Mycophenolic acid inhibits replication of Type 2 Winnipeg, a cerebrospinal fluid-derived reovirus isolate

Abstract

Background: The role of reoviruses in human disease is uncertain. Most identified cases are sporadic and asymptomatic or produce minor upper respiratory or gastrointestinal symptoms. In November 1997, a reovirus was isolated from the cerebrospinal fluid of a severe combined immune deficient infant in Winnipeg, Manitoba. RNA characterization and sequencing studies demonstrated this reovirus isolate to be unique. Thus, the virus was named Type 2 Winnipeg (T2W).

Objective: Mycophenolic acid (MPA), a drug primarily used as an immunosuppressive agent, was assessed in the capacity to inhibit T2W viral growth.

Methods: The effects of MPA on viral growth were determined by plaque reduction assays. Cells were treated with different MPA concentrations, infected with T2W and incubated at 37 degrees C for 0 h to 72 h. Virus titres were determined and compared with untreated controls.

Results: Production of infectious T2W progeny decreased more than 99% at 3 microg/mL MPA compared with untreated controls. Inhibition was not caused by cell toxicity because there was no difference in cell viability. The 50% cell toxic dose was 30 microg/mL MPA.

Conclusions: MPA was able to inhibit viral growth of the novel reovirus T2W. Although MPA is usually used as an immunosuppressive agent, and despite the fact that T2W was isolated from an immunocompromised patient, these results suggest that MPA could have been used as a possible treatment at subimmunosuppressive doses. Animal studies to better define the antiviral and immunosuppressive activities of MPA (and its prodrug mycophenolate mofetil) appear warranted.

Keywords: Mycophenolic acid; Reovirus; Severe combined immune deficiency.

Figure 1

Effect of mycophenolic acid (MPA) on Type 2 Winnipeg (T2W). A Effect of MPA on production of infectious T2W reovirus progeny. L929 cells were pretreated with concentrations of MPA ranging from 0 µg/mL to 100 µg/mL for 1 h before infection with T2W at a multiplicity of infection of 0.12 plaque forming units(PFU)/cell. After virus adsorption, cells were overlaid with fresh minimal essential media containing the appropriate concentrations of MPA and incubated at 37°C. Virus was harvested between 65 h and 72 h postinfection and viral titre was determined. The data represent the average of a minimum of two experiments (n=2) and the error bars represent 1 SD. B Measurement of the cytotoxic effects of MPA on L929 cells. L929 cells were added to a 96-well plate (5x10³ cells/well) and incubated with various concentrations of MPA for 24 h. Cellular response to MPA was measured with the Roche Cell Proliferation Reagent WST-1 (Roche Applied Science, Canada) and plotted against MPA concentration. The data represent the average of a minimum of five experiments (n=5) and the error bars represent 1 SD. TD₅₀ 50% toxic dose

Figure 2

Production of Type 2 Winnipeg virus progeny over time in the presence of mycophenolic acid (MPA). L929 cells were pretreated with 0 µg/mL or 3 µg/mL MPA for 1 h before infection with Type 2 Winnipeg at a multiplicity of infection of 0.12 plaque forming units (PFU)/cell. After virus adsorption, cells were overlaid with fresh minimal essential media that contained either 0 µg/mL (▭)or 3 µg/mL (•) MPA and incubated at 37°C. Virus was harvested at indicated times between 0 h and 72 h postinfection and viral titre was determined. The data represent the average of a minimum of two experiments (n=2) and the error bars represent 1 SD