Evaluating Recombinant ORFV Viruses' Replication and Gene Expression

Replication kinetics of ORFV^Δ121cH6/1 or ORFV^Δ121cH8/1 recombinant viruses were assessed through single- and multi-step growth curves in OFTu and STu cells and compared to the parental OV-IA82, as previously described (5 (link)). Briefly, for the multistep and single-step growth curves, the cells were infected at 0.1 MOI and 10 MOI, respectively, and subsequently harvested at 6, 12, 24, 48, and 72 hours post-infection (hpi). The controls were mock-infected OFTu and STu collected at 0 hpi. Viral titers were determined at each time point by limiting dilution and expressed as tissue culture infectious dose 50 (TCID₅₀) per milliliter.
To evaluate the expression of heterologous genes by the recombinant viruses, OFTu cells were infected with either ORFV^Δ121cH6/1 or ORFV^Δ121cH8/1, and evaluated by indirect IFA, flow cytometry, and Western blot, as previously described (49 (link)). Anti-FLAG tag epitope monoclonal mouse antibody (Sigma-Aldrich) was used for the detection of the fusion cH6/1- or cH8/1-flag proteins in all assays.
The stability of both cH6/1 and cH8/1s genes inserted into the ORFV121 locus was evaluated during 10 serial passages of the respective recombinant viruses in OFTu cells by assessing expression through IFA by using a monoclonal anti-FLAG M2 mouse antibody (1:500, F1804-5MG, Sigma-Aldrich), as previously described (49 (link)).