Inhibition of Newcastle disease virus replication by RNA interference targeting the matrix protein gene in chicken embryo fibroblasts.
ABSTRACT Newcastle disease (ND) is an infectious viral disease of birds caused by the Newcastle disease virus (NDV), also known as avian paramyxovirus type 1 (AMPV-1), which leads to severe economic losses in the poultry industry worldwide. In this study, the application of RNA interference (RNAi) for inhibiting the replication of NDV in cell culture by targeting the viral matrix protein gene (M) is described. Two M-specific shRNA-expressing plasmid constructs, named pS(M641) and pS(M827), were evaluated for antiviral activity against the NDV strain NA-1 by cytopathic effects (CPE), virus titration and real-time RT-PCR. After 36h of infection, both pS(M641) and pS(M827) reduced virus titers by 79.4- and 31.6-fold, respectively, and they down-regulated mRNA expression levels of the matrix protein gene M by 94.6% and 84.8%, respectively, in chicken embryo fibroblast (CEF) cells, while only pS(M641) significantly decreased CPE, compared to the control group. These results indicated that the M gene 641 and 827 sites represent potential antiviral therapy targets, and RNAi targeting of the M gene could not only represent an effective treatment in Newcastle disease but also aid as a method for studying the replication of NDV.
Article: RNA interference targeting virion core protein ORF095 inhibits Goatpox virus replication in Vero cells.[show abstract] [hide abstract]
ABSTRACT: 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. 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. 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.Virology Journal 02/2012; 9:48. · 2.34 Impact Factor