Nitazoxanide and Tizoxanide Inhibit EHV-1 and Influenza Type A Virus Replication in Vitro
ACVIM 2008
R.J. Callan; L.V. Ashton; L.S. Goehring
Colorado State University College of Veterinary Medicine and Biological Sciences
Fort Collins, CO, USA

There is a need for development of specific antiviral treatment of equine viral diseases. Nitazoxanide (NTZ, Navigator) and its metabolite, tizoxanide (TIZ), are thiazolide compounds that demonstrate antimicrobial activity against a variety of protozoal, bacterial, and viral organisms. The hypothesis for this study is that NTZ and TIZ will decrease replication of equine herpesvirus type 1 (EHV-1) and influenza type A viruses in cell culture. The purpose of the study was to determine the antiviral effective concentrations of NTZ and TIZ that result in 50% (EC50) and 90% (EC90) reduction of equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) production in culture supernatants. In addition, the cytotoxicity and selectivity index of the compounds on cell culture were determined.

Three EHV-1 and three EIV isolates were cultured on RK-13 or Madin-Darby Canine Kidney (MDCK) cells, respectively, in the presence of varying concentrations of NTZ and TIZ. In vitro cytotoxicity concentration (CC50) of NTZ and TIZ was determined as the concentration resulting in a 50% decrease in viability of uninfected cells at 96 hours incubation using an alamar blue reduction assay. Cells were grown in 96 well plates and infected with 7 plaque forming units of virus in the presence of varying concentrations of compound. Viral titers of culture supernatants were determined at 96 hours by plaque assay or TCID50. The EC50 and EC90 for the compounds were determined by linear regression of supernatant viral titers. The selectivity index was calculated as the EC50/CC50.

The CC50 of NTZ and TIZ for RK-13 cells was 15 µM and 25 µM, respectively. The CC50 of NTZ and TIZ for MDCK cells was 65 µM and 130 µM, respectively. The mean EC50 and EC90 and the SI for EHV-1 and EIV are listed in the table below.





EC50 (µM)

EC90 (µM)


EC50 (µM)

EC90 (µM)
















NTZ and its metabolite TIZ show antiviral activity against EHV-1 and EIV. Both compounds were more effective at inhibiting EIV replication than EHV-1. The in vitro results indicate that NTZ may be an effective drug for the prevention or treatment of EHV-1 and EIV infection in horses. Based on these results, further in vivo testing of NTZ for the control or treatment of EHV-1 or EIV in horses is warranted.

Speaker Information
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Robert Callan, DVM, MS, PhD, DACVIM
Colorado State University
Ft. Collins, CO

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