Optimization of chemical induction for evaluation of endogenous retroviruses in different species

Chemical inducers such as 5-azacytidine (AzaC) and 5’iodo-2’deoxyuridine (lUdR) have been used to discover and characterize endogenous retroviruses from rodent and avian speciecs, for example KBALB mouse cells. We found that induction conditions that have been optimized for mouse cells were not successful in inducing retrovirus from cell lines of other species, including nonhuman primates. Therefore, we developed a stepwise strategy based upon identification of critical parameters for endogenous retrovirus induction in mouse cells for optimizing induction conditions for non-murine cells. Using this approach, we have determined optimum conditions for investigating inducible endogenous retroviruses from Vero cells, which are of African green monkey (AGM) origin, a species that has never been reported to produce endogenous retroviruses [1]. Materials and methods Based upon a step-wise induction strategy [2], Vero cell growth characteristics such as growth curve, population doubling time, and cell cycle phase were determined; drug dose range was obtained by evaluating cell toxicity and cell recovery using different drug concentrations (lUdR, 50- 3,200 ug/ml; AzaC, 0.2125- 40 ug/ml; and NaBut, 1- 6 mM). Cellular RNAs were tested for endogenous retrovirus activation using virus-specific PCR assays and filtered supernatants were analyzed for virus particle production using a highly-sensitive RT assay and PCR assays [1]. Infectivity studies were done using various cell lines from different species based upon their susceptibility to known retrovirus infections. Cellular RNAs from untreated and AzaC-treated cells will be

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