Broad spectrum antiviral fraction from the lichen Ramalina farinacea

Abstract

Extracts from the lichen Ramalina farinacea (RF) have previously been shown to be antibacterial, antifungal and most recently, inhibitory to lentiviral and adenoviral vectors. We now determined the antiviral activity of subfractions of the ethylacetate-soluble fraction of RF against a broad spectrum of wild-type viruses, including human immunodeficiency virus type1 (HIV-1), herpes simplex type1 (HSV-1) and respiratory syncytial virus (RSV). Wild-type HIV-1, HSV-1 or RSV were pre-incubated with various concentrations of the ET4 subfraction for 30 minutes 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 the beta-galactosidase expression of the respective indicator cell lines in single round infection assays while the anti-RSV activity was determined via an immunoflourescent technique, employing monoclonal mouse antibody against the P-protein of RSV (as 1° antibody) and a peroxidase-conjugated rabbit antimouse IgG (as 2° antibody). 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 RT assay. ET4 has previously been shown to strongly inhibit the infectivity of lentiviral, retroviral and adenoviral vectors. The present result also shows very strong inhibition of HIV-1, HSV-1 and RSV, with the 50% antiviral concentrations (IC50) of 0.33, 6.09 and 3.65µg/ml respectively. Time-of-addition studies suggest that both entry and post-entry steps of the HIV-1 replication cycle are targeted. Furthermore, ET4 inhibited HIV-1 RT with an IC50 of 0.022µg/ml. ET4 may represent a novel source for lead phyto-antiviral agents. Further fractionation will reveal whether the broad spectrum antiviral activity results from a single compound or from different compounds present in ET4. Acknowledgements: The support of the Alexander von Humboldt Foundation to ECO is gratefully acknowledged. State Committee for Scientific Research, Grant No PBZ-KBN 112/P06/2005