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

Abstract

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.

Keywords: E119V; H7N9; I222K; I222R; R292K; ferrets; influenza virus; mice; oseltamivir; peramivir.

Figure 1.

Replication kinetics of influenza A(H7N9) isolated from a patient in Taiwan. Madin-Darby canine kidney (MDCK; A) or MDCK-SIAT1 (B) cells were infected with each plaque-purified virus at a multiplicity of infection of 0.0001. Virus-containing supernatants were collected at the indicated time points, and virus titers were determined in MDCK-SIAT1 cells. The statistically significant difference between wild-type (WT) and R292K viruses are shown (C). Dotted lines indicate the limit of virus titer detection. *P = .0175; **P < .0001. Abbreviation: TCID₅₀, median tissue culture infective doses.

Figure 2.

Virulence of Taiwan/1(H7N9) neuraminidase variants in mice. Animals (n = 4/group) were intranasally inoculated with 10¹, 10², 10³, 10⁴, or 10⁵ median tissue culture infective doses (TCID₅₀) of indicated viruses. Body weight change (A, C, E, G, and I) and survival (B, D, F, H, and J) were monitored daily. Mice that lost ≥25% (dotted line) of their baseline weight were humanely euthanized. Each value (A, C, E, G, and I) represents the average percentage loss of body weight (±SD). Abbreviation: SD, standard deviation.

Figure 3.

Replicative fitness of Taiwan/1(H7N9) neuraminidase variants in ferrets. Animals (n = 3–4/group) were intranasally inoculated with 10⁶ median tissue culture infective doses (TCID₅₀) of the indicated viruses. Nasal washes were collected daily for 10 days, and virus titers (A) (*P ≤ .004), inflammatory cell counts (B), and protein concentrations (C) were determined. The body weight change (D) and temperature (E) were recorded daily. The statistically significant difference in nasal wash virus titers between wild-type and R292K viruses is shown. Dotted lines indicate limit of virus titer detection. SDs and P values are not shown if <3 animals shed virus. Abbreviation: SD, standard deviation.