Intracellular ATP and Total Adenylate Concentrations Are Critical Predictors of Reovirus Productivity From Vero Cells A. Burgener, 1 K. Coombs, 2 M. Butler 1 1 Department of Microbiology, Buller Bldg., University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada; telephone: 204-474-6543; fax: 204-474-7603; e-mail: butler@cc.umanitoba.ca 2 Department of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada Received 15 April 2005; accepted 29 December 2005 Published online 28 March 2006 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/bit.20873 Abstract: The productivity of reovirus type-3 Dearing was studied in cultures of Vero cells in serum-free media. Viral productivity was dependent upon the metabolic state of the cells rather than the phase of growth at which the cells were infected. Cells at different energy states were established by 24-h incubation in nutrient-depleted media. This resulted in variable intracellular nucleotide concentrations but high cellular viability was maintained. Of the nucleotides analyzed at the time of infection only the intracellular [ATP] and total adenylate nucleotides were positively correlated with viral productivity. The correlated data followed a sigmoidal plot with an equation defined by polynomial regression analysis. Apparent threshold values of 3.2 fmol/cell and 3.3 fmol/cell were established for ATP and total adenylate, respectively, at which the viral production was 50% the maximal value. Cultures with lower ATP and total adenylate levels at the time of infection resulted in as much as a 95% reduction in overall viral titer compared to the control. The adenylate energy charge (AEC) showed a negative correlation with viral production with an AEC value >0.97 resulting in low virus productivity. Intracellular ATP or total adenylate concentration at the point of infection may be used as a predictor of viral yield in bioprocesses designed for virus/ vaccine production. ß 2006 Wiley Periodicals, Inc. Keywords: Vero; reovirus; ATP; adenylate; energy charge INTRODUCTION Recent work has shown the potential for using the mammalian reovirus as an oncolytic (anti-cancer) agent (Coffey et al., 1998). The virus is able to exploit an activated Ras pathway which is common to 30% of human cancers. Presently reovirus is being tested to treat various forms of cancer including brain, prostate, and glioma (Oncolytics Biotech, Inc., Calgary, Alberta, Canada). It is also being tested in the treatment of breast cancer showing early success (Hirasawa et al., 2003; Norman et al., 2002). These findings suggest the need to produce large quantities of reovirus for human therapeutic use. The mammalian reoviruses are members of the Orthor- eovirus genus of the family Reoviridae. The Reoviridae include a number of important human and animal pathogens such as rotavirus (Estes, 2001; Kapikian et al., 2001) and orbivirus (Roy, 2001). Rotavirus infection is estimated to be directly responsible for up to 2 million deaths each year, and despite intensive efforts, the availability of an effective vaccine has been elusive (Lang et al., 1996). The first available vaccine (RotaShield) was withdrawn from the market in 1999 due to a high incidence of secondary compli- cations (Murphy et al., 2001). However, an attenuated human rotavirus vaccine (Rotarix) has recently become available and promises to be effective for the on-going world-wide Rotavirus Vaccine Program (www.rotavirusvaccine.org). Reovirus, the prototype of this family, remains an effective model for studying the characteristics of the double stranded RNAviruses (Nibert and Schiff, 2001; Tyler, 2001). Reovirus is produced routinely in either monolayer or stirred cultures of mouse L929 fibroblasts in a serum-containing medium (Jung et al., 2004; Nibert et al., 1996). However, serum- containing culture systems are becoming undesirable for the large-scale production of vaccines. There are a number of disadvantages of serum supplementation including batch-to- batch variation in composition, the high protein content which hinders product purification and the potential for viral, mycoplasma or prion contamination. The recent threat to human health caused by the undefined agents of bovine spongiform encephalopathy (BSE) is likely to limit the continued use of bovine serum in culture processes used for the synthesis of health-care products such as viral vaccines (Asher, 1999). Furthermore, the L929 cell line is not currently accepted by the WHO for the production of human vaccines. An alternative approach is to use a cell line commonly used for the production of human vaccines, such as the Vero cell line. Vero cells have been accepted for viral vaccine production under specified regulatory guidelines and are ß 2006 Wiley Periodicals, Inc. Correspondence to: M. Butler