R292K Substitution and Drug Susceptibility of Influenza A(H7N9) Viruses

Sleeman, Katrina; Guo, Zhu; Barnes, John; Shaw, Michael; Stevens, James; Gubareva, Larisa V.

doi:10.3201/eid1909.130724

Cited by 63 publications

(62 citation statements)

References 12 publications

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“…The R292K substitution in H7N9 viruses is the most commonly reported marker associated with highly reduced inhibition by oseltamivir in the NI assay (32,33). R292K has been detected 1 to 9 days after initiating treatment (3,4,32).…”

Section: Discussionmentioning

confidence: 99%

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

Marjuki

Mishin

Chesnokov

et al. 2015

J Virol

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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.

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Section: Discussionmentioning

confidence: 99%

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

Marjuki

Mishin

Chesnokov

et al. 2015

J Virol

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“…Genotypic assays alone are not sufficient for antiviral surveillance studies, and a phenotypic assay is still needed to identify novel substitutions that reduce inhibition or to identify the impact of a substitution in a specific genetic background. Different substitutions in NA result in different levels of NA activity in buffers of different pHs (65,66). It might be possible to reduce the masking effect through simple modifications of the NI assay, although more studies are necessary to examine this effect on the breadth of NA substitutions in influenza B viruses.…”

Section: Discussionmentioning

confidence: 99%

Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

Burnham

Armstrong

Webster

et al. 2015

J Virol

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Influenza A and B viruses are human pathogens that are regarded to cause almost equally significant disease burdens. Neuraminidase (NA) inhibitors (NAIs) are the only class of drugs available to treat influenza A and B virus infections, so the development of NAI-resistant viruses with superior fitness is a public health concern. The fitness of NAI-resistant influenza B viruses has not been widely studied. Here we examined the replicative capacity and relative fitness in normal human bronchial epithelial (NHBE) cells of recombinant influenza B/Yamanashi/166/1998 viruses containing a single amino acid substitution in NA generated by reverse genetics (rg) that is associated with NAI resistance. The replication in NHBE cells of viruses with reduced inhibition by oseltamivir (recombinant virus with the E119A mutation generated by reverse genetics [rg-E119A], rg-D198E, rg-I222T, rg-H274Y, rg-N294S, and rg-R371K, N2 numbering) or zanamivir (rg-E119A and rg-R371K) failed to be inhibited by the presence of the respective NAI. In a fluorescence-based assay, detection of rg-E119A was easily masked by the presence of NAI-susceptible virus. We coinfected NHBE cells with NAI-susceptible and -resistant viruses and used next-generation deep sequencing to reveal the order of relative fitness compared to that of recombinant wild-type (WT) virus generated by reverse genetics (rg-WT): rg-H274Y > rg-WT > rg-I222T > rg-N294S > rg-D198E > rg-E119A Ͼ Ͼ rg-R371K. Based on the lack of attenuated replication of rg-E119A in NHBE cells in the presence of oseltamivir or zanamivir and the fitness advantage of rg-H274Y over rg-WT, we emphasize the importance of these substitutions in the NA glycoprotein. Human infections with influenza B viruses carrying the E119A or H274Y substitution could limit the therapeutic options for those infected; the emergence of such viruses should be closely monitored. Annual vaccination is an effective method for controlling influenza disease. The current FDA-approved quadrivalent seasonal influenza vaccine includes both antigenically distinct hemagglutinin (HA) lineages of influenza B virus (i.e., Yamagata and Victoria) (8, 9). Antiviral treatment is another option for the control of influenza, and the neuraminidase (NA) inhibitors (NAIs) are currently the only class of antivirals approved for prophylaxis and treatment of influenza B virus infections. NAIs limit influenza

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“…The effects of NA inhibitors against H7N9 virus have been examined in mice, but effects of NA inhibitors in mice infected with H7N9 virus were controversial in previous studies (4,(9)(10)(11). Furthermore, many patients infected with H7N9 virus have been treated with NA inhibitors, but clinical outcomes after treatment were not always satisfactory, probably due to late initiation of antiviral therapy, coexisting medical complications, and emergence of a virus that is resistant to antiviral drugs (3,10,(12)(13)(14). However, the frequency of emergence of a resistant virus has not been determined in human patients.…”

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confidence: 99%

Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates

Itoh

Shichinohe

Nakayama

et al. 2015

Antimicrob Agents Chemother

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viruses with low sensitivity to the NA inhibitors were detected was much higher than that of macaques in which variant H5N1 highly pathogenic influenza virus was detected after treatment with one of the NA inhibitors in our previous study. The virus with R289K in NA was reported in samples from human patients, whereas that with I219T in NA was identified for the first time in this study using macaques, though no variant H7N9 virus was reported in previous studies using mice. Therefore, the macaque model enables prediction of the frequency of emerging H7N9 virus resistant to NA inhibitors in vivo. Since H7N9 strains resistant to NA inhibitors might easily emerge compared to other influenza viruses, monitoring of the emergence of variants is required during treatment of H7N9 influenza virus infection with NA inhibitors.

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R292K Substitution and Drug Susceptibility of Influenza A(H7N9) Viruses

Cited by 63 publications

References 12 publications

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

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

Competitive Fitness of Influenza B Viruses with Neuraminidase Inhibitor-Resistant Substitutions in a Coinfection Model of the Human Airway Epithelium

Emergence of H7N9 Influenza A Virus Resistant to Neuraminidase Inhibitors in Nonhuman Primates

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