T-705 (Favipiravir) Induces Lethal Mutagenesis in Influenza A H1N1 Viruses In Vitro

ABSTRACT Several novel anti-influenza compounds are in various phases of clinical development. One of these, T-705 (favipiravir), has a mechanism of action that is not fully understood but is suggested to target influenza virus RNA-dependent RNA polymerase. We investigated the mechanism of T-705 activity against influenza A (H1N1) viruses by applying selective drug pressure over multiple sequential passages in MDCK cells. We found that T-705 treatment did not select specific mutations in potential target proteins, including PB1, PB2, PA, and NP. Phenotypic assays based on cell viability confirmed that no T-705-resistant variants were selected. In the presence of T-705, titers of infectious virus decreased significantly (P < 0.0001) during serial passage in MDCK cells inoculated with seasonal influenza A (H1N1) viruses at a low multiplicity of infection (MOI; 0.0001 PFU/cell) or with 2009 pandemic H1N1 viruses at a high MOI (10 PFU/cell). There was no corresponding decrease in the number of viral RNA copies; therefore, specific virus infectivity (the ratio of infectious virus yield to viral RNA copy number) was reduced. Sequence analysis showed enrichment of G→A and C→T transversion mutations, increased mutation frequency, and a shift of the nucleotide profiles of individual NP gene clones under drug selection pressure. Our results demonstrate that T-705 induces a high rate of mutation that generates a nonviable viral phenotype and that lethal mutagenesis is a key antiviral mechanism of T-705. Our findings also explain the broad spectrum of activity of T-705 against viruses of multiple families.

[1]  N. Dimmock,et al.  Defective interfering viruses and their potential as antiviral agents , 2010, Reviews in medical virology.

[2]  R. Webster,et al.  Combination chemotherapy, a potential strategy for reducing the emergence of drug-resistant influenza A variants. , 2006, Antiviral research.

[3]  E. Domingo,et al.  An interfering activity against lymphocytic choriomeningitis virus replication associated with enhanced mutagenesis. , 2010, The Journal of general virology.

[4]  J. Neyts,et al.  Favipiravir (T-705) inhibits in vitro norovirus replication. , 2012, Biochemical and biophysical research communications.

[5]  L. Gubareva,et al.  Antiviral agents for the treatment and chemoprophylaxis of influenza --- recommendations of the Advisory Committee on Immunization Practices (ACIP). , 2011, MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports.

[6]  D. Smee,et al.  Intracellular metabolism of favipiravir (T-705) in uninfected and influenza A (H5N1) virus-infected cells. , 2009, The Journal of antimicrobial chemotherapy.

[7]  Vasiliy P. Mishin,et al.  In Vitro Antiviral Activity of Favipiravir (T-705) against Drug-Resistant Influenza and 2009 A(H1N1) Viruses , 2010, Antimicrobial Agents and Chemotherapy.

[8]  R. K. Robins,et al.  Broad-Spectrum Antiviral Activity of Virazole: 1-f8- D-Ribofuranosyl- 1,2,4-triazole- 3-carboxamide , 1972, Science.

[9]  W. Parker,et al.  Metabolism and antiviral activity of ribavirin. , 2005, Virus research.

[10]  D. Engelthaler,et al.  In Vitro System for Modeling Influenza A Virus Resistance under Drug Pressure , 2010, Antimicrobial Agents and Chemotherapy.

[11]  D. Smee,et al.  Efficacy of Orally Administered T-705 on Lethal Avian Influenza A (H5N1) Virus Infections in Mice , 2006, Antimicrobial Agents and Chemotherapy.

[12]  L. Philipson,et al.  Inhibition of Influenza Virus Ribonucleic Acid Polymerase by Ribavirin Triphosphate , 1977, Antimicrobial Agents and Chemotherapy.

[13]  M. Mendenhall,et al.  In vitro and in vivo activities of T-705 against arenavirus and bunyavirus infections. , 2007, Antimicrobial agents and chemotherapy.

[14]  D. von Laer,et al.  A-to-G Hypermutation in the Genome of Lymphocytic Choriomeningitis Virus , 2006, Journal of Virology.

[15]  H. Narita,et al.  In Vitro and In Vivo Activities of Anti-Influenza Virus Compound T-705 , 2002, Antimicrobial Agents and Chemotherapy.

[16]  V. Simon,et al.  Broad-spectrum antiviral that interferes with de novo pyrimidine biosynthesis , 2011, Proceedings of the National Academy of Sciences.

[17]  Marco Vignuzzi,et al.  Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications. , 2005, Virus research.

[18]  Yoshihiro Kawaoka,et al.  T-705 (favipiravir) activity against lethal H5N1 influenza A viruses , 2009, Proceedings of the National Academy of Sciences.

[19]  D. Smee,et al.  T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections , 2009, Antiviral Research.

[20]  L. Finelli,et al.  Emergence of a novel swine-origin influenza A (H1N1) virus in humans. , 2009, The New England journal of medicine.

[21]  Identification of a Ribavirin‐resistant NS5B mutation of hepatitis C virus during ribavirin monotherapy , 2003, Hepatology.

[22]  E. Domingo,et al.  Lethal mutagenesis of viruses. , 2011, Current opinion in virology.

[23]  R. K. Robins,et al.  THE RELATIONSHIP BETWEEN THE METABOLISM OF RIBAVIRIN AND ITS PROPOSED MECHANISM OF ACTION , 1977, Annals of the New York Academy of Sciences.

[24]  L. Grohskopf,et al.  Immunization of health-care personnel: recommendations of the Advisory Committee on Immunization Practices (ACIP). , 2011, MMWR. Recommendations and reports : Morbidity and mortality weekly report. Recommendations and reports.

[25]  G. Pugliese,et al.  Severe Streptococcus pyogenes Infections, United Kingdom, 2003–2004 , 2008, Emerging infectious diseases.

[26]  D. Smee,et al.  T-705 (Favipiravir) Inhibition of Arenavirus Replication in Cell Culture , 2010, Antimicrobial Agents and Chemotherapy.

[27]  M. Vignuzzi,et al.  Arbovirus high fidelity variant loses fitness in mosquitoes and mice , 2011, Proceedings of the National Academy of Sciences.

[28]  James M Aramini,et al.  Structures of influenza A proteins and insights into antiviral drug targets , 2010, Nature Structural &Molecular Biology.

[29]  B. Bass,et al.  Preferential selection of adenosines for modification by double‐stranded RNA adenosine deaminase. , 1994, The EMBO journal.

[30]  Pan Xin,et al.  Novel Swine-Origin Influenza A(H1N1) Virus , 2009 .

[31]  E. Domingo,et al.  RNA virus fitness , 1997, Reviews in medical virology.

[32]  J. Mullins,et al.  Lethal mutagenesis of HIV with mutagenic nucleoside analogs. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[33]  Nobuhiko Nomura,et al.  Mechanism of Action of T-705 against Influenza Virus , 2005, Antimicrobial Agents and Chemotherapy.

[34]  D. Smee,et al.  Error-prone replication of West Nile virus caused by ribavirin. , 2005, Antiviral research.

[35]  R. J. Bauer,et al.  Mechanism of action of 1- -D-ribofuranosyl-1,2,4-triazole-3-carboxamide (Virazole), a new broad-spectrum antiviral agent. , 1973, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Fried,et al.  Side effects of therapy of hepatitis C and their management , 2002, Hepatology.

[37]  David A. Steinhauer,et al.  Mutation Frequencies at Defined Single Codon Sites in Vesicular Stomatitis Virus and Poliovirus Can Be Increased Only Slightly by Chemical Mutagenesis , 1990, Journal of virology.

[38]  V. Stollar,et al.  Sindbis virus mutants resistant to mycophenolic acid and ribavirin. , 1987, Virology.

[39]  Julie K. Pfeiffer,et al.  Ribavirin Resistance in Hepatitis C Virus Replicon-Containing Cell Lines Conferred by Changes in the Cell Line or Mutations in the Replicon RNA , 2005, Journal of Virology.

[40]  J. Oxford,et al.  The H274Y mutation in the influenza A/H1N1 neuraminidase active site following oseltamivir phosphate treatment leave virus severely compromised both in vitro and in vivo. , 2002, Antiviral research.

[41]  R. Webster,et al.  Drugs in Development for Influenza , 2010, Drugs.

[42]  D. Feigelstock,et al.  Selection of hepatitis C virus resistant to ribavirin , 2011, Virology Journal.

[43]  N. Khardori.  Emergence of a Novel Swine-Origin Influenza A (H1N1) Virus in Humans , 2009 .

[44]  Julie K. Pfeiffer,et al.  A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[45]  A. Monto,et al.  The role of antivirals in the control of influenza. , 2003, Vaccine.

[46]  Baek Kim,et al.  Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex , 2010, PloS one.

[47]  M. Vignuzzi,et al.  Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds , 2010, PLoS pathogens.

[48]  P. Palese,et al.  Modulation of influenza virus replication by alteration of sodium ion transport and protein kinase C activity , 2008, Antiviral Research.

[49]  D. Smee,et al.  Activity of T-705 in a Hamster Model of Yellow Fever Virus Infection in Comparison with That of a Chemically Related Compound, T-1106 , 2008, Antimicrobial Agents and Chemotherapy.

[50]  Pedro R. Lowenstein,et al.  Response of Foot-and-Mouth Disease Virus to Increased Mutagenesis: Influence of Viral Load and Fitness in Loss of Infectivity , 2000, Journal of Virology.

[51]  N. Cox,et al.  Characterization of an avian influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. , 1998, Science.

[52]  C. Cameron,et al.  RNA virus error catastrophe: Direct molecular test by using ribavirin , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[53]  Ying-Ta Wu,et al.  High-throughput identification of compounds targeting influenza RNA-dependent RNA polymerase activity , 2010, Proceedings of the National Academy of Sciences.