Neuraminidase Mutations Conferring Resistance to Oseltamivir in Influenza A(H7N9) Viruses

Abstract

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 twicedaily treatment for 5 days. Treatment of ferrets infected with wild-type virus resulted in a modest dose-dependent reduction (0.7 to 1.5 log 10 50% tissue culture infectious dose [TCID 50 ]) 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. IMPORTANCEThis 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.

Comparison of the Efficacy of N9 Neuraminidase-Specific Monoclonal Antibodies against Influenza A(H7N9) Virus Infection

“…Because the novel A(H7N9) virus is resistant to M2 blockers, oseltamivir, an NA inhibitor, is commonly used to treat patients with A(H7N9) infection (1,29,30). A number of oseltamivir-resistant clinical A(H7N9) isolates have been identified.…”

“…A number of oseltamivir-resistant clinical A(H7N9) isolates have been identified. Mutations in the NA that are associated with antiviral resistance in these viruses include E119V, I222R or I222K, A246T, and R292K (8,29,31,32). We rescued H6N9 AH/13 viruses that contained 119V, 222R, 246T, or 292K in the NA and tested the ability of MAbs 1E8, 2F6, 11B2, and 10F4 to inhibit their NA activity.…”

Detection of oseltamivir‐resistant zoonotic and animal influenza A viruses using the rapid influenza antiviral resistance test

“…Amino acid substitutions known to reduce susceptibility to oseltamivir E119V, I222K/R, H274Y, R292K, and R371K (N2 numbering) have been detected in the NA of A(H7N9) viruses isolated from humans . In addition, I222T was detected in an A(H7N9) virus isolated from a non‐human primate after oseltamivir treatment .…”

H7N9 avian influenza A virus in China: a short report on its circulation, drug resistant mutants and novel antiviral drugs

“…NAI treatment of wild-type H7N9 infected ferrets resulted in a modest dose-dependent reduction of viral titers in nasal washes, while only a small reduction of viral titers was detected in nasal washes of ferrets infected with the NA-I222K virus [31]. However, NAI treatment failed to inhibit the replication of H7N9 viruses with the NA-R292K or NA-E119V substitutions [33,34]. Recent studies using NAIs and H7N9 virus infections were performed in ducks [28,35].…”