RESUMO
BACKGROUND: Encephalomyocarditis virus (EMCV) can cause myocarditis, respiratory failure, reproductive failure, and sudden death in pre-weaned piglets, which has been isolated in China. EMCV VP1 protein was one of the most important structural proteins and played an important role in the protective immunity. In this study, 10 monoclonal antibodies (McAbs) against EMCV VP1 were screened and identified. RESULTS: Epitope mapping results indicated that McAbs (6E11, 7A7, 7C9) specifically recognized the linear epitopes V(2)ENAEK(7), McAbs (1D1, 2A2, 5A1, 5A11, 5G1) recognized the epitope F(19)VAQPVY(25), and McAbs 1G8 and 3A9 recognized P(42)IGAFTVK(49). Protein sequence alignment of VP1 with 16 EMCV isolates indicated that the epitope F(19)VAQPVY(25) was conserved in all the reference strains. The epitopes P(42)IGAFTVK(49) and V(2)ENAEK(7) only had 1 or 2 variable amino acid among the reference strains. The 3D model analysis results showed that these epitopes presented as spheres were shown within the context of the complete particle. CONCLUSIONS: In this study, ten McAbs against EMCV VP1 were developed and three B-cells epitopes (2-7aa, 19-25aa and 42-49aa) were defined in VP1. All the results herein will promote the future investigations into the function of VP1 of EMCV and development of diagnostic methods of EMCV.
Assuntos
Antígenos Virais/imunologia , Proteínas do Capsídeo/imunologia , Vírus da Encefalomiocardite/imunologia , Epitopos de Linfócito B/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Antígenos Virais/isolamento & purificação , Proteínas do Capsídeo/isolamento & purificação , Infecções por Cardiovirus/virologia , China , Vírus da Encefalomiocardite/isolamento & purificação , Mapeamento de Epitopos , Suínos , Doenças dos Suínos/virologiaRESUMO
Encephalomyocarditis virus (EMCV) can infect many host species and cause acute myocarditis and sudden death in preweaned piglets. In this study, an EMCV strain (NJ08) was isolated from newborn pigs with clinical signs on a pig farm in mideastern China. It was identified by indirect immunofluorescence assay and reverse-transcription polymerase chain reaction. Experiments showed that the isolate could cause severe clinical symptoms and pathological changes in mice but no obvious clinical and pathological changes in commercial piglets. Complete genomic sequencing showed that the NJ08 strain was 78.3% to 100% identical with other isolates in regions coding for various proteins. Phylogenetic analysis showed that the NJ08 isolate belonged to subgroup Ia. This study confirmed that an EMCV isolate from pigs could be fatal to mice and provided new epidemiologic data on EMCV in China.
Assuntos
Infecções por Cardiovirus/veterinária , Vírus da Encefalomiocardite/isolamento & purificação , Doenças dos Suínos/virologia , Animais , Animais Recém-Nascidos , Sequência de Bases , Bioensaio/veterinária , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Infecções por Cardiovirus/virologia , China , Vírus da Encefalomiocardite/genética , Vírus da Encefalomiocardite/patogenicidade , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/veterinária , RNA Viral/química , RNA Viral/genética , Alinhamento de Sequência , SuínosRESUMO
Encephalomyocarditis virus (EMCV) infection can cause myocarditis and sudden death in pre-weaned piglets and severe reproductive failure in sows. There are no specific antiviral drugs for the treatment of the virus infection. In this study, four recombinant adenoviruses expressing small interfering RNAs (siRNAs) targeting to 1D or 3AB protein genes of EMCV were constructed and their inhibition efficiency on the replication of EMCV was evaluated in both Marc-145 cells and mice. The results showed that the rAd5 expressing siRNAs (rAd5) could inhibit EMCV replication in Marc-145 cells in protein and mRNA levels, as well as the virus yield by approximately 100-1000 times. And the inhibition of viral replication was sustained for 72h and dose-dependent. Animal experiment results showed that EMCV VP1 mRNA level in the brain of mice in the rAd5 groups were obviously lower than those in rAd-G1 and challenge control groups. The virus yields in rAd-1D-2 and rAd-3AB-1 groups were markedly decreased by more than 90.0%. The survival rates of mice in rAd-1D-2 group were significantly higher than those in challenge control groups. Furthermore, the survival mice only showed minor microscopic lesions in brain and minor edema of nerve cell, which was obviously slighter than those in challenge control groups. These results indicated that siRNAs mediated by the adenovirus could provide protective efficacy against EMCV challenge in mice. It might provide a potential strategy for combating EMCV.
Assuntos
Adenoviridae/genética , Infecções por Cardiovirus/terapia , Vírus da Encefalomiocardite/genética , RNA Interferente Pequeno/genética , RNA Viral/genética , Animais , Encéfalo/patologia , Encéfalo/virologia , Edema Encefálico/patologia , Edema Encefálico/virologia , Proteínas do Capsídeo/genética , Infecções por Cardiovirus/virologia , Linhagem Celular , Modelos Animais de Doenças , Vírus da Encefalomiocardite/fisiologia , Feminino , Genes Virais , Engenharia Genética/métodos , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos/genética , Plasmídeos/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , Fatores de Tempo , Carga Viral , Replicação ViralRESUMO
Encephalomyocarditis virus (EMCV) could infect many host species and cause acute myocarditis and sudden death in pre-weaned piglets. It was necessary to develop new antiviral strategies for the treatment of the virus infection. Here, four plasmids expressing shRNA (small hairpin RNA) targeted to 1D or 3AB protein genes of EMCV were constructed and their inhibition efficiency on the replication of EMCV was evaluated in both BHK21 cells and mice. The results showed that three out of those four shRNA constructs could significantly inhibit EMCV replication in BHK21 cells on the levels of viral RNA and protein. Moreover, it was found that the shRNAs could suppress significantly the load of EMCV in the brain tissue of the mice pretreated with the constructs for 6-24 h. The clinical signs and pathological lesions of the mice in the groups inoculated with the shRNA constructed were milder obviously, compared with those in pSUPER-mN3 and challenge control groups. The survival rates of mice inoculated with pSUPER-3AB-1, pSUPER-3AB-2, and pSUPER-1D-1 for 12 h was 100, 80, and 40%, respectively, while, in the control groups it was only 20%. It indicated that the vector-based shRNA targeting to 3AB and 1D genes might be a potential anti-EMCV strategy.