLPs could elicit anti-PCV2b and anti-FMDV antibody response in mice and guinea pigs without inducing antibodies against decoy epitope. Moreover, the Capfb-VLPs could enhance the percentage and activation of B cells in lymph nodes when the mice were stimulated with inactivated FMDV or PCV2b. These data suggested that the Capfb-VLPs could be an efficacious candidate antigen for developing a novel PCV2b-FMDV bivalent vaccine.


Assuntos
Circovirus/imunologia , Vírus da Febre Aftosa/imunologia , Proteínas Recombinantes/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Infecções por Circoviridae/prevenção & controle , Infecções por Circoviridae/veterinária , Circovirus/patogenicidade , Epitopos de Linfócito B/imunologia , Escherichia coli/genética , Febre Aftosa/prevenção & controle , Vírus da Febre Aftosa/patogenicidade , Cobaias , Camundongos Endogâmicos ICR , Microscopia Eletrônica de Transmissão , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Vacinas Virais/genética , Vírion/imunologia
20.
Benef Microbes ; 9(6): 951-961, 2018 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-30232907

RESUMO

In our previous study we confirmed an antiviral activity of probiotic Lactobacillus reuteri L26 which was mediated by stimulation of local intestinal immunity. The aim of this paper was to evaluate the influence of L. reuteri L26 on the systemic immune response in gnotobiotic mice infected with porcine circovirus type 2 (PCV2). A total of 30 germ-free mice were divided into 3 groups and animals in noninfected and infected control groups (NC and IC; n=10) received sterile de Man-Rogosa-Sharpe broth for 7 days and animals in experimental group L+PCV (n=10) were inoculated with L. reuteri L26. Subsequently, mice in L+PCV and IC groups were infected with PCV2; however, mice in the control group received virus cultivation medium (mock). The results showed an increase of percentage of cytotoxic cells (CD8+ and CD49b+CD8-) and oxidative burst of phagocytes, up-regulation of the gene expression of RANTES, granulocyte-macrophage colony-stimulating factor, interferon-γ and immunoglobulin A in blood above all in the later phase of infection (14 dpi) in L+PCV group accompanied by higher load of PCV2 in the serum. These findings indicate that L. reuteri L26 has a potential to induce systemic immune reaction, but in gnotobiotic mice immune stimulation can increase virus replication.


Assuntos
Infecções por Circoviridae/prevenção & controle , Circovirus/imunologia , Fatores Imunológicos/administração & dosagem , Lactobacillus reuteri/imunologia , Probióticos/administração & dosagem , Animais , Infecções por Circoviridae/imunologia , Citocinas/análise , Vida Livre de Germes , Imunoglobulina A/sangue , Lactobacillus reuteri/crescimento & desenvolvimento , Camundongos , Fagócitos/imunologia , Linfócitos T Citotóxicos/imunologia
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