Design and Preparation of shRNA Molecules
In this study, first of all, the shRNAs targeting sequences ORF025-DNA pol gene were designed by means of three online design algorithm, namely: BLOCK-iTRNAi Designer (http://rnaide signer.lifetechnologies.com/rnaiexpress/),WIsiRNA Selection Program (http://sirna.wi.mit.edu/home.php) and siRNA Wizard Software (www.invivogen.com/sirna-wizard). Having more precisely investigated the sequence and structural shRNA design rules, we selected the most potent ones [7,27-29]. Apart from considering shRNA design tools, the proposed shRNAs were manually surveyed according to the parameters suggested by TomTuschl's rules , Mcintyre et al.  and Taxman et al. .
A BLAST search needs to be performed in order to eliminate shRNAs having much homology with sheep genomic and transcripts database (http://blast.ncbi.nlm.nih.gov/), and fortunately no homologies were observed.
Production of lentivector expressing shRNAs and ORFV- DNA polymerase
Three shRNA molecules were designed based on the ORFV-OV-SA00- DNA pol gene (accession number AY386264.1). Synthesized shRNAs were cloned separately into plasmids pCDH-CMV-MCS-EF1-cGFP-T2A-Puro to the downstream of the CMV promoter (which was kindly provided from Bonbiotech company). The map of the vector is illustrated in figure3. Furthermore, DnaQ_like exonuclease domain of ORFV-DNA pol gene was synthesized and cloned in the same lentivector. The lentivector used in this study, contained the CopGFP gene under the control of an EF1 promoter in order to pursue shRNA transfection efficiency. It is worth mentioning that the lentivector contained the gene of puromycin resistance cassette as well. All chemically-synthesized oligonucleotides were ligated into the lentivector which had been digested by EcoRI and BamHI. Afterwards, lentivectors ligated in the previous step were transformed into the Escherichia coli strain DH5α. After verifying the accuracy of cloning by sequencing, the accurate clones were used for transfection. As a mock (no shRNA GFP lentivector),we used pEZX-MR03 that contained the EGFP gene under the control of a CMV promoter lacking any significant shRNA sequences .
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Cells and virus propagation
MDBK cells (NBL-1; ATCC Number: CCL-22) were cultured in DMEM (Gibco, America, Catalog No. 116- 12800) with 10% FBS (Gibco, America, Catalog No. 106- 10270) 2 mM L-glutamine, 1X penicillin streptomycin (Sigma, America Catalog No. 116- 12800) as well as 2.5 mg/L amphotericin B at 37˚C with 5 % CO2 incubator . ORFV-SA00 standard strain was kindly provided by Dr. Asgari (Agricultural Research Service, United States Department of Agriculture, Greenport, New York). The titer of the virus stock was 8.91× 104 TCID50/ml. ORFV-SA00 was used at 100TCID50 for in vitro infection experiments .
Packaging of lentiviruses
In the present study, third-generation lentivirus packaging systems was applied in order to generate designed shRNAs and ORFV DnaQ_like expressing lentiviral vectors.
Having cultured HEK 293 T cells (ATCC Number: CRL-1573), we co-transfected the cells with a mixture of three vectors namely: 21 µg psPAX packaging vector ,10.5 µg pMD2.G encoding the vesicular stomatitis virus glycoprotein (VSV-G) envelope and 21 µg pCDH (or pEZX-MR03 as a mock) transfer vector by means of Ca-Po4 reagent according to the bonbiotech (Iran) company’s guidelines. The co-transfection was performed in 10-cm plates similar to the conditions mentioned in manufacturer’s instructions in order to gain a confluency of approximately 70-80%.
48 and 72h after transfection, the GFP expression in transfected cells was monitored and photographed by a fluorescent microscope (Fig.4a). On the other hand, at the same time, the supernatant from these plates were collected and cleared by centrifugation (1000 g, 15 min) and were kept in -70 °C until the infection of MDBK cells .
Preparation of MDBK cells expressing ORFV DnaQ_like and its challenge with shRNA
To prepare MDBK cells expressing ORFV DnaQ_like, an amount of 105 trypsinized MDBK cells were infected in a 6-well plate by lentiviral vectors (MOI = 0.8), diluted in 1mL of DMEM medium supplemented by 10% FBS. The medium was changed with 2ml of DMEM after 12 h of infection. At 48 h and 72 h after infection, MDBK cells were observed using a fluorescent microscope . Infected cells were identified using RT-PCR. For RT-PCR, RNA was isolated from infected cells and then underwent a DNase treatment. Then, RT- PCR was carried out using the following primers: Forward: 5'- GGGACCGAGACAGTCAACTT -3' and Reverse: 5'- GGTCCCGTTGTTGTTGTTGA -3'. The PCR thermal cycle programs include denaturation at 95 º C for 2 min followed by 30 cycles at 95 º C for 30 s, 52 º C for 30 s, and 72 º C for 30 s, followed by a final extension at 72 º C for 5 min. The positive (MDBK infected with ORFV) and negative (MDBK cells without any infection) controls were used in each test.
To infect MDBK cells expressing ORFV DnaQ_likeby lentiviruses, the cells (90% confluent) were infected in a 6-well plate by lentiviral vectors (MOI = 0.8), diluted in 1ml of DMEM medium supplemented by 3% FBS. The medium was changed with DMEM supplemented by 10% FBS and 1X penicillin streptomycin after 12 h of infection. The cells were re-infected after 12 h post changing medium with the same method and MDBK cells were maintained in DMEM, 10%FBS containing 1X penicillin streptomycin (Sigma). For each shRNA, three wells were considered .
Infection /challenge of lentivirus expressing shRNAs with ORFV
3*105MDBK cells per 6-well plates were seeded and cultured with the conditions mentioned in the previous part. The next day, after removing the culture medium, lentiviruses were inoculated into wells. For each shRNA, three wells were considered. To achieve more infecting efficiency, 24 h after the first infection with lentivirus, the infection repeated like that of the previous step.
48 h after the first infection, MDBK cells were monitored under a fluorescent microscope to evaluate GFP expression (Fig. 4b). In case of observing the appropriate GFP expression, the challenge with ORFV was performed. After 72 h, cells were monitored in terms of phenotypes and the development of CPE .MDBK cells infected with ORFV were considered as positive controls and MDBK cell infected with mock vector or without any viral infection were considered as negative controls .
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To determine the changes in CPE as well as the effects and reduction of ORFV titer,TCID50 test was performed according to the protocol after challenging with lentiviruses expressing shRNAs, . TCID50 was performed in 96-well plates with three replicates per dilution and in triplicate for each infection condition. Finally, viral titers were calculated based on the Spearman-Karber formula .
Total RNA was extracted from MDBK cells using the RNeasy mini kit (Qiagen, Crawley, UK), 72h after the infection. The on-column DNase digestion (Qiagen, Crawley, UK) was used to remove contamination off DNA. The cDNA was synthesized from 1μg total RNA by reverse transcriptase enzyme.
Reverse-transcription performed by Superscript II (Invitrogen) and oligo(dT) for 1 h at 42 ºC. RT-qPCR was performed using Power SYBR Green Mastermix (Applied Biosystems, Warrington, UK). The primers were designed using the GenScript real-time RT-PCR Primer Design web tool. The primer sequences were screened using a BLAST search to confirm its specificity and the PCR products were run on an agarose gel to confirm that products of the expected size were detected. The sequences of forward and reverse primers were as follows: Forward: 5´- GGGACCGAGACAGTCAACTT- 3´, Reverse: 5´- GGTCCCGTTGTTGTTGTTGA- 3´, Bos Taurus GAPDH Forward: 5’ - TGAGGACCAGGTTGTCTCCT- 3’and Bos Taurus GAPDH Reverse: 5’- CACCCTGTTGCTGTAGCCAAAT– 3’. The reactions were analyzed upon an iQ5 real-time RT-PCR detection system. The PCR reaction was started with 30 s minute denaturation at 95 °C. 40 cycles of 95°C for 30 s, 52°C for 45 s and 72°C for 1 min were carried out. Cycle threshold (Ct) values were normalized to GAPDH, and a relative ORFV RNA level was done by the ∆∆Ct method .The fold-change values relative to control were multiplied by 100 to obtain the reduction of percentage in ORFV replication. Experiments were performed in triplicate. The ampliﬁcation was identified by melting curve profile.