Abstract:

Objective To investigate the activity and mechanism of acetylshikonin (AS) against herpes simplex virus type 2 (HSV-2). Methods By acyclovir (ACV) being used as positive control agent, we examined the cell viability with CCK-8 in order to determine the maximum safe concentration of AS to Vero cell. Cytopathogenic effect method and plaque-reduction neutralization assay were used to detect the antiviral activity of AS against HSV-2. RT-qPCR incubated with TCID₅₀ was used to determine viral load to evaluate the inhibitory effect of AS on different stages of HSV-2 replication cycle. The effect of AS on virion morphology was visualized under the transmission electron microscope. Results The maximal atoxic concentration of AS to Vero cell was 3.785 μmol/L, and AS significantly inhibited the replication of virus and reduced plaque formation at the safe concentration of 1.678-3.785 μmol/L. Meanwhile, the replication of HSV-2 virus was inhibited by AS with an 50% effective concentration (EC₅₀) value at 0.334 μmol/L. The virus titer of the treatment group was lower than that of the corresponding virus infection control group at 12 h and 36 h of release stage (13.43±10.04 vs. 127.00±0.32, 3.47±0.55 vs. 5.80±0.12, respectively), the difference was statistically significant (t=8.359, 4.161; both P<0.05), and the virus replication load of the treatment group was lower than that of the corresponding virus infection control group at 12 h, 24 h and 36 h of co-incubation with HSV-2 (0.24±0.09 vs. 1.35±0.07, 4.46±0.06 vs. 6.75±0.04, 2.70±0.04 vs. 5.27±0.10, respectively), the difference was statistically significant (t=9.920, 33.360, 24.020; all P<0.05). Besides, treatment with AS resulted in abnormal morphology of certain HSV-2 virions, and the ultrastructure of HSV-2 changed gradually with added AS concentration. Conclusion The results reported here indicated that AS possessed a certain antiviral effect on HSV-2. The potential mechanism is to exert antiviral effect by directly destroying the complete structure of the virus particles.

Key words: Acetylshikonin, Herpes simplex virus type 2, Antiviral effect, Inactivation effect