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In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

ABSTRACT: Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications.

SUBMITTER: Wang G

PROVIDER: S-EPMC4010719 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

Wang Gaili G He Wenqi W Song Deguang D Li Jida J Bao Yingfu Y Lu Rongguang R Bi Jingying J Zhao Kui K Gao Feng F

Archives of virology 20131101 5

Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interf ...[more]

PMID: 24178308

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Project description: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.

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In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

Publications

In vitro RNA interference targeting the DNA polymerase gene inhibits orf virus replication in primary ovine fetal turbinate cells.

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