Detection of Molecular Markers of Antiviral Resistance in Influenza A (H5N1) Viruses Using a Pyrosequencing Method

Abstract

Resistance of influenza viruses to antiviral drugs can emerge following medication or may result from natural variation. Two classes of anti-influenza virus drugs targeting either the M2 protein (amantadine and rimantadine) or neuraminidase (NA; oseltamivir and zanamivir) are currently licensed. These drugs are expected to be important in controlling the early stages of a potential pandemic. In the present study, we describe how a pyrosequencing method can be used to rapidly detect established molecular markers of resistance to M2 blockers and NA inhibitors in influenza A (H5N1) viruses. The residues L26, V27, A30, S31, and G34 in the M2 protein were targeted for pyrosequencing. The NA residues for pyrosequencing analysis included the established markers of drug resistance (H274 and N294), as well as residues of less certain relevance (V116, I117

Competitive Fitness of Oseltamivir-Sensitive and -Resistant Highly Pathogenic H5N1 Influenza Viruses in a Ferret Model

“…S1 in the supplemental material). We were interested in the existence of potential hostdependent NA mutations at the catalytic (R292) and framework (E119, I222, and N294) sites and in close proximity to the NA enzyme active site (V116, I117, Q136, K150, and Y252) because mutations at these residues either confer resistance and cross-resistance to oseltamivir and zanamivir (12,24,26,35) or were linked to reduced drug susceptibility in avian and human viruses carrying N1 NA (10). In most groups, host-dependent NA mutations were detected in 2 of 3 or 3 of 3 animals.…”

The Evolving Threat of Influenza Viruses of Animal Origin and the Challenges in Developing Appropriate Diagnostics

“…These drugs are experimentally effective against most animal influenza viruses. Molecular assays (e.g., real-time RT-PCR assay and pyrosequencing) for specific mutations known to confer antiviral resistance (e.g., H275Y in N1, E119V in N2, and S31N in M2) have been established (39,40 ), but these assays are unlikely to be useful for detecting such mutations in a pandemic strain that is yet to emerge. Furthermore, viruses containing resistant mutations located at other positions/segments would not be picked up by these assays.…”

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

“…They inhibit virus replication by mimicking the natural substrate of the influenza neuraminidase (the sialic acid receptors), and bind to the active site, preventing neuraminidase from cleaving host-cell receptors or releasing new viruses [1]. Generally, resistance of influenza viruses to antiviral drugs can emerge following medication or may result from natural variation [3]. In case of NAIs, different resistant mechanisms have been observed.…”