2.1 Cell lines and viruses:
MDBK cells (ATCC Number: CCL-22) and Human embryonic kidney 293T cells (ATCC Number: CRL-1573) cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco CO) supplemented with 10% Fetal bovine serum (FBS; Gibco CO) and 1% penicillin/streptomycin (Pen-Strep, Sigma CO). IBRV was kindly provided by Dr. Darounkola (Institute of Virology, Faculty of Veterinary Medicine, University of Zürich) and was propagated in MDBK cells
2.2 Design of anti- BHV1-UL25 shRNA molecules
For a better efficiency, shRNA molecules must be designed to disrupt expression of conserved regions of the viral target gene. So by using NCBI database, the sequence of BHV-1-UL25 gene (Accession number: AJ004801.1) was obtained and was aligned with the sequence of this gene in the other strains of virus by CLUSTAL OMEGA online software. Therefore the most homologous regions were selected for further analyzes. Several shRNA molecules were designed using three online web servers such as WI siRNA selection program, BLOCK-IT RNAi designer and www.invivogen.com/siRNA-wizard. The sequences of proposed shRNA molecules were matched to the target gene sequence to find those which target the conserved regions. Since it is important that shRNA molecules should easily access to the UL25 mRNA, CLC Genomic Work brench software was applied to predict the secondary structure RNA of UL25 gene and the position of shRNA molecules that are related to the conserved regions in this structure. Selected sequences were submitted to a BLAST search against the cattle genome sequence to ensure that the host genome was not targeted. After the subsequent investigation of optimal CG percentage and off-target effects, three shRNA sequences were finalized. The sequences of the cut-off site of the BamHI and EcoRI restriction enzymes were placed on both sides of shRNA sequences and TCAAGAG were placed as a loop sequence. Afterwards, the oligonucleotides were synthesized by Bioneer Co.
2.3.Preparation of anti- BHV1-UL25 shRNAs
The proposed shRNA molecules were synthesized as single-strand oligoes by Bioneer Co. and were annealed with each other. In order to annealing, a mixture of 1µL of sense oligo (200 µM), 1µL of antisense oligo (200 µM), and 2 µl of sterile deionized water were mixed, then the mixture was placed in a boiling water at 95 ° C for 4 minutes to remove all possible secondary structures. Finally, it was placed at room temperature for 10 minutes to form the desired double strands.
2.4. Production of lentivectors expressing anti- BHV1-UL25 shRNAs
2.4.1. Lentiviral Plasmaids: In the present study, psPAX2 (10703 bp); a packaging construct that containing gag and pol genes, pMD2G; an env (VSV-G) plasmid (5824 bp) and a self-inactivating (SIN) transfer vector plasmid pCDH-CMV-MCS-EF1-CGFP-T2A-Puro (CD513B-1,8220 bp) were used to generate third-generation lentiviral vectors. pEZX-MR03 containing eGFP gene (7945 bp) was applied as control plasmid (all the plasmid were kindly provided from Bonbiotech company(Iran)).
2.4.2. Production of Lentiviral transfer Plasmaid: To construct lentiviral plasmids carrying shRNA sequences, pCDH-CMV-MCS-EF1-CGFP-T2A-Puro lentiviral plasmid was double- digested with BamHI (Roche, Germany,) and EcoRI (Roche, Germany). The Ligation reaction was carried out with T4 10X ligation buffer (Fermentas, Germany, Cat. No.: B69), T4 DNA ligase (10U) (Fermentas, Germany), synthetized shRNA oligos and lentiviral plasmid and were transformed in the E.coli DH5α. Then, colony PCR reaction was performed using the general primers of pCDH-CMV-MCS-EF1-cGFP-T2A-Puro lentiviral plasmid. The sequences of primers were as follows: CMV-F: AATGGGCGGTAGGCGTGTA -3'and EF1-R: 5'- GGACTGTGGGCGATGTG -3'. The PCR thermal cycle program was consisted of denaturation at 95 º C for 5 min followed by 30 cycles at 95 º C for 30 s, 55 º C for 40 s and 72 º C for 60 s, followed by a final extension at 72 º C for 10 minutes. Finally the recombinant lentiviral plasmids were sequenced by Bioneer Co.
2.4.3. Co- transfection of lentiviral plasmids: After two passages of HEK 293 T cells the cells were co-transfected with a mixture of 21 µg psPAX,10.5 µg pMD2.G and 21 µg pCDH (or pEZX-MR03 as a mock) using Ca-Po4 reagent according to the bonbiotech 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% (Zufferey et al., 2000). 48 and 72 h post transfection, lentivectors in the supernatant were harvested by centrifugation (1000 g, 15 min) and were kept in -70 °C until the infection of MDBK cells. The transfected HEK- 293T cells were photographed under a fluorescent microscope for monitoring EGFP expression.
2.5. Lentivector transduction, challenge with BHV-1:
MDBK monolayers (5*103 cells in 96-well plate, 70% confluency) were propagated and inoculated with 280 µL of lentiviruses expressing shRNAs (MOI=0.5), diluted in 1mL of DMEM medium supplemented by 3% FBS. After 12 hours, the medium of each well was replaced with fresh DMEM supplemented with 3% FBS. At 48 h and 72 h after infection, MDBK cells were observed using a fluorescent microscope and EGFP expression was monitored for confirmation of shRNA- expression.
Subsequently, transduced MDBK cells were infected by BHV-1 and monitored for the development of cytopathic effects. Finally TCID50 titers were calculated for the wells that infected with the BHV-1 and shRNA expressing lentivectors and positive (cells infected with only BHV-1 or BHV-1 and scrambled lentivector) and negative controls (cells infected with scrambled lentivector or without viral infection).