Neuraminidase Mutations Conferring Resistance to Oseltamivir in Influenza A(H7N9) Viruses.
Human infections by avian influenza A(H7N9) virus entail substantial morbidity and mortality. Treatment of infected patients with the neuraminidase (NA) inhibitor oseltamivir was associated with emergence of viruses carrying NA substitutions. In the NA inhibition (NI) assay, R292K conferred highly reduced inhibition by oseltamivir, while E119V and I222K each caused reduced inhibition. To facilitate establishment of laboratory correlates of clinically relevant resistance, experiments were conducted in ferrets infected with virus carrying wild-type or variant NA genes recovered from the A/Taiwan/1/2013 isolate. Oseltamivir treatment (5 or 25 mg/kg of body weight/dose) was given 4 h postinfection, followed by twice-daily treatment for 5 days. Treatment of ferrets infected with wild-type virus resulted in a modest dose-dependent reduction (0.7 to 1.5 log10 50% tissue culture infectious dose [TCID50]) in nasal wash viral titers and inflammation response. Conversely, treatment failed to significantly inhibit the replication of R292K or E119V virus. A small reduction of viral titers was detected on day 5 in ferrets infected with the I222K virus. The propensity for oseltamivir resistance emergence was assessed in oseltamivir-treated animals infected with wild-type virus; emergence of R292K virus was detected in 3 of 6 ferrets within 5 to 7 days postinfection. Collectively, we demonstrate that R292K, E119V, and I222K reduced the inhibitory activity of oseltamivir, not only in the NI assay, but also in infected ferrets, judged particularly by viral loads in nasal washes, and may signal the need for alternative therapeutics. Thus, these clinical outcomes measured in the ferret model may correlate with clinically relevant oseltamivir resistance in humans.


OBJECTIVE
This report provides more evidence for using the ferret model to assess the susceptibility of influenza A(H7N9) viruses to oseltamivir, the most prescribed anti-influenza virus drug. The information gained can be used to assist in the establishment of laboratory correlates of human disease and drug therapy. The rapid emergence of viruses with R292K in treated ferrets correlates well with the multiple reports on this NA variant in treated human patients. Our findings highlight the importance of the discovery and characterization of new antiviral drugs with different mechanisms of action and the use of combination treatment strategies against emerging viruses with pandemic potential, such as avian H7N9 virus, particularly against those carrying drug resistance markers.
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Henju
H
Marjuki
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Vasiliy P
VP
Mishin
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA Battle Memorial Institute, Atlanta, Georgia, USA.
Anton P
AP
Chesnokov
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA Battle Memorial Institute, Atlanta, Georgia, USA.
Juan A
JA
De La Cruz
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Charles T
CT
Davis
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Julie M
JM
Villanueva
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA.
Alicia M
AM
Fry
Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, USA lgubareva@cdc.gov.
Larisa V
LV
Gubareva
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D004195:Disease Models, Animal; D024882:Drug Resistance, Viral / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005289:Ferrets; D005814:Genes, Viral; D006801:Humans; D064766:Influenza A Virus, H7N9 Subtype / Q000187:drug effects* / Q000235:genetics* / Q000502:physiology; D007251:Influenza, Human / Q000188:drug therapy* / Q000821:virology*; D008297:Male; D009154:Mutation; D009439:Neuraminidase / Q000235:genetics*; D009976:Orthomyxoviridae Infections / Q000188:drug therapy / Q000821:virology; D053139:Oseltamivir / Q000494:pharmacology*; D012141:Respiratory Tract Infections / Q000188:drug therapy / Q000473:pathology / Q000821:virology; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D004791:Enzyme Inhibitors; D053139:Oseltamivir; D009439:Neuraminidase