SUSCEPTIBILITY OF EPSTEIN BARR VIRUS (EBV) STRAINS TO CANDIDATE ANTIVIRAL DRUGS

Background:  EBV is associated with a wide spectrum of diseases, including infectious mononucleosis (IM) and post-transplant lymphoproliferative disorder. Although the use of antiviral agents in the treatment of EBV-related diseases is controversial, we have shown that valacyclovir therapy reduced oral excretion of EBV and possibly produced a clinical benefit in IM (J Clin Virol 2007; 39:16-21).  In order to choose the optimum drug for further clinical studies, we developed an in vitro assay for susceptibility of EBV to candidate drugs (ICAAC 2007). Here we describe several modifications of this assay, which improve its performance and through-put.  We also report for the first time, to our knowledge, the drug susceptibilities of isolates from patients with IM.

Methods:  The assay modifications were done using the EBV producer cell line P3HR1 (originally derived from a Burkitt-lymphoma patient). P3HR1 cells were grown in the presence of varying concentrations of acyclovir (ACV), ganciclovir (GCV) or H2G and 20 ng/mL TPA (12-O-tetradecanoyl-phorbol-13-acetate), which induces replication of EBV DNA. Instead of allowing the cells to be exposed to TPA for 7 days, as we had done previously, we washed them in PBS and resuspended them in fresh media with the same drug concentration but without TPA on day 2.  On day 7, total cellular DNA was extracted (Qiagen mini Blood kit) from one million cells, and EBV DNA was detected using an in-house quantitative real-time PCR method that amplifies a 71-bp portion of the EBNA1 gene. In order to increase the through-put of the assay, 24-well plates were used instead of flasks, and a defined volume (200 ml) rather than a defined number (1 million) of cells was extracted.  For each drug, the concentration required to reduce EBV DNA in TPA-stimulated P3HR1 cells by 50% (IC50) was determined by 3 independent experiments. Each experiment was performed in duplicate or triplicate.  Patient EBV isolates were tested in a similar manner after lymphocyte cell lines were established by serial passages in the presence of cyclosporin.

Results:  We demonstrated enhanced viability of the P3HR1 cells when TPA was removed on Day 2 as compared with TPA being in contact with the cells for all 7 days.  The ratio of viable to nonviable cells on day 7 was improved from 1.55+/-0.04 to 8.7+/-0.9.  Using this assay modification, the IC50s of each drug (and SDs) were ACV, 9 (3) mM; GCV, 3 (11) mM; and H2G, 3 (2) mM. EBV isolated from IM patients had similar drug susceptibilities. We also showed that whether the assay was set up in flask or plate format, the results were virtually identical.

Conclusions:  We have demonstrated the susceptibility of EBV to various antiviral drugs using a relatively simple in vitro assay. For the first time we have shown the susceptibility of EBV strains from patients with IM. This in vitro method may be useful for monitoring development of resistance when immunocompromised hosts with serious EBV diseases are treated with antiviral agents.