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

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.


Introduction
Orf, also known as contagious ecthyma or cutaneous pustular dermatitis, is an acute contagious disease of sheep, goats, wild ruminants, and humans worldwide. It is caused by orf virus (ORFV), which is an epitheliotropic parapoxvirus, the prototype member of the genus Parapoxvirus, family Poxviridae [1]. ORFV generally induces proliferative and self-limiting lesions around the mouth, teats and skin of affected animals, especially in young lambs [2].The mortality rate is as high as 93 % in kids when lesions of the lips and udders prevent them from receiving nutrition, thus leading to rapid emaciation [3,4]. Outbreaks of orf have occurred in many countries where substantial numbers of sheep and goats are raised [5]. The disease not only causes huge economic losses to the sheep industry but also, as a zoonotic disease, poses a threat to human health [2].
The ORFV genome is an approximately 140-kbp double-stranded DNA containing at least 132 putative genes [6]. Like other poxviruses, the ORFV genome contains a large central coding region (ORFs 009 to 111), which includes homologues of conserved poxvirus genes involved in basic replicative mechanisms and structure and morphogenesis of intracellular mature and extracellular enveloped virions, bounded by two identical inverted terminal repeat regions [7]. ORF025 encodes the DNA polymerase (DNA Pol), which is the core enzyme in the process of ORFV replication and catalyzes the replication of the viral genome.
RNA interference (RNAi) is a conserved gene-silencing mechanism that is induced by 19-to 27-nucleotide (nt) small interfering RNA (siRNA) molecules that are homologous to the target genes [8,9]. RNAi technology is not only a powerful tool for functional genomics studies but is also a potentially useful antiviral method, and it is increasingly being used to inhibit the replication of viral pathogens [10]. Therefore, it has been suggested that the preferred method for controlling the virus is to interfere with replication of the viral genome and the expression of viral genes. We designed specific siRNAs to target the DNA polymerase genes of ORFV in order to test whether RNAi could selectively target ORFV viral DNAs. This study provides not only an experimental basis for the development of a new anti-ORFV strategy but also a new approach for studying ORFV infection and replication.

Cells and virus propagation
Primary ovine fetal turbinate (OFTu) cells were cultured in minimal essential medium (MEM; (Hyclone) with 10 % fetal bovine serum (FBS; Hyclone), 2 mM L-glutamine, and 100 U of penicillin, 100 lg of streptomycin, and 20 lg of nystatin per ml in a 37°C, 5 % CO 2 incubator. Orf virus was isolated from scabs collected from lesions of a 6-weekold small-tailed Han sheep showing typical symptoms of orf virus infection in November 2008 in Jilin Province, China [2]. When 90 % of the virus-infected cells showed a cytopathic effect (CPE), the cultures were collected after undergoing three freeze-thaw cycles.
Isolation of genomic DNA DNA was extracted from CPE-positive cell cultures using a Takara MiniBEST Viral DNA Extraction Kit (Takara, Dalian, China) according to the manufacturer's instructions and was used as template in PCR.

PCR amplification
The ORF025 gene was amplified by PCR from DNA extracted from CPE-positive cell cultures. The complete ORF025 (DNA polymerase) gene sequence was divided into three sections called ''DNA polymerase I'', ''DNA polymerase II'' and ''DNA polymerase III'' and specific primers were designed using Primer Premier 5.0 software based on published ORFV (ORFV-OV-SA00 strain) genomic sequences available in the NCBI GenBank database (accession number AY386264.1). These primers were custom synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China. The six primer sets used in this study were specific for ORF025 ( Table 1). The PCRs for DNA polymerase I had an initial denaturation step of 95°C for 2 min, followed by 35 cycles of 94°C for 45 s, 59°C for 45 s, 72°C for 45s, and a final extension of 72°C for 10 min. The PCR for DNA polymerase II had an initial denaturation step of 95°C for 2 min followed by 30 cycles of 94°C for 45 s, 58°C for 45 s, and 72°C for 1 min, and a final extension at 72°C for 10 min. The PCR for DNA polymerase III had an initial denaturation step of 95°C for 2 min, followed by 30 cycles of 94°C for 30 s, 55°C for 30 s, and 72°C for 150 s, and a final extension at 72°C for 10 min. PCR was carried out in 25-lL reaction mixtures containing 2.5 lL PCR buffer, 200 mM each dNTP, 0.2 mM each oligonucleotide primer, 100 ng of each DNA sample, 0.5 U rTaq DNA polymerase (Takara, Dalian, China), and 10 lL sterilized water. Amplicons were visualized by electrophoresis in 1 % agarose gels and documented using a gel documentation system.

Cloning and DNA sequencing
The PCR products of the DNA polymerase I, II, and III fragments were cloned into the pMD-18T vector (Takara, Dalian, China) and used to transform E. coli DH5a. At least six positive clones of each selected amplification product were sequenced at Shanghai Sangon Biological Engineering Technology and Services Co., Ltd., China. The three sequences were combined to obtain the complete sequence of ORF025.

ORFV siRNA preparation
Based on the complete sequences of ORF025, four pairs of siRNAs, D704, D1017, D1388 and negative siRNA control (scrambled siRNA) were designed and synthesized by Shanghai GenePharma Co., Ltd. The sequences are shown in Table 2.

Cell culture and transfection
OFTu cells were cultured in 24-well plates at a concentration of 1.0 9 10 5 cells/well and incubated in a CO 2 A standard curve for the determination of ORFV genome copy numbers was created by real-time PCR of standard plasmid pMT-B2L preparations at serial dilutions of 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 and 10 7 copies/lL. The specificity of the realtime PCR was confirmed by sequencing the product.

PCR amplification of the orf virus DNA polymerase gene
Specific products of the expected size (888 bp, 1001 bp and 1752 bp; Fig. 1) were obtained when a sample extracted from CPE-positive OFTu cells was amplified by PCR. The nucleotide sequences of these amplicons were combined to obtain the complete sequence of the ORFV 025 gene (3039 bp).

CPE analysis
Transfection complexes (siRNAs and transfection reagent) were completely removed after transfection for 6 h, and 100 TCID 50 of orf virus suspensions was added to each well. Untransfected cells were used as a control. At 48 h after orf virus infection, observation of CPE indicated siRNA704, siRNA1017 and siRNA1388 appeared to inhibit virus replication to varying degrees (Fig. 2).

Examination of siRNA effect by infectious virus assay
To further investigate the inhibitory effect of siRNAs, the microtiter method was used for titration of orf virus at 48h postinfection, and the results (Fig. 3) showed that, in control cells transfected with scrambled siRNA, titers reached 1 9 10 8.7 /0.1 ml (TCID 50 ) at 48 h postinfection,

Examination of siRNA effect by real-time PCR
To quantify the effect of siRNA on viral replication, the viral genome copy number was determined at 48 h postinfection by real-time quantitative PCR, using serially diluted plasmid pMD-B2L as a standard. The correlation coefficient (R 2 ) of the standard curve was 0.991, and PCR amplification efficiencies (E) were [0.96, with only one peak in all dissociation curves, thereby demonstrating the specificity and reliability of the analysis. The results indicated a significant reduction in the relative expression level of the viral genome per nanogram of total DNA, by 84 %, 89 % and 73 %, respectively, compared to the mock group at 48 h postinfection, when siRNA704, siRNA1017 and siRNA1388, respectively, were used (Fig. 4).

Discussion
During RNAi, which is an RNA-mediated pathway of gene silencing mediated by small RNA molecules, introduction of double-stranded RNA (dsRNA) encoding a subsequence of a gene leads to reduction in expression of the corresponding gene in various organisms, including animals and plants [11]. Long double-stranded RNA is cleaved by an endonuclease (Dicer) into 21-to 25-nt fragments (small interfering RNA; siRNA), and these oligonucleotides can serve as guide sequences to direct a multicomponent nuclease to a specific messenger RNA to destroy it [12]. Studies of the antiviral activity of siRNA have shown that it is effective in the inhibition of viral infection and modulation of viral replication in cultured mammalian cells.
Examples include HIV-1 [13], severe acute respiratory syndrome virus [14], hepatitis C virus [15], influenza virus [16], swine fever virus (Porntrakulpipat et al., 2010), goatpox virus [9], and Newcastle disease virus [17]. RNAi is increasingly being used to inhibit the replication of viral pathogens and is a promising approach for antiviral therapy in mammals. siRNAs can be introduced into mammalian cells by in vitro transcription or by transfection with chemically synthesized siRNAs to achieve effective and very rapid silencing of a target gene, although this approach is limited to cells that can be transfected at high rates, and the effects are transient [8]. The DNA polymerase gene, which is located in the central region of the genome of ORFV, is highly conserved within species and is a highly conserved gene that codes for the most conserved nonstructural protein, which catalyzes the replication of the viral genome and plays a primary role in viral replication [18].
In the present study, the complete genome sequences of the ORFV Jilin isolate was determined. Three pairs of siRNAs were then synthesized based on the complete open reading frame of the DNA polymerase gene of the ORFV Jilin isolate. These siRNAs were then imported into cells by in vitro transfection, and the efficiency of inhibition of viral replication was tested using three assays: CPE, TCID 50 determination and real-time PCR. Our results demonstrate that the three siRNAs were highly capable of inhibiting viral DNA genome replication. Based on the replication kinetics of ORFV in OFTu cells prior to siRNA transfection and the peak period of viral proliferation (data not shown), the cell cultures for viral titration and DNA extraction were harvested at 48 h postinfection.
At 48 h after virus infection, differences in CPE indicated that siRNA704, siRNA1017 and siRNA1388 inhibit virus replication to varying degrees (Fig. 2). To further examine the level of viral inhibition, we tested the viral titers of infected cells by TCID 50 assay. The results showed that siRNAs caused an approximately 59-to 199-fold decrease in virus yield. Furthermore, the results of realtime PCR indicated that approximately 73-89 % less viral DNA was present in cells transfected with the siRNA than in control cells.
In summary, the use of RNAi directed against the ORFV DNA polymerase gene could effectively inhibit viral replication. Based on the present data and the advantages of siRNA technology, we propose that siRNAs targeting the DNA polymerase gene may be used as a tool to study ORFV replication and pathogenesis in future studies.