Enhanced Genetic Characterization of Influenza A(H3N2) Viruses and Vaccine Effectiveness by Genetic Group, 2014-2015.

BACKGROUND: During the 2014-2015 US influenza season, expanded genetic characterization of circulating influenza A(H3N2) viruses was used to assess the impact of the genetic variability of influenza A(H3N2) viruses on influenza vaccine effectiveness (VE). METHODS: A novel pyrosequencing assay was used to determine genetic group, based on hemagglutinin (HA) gene sequences, of influenza A(H3N2) viruses from patients enrolled at US Influenza Vaccine Effectiveness Network sites. VE was estimated using a test-negative design comparing vaccination among patients infected with influenza A(H3N2) viruses and uninfected patients. RESULTS: Among 9710 enrollees, 1868 (19%) tested positive for influenza A(H3N2) virus; genetic characterization of 1397 viruses showed that 1134 (81%) belonged to 1 HA genetic group (3C.2a) of antigenically drifted influenza A(H3N2) viruses. Effectiveness of 2014-2015 influenza vaccination varied by influenza A(H3N2) virus genetic group from 1% (95% confidence interval [CI], -14% to 14%) against illness caused by antigenically drifted influenza A(H3N2) virus group 3C.2a viruses versus 44% (95% CI, 16%-63%) against illness caused by vaccine-like influenza A(H3N2) virus group 3C.3b viruses. CONCLUSIONS: Effectiveness of 2014-2015 influenza vaccination varied by genetic group of influenza A(H3N2) virus. Changes in HA genes related to antigenic drift were associated with reduced VE.

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

UNLABELLED: 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. IMPORTANCE: 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.

Characterization of drug-resistant influenza A(H7N9) variants isolated from an oseltamivir-treated patient in Taiwan.

BACKGROUND: Patients contracting influenza A(H7N9) infection often developed severe disease causing respiratory failure. Neuraminidase (NA) inhibitors (NAIs) are the primary option for treatment, but information on drug-resistance markers for influenza A(H7N9) is limited. METHODS: Four NA variants of A/Taiwan/1/2013(H7N9) virus containing a single substitution (NA-E119V, NA-I222K, NA-I222R, or NA-R292K) recovered from an oseltamivir-treated patient were tested for NAI susceptibility in vitro; their replicative fitness was evaluated in cell culture, mice, and ferrets. RESULTS: NA-R292K led to highly reduced inhibition by oseltamivir and peramivir, while NA-E119V, NA-I222K, and NA-I222R caused reduced inhibition by oseltamivir. Mice infected with any virus showed severe clinical signs with high mortality rates. NA-I222K virus was the most virulent in mice, whereas virus lacking NA change (NA-WT) and NA-R292K virus seemed the least virulent. Sequence analysis suggests that PB2-S714N increased virulence of NA-I222K virus in mice; NS1-K126R, alone or in combination with PB2-V227M, produced contrasting effects in NA-WT and NA-R292K viruses. In ferrets, all viruses replicated to high titers in the upper respiratory tract but produced only mild illness. NA-R292K virus, showed reduced replicative fitness in this animal model. CONCLUSIONS: Our data highlight challenges in assessment of the replicative fitness of H7N9 NA variants that emerged in NAI-treated patients.

An investigational antiviral drug, DAS181, effectively inhibits replication of zoonotic influenza A virus subtype H7N9 and protects mice from lethality.

Human infections caused by avian influenza A virus type subtype H7N9 have been associated with substantial morbidity and mortality. Emergence of virus variants carrying markers of decreased susceptibility to neuraminidase inhibitors was reported. Here we show that DAS181 (Fludase), an antiviral drug with sialidase activity, potently inhibited replication of wild-type influenza A(H7N9) and its oseltamivir-resistant R292K variants in mice. A once-daily administration initiated early after lethal infection hampered body weight loss and completely protected mice from lethality. We observed a time-dependent effect for 24-72-hour delayed DAS181 treatments on morbidity and mortality. The results warrant further investigation of DAS181 for influenza treatment.