Abstract

BackgroundGoatpox 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.ResultsFour 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.ConclusionsThis 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.

Highlights

  • Goatpox is an economically important disease in goat and sheep-producing areas of the world

  • Transient cellular transfection and analysis of the targeted gene and enhanced green fluorescent protein (EGFP) expression in BHK-21 cells Different short interfering RNA (siRNA) suppressed the expression of fusion green fluorescent protein in BHK-21 cells is different

  • The results showed that the number of EGFP-expressing cell was markedly reduced in the sample transfected with homologous siRNAs than sample transfected with heterologous siRNAs or non-transfected (Figure 3A)

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Summary

Introduction

Goatpox is an economically important disease in goat and sheep-producing areas of the world. GTPV is a member of the Genus Capripoxvirus of the family Poxviridae [1], which includes the Sheeppox virus (SPPV) and the Lumpy Skin Disease Virus (LSDV) of cattle. Both sheeppox and goatpox are endemic in Africa, the Middle East and many countries in Asia, and the diseases caused by these viruses have a significant economic impact on the livestock industry in Africa and Asia [2]. Hairpin expression vectors have been used to induce gene silencing in a large number of studies on viruses [11,22,23,24,25,26]

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