Dynamic residue interaction network analysis of the oseltamivir binding site of N1 neuraminidase and its H274Y mutation site conferring drug resistance in influenza A virus

Background Oseltamivir (OTV)-resistant influenza virus exhibits His-to-Tyr mutation at residue 274 (H274Y) in N1 neuraminidase (NA). However, the molecular mechanisms by which the H274Y mutation in NA reduces its binding affinity to OTV have not been fully elucidated. Methods In this study, we used dynamic residue interaction network (dRIN) analysis based on molecular dynamics simulation to investigate the correlation between the OTV binding site of NA and its H274Y mutation site. Results dRIN analysis revealed that the OTV binding site and H274Y mutation site of NA interact via the three interface residues connecting them. H274Y mutation significantly enhanced the interaction between residue 274 and the three interface residues in NA, thereby significantly decreasing the interaction between OTV and its surrounding loop 150 residues. Thus, we concluded that such changes in residue interactions could reduce the binding affinity of OTV to NA, resulting in drug resistant influenza viruses. Using dRIN analysis, we succeeded in understanding the characteristic changes in residue interactions due to H274Y mutation, which can elucidate the molecular mechanism of reduction in OTV binding affinity to influenza NA. Finally, the dRIN analysis used in this study can be widely applied to various systems such as individual proteins, protein-ligand complexes, and protein-protein complexes, to characterize the dynamic aspects of the interactions.

Oseltamivir-Resistant Influenza A (H1N1) Virus Strain with an H274Y Mutation in Neuraminidase Persists without Drug Pressure in Infected Mallards

ABSTRACTInfluenza A virus (IAV) has its natural reservoir in wild waterfowl, and emerging human IAVs often contain gene segments from avian viruses. The active drug metabolite of oseltamivir (oseltamivir carboxylate [OC]), stockpiled as Tamiflu for influenza pandemic preparedness, is not removed by conventional sewage treatment and has been detected in river water. There, it may exert evolutionary pressure on avian IAV in waterfowl, resulting in the development of resistant viral variants. A resistant avian IAV can circulate among wild birds only if resistance does not restrict viral fitness and if the resistant virus can persist without continuous drug pressure. In thisin vivomallard (Anas platyrhynchos) study, we tested whether an OC-resistant avian IAV (H1N1) strain with an H274Y mutation in the neuraminidase (NA-H274Y) could retain resistance while drug pressure was gradually removed. Successively infected mallards were exposed to decreasing levels of OC, and fecal samples were analyzed for the neuraminidase sequence and phenotypic resistance. No reversion to wild-type virus was observed during the experiment, which included 17 days of viral transmission among 10 ducks exposed to OC concentrations below resistance induction levels. We conclude that resistance in avian IAV that is induced by exposure of the natural host to OC can persist in the absence of the drug. Thus, there is a risk that human-pathogenic IAVs that evolve from IAVs circulating among wild birds may contain resistance mutations. An oseltamivir-resistant pandemic IAV would pose a substantial public health threat. Therefore, our observations underscore the need for prudent oseltamivir use, upgraded sewage treatment, and surveillance for resistant IAVs in wild birds.

Simultaneous detection and subtyping of H274Y-positive influenza A (H1N1) using pyrosequencing

Introduction: We investigated the frequency of H274Y-positive swine-origin 2009 A (H1N1) influenza virus outbreak in Thailand during May-August 2009. Methodology: This study sought to find Oseltamivir resistance mutation H274Y by using pyrosequencing. Results: From 8,710 real-time RT-PCR swine-origin 2009 A(H1N1) influenza virus-positive specimens, 100 randomly selected samples identified one such virus with H274Y mutation using pyrosequencing. Conclusions: The patient probably acquired oseltamivir resistance from natural variation, since he had never received that form of treatment before and recovered from influenza-like symptoms without using anti-influenza drugs.

Prophylactic Activity of Intramuscular Peramivir in Mice Infected with a Recombinant Influenza A/WSN/33 (H1N1) Virus Containing the H274Y Neuraminidase Mutation

ABSTRACT Peramivir is a neuraminidase (NA) inhibitor (NAI) under development that must be administered by the systemic route. The prophylactic activity of intramuscular (IM) peramivir was evaluated with mice infected with wild-type (WT) and oseltamivir-resistant (H274Y NA mutant) recombinant influenza A/WSN/33 (H1N1) viruses. Treatment regimens consisted of IM injections starting 1 h before viral challenge that were single (45 mg/kg or 90 mg/kg) or multiple (45 mg/kg daily for 5 days). All peramivir regimens prevented mortality and weight loss while significantly reducing lung viral titers (LVT) in mice infected with the WT virus. For animals infected with the H274Y mutant, the multiple-dose regimen completely prevented mortality and was associated with significant reduction in weight loss and LVT compared to untreated animals. In contrast, both single-treatment regimens reduced mortality and weight loss but did not significantly reduce LVT. Although further experiments using different influenza A/H1N1 virus strains and other animal models are needed, our results suggest that 5-day IM peramivir therapy may be considered a prophylactic alternative to control influenza infections caused by oseltamivir-resistant viruses with the H274Y mutation.