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1.
Mol Immunol ; 136: 55-64, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34087624

RESUMO

Pseudorabies virus (PRV) is an enveloped double-stranded DNA virus that is the etiological agent of Aujeszky's disease in pigs. Vaccination is currently available to prevent PRV infection, but there is still an urgent need for new strategies to control this infectious disease. Histone deacetylases (HDACs) are epigenetic regulators that regulate the histone tail, chromatin conformation, protein-DNA interaction and even transcription. Viral transcription and protein activities are intimately linked to regulation by histone acetyltransferases and HDACs that remodel chromatin and regulate gene expression. We reported here that genetic and pharmacological inhibition of HDAC1 significantly influenced PRV replication. Moreover, we demonstrated that inhibition of HDAC1 induced a DNA damage response and antiviral innate immunity. Mechanistically, the HDAC1 inhibition-induced DNA damage response resulted in the release of double-strand DNA into the cytosol to activate cyclic GMP-AMP synthase and the downstream STING/TBK1/IRF3 innate immune signaling pathway. Our results demonstrate that an HDAC1 inhibitor may be used as a new strategy to prevent Aujeszky's disease in pigs.


Assuntos
Herpesvirus Suídeo 1/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Pseudorraiva/tratamento farmacológico , Células 3T3 , Animais , Linhagem Celular , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Células HEK293 , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Histona Desacetilase 1/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Imunidade Inata/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Pseudorraiva/imunologia , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/genética , Suínos , Doenças dos Suínos/virologia , Replicação Viral/efeitos dos fármacos
2.
J Virol ; 95(16): e0076021, 2021 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-34037418

RESUMO

Pseudorabies virus (PRV) is the causative pathogen of Aujeszky's disease in pigs. Although vaccination is currently applied to prevent the morbidity of PRV infection, new applications are urgently needed to control this infectious disease. Poly(ADP-ribose) polymerase 1 (PARP1) functions in DNA damage repair. We report here that pharmacological and genetic inhibition of PARP1 significantly influenced PRV replication. Moreover, we demonstrate that inhibition of PARP1 induced DNA damage response and antiviral innate immunity. Mechanistically, PARP1 inhibition-induced DNA damage response resulted in the release of double-stranded DNA (dsDNA) into the cytosol, where dsDNA interacted with cyclic GMP-AMP (cGAMP) synthase (cGAS). cGAS subsequently catalyzed cGAMP production to activate the STING/TBK1/IRF3 innate immune signaling pathway. Furthermore, challenge of mice with PARP1 inhibitor stimulated antiviral innate immunity and protected mice from PRV infection in vivo. Our results demonstrate that PARP1 inhibitors may be used as a new strategy to prevent Aujeszky's disease in pigs. IMPORTANCE Aujeszky's disease is a notifiable infectious disease of pigs and causes economic losses worldwide in the pig industry. The causative pathogen is PRV, which is a member of the subfamily Alphaherpesvirinae of the family Herpesviridae. PRV has a wide range of hosts, such as ruminants, carnivores, and rodents. More seriously, recent reports suggest that PRV can cause human endophthalmitis and encephalitis, which indicates that PRV may be a potential zoonotic pathogen. Although vaccination is currently the major strategy used to control the disease, new applications are also urgently needed for the pig industry and public health. We report here that inhibition of PARP1 induces DNA damage-induced antiviral innate immunity through the cGAS-STING signaling pathway. Therefore, PARP1 is a therapeutic target for PRV infection as well as alphaherpesvirus infection.


Assuntos
Antivirais/imunologia , Dano ao DNA/imunologia , Imunidade Inata/efeitos dos fármacos , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Pseudorraiva/tratamento farmacológico , Animais , Antivirais/farmacologia , Linhagem Celular , Herpesvirus Suídeo 1/efeitos dos fármacos , Herpesvirus Suídeo 1/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Camundongos , Nucleotidiltransferases/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pseudorraiva/imunologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Suínos , Replicação Viral/efeitos dos fármacos
3.
Vet Microbiol ; 258: 109104, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34004569

RESUMO

Pseudorabies is a highly infectious disease with severe clinical symptoms, causing acute death in infected pigs and leading to substantial economic losses among swine producers. In this study, a vaccine candidate strain in which the protein kinase UL13 gene was deleted was constructed with the CRISPR/Cas9 system based on the recombinant pseudorabies virus (PRV) ZJ01-ΔgI/gE/TK. Pigs immunized with ZJ01-ΔgI/gE/TK or ZJ01-ΔgI/gE/TK/UL13 produced high levels of anti-gB antibodies and virus-neutralizing antibodies. ZJ01-ΔgI/gE/TK/UL13 provided greater protective efficacy against challenge with PRV variant strain ZJ01 than did Bartha-K61 or ZJ01-ΔgI/gE/TK. The pigs vaccinated with ZJ01-ΔgI/gE/TK/UL13 excreted significantly less virus than those vaccinated with Bartha-K61 or ZJ01-ΔgI/gE/TK. The viral loads in the lungs of pigs treated with ZJ01-ΔgI/gE/TK/UL13 were lower than those in pigs treated with ZJ01-ΔgI/gE/TK after challenge with PRV variant strain ZJ01. These data indicated that ZJ01-ΔgI/gE/TK/UL13 had greater protective efficacy and safety than the commercial ZJ01-ΔgI/gE/TK and Bartha-K61 vaccines, and could be developed as a promising vaccine candidate for the prevention and control of this disease.


Assuntos
Herpesvirus Suídeo 1/genética , Vacinas contra Pseudorraiva/imunologia , Pseudorraiva/virologia , Doenças dos Suínos/prevenção & controle , Animais , Linhagem Celular , Interferon beta/genética , Interferon beta/metabolismo , Pseudorraiva/imunologia , Testes Sorológicos , Suínos , Doenças dos Suínos/virologia
4.
BMC Vet Res ; 17(1): 164, 2021 Apr 14.
Artigo em Inglês | MEDLINE | ID: mdl-33853597

RESUMO

BACKGROUND: Since 2011, numerous highly virulent and antigenic variant viral strains have been reported in pigs that were vaccinated against the swine pseudorabies virus. These infections have led to substantial economic losses in the Chinese swine industry. RESULTS: This study, constructed a novel recombinant vaccine strain with gI/gE deletion (PRV-GD2013-ΔgI/gE) by overlapping PCR and homologous recombination technology. The growth curves and plaque morphology of the recombinant virus were similar to those of the parental strain. However, PRV-GD2013-ΔgI/gE infection was significantly attenuated in mice compared with that of PRV-GD2013. Two-week-old piglets had normal rectal temperatures and displayed no clinical symptoms after being inoculated with 105 TCID50 PRV-GD2013-ΔgI/gE, indicating that the recombinant virus was avirulent in piglets. Piglets were immunized with different doses of PRV-GD2013-ΔgI/gE, or a single dose of Bartha-K61 or DMEM, and infected with PRV-GD2013 at 14 days post-vaccination. Piglets given high doses of PRV-GD2013-ΔgI/gE showed no obvious clinical symptoms, and their antibody levels were higher than those of other groups, indicating that the piglets were completely protected from PRV-GD2013. CONCLUSIONS: The PRV-GD2013-ΔgI/gE vaccine strain could be effective for immunizing Chinese swine herds against the pseudorabies virus (PRV) strain.


Assuntos
Vacinas contra Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Doenças dos Suínos/virologia , Animais , Linhagem Celular , Cricetinae , Feminino , Deleção de Genes , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Recombinação Homóloga , Camundongos Endogâmicos BALB C , Reação em Cadeia da Polimerase , Pseudorraiva/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/prevenção & controle , Vacinas Sintéticas/virologia , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia
5.
J Vet Sci ; 22(2): e20, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33774936

RESUMO

BACKGROUND: Pseudorabies virus (PRV) infection leads to high mortality in swine. Despite extensive efforts, effective treatments against PRV infection are limited. Furthermore, the inflammatory response induced by PRV strain GXLB-2013 is unclear. OBJECTIVES: Our study aimed to investigate the inflammatory response induced by PRV strain GXLB-2013, establish an inflammation model to elucidate the pathogenesis of PRV infection further, and develop effective drugs against PRV infection. METHODS: Kunming mice were infected intramuscularly with medium, LPS, and different doses of PRV-GXLB-2013. Viral spread and histopathological damage to brain, spleen, and lung were determined at 7 days post-infection (dpi). Immune organ indices, levels of reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines, as well as levels of activity of COX-2 and iNOS were determined at 4, 7, and 14 dpi. RESULTS: At 105-106 TCID50 PRV produced obviously neurological symptoms and 100% mortality in mice. Viral antigens were detectable in kidney, heart, lung, liver, spleen, and brain. In addition, inflammatory injuries were apparent in brain, spleen, and lung of PRV-infected mice. Moreover, PRV induced increases in immune organ indices, ROS and NO levels, activity of COX-2 and iNOS, and the content of key pro-inflammatory cytokines, including interleukin (IL)-1ß, IL-6, tumor necrosis factor-α, interferon-γ and MCP-1. Among the tested doses, 10² TCID50 of PRV produced a significant inflammatory mediator increase. CONCLUSIONS: An inflammatory model induced by PRV infection was established in mice, and 10² TCID50 PRV was considered as the best concentration for the establishment of the model.


Assuntos
Herpesvirus Suídeo 1/fisiologia , Inflamação/veterinária , Pseudorraiva/imunologia , Animais , Modelos Animais de Doenças , Inflamação/imunologia , Inflamação/fisiopatologia , Inflamação/virologia , Pseudorraiva/fisiopatologia , Pseudorraiva/virologia , Sus scrofa , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/fisiopatologia , Doenças dos Suínos/virologia
6.
J Vet Sci ; 22(2): e23, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33774939

RESUMO

BACKGROUND: Pseudorabies (PR), caused by the pseudorabies virus (PRV), is an endemic disease in some regions of China. Although there are many reports on epidemiological investigations into pseudorabies, information on PRV gI antibody dynamics in one pig farm is sparse. OBJECTIVES: To diagnose PR and analyze the course of PR eradication in one pig farm. METHODS: Ten brains and 1,513 serum samples from different groups of pigs in a pig farm were collected to detect PRV gE gene and PRV gI antibody presence using real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: The July 2015 results indicated that almost all brain samples were PRV gE gene positive, but PRV gI antibody results in the serum samples of the same piglets were all negative. In the boar herd, from October 2015 to July 2018 three positive individuals were culled in October 2015, and the negative status of the remaining boars was maintained in the following tests. In the sow herd, the PRV gI antibody positive rate was always more than 70% from October 2015 to October 2017; however, it decreased to 27% in January 2018 but increased to 40% and 52% in April and July 2018, respectively. The PRV gI antibody positive rate in 100-day pigs markedly decreased in October 2016 and was maintained at less than 30% in the following tests. For 150-day pigs, the PRV gI antibody positive rate decreased notably to 10% in April 2017 and maintained a negative status from July 2017. The positive trend of PRV gI antibody with an increase in pig age remarkably decreased in three tests in 2018. CONCLUSIONS: The results indicate that serological testing is not sensitive in the early stage of a PRV infection and that gilt introduction is a risk factor for a PRV-negative pig farm. The data on PRV gI antibody dynamics can provide reference information for pig farms wanting to eradicate PR.


Assuntos
Anticorpos Antivirais/imunologia , Pseudorraiva/diagnóstico , Pseudorraiva/imunologia , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/imunologia , Criação de Animais Domésticos/métodos , Animais , China , Feminino , Masculino , Sus scrofa , Suínos
7.
Biomed Environ Sci ; 33(6): 444-447, 2020 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-32641207

RESUMO

Pseudorabies virus (PRV), a veterinary pathogen that infects domestic animals as well as wild animals such as wild boar and feral swine, was recently reported to infect human and led to endophthalmitis and encephalitis. A retrospective seroepidemiologic survey was conducted using 1,335 serum samples collected from patients with encephalitis and ELISA positive rates were 12.16%, 14.25%, and 6.52% in 2012, 2013, and 2017, respectively. The virus neutralizing antibody titers of positive samples correlated well with ELISA results. The pseudorabies virus antibody positive rate of patients with encephalitis were higher than that of healthy people in 2017. The above results suggest that some undefined human encephalitis cases may be caused by PRV infection.


Assuntos
Anticorpos Antivirais/sangue , Encefalite/imunologia , Herpesvirus Suídeo 1/imunologia , Pseudorraiva/imunologia , Adulto , Animais , China , Encefalite/virologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prevalência , Pseudorraiva/sangue , Pseudorraiva/virologia , Estudos Retrospectivos , Estudos Soroepidemiológicos , Adulto Jovem
8.
Virol J ; 17(1): 19, 2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-32014014

RESUMO

BACKGROUND: Porcine parvovirus (PPV) and pseudorabies virus (PRV) are the important etiological agents of swine infectious diseases, resulting in huge economic losses to the Chinese swine industry. Interleukin-6 (IL-6) has the roles to support host immune response to infections as a pleiotropic cytokine. It is essential to construct a live attenuated vaccine-based recombinant PRV that expresses PPV VP2 protein and porcine IL-6 for prevention and control of PRV and PPV. METHODS: The recombinant plasmid, pGVP2-IL6, was constructed by porcine IL-6 gene substituting for EGFP gene of the PRV transfer plasmid pGVP2-EGFP containing VP2 gene of PPV. Plasmid pGVP2-IL6 was transfected into swine testicle cells pre-infected with the virus rPRV-VP2-EGFP strain through homologous recombination and plaque purification to generate a recombinant virus rPRV-VP2-IL6. The recombinant PRV was further identified by PCR and DNA sequencing, and the expression of the VP2 protein and porcine IL-6 was analyzed by reverse transcription-PCR (RT-PCR) and Western blot. The virus titer was calculated according to Reed and Muench method. The immunogenicity of the recombinant virus was preliminarily evaluated in mice by intramuscular administration twice with the rPRV-VP2-IL6 at 4-week intervals. RESULTS: A recombinant virus rPRV-VP2-IL6 was successfully constructed and confirmed in this study. The properties of rPRV-VP2-IL6 were similar to the parental virus HB98 in terms of growth curve, morphogenesis and virus plaque sizes, and rPRV-VP2-IL6 was proliferated in different cell types. It induced specific antibodies against PPV as well as a strong increase of PPV-specific lymphocyte proliferation responses in mice immunized with rPRV-VP2-IL6, and provided partial protection against the virulent PPV challenge. rPRV-VP2-IL6 also induced a high level of neutralizing antibodies against PRV, and significantly reduced the mortality rate of (1 of 10) following virulent PRV challenge compared with the control (10 of 10). CONCLUSIONS: The recombinant rPRV-VP2-IL6 might be a potential candidate vaccine against PRV and PPV infections in pigs.


Assuntos
Antígenos Virais/genética , Proteínas do Capsídeo/genética , Herpesvirus Suídeo 1/genética , Interleucina-6/genética , Infecções por Parvoviridae/veterinária , Pseudorraiva/prevenção & controle , Vacinas Virais/imunologia , Animais , Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Esquema de Medicação , Feminino , Herpesvirus Suídeo 1/imunologia , Imunogenicidade da Vacina , Injeções Intramusculares , Interleucina-6/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Infecções por Parvoviridae/imunologia , Infecções por Parvoviridae/prevenção & controle , Pseudorraiva/imunologia , Suínos , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Vacinas Virais/genética
9.
Biosens Bioelectron ; 155: 112101, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32090873

RESUMO

A novel time-resolved fluorescence blocking lateral flow immunoassay (TRF-BLFIA) was developed for on-site differential diagnosis of pseudorabies virus (PRV)-infected and vaccinated pigs using europium nanoparticles (EuNPs)-labeled virion antigens and high titer PRV gE monoclonal antibodies (PRV gE-mAb). Upon application of a positive serum sample, the specific epitopes of gE protein on the EuNPs-PRV probe were blocked, inhibiting binding to the PRV gE-mAb on the T line, resulting in low or negligible fluorescence signal, whereas when a negative sample was applied, EuNPs-PRV probes would be able to bind the antibody at the T line, leading to high fluorescence signal. Under optimized conditions, TRF-BLFIA provided excellent sensitivity and selectivity. When testing swine clinical samples (n = 356), there was 96.1% agreement between this method and a most widely used commercial gE-ELISA kit. Moreover, our method was rapid (15 min), cost-efficient and easy to operate with simple training, allowing for on-site detection. Thus, TRF-BLFIA could be a practical tool to differentially diagnose PRV-infected and vaccinated pigs.


Assuntos
Antígenos Virais , Európio , Imunofluorescência , Herpesvirus Suídeo 1/imunologia , Nanopartículas Metálicas , Pseudorraiva/diagnóstico , Doenças dos Suínos/diagnóstico , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Pseudorraiva/imunologia , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Suínos , Doenças dos Suínos/imunologia
10.
J Virol ; 94(6)2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31896600

RESUMO

Differentiating infected from vaccinated animals (DIVA) strategies have been central enabling techniques in several successful viral disease elimination programs. However, owing to their long and uncertain development process, no DIVA-compatible vaccines are available for many important diseases. We report herein a new DIVA strategy based on hybrid protein-peptide microarrays which can theoretically work with any vaccine. Leading from our findings from peste des petits ruminants (PPR) virus, we found 4 epitope-containing short peptides (ECSPs) which have distinct IgG serodynamics: anti-ECSP IgGs only exist for 10 to 60 days postvaccination (dpv), while anti-protein IgGs remained at high levels for >1,000 dpv. These data enabled the design of a DIVA diagnostic microarray containing 4 ECSPs and 3 proteins, which, unlike competitive enzyme-linked immunosorbent assay (cELISA) and virus neutralization tests (VNTs), enables ongoing monitoring of serological differences between vaccinated individuals and individuals exposed to the pathogen. For 25 goats after 60 dpv, 13 were detected with positive anti-ECSP IgGs, indicating recent infections in vaccinated goat herds. These DIVA diagnostic microarrays will almost certainly facilitate eradication programs for (re)emerging pathogens and zoonoses.IMPORTANCE Outbreaks of infectious diseases caused by viruses, such as pseudorabies (PR), foot-and-mouth disease (FMD), and PPR viruses, led to economic losses reaching billions of dollars. Both PR and FMD were eliminated in several countries via large-scale vaccination programs using DIVA-compatible vaccines, which lack the gE protein and nonstructural proteins, respectively. However, there are still extensive challenges facing the development and deployment of DIVA-compatible vaccines because they are time-consuming and full of uncertainty. Further, the negative marker strategy used for DIVA-compatible vaccines is no longer functional for live-attenuated vaccines. To avoid these disadvantageous scenarios, a new strategy is desired. Here, we made the exciting discovery that different IgG serodynamics can be monitored when using protein-based assays versus arrays comprising ECSPs. This DIVA microarray strategy should, in theory, work for any vaccine.


Assuntos
Anticorpos Antivirais/imunologia , Epitopos/química , Imunoglobulina G/imunologia , Peptídeos/química , Peste dos Pequenos Ruminantes/imunologia , Vírus da Peste dos Pequenos Ruminantes/imunologia , Análise Serial de Proteínas , Vacinação , Animais , Febre Aftosa/imunologia , Febre Aftosa/prevenção & controle , Cabras , Peste dos Pequenos Ruminantes/prevenção & controle , Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Vacinas Virais/imunologia
11.
Vet Microbiol ; 240: 108543, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31902487

RESUMO

Since 2011, to control the spread of pseudorabies (PR), US7/US8/UL23-deleted recombinant PRV (rPRV) vaccines based on current variants have been developed. The vaccines can provide effective immune protection to pigs, but fur-bearing animals, such as dogs, foxes, and minks, are increasingly infected by PRV due to consuming contaminated raw meat or offal from immunized pigs. It is suspected that the attenuated PRV vaccine strain is not safe for these fur-bearing animals. To confirm this, we construct a US7/US8/UL23-deleted and a US7/US8/UL23/US3-deleted rPRV based on PRV GL isolated from fox using the CRISPR/Cas9 method. Growth kinetics in vitro and pathogenicity in dogs were compared between the wild type and both rPRVs. The results showed that the growth kinetics of wild-type PRV and US7/US8/UL23-deleted rPRV were faster than those of US7/US8/UL23/US3-deleted recombinant PRV from 24 h to 48 h post infection. Moreover, PRV GL- and rPRVdelUS7/US8/UL23-infected cells formed cell-cell fusion, but the rPRVdelUS7/US8/UL23/US3-infected cells did not. Dogs challenged with wild-type PRV or US7/US8/UL23-deleted rPRV showed obvious nervous symptoms, and all the dogs died, but the group challenged with the US7/US8/UL23/US3-deleted rPRV did not show any nervous symptoms, and all the dogs survived for the duration of the experiment. Tissue viral load analyses also showed that the virulence of the US7/US8/UL23/US3-deleted rPRV was significantly reduced in dogs. This study provides evidence that the US7/US8/UL23-deleted rPRV variant still exhibits high virulence for dogs and also highlights the role of the US3 gene in the pathogenicity of PRV in dogs and provides a strategy for developing a safer vaccine.


Assuntos
Deleção de Genes , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/patogenicidade , Pseudorraiva/virologia , Vacinas Antirrábicas/imunologia , Proteínas Virais/genética , Animais , Anticorpos Antivirais/sangue , Sistemas CRISPR-Cas , Cães , Herpesvirus Suídeo 1/crescimento & desenvolvimento , Pseudorraiva/imunologia , Vacinas Antirrábicas/administração & dosagem , Vacinas Antirrábicas/genética , Vacinação , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/imunologia , Proteínas do Envelope Viral/genética , Virulência
12.
Comp Immunol Microbiol Infect Dis ; 68: 101400, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31794953

RESUMO

Newborn humans and animals are highly susceptible to viral infections. The Aujeszky´s disease virus (ADV) is a porcine herpes virus 1 which infects the respiratory tract and is lethal during the first weeks of life. Current intramuscular vaccines, applied at weaning, induce poor mucosal immunity and frequently fail to prevent and control the disease. Additionally, early vaccination has not been studied thoroughly. Therefore, we studied a systemic/mucosal route of immunization using an inactivated ADV vaccine in two-and fourteen-day-old groups of unweaned SPF miniature Vietnamese pigs, measuring the anti ADV antibody (ELISA) and cytokine (qPCR) responses in systemic and mucosal samples. The results showed that the serum ADV-specific IgG response was higher in the 14-day groups. However, the nasal IgA responses were similar in immunized groups, although the response in saliva was higher in the 2-day old group. Moreover, in vitro ADV stimulated peripheral blood mononuclear cells and lung cells from immunized pigs showed higher IFN-γ mRNA production in the 14-day old group than in younger animals and similar levels of IL-4 and IL-10 transcripts. Our data suggest that early mucosal immunization induce humoral and cellular systemic and mucosal immune responses against ADV in young pigs and younger animals may have compensatory mechanisms to overcome early immaturity and maternal-driven immune interference. Therefore, early protection in susceptible animals could be induced using this immunization protocol, opening the possibility for its application against other viral pathogens of pigs and for traslational studies in humans.


Assuntos
Anticorpos Antivirais/sangue , Imunidade nas Mucosas , Pseudorraiva/prevenção & controle , Doenças dos Suínos/prevenção & controle , Vacinas Virais/imunologia , Animais , Animais Recém-Nascidos , Citocinas/imunologia , Herpesvirus Suídeo 1 , Imunidade Celular , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Interferon gama/imunologia , Pseudorraiva/imunologia , Organismos Livres de Patógenos Específicos , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vacinas de Produtos Inativados/administração & dosagem , Vacinas de Produtos Inativados/imunologia , Vacinas Virais/administração & dosagem
13.
Antiviral Res ; 173: 104652, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31751590

RESUMO

Both classical swine fever (CSF) and pseudorabies are highly contagious, economically significant diseases of swine in China. Although vaccination with the C-strain against classical swine fever virus (CSFV) is widely carried out and severe outbreaks of CSF seldom occur in China, CSF is sporadic in many pig herds and novel sub-subgenotypes of CSFV endlessly emerge. Thus, new measures are needed to eradicate CSFV from Chinese farms. The emergence of a pseudorabies virus (PRV) variant also posed a new challenge for the control of swine pseudorabies. Here, the recombinant PRV strain JS-2012-ΔgE/gI-E2 expressing E2 protein of CSFV was developed by inserting the E2 expression cassette into the intergenic region between the gG and gD genes of the gE/gI-deletion PRV variant strain JS-2012-ΔgE/gI. The recombinant virus was stable when passaged in vitro. A single vaccination of JS-2012-ΔgE/gI-E2 via intramuscular injection fully protected against lethal challenges of PRV and CSFV. Vaccination of piglets with the recombinant JS-2012-ΔgE/gI-E2 in the presence of high levels of maternally derived antibodies (Abs) to PRV can provide partial protection against lethal challenge of CSFV. Vaccination of the recombinant PRV JS-2012-ΔgE/gI-E2 strain did not induce the production of Abs to the gE protein of PRV or to the CSFV proteins other than E2. Thus, JS-2012-ΔgE/gI-E2 appears to be a promising recombinant marker vaccine candidate against PRV and CSFV for the control and eradication of the PRV variant and CSFV.


Assuntos
Peste Suína Clássica/prevenção & controle , Expressão Gênica , Vetores Genéticos/genética , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Pseudorraiva/prevenção & controle , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Peste Suína Clássica/imunologia , Peste Suína Clássica/patologia , Vírus da Febre Suína Clássica/genética , Vírus da Febre Suína Clássica/imunologia , Ordem dos Genes , Herpesvirus Suídeo 1/patogenicidade , Pseudorraiva/imunologia , Pseudorraiva/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Suínos , Doenças dos Suínos/prevenção & controle , Vacinação , Vacinas Virais/genética , Vacinas Virais/imunologia
14.
Virology ; 536: 49-57, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31400549

RESUMO

Molecular adjuvants are vaccine delivery vehicle to increase specific antigens effectiveness. Herein, we concentrated on IgG Fc, an effective molecular adjuvant, to develop novel pseudorabies virus (PRV) subunit vaccines. Two major protective antigen genes of PRV were constructed and linked into the mouse IgG Fc fragment. The gD, gD-IgG2aFc, gB and gB-IgG2aFc proteins were expressed using a baculovirus system. Mice intranasally immunized with gD-IgG2aFc or gB-IgG2aFc subunit vaccine exhibited significantly higher PRV-specific antibodies, neutralizing antibodies and intracellular cytokines than the mice intranasally immunized with gD or gB subunit vaccine. Moreover, no histopathological lesions were observed in mice immunized with gB-IgG2aFc subunit vaccine via histopathology examination. Further, the gB-IgG2aFc subunit vaccine was efficient for PRV infection compared with live attenuated vaccine. Overall, these results suggest that IgG2a Fc fragment, as a potential molecular adjuvant, fused with PRV antigen might be a promising and efficient PRV vaccine candidate.


Assuntos
Herpesvirus Suídeo 1/efeitos dos fármacos , Fragmentos Fc das Imunoglobulinas/biossíntese , Vacinas contra Pseudorraiva/biossíntese , Pseudorraiva/prevenção & controle , Proteínas Recombinantes de Fusão/biossíntese , Proteínas do Envelope Viral/biossíntese , Adjuvantes Imunológicos/administração & dosagem , Adjuvantes Imunológicos/biossíntese , Adjuvantes Imunológicos/genética , Animais , Anticorpos Antivirais/biossíntese , Baculoviridae/genética , Baculoviridae/metabolismo , Citocinas/genética , Citocinas/imunologia , Células Epiteliais/patologia , Células Epiteliais/virologia , Feminino , Glicoproteínas/administração & dosagem , Glicoproteínas/biossíntese , Glicoproteínas/genética , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Herpesvirus Suídeo 1/patogenicidade , Imunização , Fragmentos Fc das Imunoglobulinas/administração & dosagem , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/administração & dosagem , Imunoglobulina G/biossíntese , Imunoglobulina G/genética , Rim/patologia , Rim/virologia , Camundongos , Camundongos Endogâmicos BALB C , Pseudorraiva/imunologia , Pseudorraiva/mortalidade , Pseudorraiva/virologia , Vacinas contra Pseudorraiva/administração & dosagem , Vacinas contra Pseudorraiva/genética , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Análise de Sobrevida , Suínos , Vacinas de Subunidades , Proteínas do Envelope Viral/administração & dosagem , Proteínas do Envelope Viral/genética
15.
Microb Cell Fact ; 18(1): 103, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31170996

RESUMO

BACKGROUND: Pseudorabies caused by pseudorabies virus (PRV) mainly infects the swine and seriously threatens the biosafety of the other animals, including humans. Since 2011, the outbreaks of PRV mutants have caused enormous economic losses in the swine industry, and traditional vaccines cannot offer enough protection. PRV can transmit by direct contact, aerosol transmission and pollutants. PRV mainly transmit through the nasal mucosa. After infecting the nasal epithelial cells, PRV can quickly infect the olfactory nerve and establish a potential infection of sensory neurons. Therefore, nasal immunity can effectively prevent viral colonization infection. Recombinant Bacillus subtilis has been widely used to deliver antigen and achieve adequate protective immune responses. RESULTS: The present study successfully constructed recombinant Bacillus subtilis (B. subtilis) expressing the dominant antigen regions of PRV gC and gD proteins (named B. subtilis-gCa and B. subtilis-gDa). Furtherly, we evaluated the immunogenicity of the two recombinant B. subtilis in mice. The mice intranasal administration with B. subtilis-gCa and B. subtilis-gDa effectively stimulated IgA and IgG immune responses, further regulated specific T lymphocytes proliferative response by IFN-γ and IL-10, and ultimately produced high titers of neutralizing antibodies against PRV infection. In particular, B. subtilis-gDa possessed more excellent immune effect than B. subtilis-gCa in mice. CONCLUSIONS: These results suggested that B. subtilis-gCa and B. subtilis-gDa could trigger high levels of mucosal and systemic immune responses and would be potential candidates for developing PRV vaccines.


Assuntos
Bacillus subtilis , Herpesvirus Suídeo 1/imunologia , Pseudorraiva/prevenção & controle , Vacinas Virais/administração & dosagem , Administração Intranasal , Animais , Bacillus subtilis/genética , Bacillus subtilis/imunologia , Feminino , Imunidade nas Mucosas , Camundongos Endogâmicos BALB C , Pseudorraiva/imunologia , Vacinas Virais/genética , Vacinas Virais/imunologia
16.
Vet Microbiol ; 234: 83-91, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31213277

RESUMO

Since 2011, there have been outbreaks of pseudorabies (PR) in several pig farms despite vaccination coverage, which causes substantial economic loss to the swine industry in China. The emergence of a pseudorabies virusvariant strain with high virulence and antigenic variation (e.g., PRV ZJ01), is considered to be the primary cause. In this study, truncated gB, gC, and gE of PRV ZJ01 was expressed and used to generate seven monoclonal antibodies (mAbs) against gB, gC, or gE. An indirect immunofluorescence assay (IFA) revealed that these mAbs were specific against PRV. Subsequently we identified the B cell epitopes recognized by these mAbs by Western blot. The mAbs 5A2 and 6G5 against gB recognized the same B cell linear epitope at 576SAVATAA582, the mAb 5D10 against gC recognized the B cell linear epitope at 134GETFE138, mAb 7C5 against gC recognized the B cell linear epitope at 143RRGRFRSPDAD153, and mAbs 3E1, 3H8, and 4D2 against gE recognized the same B cell linear epitope at 151IGDYL155 of gE. Biological information analysis showed that these B cell linear epitopes are highly conserved among different PRV isolates and the epitope 143RRGRFRSPDAD153 with a high antigenic index and high hydrophilicity, fully exposed on the surface of the gC, is likely to be an important B cell epitope. These mAbs and their defined epitopes may provide useful tools for the study of the structure and function of the PRV protein, analysis of antigenic epitope characteristics, and establishment of antibody detection methods.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos de Linfócito B/imunologia , Pseudorraiva/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Linhagem Celular , Feminino , Hibridomas , Camundongos , Camundongos Endogâmicos BALB C , Suínos
17.
Vet Res ; 50(1): 9, 2019 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-30717799

RESUMO

As a key cellular transcription factor that plays a central role in cellular responses to a broad range of stress factors, p53 has generally been considered as a host cell restriction factor for various viral infections. However, the defined roles of p53 in pseudorabies virus (PRV) replication, pathogenesis, and host responses remain unclear. In the present study, we initially constructed a p53 overexpressing a porcine kidney epithelial cell line (PK-15) to detect the effect of p53 on PRV replication in vitro. The results show that viral glycoprotein B (gB) gene copies and the titers of virus were significantly higher in p53 overexpressing PK-15 cells than in PK-15 and p53 inhibitor treated p53 overexpressing PK-15 cells. A similar result was also found in the p53 inhibitor PFT-α-treated PK-15 cells. We then examined the effects of p53 on PRV infection in vivo by using p53-knockout (p53-/-) mice. The results show that p53 knockout not only led to significantly reduced rates of mortality but also to reduced viral replication and development of viral encephalitis in the brains of mice following intracranial inoculation. Furthermore, we examined the effect of p53 knockout on the expression of the reported host cell regulators of PRV replication in the brains of mice by using RNA sequencing. The results show that p53 knockout downregulated the interferon (IFN) regulator genes, chemokine genes, and antiviral genes after PRV infection. This finding suggests that p53 positively regulates viral replication and pathogenesis both in vitro and in vivo. These findings offer novel targets of intrinsic host cell immunity for PRV infection.


Assuntos
Herpesvirus Suídeo 1/fisiologia , Herpesvirus Suídeo 1/patogenicidade , Imunidade Inata , Pseudorraiva/imunologia , Doenças dos Suínos/imunologia , Proteína Supressora de Tumor p53/genética , Replicação Viral , Animais , Linhagem Celular , Interações Hospedeiro-Patógeno , Pseudorraiva/fisiopatologia , Pseudorraiva/virologia , Suínos , Doenças dos Suínos/fisiopatologia , Doenças dos Suínos/virologia , Proteína Supressora de Tumor p53/metabolismo , Virulência
18.
J Virol ; 93(7)2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30700600

RESUMO

Natural killer (NK) cells are components of the innate immunity and are key players in the defense against virus-infected and malignant cells. NK cells are particularly important in the innate defense against herpesviruses, including alphaherpesviruses. Aggravated and life-threatening alphaherpesvirus-induced disease has been reported in patients with NK cell deficiencies. NK cells are regulated by a diversity of activating and inhibitory cell surface receptors that recognize specific ligands on the plasma membrane of virus-infected or malignant target cells. Although alphaherpesviruses have developed several evasion strategies against NK cell-mediated attack, alphaherpesvirus-infected cells are still readily recognized and killed by NK cells. However, the (viral) factors that trigger NK cell activation against alphaherpesvirus-infected cells are largely unknown. In this study, we show that expression of the gB glycoprotein of the alphaherpesvirus pseudorabies virus (PRV) triggers NK cell-mediated cytotoxicity, both in PRV-infected and in gB-transfected cells. In addition, we report that, like their human and murine counterpart, porcine NK cells express the activating receptor paired immunoglobulin-like type 2 receptor beta (PILRß), and we show that gB expression triggers increased binding of recombinant porcine PILRß to the surfaces of PRV-infected cells and gB-transfected cells.IMPORTANCE Natural killer (NK) cells display a prominent cytolytic activity against virus-infected cells and are indispensable in the innate antiviral response, particularly against herpesviruses. Despite their importance in the control of alphaherpesvirus infections, relatively little is known about the mechanisms that trigger NK cell cytotoxicity against alphaherpesvirus-infected cells. Here, using the porcine alphaherpesvirus pseudorabies virus (PRV), we found that the conserved alphaherpesvirus glycoprotein gB triggers NK cell-mediated cytotoxicity, both in virus-infected and in gB-transfected cells. In addition, we report that gB expression results in increased cell surface binding of porcine paired immunoglobulin-like type 2 receptor beta (PILRß), an activating NK cell receptor. The interaction between PILRß and viral gB may have consequences that stretch beyond the interaction with NK cells, including virus entry into host cells. The identification of gB as an NK cell-activating viral protein may be of importance in the construction of future vaccines and therapeutics requiring optimized interactions of alphaherpesviruses with NK cells.


Assuntos
Glicoproteínas/imunologia , Herpesvirus Suídeo 1/imunologia , Células Matadoras Naturais/imunologia , Glicoproteínas de Membrana/imunologia , Pseudorraiva/imunologia , Receptores de Células Matadoras Naturais/imunologia , Proteínas Virais/imunologia , Animais , Linhagem Celular , Humanos , Rim/virologia , Camundongos , Coelhos , Suínos , Internalização do Vírus
19.
BMC Vet Res ; 15(1): 2, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606159

RESUMO

BACKGROUND: Since 2011, pseudorabies caused by a variant PRV has re-emerged in many Chinese Bartha-K61-vaccinated pig farms. An efficacious vaccine is necessary to control this disease. We described the construction of a gD&gC-substituted pseudorabies virus (PRV B-gD&gCS) from the Bartha-K61 (as backbone) and AH02LA strain (as template for gD and gC genes) through bacterial artificial chromosome (BAC) technology using homologous recombination. The growth kinetics of PRV B-gD&gCS was compared with Bartha-K61. Its safety was evaluated in 28-day-old piglets. Protection efficacy was tested in piglets by lethal challenge with AH02LA at 7 days post vaccination, including body temperature, clinical symptoms, virus shedding, mortality rate, and lung lesions. RESULTS: The results showed that a BAC clone of Bartha-K61 and a B-gD&gCS clone were successfully generated. The growth kinetics of PRV B-gD&gCS strain on ST (Swine testicular) cells was similar to that of the Bartha-K61 strain. No piglets inoculated intramuscularly with PRV B-gD&gCS strain exhibited any clinical symptoms or virus shedding. After AH02LA challenge, all piglets in PRV B-gD&gCS and Bartha-K61 groups (n = 5 each) survived without exhibiting any clinical symptoms and high body temperature. More importantly, PRV B-gD&gCS strain completely prevented virus shedding in 2 piglets and reduced virus shedding post challenge in the other 3 piglets as compared with Bartha-K61 group. CONCLUSIONS: Our results suggest that PRV B-gD&gCS strain is a promising vaccine candidate for the effective control of current severe epidemic pseudorabies in China.


Assuntos
Herpesvirus Suídeo 1/imunologia , Vacinas contra Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Doenças dos Suínos/prevenção & controle , Animais , Animais Recém-Nascidos/imunologia , Animais Recém-Nascidos/virologia , China , Variação Genética/genética , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/fisiologia , Pseudorraiva/imunologia , Suínos , Doenças dos Suínos/imunologia , Doenças dos Suínos/virologia , Vacinas Sintéticas , Eliminação de Partículas Virais
20.
Acta Virol ; 62(4): 455-458, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30472877

RESUMO

Wild boar is an important reservoir of Aujeszky's disease virus (ADV). There is concern that transmission of this virus from wild boar to domestic pigs is possible. The aim of this work was to compare the antibody response produced by single dose of a gE-deleted ADV vaccine in wild boar to revaccinated animals, to assess if simple single-dose vaccination plans should be examined as a possible control measure against ADV in wild boar. Twenty-five wild boar (ages ranging between 2.5 to 5 months) were included in this study and distributed in three different groups: a control group (n = 5), a single-dose group (10 animals vaccinated only with one dose (day 0)) and a revaccinated group [10 animals vaccinated (day 0) and revaccinated (day 28)]. Mean antibody titers against ADV were determined in three groups using an ELISA assay at three different time points [day 0 (pre-vaccination), 28 (post 1st dose) and 56 (post 2nd dose)]. At day 28, single-dose and revaccinated groups showed a significant increment of antibody titers whereas antibodies in the control group remained stable. At day 56, revaccinated animals did not show a significant increment and antibody titers were similar to those found in animals vaccinated with one dose. These results indicate that vaccination with one dose produces a similar early antibody response to revaccination and therefore, should be examined as a possible control measure against ADV in wild boar. Keywords: Aujeszky's disease; serology; vaccination; wild boar.


Assuntos
Anticorpos Antivirais , Herpesvirus Suídeo 1 , Pseudorraiva , Sus scrofa , Doenças dos Suínos/imunologia , Animais , Anticorpos Antivirais/imunologia , Formação de Anticorpos , Imunização Secundária , Pseudorraiva/imunologia , Pseudorraiva/prevenção & controle , Pseudorraiva/virologia , Suínos , Doenças dos Suínos/prevenção & controle , Doenças dos Suínos/virologia , Vacinação/veterinária
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