RESUMO
The major immunogenic proteins (E(rns), E2 and NS3) of classical swine fever virus (CSFV) (Shimen strain) were expressed in E. coli and purified by affinity chromatography. The recombinant antigens were applied to develop multiple enzyme-linked immunosorbent assays (ELISAs) for the detection of specific antibodies in pig sera. Optimum cut-off values were determined by receiver operating characteristic (ROC) analysis after testing 201 sera of vaccinated pigs and 64 negative sera of unvaccinated piglets. The multiple ELISAs were validated with 265 pig sera yielding high sensitivity and specificity in comparison with the virus neutralization results. The results demonstrated that multiple ELISAs can be a valuable tool for the detection of CSFV infection and serological surveys in CSFV-free countries or for the evaluation of the antibody responses in pigs induced by a live attenuated C-strain vaccination.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Febre Suína Clássica/imunologia , Peste Suína Clássica/diagnóstico , Técnicas de Laboratório Clínico/métodos , Medicina Veterinária/métodos , Virologia/métodos , Animais , Antígenos Virais/genética , Antígenos Virais/isolamento & purificação , Cromatografia de Afinidade , Ensaio de Imunoadsorção Enzimática/métodos , Escherichia coli/genética , Expressão Gênica , Curva ROC , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Sensibilidade e Especificidade , Suínos , Proteínas Estruturais Virais/genética , Proteínas Estruturais Virais/isolamento & purificaçãoRESUMO
E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) and has an unusual feature of RNase activity. In the present study, we demonstrate that E(rns) counteracts Newcastle disease virus (NDV)-mediated induction of IFN-beta. For this purpose, E(rns) fused to the enhanced green fluorescent protein (EGFP) was transiently expressed in porcine kidney 15 (PK15) cells. In luciferase activity assay, E(rns)-EGFP was found to prevent IFN-beta promoter-driven luciferase expression and block the induction of IFN-beta promoter mediated by NDV in a dosedependent manner. Through IFN-specific semi-quantitative RT-PCR detection, obvious decrease of IFN-beta mRNA in NDV-infected PK15 cells was observed in the presence of E(rns)-EGFP. In contrast, EGFP alone showed none of this block capacity. In addition, E(rns)-EGFP mutations with RNase inactivation were also found to block NDV-mediated induction of IFN-beta. These evidences establish a novel function for CSFV E(rns) glycoprotein in counteraction of the IFN-beta induction pathway.
Assuntos
Vírus da Febre Suína Clássica/metabolismo , Interferon beta/genética , Vírus da Doença de Newcastle/fisiologia , Proteínas do Envelope Viral/metabolismo , Animais , Linhagem Celular , Vírus da Febre Suína Clássica/genética , Regulação da Expressão Gênica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinética , Luciferases/genética , Luciferases/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Ribonucleases/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Suínos , Transfecção , Proteínas do Envelope Viral/genéticaRESUMO
E(rns) is an envelope glycoprotein of classical swine fever virus (CSFV) with unusual RNase activity. Recently, E(rns) was found to have a new function of counteracting the beta-interferon (IFN-beta) induction pathway. In this study, wildtype ErnsSM and two mutated E(rns) proteins ErnsH297k and ErnsH346k were expressed in insect cells and purified for RNase activity and function analysis. RNase activity assay in vitro demonstrated that only wildtype E(rns) protein had RNase activity. However, both wildtype ErnsSM and the two mutated E(rns)ErnsH297k and ErnsH346k as exogenous proteins had a block effect on Newcastle disease virus (NDV)-mediated IFN-beta promoter induction.