Comparative sequence and structural analysis of the ORF095 gene, a vaccinia virus A4L homolog of capripoxvirus in sheep and goats.

Sheeppox and goatpox are important transboundary animal viral diseases of sheep and goats caused by sheeppox virus (SPPV) and goatpox virus (GTPV), respectively, of the genus Capripoxvirus, family Poxviridae. Among the proteins encoded by the capripoxvirus (CaPV) genome, ORF095 (vaccinia virus A4L homolog) is an immunodominant virion core protein that plays a pivotal role in virus assembly and morphogenesis. In the present study, sequence analysis of the ORF095 genes of 27 SPPV and GTPV isolates or field samples from different geographical regions of India was performed, and structure was prediction was done by homology modeling. A multiple sequence alignment of different CaPV isolates revealed that CaPV-A4L is highly conserved, with several species-specific signature residues, namely A93, A216, A315, G136 and G146 in GTPV, G47, A63, A168 and A276 in SPPV, and G48 and C98 in lumpy skin disease virus (LSDV). Phylogenetically, the CaPV isolates were separated into three major clusters, GTPV, SPPV and LSDV, based on the complete coding sequence of the CaPV-A4L gene. Genus-specific clustering of poxviruses was observed in phylogenetic analysis based on A4L protein homologs of chordopoxviruses. A secondary structure prediction showed the presence of six α-helices and one ß-sheet as well as some coils. The signature residues identified here are potentially useful for genotyping, and the predicted characteristics of the CaPV-A4L protein make it an ideal candidate for use as an immunogenic or diagnostic antigen for the development of immunoassays in  the sero-evaluation of CaPV in target hosts.

Circulation of orf viruses containing the NZ7-like vascular endothelial growth factor (VEGF-E) gene type in India.

Orf, a poxviral skin infection of small ruminants is caused by orf virus (ORFV) of the genus Parapoxvirus of the Poxviridae family. Vascular endothelial growth factor (VEGF) is an important virulence factor that is responsible for proliferative lesions in parapoxviral infections. VEGF gene shows high intra- and inter-species variability. Two variants of VEGF have been described globally in ORFV, viz. NZ2- and NZ7-like. In the present study, ORFV isolates of different geographic regions of India were analysed on the basis of the VEGF gene. Indian ORFV isolates showed 95.7-100 % nucleotide (nt) and 78.4-99.3 % amino acid (aa) identity with each other, except ORFV-Assam/LK/14 and ORFV-Meghalaya/03 which shared 85.1-88.35 % and 79.1-81.8 % identity, at nt and aa levels, respectively with other Indian ORFV isolates. All Indian ORFVs under the study demonstrated 83.5-99.1 % nt and 80.5-97.9 % aa identity with NZ7-like VEGF as compared to 41.2-44.8 % nt and 30.7-38.4 % aa identity with NZ2-like VEGF on comparison with global ORFV strains. Phylogenetic analysis based on the VEGF gene showed two clusters of ORFV in which the Indian ORFVs clustered with NZ7-like VEGF from global ORFV strains, mostly from China. Despite the considerable variation, VEGF protein from Indian ORFV strains showed conserved VEGF homology domain with eight cysteine residues. Homology modeling of Indian ORFV strains predicted the presence of extended Loop 3 similar to NZ7-like VEGF. Therefore, the present study showed the circulation of ORFV strains with comparatively less variable NZ7-like VEGF in India which implicates its importance in the epidemiology of ORFV infections in the country.

Poxviral E3L ortholog (Viral Interferon resistance gene) of orf viruses of sheep and goats indicates species-specific clustering with heterogeneity among parapoxviruses.

Orf is a contagious disease posing a serious threat to animal and human health. E3L is one of the evolutionarily acquired immunomodulatory proteins present in orf virus (ORFV) and is responsible for conferring resistance to interferons among poxviruses. Genetic analysis of ORFV isolates of different geographical regions including Indian subcontinent targeting viral interferon resistance (VIR) gene (a homolog of vaccinia virus E3L gene) revealed a high percentage of identity among themselves and other ORFV isolates at both nt and aa levels as compared to low identity among parapoxviruses (PPVs). Phylogenetic analysis showed species-specific clustering among PPVs along with sub-clusters based on host species of origin among ORFVs infecting sheep and goats. Conserved amino acids in N-terminal Z-DNA binding domain and C-terminal ds RNA binding domain of VIR proteins of PPVs corresponding to ORFV VIR positions namely N37, Y41, P57, and W59 (necessary for Z-DNA binding) and E116, F127, F141, and K160 (necessary for dsRNA binding) were found. Further, the predicted protein characteristics and homology model of VIR protein of ORFV showed high structural conservation among poxviruses. This study on E3L genetic analysis of ORFV isolates may provide a better understanding of the molecular epidemiology of circulating strains in India and neighboring countries. Also, E3L deleted or mutated ORFV may be an as vaccine candidate and/or compounds blocking E3L may prove as an effective method for treating broad spectrum poxviral infections, suggesting a wider application in control of poxvirus infections.

Prokaryotic expression, purification and evaluation of goatpox virus ORF117 protein as a diagnostic antigen in indirect ELISA to detect goatpox.

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.

Molecular evidence and phylogenetic analysis of orf virus isolates from outbreaks in Tripura state of North-East India.

This study describes the first confirmed report of contagious ecthyma in Black Bengal goats from Tripura state, a North-Eastern state of India situated at the Indo-Bangladesh border. Outbreaks were characterized by the high rates of morbidity (58-67%), low mortality (8-10%) and case fatality (11-15%). The etiology of the outbreaks was confirmed as orf virus (ORFV) by standard virological/serological and molecular techniques including sequence analysis of B2L, a major envelop protein gene of genus Parapoxvirus. Sequence and phylogenetic analysis based on B2L gene of ORFV isolates from Tripura revealed that they were closely related to each other and also to other Indian isolates, in particular to ORFV-Shahjahanpur 82/04 isolate from North India. They revealed several specific nucleotide/amino acid substitutions, namely G299A (G100D), G660A, C705T, C795T (N267D) and G872A (R291H) which may be of notable epidemiological significance. This report necessitates the systematic investigation of orf outbreaks in susceptible populations including wild species particularly at transboundary regions by use of rapid diagnostics to control the infection by deploying an effective vaccine/therapeutics and better managemental practices.

Seroprevalence of Capripoxvirus infection in sheep and goats among different agro-climatic zones of Odisha, India.

AIM: The study was undertaken to assess the prevalence of antibodies to Capripoxviruses among small ruminants of Odisha, India. MATERIALS AND METHODS: A total of 500 random serum samples collected from 214 sheep and 286 goats across 10 agro-climatic zones of Odisha, were screened using whole virus antigen-based indirect ELISA for antibodies against Capripoxviruses. Results were analyzed by suitable statistical methods. RESULTS: Screening of 500 serum samples showed seropositivity of 8.88% and 31.47% in sheep and goats, respectively, for Capripoxviruses. The prevalence rate according to agro-climatic zone ranged from 0% (North Eastern coastal plain zone) to 48.57% (North central plateau zone) for goat pox, and 0% (Western undulating zone and North central plateau) to 22.22% (South Eastern ghat zone) for sheep pox. The difference in prevalence rates among the various agro-climatic zones was statistically significant (p<0.05) for goats, but not for sheep. Antibody prevalence rates among various districts were recorded to be the highest in Jagatsinghpur (30%) for sheep pox and Dhenkanal (80%) for goat pox. CONCLUSIONS: The study revealed serological evidence of Capripoxvirus infection in sheep and goat populations in the study area, in the absence of vaccination. Systematic investigation, monitoring, and reporting of outbreaks are necessary to devise control strategies.

Genetic analysis of L1R myristoylated protein of Capripoxviruses reveals structural homogeneity among poxviruses.

Sheeppox virus (SPPV) and goatpox virus (GTPV) are members of the genus Capripoxvirus (CaPV) of the family Poxviridae. CaPVs are responsible for important contagious diseases of small ruminants that are enzootic to the Indian sub-continent, Central and Northern Africa and the Middle East. In the present study, the sequence and phylogenetic analysis of the L1R gene of sixteen CaPV isolates (seven SPPV and nine GTPV) from India were performed along with 3D homology modeling of the L1R protein. L1R is a myristoylated protein responsible for virion assembly and being present on intracellular mature virion (IMV) surface, it is also a potent target for eliciting neutralizing antibodies. Sequence analysis of CaPV L1R gene revealed an ORF of 738bp with >99% and >96% identity within species and between species, respectively, at both nucleotide as well as amino acid levels. Phylogenetic analysis displayed distinct clusters of members of genus Capripoxvirus, as GTPV, SPPV and LSDV. L1R at the protein level showed various species-specific signature residues that may be useful for future grouping or genotyping of CaPV members. CaPV L1R was predicted to possess myristoylation motif GAAASIQTTVNTLNEKI and a potential N-glycosylation site at amino acid residue 50 (Asn). Despite of different host specificity in poxviruses, comparative sequence analysis of L1R proteins revealed highly conserved nature with presence of myristoylation motif (GXXXS) and six cysteine residues forming three disulfide bonds among all poxviruses. The conserved and immunogenic nature of the CaPV L1R gene may prove to be a potential candidate/target for developing molecular diagnostics including recombinant protein based assays and prophylactics for the control of CaPV diseases in tropical countries like India.

Expression and evaluation of recombinant P32 protein based ELISA for sero-diagnostic potential of capripox in sheep and goats.

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.