It was previously reported that C6/36 cell cultures persistently-infected with AalDNV showed markedly reduced disease severity when super-challenged with Dengue virus serotype 2 (DEN-2). A possible explanation for the effect was viral competition or viral interference. An alternative explanation was the possible production of a low-molecular-weight antiviral peptide similar to that previously reported from C6/36 cells persistently infected with DEN-2. In this study we isolated and characterized a soluble anti-DEN-2 factor(s) in the cell-free and virus-free supernatant solution (CVFS) from C6/36 cells persistently infected with AalDNV. Specifically, naïve monkey (Vero) cell cultures pre-treated with CVFS for 48 h prior to challenge with a DEN-2 stock showed viral titers reduced by 2 logs when compared to Vero cells exposed to CVFS from uninfected C6/36 cells. Protease treatment of the CVFS removed its anti-DEN-2 activity. The active soluble factor(s) had a low molecular-weight of less than 3 kDa and was a polar compound with a negative charge. Since pre-incubation for 48-hr was required to obtain maximum protection against DEN-2, the protective mechanism appeared to be inductive elicitation of a host-cell defense reaction by a prophylactic, anti-DEN-2 peptide cytokine