Oral Presentation 9th Australasian Virology Society Meeting 2017

Influenza virus susceptibility and likelihood of resistance to the antiviral drug Nitazoxanide (#29)

Paulina Koszalka 1 2 , Danielle Tilmanis 2 , Jean-Francois Rossignol 3 , Aeron Hurt 1 2
  1. University of Melbourne, Melbourne, VIC, Australia
  2. WHO Collaborating Centre for Reference and Research on Influenza, VIDRL, Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
  3. Romark Laboratories, Tampa, Florida, USA

Nitazoxanide (NTZ) is a repurposed drug currently in phase III clinical trials for the treatment of uncomplicated influenza. It is a host-targeted influenza antiviral that is thought to inhibit the endoplasmic reticulum protein ERp57, consequently affecting the maturation and trafficking of the influenza haemagglutinin protein, which is essential for viral replication. With the potential licensure of NTZ, it is important to establish the susceptibility of circulating viruses to the drug, and in addition, the potential for the emergence of viral resistance. A cell culture-based focus reduction assay was developed to determine the susceptibility of 210 circulating seasonal influenza viruses, including A(H1N1)pdm09, A(H3N2), B (Yamagata/Victoria) lineage viruses, and neuraminidase inhibitor (NAI) resistant viruses, to tizoxanide (TIZ) the active metabolic form of NTZ. TIZ was effective against all influenza subtypes tested with median EC50 values (± IQR) of 0.48 µM (0.33-0.71), 0.62 µM (0.56-0.75), 0.66 µM (0.62-0.69), and 0.60 µM (0.51-0.67) obtained for A(H1N1)pdm09, A(H3N2), B(Victoria lineage), and B(Yamagata lineage) influenza viruses respectively.  To study the propensity for the development of TIZ resistance in vitro, six influenza viruses including A(H1N1)pdm09, A(H3N2) and B viruses, were  serially passaged ten times in the absence or presence of increasing concentrations of TIZ up to 20 µM. All viruses were tested for TIZ susceptibility and the haemagglutinin, neuraminidase and matrix genes were sequenced using Sanger sequencing. None of the six viruses had any significant changes in TIZ susceptibility following serial passage in TIZ. Sequencing revealed two haemagglutinin mutations, H451K and T214A, which were unique to the TIZ passaged viruses, however, these mutations did not confer altered virus TIZ susceptibility. Our results demonstrate that currently circulating NAI-sensitive and NAI-resistant viruses are susceptible to TIZ and that resistance to this drug is unlikely to develop, possibly because the drug targets a host protein, rather than viral protein.