Identification of Anti-Epstein-Barr Virus Compounds in Extracts from Soil-dwelling Microorganisms

Abstract

Title: Identification of Anti- Epstein-Barr Virus Compounds in Extracts from Soil-dwelling Microorganisms

Introduction: Epstein-Barr virus infects more than 90% of the human population and establishes latency in resting memory B-cells. It is associated with infectious mononucleosis (IM) and many other malignant and autoimmune diseases. There is currently no available effective and safe therapy for the treatment of EBV. Given that natural products possess antimicrobial properties, our group previously examined secondary metabolites secreted by several soil-dwelling bacterial isolates for their efficacy in inhibiting EBV replication in vitro. Natural products from the QSB-12 strain grown in the Vegetative medium and BM-12 strain grown in the A medium had shown an effective anti-EBV activity with no significant cytotoxicity. The complexity and nature of these crude extracts, and their mechanism of action remain to be examined. Therefore, the main objective of this study was to fractionate and purify these crude extracts to isolate effective anti-EBV compounds with minimal cytotoxicity.

Methods: Soil samples were taken from Qsaybeh (QSB) and Beit-Meri (BM) regions in Lebanon. Bacterial isolates were inoculated into different productive culture media for secondary metabolite extraction. To assess the antiviral effects of the crude extracts, 0.1 x 106 P3HR-1 producer cells were incubated per well in a 96-well culture plate with 35 ng/mL Phorbol 12-myristate 13-acetate (PMA) and either of two concentrations (0.008 mg/mL and 0.08 mg/mL) of the crude extracts for 5 days, at 37°C in a humidified incubator. The extent of viral replication (EBV load) was assessed by quantitative real-time PCR. Additionally, the cytotoxic effect of the extracts was assayed using either 0.4% trypan blue applied to cell counting slides and read with an automated cell counter or adding 0.1μg/mL of the DAPI stain and subsequent reading with a BD FACSAria flow cytometer. Cytotoxicity was assessed after an incubation with the extracts under the same conditions mentioned above but excluding PMA, the inducer of EBV production. To fractionate the crude extracts, liquid-liquid partitioning, thin layer chromatography (TLC), and column chromatography were performed, and the resulting fractions were tested for their anti-EBV abilities and cytotoxicities as described for the crude extracts.

Results: Crude extracts from the QSB-12 bacterial isolate grown in the Vegetative (Veg) medium showed a very high reduction in the EBV load in cell-free supernatants with minimal cytotoxicity. QSB-12 16S rRNA sequencing showed that the genus of the QSB-12 bacterium is Pseudomonas. Liquid-liquid partitioning of the crude extracts prepared from the QSB-12 Veg scale-up led to the preparation of 4 fractions: water, hexane, ethyl acetate, and chloroform. The ethyl acetate and chloroform fractions were able to decrease the EBV load, at the two concentrations tested, by 55-folds (p=0.001), with no cytotoxicity observed for the lower concentration (0.008 mg/mL) tested. TLC and column chromatography resulted in the preparation of three subfractions from the ethyl acetate fraction and four from the chloroform fraction. The three ethyl acetate subfractions decreased the EBV load at a concentration of 0.08 mg/mL (p=0.002). Two of the ethyl acetate subfractions used at a concentration of 0.08 mg/mL exerted a significant cytotoxicity, indicating that one ethyl acetate subfraction is safe and effective in decreasing the EBV load in vitro. On the other hand, three of the four chloroform subfractions resulted in a significant decrease in the EBV load at a concentration of 0.08 mg/mL (p=0.01). Only one of the four chloroform subfractions exerted a significant cytotoxicity at a concentration of 0.08 mg/mL, indicating that three chloroform subfractions are safe and two out of them are effective in decreasing the EBV load in vitro.

Conclusion: Our findings suggest that one or more compounds with an anti-EBV activity are present in fractions from secondary metabolites released by soil-dwelling bacteria. The nature and structure of these compounds, and their respective modes of action remain to be assessed.