In the present study, our findings demonstrate that expression of cIFN-a following a single i.n. administration of DEF201 offers a strong protective effect in hamsters against challenge with PICV that included limiting liver disease and inducing an antiviral state that inhibited systemic and tissue viral replication. The lack of significant antiviral activity elicited by the rAd EV control virus suggests that the enhanced antiviral response produced by DEF201 is largely due to the expression of the cIFN-a gene. The weak stimulatory effect seen in 1 of the 3 experiments was not surprising considering the number of host systems that play a role in sensing the adenovirus vector [24] ; however, the effect was short-lived. In contrast, the enhancement of the host antiviral defenses by DEF201 was long-lasting with a 14-day pre-PICV challenge prophylactic window. Moreover, DEF201 was effective when given 1-2 days post-PICV infection. These data also suggest that sufficient viral replication may be necessary to elicit an adaptive immune response that confers lasting protective immunity, as, for the most part, only re-challenged animals from the 24 h DEF201 pretreatment group succumbed to a second challenge with a lethal PICV inoculum. Presumably, the robust innate immunity and antiviral state induced by the DEF201 pretreatment rapidly controlled the ,5 pfu challenge dose obviating the development of the adaptive immune response and immunological memory.