Figure 3

Kinetics of action of SCH 16 in relation to the replicative cycle of JEV in PS cells. Panel A represents the results of the experiments wherein the addition of the drug SCH 16 to virus infected PS cells was staggered (refer to Material & Methods for details). X axis represents the various time points at which SCH 16 was added after adsorption of JEV onto PS cells. Note that there was no virus yield (represented as Log TCID₅₀/ml on Y axis) in drug treated cells (blue triangle) until 8 hours post infection after which virus yield steadily increased to attain levels similar to that obtained in untreated cells (pink sphere). The Y' axis represents the optical density values obtained in the JEV antigen capture ELISA. Soluble JEV antigen was measured in the supernatant fluids obtained at 48 hrs after the experiment (refer to Material & Methods for details) in both drug treated (black square) and untreated (red diamond) cells. Panel B depicts the detection of JEV specific antigen using an immunofluorescent assay. Note the presence of bright immunofluorescence in the JEV infected monolayers (virus control). It can also be observed that JEV infected mono layers treated with SCH 16 were positive for viral antigen at 10, 12 and 14 hours post infection whilst viral antigen was undetectable by immunofluorescence at 0, 2, 4, 6 and 8 hrs post infection respectively (400×). Panel C: The amplification plots obtained in Real Time PCR depicting the detection of JEV RNA in the untreated cells and SCH 16 treated cells at varying time points post-infection. Panel C-1 depicts the typical amplification plot (fluorescence vs cycle number) obtained by the real time PCR with the RNA extracted from the virus infected untreated cells at varying time points. Note that JEV RNA was detected at all time points. In contrast JEV RNA was undetectable at 0, 2,4, and 8 hrs in the SCH 16 treated cells (Panel C2). Panel D represents the results of the experiments wherein the minimum time required for SCH 16 to exert antiviral activity was evaluated (refer to Materials & Methods for details). SCH 16 was added to all monolayers 2 hrs post virus adsorption and removed from the mono layers at periodic intervals. X-axis represents the various time points when SCH 16 was removed after JEV entry into PS cells. Note that virus yield (represented as Log TCID₅₀/ml on Y axis) in drug treated cells (blue triangle) steadily declined from 0 hrs post infection until 8 hours post infection after which there was no virus production noted in drug treated cells. On the contrary virus yields continued to be high in untreated cells (pink sphere) at all time points. The Y' axis represents the optical density values obtained in the JEV antigen capture ELISA. Soluble JEV antigen was measured in the supernatant fluids obtained at 48 hrs after infection (refer to Materials & Methods for details) in both drug treated (black square) and untreated (red diamond) cells. Panel E: Effect of duration of antiviral action of SCH 16 on JEV replication post-infection. SCH 16 was added to JEV infected PS cell monolayer at 0 hours post – adsorption and the inoculums were removed at different time points post-infection (0 to 14 hrs). The monolayer was stained using JEV specific monoclonal antibodies by IFA at 48 hours (400×). Presence of cell bound antigen can be appreciated upon the removal of SCH 16 in the early hours (up to 4 hours) of viral replicative cycle, while viral antigen was not detected when SCH 16 was retained with the infected monolayer for longer duration (8 hours andmore). Panel F: The amplification plots obtained in Real Time PCR depicting the detection of JEV RNA in the infected cells treated with SCH 16 at 0 hours and inoculums removed at varying time points (refer to Materials and methods for details). Panel F-1 depicts the typical amplification plot (fluorescence vs cycle number) obtained by the real time PCR with the RNA extracted from the virus infected untreated cells at varying time points. Note that JEV RNA was detected at all time points. In contrast JEV RNA was detectable only at 0 and 4 hrs in the SCH 16 treated cells and undetectable beyond 8 hrs (Panel F2).