Background: An ethylacetate-soluble fraction (ET4) from the lichen Ramalina farinacea has previously been shown to inhibit the infectivity of lentiviral and adenoviral vectors, as well as wild-type HIV-1. We now determined the antiviral activity of ET4 against other wild-type viruses, including the herpes simplex virus type 1 (HSV-1) and the respiratory syncytial virus (RSV). Methods: Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of ET4 for 30 min at 37°C before adding to P4CCR5 indicator cell line (HIV-1), ELVIS TM indicator cell line (HSV-1) or HEp2 cell line (RSV) in 96-well microtitre plates. Controls contain virus alone without ET4. The anti-HIV and anti-HSV activities were quantified by estimating beta-galactosidase expression of the respective indicator cell lines while the anti-RSV activity was determined via an immunofluorescent technique, employing monoclonal mouse antibody against the P-protein of RSV. Toxicity of ET4 to cell lines was evaluated in parallel using either the BrdU incorporation method or the MTT method. The effect of ET4 on the enzymatic activity of HIV-1 reverse transcriptase was also evaluated using a chemiluminescent reverse transcriptase assay. Bioassay-guided fractionation of the whole methanol extract of R. farinacea involved sequential screening of HPLC fractions using a vector-based assay technique. Results:ET4 inhibited HSV-1 and RSV potently (IC50 = 6.09 and 3.65 μg/ml, respectively). Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle and the entry step of the RSV replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 reverse transcriptase with an IC50 of 0.022μg/ml. Bioassay-guided fractionation of ET4 led to the identification sub-fraction rfO, with activity against lentiviral vector and HIV-1 (RNA viruses) but not against HSV-1 (DNA virus) and sub-fraction rfM, with activity against HSV-1 but not against the lentiviral vector. Conclusions:ET4 represents a novel fraction from the lichen R. farinacea with broad spectrum antiviral activity against DNA viruses (adenovirus and HSV-1) and RNA viruses (HIV-1 and RSV). The effect against DNA and RNA viruses is mediated by different sub-fractions within R. farinacea.

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