Background This study presents preclinical data of a novel interferon (IFN)-α8 fusion protein, PF-04849285, and compares it with IFN-α2 and pegylated IFN-α2; the latter being the current standard of care for HCV. Methods The antiviral properties were evaluated in vitro using the HCV replication assay (replicon) and the general encephalomyocarditis virus assay. The binding affinity to both IFNR-subunits was assessed using surface plasmon resonance. Ex vivo experiments using cynomolgus monkey and human blood were used for the evaluation of induction of IFN-inducible biomarkers (interferon inducible protein 10 [IP-10], 2′-5′-oligoadenylate synthetase [OAS2] and interleukin-6 [IL-6]). The molecule was tested intravenously and subcutaneously in cynomolgus monkey in a single dose study for two weeks at 0.01, 1, 5 and 20 mg/kg. Each route and dose combination was given to a single male animal, blood samples were collected for evaluation of biomarkers and pharmacokinetics. The compound was also tested in cynomolgus monkey in a multiple dose study for four weeks, with a twice-a-week dosing prior to a three-week wash-out period for toxicokinetics, pharmacokinetics, and biomarker evaluation at 20, 50 or 100 mg/kg subcutaneously and 20 mg/kg intravenously. Results The molecule is 10x more potent than the pegylated IFN-α2a, with potency similar to the unmodified IFN-α2a. No unanticipated findings were observed in cynomolgus monkey when dosed up to 20 mg/kg, >10,000-fold margin over the anticipated efficacious human dose. Conclusions The biomarker and toxicological findings were consistent with a potent IFN molecule. The potency and pharmacokinetic properties of the molecule are consistent with dosing at least twice daily with the potential for monthly dosing.