Assessment and Characterization of an Anti-EBV Compound Extracted from Soil-Dwelling Bacteria

Abstract

Introduction: The Epstein Barr Virus (EBV) is thought to infect >90% of the population world-wide; the virus then establishes latency in resting memory B-cells. The virus is associated with infectious mononucleosis (IM), malignancies, autoimmune disorders and organ transplant rejection. Currently, there are no available drugs/vaccines to treat or prevent EBV efficiently. The overall objective of the study at hand is to successfully identify, purify, and isolate an active compound with limited cytoxicity that can inhibit EBV replication. Given the fact that natural products are a good and novel source of antimicrobials, our group, in a previous study identified that secondary metabolites produced by the QSB-12 strain of the Pseudomonas genus grown in the Vegetative (Veg) medium have a potent and minimally cytotoxic anti-EBV effect in vitro. The main objective of this study was to further purify the QSB-12 Veg fractions and to isolate and characterize an effective anti-EBV compound with minimal cytotoxicity in these fractions.

Methods: High-performance liquid chromatography (HPLC) was performed on the combined QSB-12-acquired ethyl acetate and chloroform subfractions that were most effective and minimally cytotoxic in our previous study. To assess for the antiviral effects of the post-HPLC fractions, 0.1 x 106 P3HR-1, EBV producer cells were incubated per well in a 96-well culture plate with 65 ng/mL Phorbol 12-myristate 13-acetate (PMA) and a concentration of 0.08 mg/mL of the fractions 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 percentage of cell viability after incubating with the fractions was assessed using the 0.4% trypan blue exclusion assay. Total live and dead cells were determined in triplicates. Cytotoxicity was assessed after an incubation with the fractions under the same conditions mentioned above but excluding PMA, the inducer of EBV production. After determining the most effective and least cytotoxic fraction, liquid chromatography-mass spectrometry (LC-MS) was performed to characterize the EBV-inhibitory compound and determine its molecular formula. Half maximal inhibitory concentration (IC50) was performed by serially diluting the fraction of interest and incubation with the EBV-producer cells followed by viral load quantification by real-time PCR. Results: Six post-HPLC fractions were assessed for their potential anti-EBV effect. Five fractions were able to significantly decrease the EBV viral load in culture supernatants. However, fraction QSB-C showed the most potent inhibition with minimal cytotoxicity. All fractions were further characterized using LC-MS. The latter identified compounds of similar mass and formula across all fractions. A compound of interest was identified with a specific molecular mass of 245.1271 for an [M+H] ion and a formula of C12H15N5O with 98% purity in the QSB-C fraction. Therefore, the IC50 of the QSB-C fraction was assessed and a value of 7.854 ± 1 µg/mL was determined to decrease the EBV viral load in culture supernatants by 50%.

Conclusion: Our findings have determined that a compound of interest produced by the QSB-12 bacterial isolate of the Psuedomonas genus has potent anti-EBV activity; this compound seems to have minimal cytotoxicity when assessed in vitro in the P3HR-1 cell line. Its 2D-structure and particular mechanism of action is yet to be fully determined; however, based on its molecular formula, and since it was assessed in EBV-positive P3HR-1 cells, the compound is likely inhibiting the stage of DNA synthesis and replication in the viral life cycle.