Early detection of endogenous retroviruses in chemically induced mouse cells

Endogenous retroviral sequences are present as an integral part of eukaryot ic genomes. Although the majority of these sequences are defective, a few c an produce infectious virus, either spontaneously upon long-term culture or by treatment with various chemical or other agents. Early, extensive studi es of retrovirus induction were done in mouse cells; however, similar studi es have not been done using state-of-the-art vir-us detection assays and wi th cells of other mammalian species. To investigate induction and detection of occult retroviruses in cells of different species, especially primate c ells that are used in production of biologics, we have initially determined the optimum conditions for retrovirus induction in chemically treated K-BA LB mouse cells using highly sensitive product-enhanced reverse transcriptas e (PERT) assays as well as transmission electron microscopy (TEM). Retrovir us induction was detected at day I post-drug treatment under all test condi tions but was optimum using 30 mug ml(-1) of 5-iododeoxyuridine (IdU) for 2 4 h. Additionally, the combination of IdU and 5-azacytidine specifically en hanced activation of type C particles. RT activity was detected by PERT ass ays in one microliter equivalent of test sample and retroviral particle pro duction was seen by TEM analysis. The induction of infectious murine leukem ia retroviruses was confirmed by infectivity assays and correlated with PER T activity. These results indicate that strategies for detection of occult viral agents should include optimization of induction conditions using mult iple viral detection assays to evaluate virus activation. (C) 2001 Elsevier Science B.V. All rights reserved.