Green = anti-DEN and Red = pseudocolor for T0-PRO-3

iodide staining of DNA (nuclei). To confirm that the DEN-2 positive cells arose from challenge with the DEN-2 stock and not from virions in the 5 kDa filtrate, naïve C6/36 cells were exposed to the 5 kDa filtrate, to wash from the upper side of the 5 kDa membrane and to unfiltered supernatant solution from the culture from which the filtrate was derived (i.e., 19th passage of a culture persistently infected with DEN-2) (Figure BI 2536 chemical structure 2). After 2 days of incubation, phase contrast microscopy revealed that the wash from the upper side of the 5 kDa membrane resulted in the most severe cytopathology (i.e., many fused giant cells) in the naïve C6/36 cells (Figure 1D and Figure 2F), while exposure to the whole, unfiltered culture filtrate (Figure 2D) gave cytopathology similar to that produced by the DEN-2 stock (i.e., fewer fused giant cells)(Figure 2B). Pre-exposure of naïve C6/36 cells to the 5 kDa filtrate reduced the severity of Dengue infection (i.e.,

no fused giant cells) (Figure 2C) and exposure to the 5 kDa filtrate in the absence of DEN-2 challenge resulted in no cytopathology (Figure 2E), i.e., morphology similar to that of unchallenged, Selleck EX527 naïve cells (Figure Interleukin-2 receptor 2A). Figure 2 Phase contrast photomicrographs of C6/36 cells at 2 days post-challenge with DEN-2. (A) Unchallenged naïve control cells. (B) Untreated C6/36 cells challenged with DEN-2 stock

and showing cytopathic, fused giant cells. (C) C6/36 cells pre-treated with the 5 kDa filtrate before challenge with the DEN-2 stock and showing fewer cytopathic, fused giant cells than the untreated cells in B. (D) C6/36 cells exposed to the whole supernatant solution from cultures persistently infected with DEN-2 and showing similar cytopathology to that in B. (E) C6/36 cells exposed to the 5 kDA filtrate (control not challenged with DEN-2) and showing no cytopathology (i.e., similar to A with no DEN-2 infection). (F) C6/36 cells exposed to the wash from the upper side of the 5 kDa membrane and showing the greatest number of selleck cytopathic giant cells (i.e., more than that in B and similar to Figure 1D). In summary, results from these tests indicated that 48 h pre-exposure of C6/36 cells to a low molecular weight substance(s) in a 5 kDa filtrate from persistently-infected cells was able to induce a protective response against DEN-2 virus infection in naïve cells.