Selectivity index, TC50 for african green monkey BSC1 cells after 20 hrs to EC50 for Vaccinia virus WR in BSC1 cells after 20 hrs by cell protection assay
Antiviral activity against Vaccinia virus WR in BSC1 cells assessed as retention of antiviral potency by measuring exogenous double stranded DNA trapping
Inhibition Assay: A rapid plate DNA synthesis assay was performed using optimized conditions. Briefly, a 1.2:1 ratio of a 20-mer oligonucleotide primer (5′-GCGAATGAATGACCGCTGAC-3′, SEQ ID No. 1) and a 5′-end biotinylated 100-mer oligonucleotide template (5′-Biotin-AGCACTATTGACATTACAGAGTCGCCTTGGCTCTCTGGCTGTTCGTTGCGGGCTCCGCG TGCGTTGGCTTCGGTCGTCCCGTCAGCGGTCATTCATTGGC-3′) were annealed and loaded into a 96-well microtiter streptavidin-coated plate (Streptawell plates, Roche Applied Science, Indianapolis, Ind., USA) at 5 pmol/well. The wells were incubated at 37 C for 90 min, and washed with 100 L PBS. The reaction was conducted in low salt buffer (20 mM Tris-HCl pH 7.4, 3 mM MgCl2, 0.1 mM EDTA, 0.5 mM DTT, 2% glycerol, 40 ug/mL BSA, 5 uM dNTPs, 1 uM digoxigenin-11-2′-deoxyuridine-5′-triphosphate (DIG-dUTP, Roche Applied Science) with 1 μL vaccinia infected cell lysate or 1 uL of in vitro translated E9 DNA polymerase. The reaction plates were incubated at 37° C