Species-specific inhibition of capripoxvirus replication by host antiviral protein kinase R.

The role of interferon (IFN)-induced protein kinase R (PKR) in capripoxvirus (CaPV)-infected cells remains unknown. In this study, we show that CaPV infection triggered PKR and eukaryotic translation initiation factor 2 alpha (eIF2α) protein phosphorylation in a dose-dependent manner, and that this leads to decreased CaPV replication. Overexpression of PKR compromised viral gene expression and inhibited sheeppox virus (SPPV) replication. Downregulation of PKR with siRNAs significantly decreased eIF2α phosphorylation and reduced the mRNA level of IFN-ß, which increased virus replication. In luciferase assays, species-different CaPVs K3L proteins inhibited sheep PKR (sPKR): goatpox virus K3L strongly inhibited sPKR and goat PKR (gPKR), but SPPV K3L only partially inhibited gPKR. These results are the first to show that SPPV infection induces phosphorylation of eIF2α through PKR activation, which then results in restriction of CaPV replication. Furthermore, our data show that CaPV K3L inhibits PKR in a species-specific manner. The results presented are consistent with the hypothesis that different levels of PKR inhibition by K3L orthologs from various viruses could potentially contribute to the host range function of K3L.

Construction of an attenuated goatpox virus AV41 strain by deleting the TK gene and ORF8-18.

Goatpox virus (GTPV) is prevalent in goats and is associated with high mortality. This virus causes fever, skin nodules, lesions in the respiratory and lymph node enlargement. Considering the safety risks and side effects of vaccination with attenuated live GPTV vaccine strain AV41, an attenuated goatpox virus (GTPV-TK-ORF), was constructed by deleting non-essential gene fragments without affecting replication and related to the virulence and immunomodulatory functions of the goatpox virus AV41 strain (GTPV-AV41) using homologous recombination and the Cre (Cyclization Recombination Enzyme)/Loxp system. The results of both in vivo and in vitro experiments demonstrated that GTPV-TK-ORF was safer than wild type GTPV-AV41, possessed satisfactory immunogenicity, and could protect goats from a virulent GTPV-AV40 infection. Moreover, the IFN-γ, GTPV-specific antibody, and neutralizing antibody levels in the GTPV-TK-ORF-immunized group were significantly higher than that in the normal saline control group following immunization (P < 0.01). Thus, GTPV-TK-ORF may be used as a potential novel vaccine and viral vector with good safety and immunogenicity.

RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells.

BACKGROUND: Goatpox is an economically important disease in goat and sheep-producing areas of the world. Many vaccine strategies developed to control the disease are not yet completely successful. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses. However, none of these studies has been attempted to study GTPV. In the interest of exploiting improved methods to control goat pox, it is participated that RNAi may provide effective protection against GTPV. In this study we show the suppression of Goatpox virus (GTPV) replication via knockdown of virion core protein using RNA interference. RESULTS: Four short interfering RNA (siRNA) sequences (siRNA-61, siRNA-70, siRNA-165 and siRNA-296) against a region of GTPV ORF095 were selected. Sense and antisense siRNA-encoding sequences separated by a hairpin loop sequence were designed as short hairpin RNA (shRNA) expression cassettes under the control of a human U6 promoter. ORF095 amplicon was generated using PCR, and then cloned into pEGFP-N1 vector, named as p095/EGFP. p095/EGFP and each of the siRNA expression cassettes (p61, p70, p165 and p296) were co-transfected into BHK-21 cells. Fluorescence detection, flow cytometric analysis, retro transcription PCR (RT-PCR) and real time PCR were used to check the efficiency of RNAi. The results showed that the ORF095-specific siRNA-70 effectively down-regulated the expression of ORF095. When Vero cells were transfected with shRNA expression vectors (p61/GFP, p70/GFP, p165/GFP and p296/GFP) and then infected with GTPV, GTPV-ORF095-70 was found to be the most effective inhibition site in decreasing cytopathic effect (CPE) induced by GTPV. The results presented here indicated that DNA-based siRNA could effectively inhibit the replication of GTPV (approximately 463. 5-fold reduction of viral titers) on Vero cells. CONCLUSIONS: This study demonstrates that vector-based shRNA methodology can effectively inhibit GTPV replication on Vero cells. Simultaneously, this work represents a strategy for controlling goatpox, potentially facilitating new experimental approaches in the analysis of both viral and cellular gene functions during of GTPV infection.

A vero cell derived combined vaccine against sheep pox and Peste des Petits ruminants for sheep.

The combined sheep pox and Peste des Petits ruminants (PPR) vaccine was prepared in lyophilized form containing recommended doses of both vaccine viruses. Safety and immunogenicity of this combined vaccine was evaluated in sheep. Sheep immunized subcutaneously with 1ml of live attenuated vaccine consisting of 10(3)TCID(50) each of sheep pox virus (SPV) Romanian Fanar (RF) strain and Peste des Petits ruminants virus (PPRV-Sungri/96 strain) were monitored for clinical and serological responses for a period of four weeks post immunization (pi) and two week post challenge (pc). Specific antibodies directed to sheep pox virus could be demonstrated by indirect ELISA and serum neutralization test (SNT). Competitive ELISA and SNT were used for demonstration of antibodies to PPR virus. All the immunized animals resisted challenge with virulent SPV or PPRV on day 30pi, while control animals developed characteristic signs of disease. Specific virus could be detected in the unvaccinated control animals after challenge but not from any of the immunized sheep. Combined vaccine was found to be safe and potent as evident from sero conversion as well as challenge studies in sheep. This indicates that component vaccines did not interfere each other and can be used in target population for economic vaccination strategies.

The current status of sheep pox disease.

Sheep are the moving banks of shepherds and their economic contribution in terms of meat, wool and skin/hide is immense. Various infectious diseases jeopardize the optimum productivity; among which sheep pox is more important as the disease restricts the export of sheep and their products besides other economic losses. Although, clinical signs are indicative of the disease but a laboratory confirmation is necessary for unequivocal diagnosis and studying epidemiology. The causative agent, sheep pox virus (SPV), is antigenically and genetically closely related to goat pox virus (GPV) and lumpy skin disease virus (LSDV), the other members of the genus capripox virus. In some countries, SPV and GPV are cross infective to small ruminants posing problem in diagnosis and epidemiology. However, recent studies have showed that the viruses are phylogenetically distinct and can be differentiated by molecular tools. Prophylaxis using attenuated vaccines is the choice of control measure as the immunity is long lasting. Detailed information on isolation, identification, pathology, epidemiology, diagnosis and prophylaxis would not only help in updating the knowledge of scientific fraternity but will be useful to the policy makers in order to formulate appropriate measures for control and eradication of the disease. This synthesis is to present an up-to-date review of the disease and its control to provide the reader with an overview of the problem.