RESUMEN
Goatpox is an economically significant transboundary viral disease of goats that is caused by goatpox virus (GTPV). This study describes the prokaryotic expression of the GTPV ORF117 protein, a homologue of vaccinia virus A27L, and evaluation of its diagnostic potential in ELISA. The GTPV ORF117 gene was cloned into the pET32a vector to express recombinant ORF117 protein (rA27L) in E. coli BL21-CodonPlus (DE3)-RIPL. The bacterial expression of the protein was confirmed by western blot analysis using anti-GTPV polyclonal antibodies that detected rA27L, which is ~ 35 kDa in size. rA27L was affinity purified under native conditions and used to assess the antibody response in an optimized indirect ELISA. The purified antigen specifically reacted with anti-GTPV and anti-SPPV serum in ELISA. A preliminary screening of random and purposive serum samples (n = 520) from sheep and goats using this optimized ELISA gave a positivity rate of 19.4 % with a diagnostic specificity of 88.7% and diagnostic sensitivity of 98.5% when compared to the gold standard serum neutralization test. Our results suggest that the indirect ELISA based on the rA27L protein has potential for serosurveillance and seromonitoring of GTPV in goats.
Asunto(s)
Antígenos Virales/análisis , Antígenos Virales/genética , Capripoxvirus/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Enfermedades de las Cabras/virología , Infecciones por Poxviridae/veterinaria , Proteínas Virales/análisis , Proteínas Virales/genética , Animales , Antígenos Virales/aislamiento & purificación , Antígenos Virales/metabolismo , Capripoxvirus/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Enfermedades de las Cabras/diagnóstico , Cabras , Infecciones por Poxviridae/diagnóstico , Infecciones por Poxviridae/virología , Proteínas Virales/aislamiento & purificación , Proteínas Virales/metabolismoRESUMEN
The study is aimed to develop and evaluate a recombinant P32 protein based ELISA for sero-monitoring and sero-surveillance using known and random/suspected serum samples for capripox infections from sheep and goats. Truncated P32 gene of goatpox virus (with an ORF of 750 bp) was expressed in E. coli BL-21 CodonPlus (DE3)-RIPL cells using pET32a vector and characterized by SDS-PAGE analysis and confirmed by western blotting as 48 kDa polyhistidine-tagged fusion protein. The protein was purified under denaturing conditions using 8M urea and characterized by SDS-PAGE and immunoblotting. The purified protein was used for optimizing ELISA in a chequerboard titration method using anti-GTPV serum as known positive. The optimized conditions were found to be 300 ng of protein/well, 1:10 dilution of antibody, 1:10000 dilution of rabbit anti-goat/sheep conjugate with 3% skim milk powder and 2% gelatin in phosphate buffer saline containing tween-20 as blocking buffer. The expressed protein was specific only for goatpox virus and sheeppox virus but did not react with related viruses of sheep and goats namely orf virus, peste de petits ruminants virus, bluetongue virus and foot and mouth disease virus. The optimized ELISA was evaluated using pre-vaccinated, post-vaccinated and also post-challenge sera. The assay was found to have a diagnostic specificity of 100/98.7% and sensitivity of 97.1/98.1% when compared to whole virus antigen based ELISA/SNT by receiver operating characteristic (ROC) analysis. The optimized ELISA is able to determine the progression of antibody response against GTPV and SPPV following vaccination and challenge in sheep and goats. The rP32 protein based ELISA was evaluated using random field serum samples (n = 1008) suspected for sheeppox and goatpox and it has shown positivity rate as 24.4%. The rP32 protein based ELISA was found to be specific and sensitive for sero-evaluation of sheeppox virus and goatpox virus following vaccination and infection in sheep and goats.
Asunto(s)
Capripoxvirus/aislamiento & purificación , Enfermedades de las Cabras/diagnóstico , Infecciones por Poxviridae/diagnóstico , Infecciones por Poxviridae/veterinaria , Enfermedades de las Ovejas/diagnóstico , Proteínas Virales/inmunología , Animales , Anticuerpos Antivirales/inmunología , Ensayo de Inmunoadsorción Enzimática , Enfermedades de las Cabras/sangre , Enfermedades de las Cabras/virología , Cabras/sangre , Cabras/virología , Infecciones por Poxviridae/sangre , Infecciones por Poxviridae/virología , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sensibilidad y Especificidad , Pruebas Serológicas , Ovinos/sangre , Ovinos/virología , Enfermedades de las Ovejas/sangre , Enfermedades de las Ovejas/virología , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
The fusion gene (ORF 117) sequences of twelve (n = 12) capripox virus isolates namely sheeppox (SPPV) and goatpox (GTPV) viruses from India were demonstrated for their genetic and phylogenetic relationship among them. All the isolates were confirmed for their identity by routine PCR before targeting ORF 117 gene for sequence analysis. The designed primers specifically amplified ORF 117 gene as 447 bp fragment from total genomic DNA extracted from all the isolates. Sequence analysis revealed a significant percentage of identity among GTPV, SPPV and between them at both nucleotide and amino acid levels. The topology of the phylogenetic tree revealed that three distinct clusters corresponding to SPPV, GTPV and lumpy skin disease virus was formed. However, SPPV Pune/08 and SPPV Roumanian Fanar isolates were clustered into GTPV group as these two isolates showed a 100 and 99.3 % identity with GTPV isolates of India at nt and aa levels, respectively. Protein secondary structure and 3D view was predicted and found that it has high antigenic index and surface probability with low hydrophobicity, and it can be targeted for expression and its evaluation to explore its diagnostic potential in epidemiological investigation in future.
