Caffeic Acid Disrupts ILHV Replication

Time-of-drug addition were performed to explore which steps of the ILHV replication cycle are blocked by caffeic acid. Briefly, CA was added to the virus and/or host cells at different times before, during, and after viral inoculation into the cells as follows (Figure 1): (1) pre-treatment of virus followed by inoculation of the treated virus into the cells investigates whether CA has virucidal or neutralizing activity; (2) pre-treatment of the cells with CA before viral inoculation explores whether this substance could block the viral receptor, inhibiting viral attachment to the host cells, or if it could induce production of antiviral host factors; (3) co-treatment of cells and virus during virus inoculation examines the function of CA during the steps of virus entry, including virucidal (neutralizing) activity and blockade of viral attachment and penetration into the cells; (4) treatment of virus-infected cells during the entire post-inoculation period investigates the antiviral effects of CA during post-entry steps such as genome translation and replication, virion assembly, and virion release from the cells. Viral infection experiments were performed in A549, HepG2, or Vero cells seeded in 24-well plates treated with CA or untreated controls. Under the different conditions described above, the cells were infected with MOI 1 of ILHV for one hour at 37 °C and revealed through the virus plaque-forming assay titration of supernatant or cell content (described in Section 2.4). Three independent experiments with quadruplicate measurements were performed. Data were analyzed by four-parameter curve fitting from a dose–response curve using GraphPad Prism (version 8.00) to calculate EC₅₀ (concentration of the compound that inhibited 50% of the infection), and the selectivity index for the compound was calculated as the CC₅₀:EC₅₀ ratio.