Deep Sequencing Reveals Mixed Infection with 2009 Pandemic Influenza A (H1N1) Virus Strains and the Emergence of Oseltamivir Resistance

Although an influenza infection is typically self-limiting and usually lasts <1 week, prolonged viral shedding is commonplace in immunosuppressed patients [1–6], such that de novo mutations can develop and accumulate over longer periods. In the case of a mixed infection, in which an individual host is infected with multiple viral variants either simultaneously or sequentially, the extended duration of influenza infection may lead to changes in the frequencies of any of the infecting variants [7] and can facilitate reassortment among them. As a consequence, immunosuppressed individuals, particularly those experiencing mixed infection, may constitute an important source of genetic and phenotypic diversity, generating variants that differ in such properties as antiviral susceptibility, virulence, and transmissibility. In March 2009, Mexico experienced outbreaks of respiratory illness caused by a novel influenza A (H1N1) virus of swine origin [8]. Subsequently, cases of infection with this virus were found both in the United States and globally, and a pandemic was officially declared by the World Health Organization on 11 June 2009. Because the pandemic virus was demonstrated to already be resistant to the M2-ion channel blockers (amantadine and rimantadine) [9], neuraminidase inhibitors were used as first-line drugs in both prophylaxis and treatment of patients infected with H1N1/2009. Although the pandemic virus remains largely susceptible to oseltamivir and zanamivir, sporadic cases of oseltamivir resistance have been reported worldwide [10–13]. The resistance mutation in all of these cases is a histidine-to-tyrosine amino acid substitution at residue 275 of the neuraminidase (NA) protein (H274Y in N2 numbering; H275Y in N1 numbering). Because of the rapid spread of H275Y-based oseltamivir resistance in seasonal H1N1 virus strains [14], even sporadic reports of this mutation in the pandemic H1N1/2009 virus are of significance. Oseltamivir resistance in virus strains infecting immunosuppressed patients was reported to emerge within a few days after treatment, rather than after prolonged therapy [15–17]. Clearly, this rapid selection for drug resistance is of major concern. From the early stages of its emergence in the human population, genomic sequence data on the pandemic H1N1/2009 virus have been made rapidly available on public databases. In most cases, the sequences generated for these analyses represent consensus sequences taken from single time points during infection, thereby providing a snapshot of the dominant virus variant infecting each patient. We have previously shown that mixed infections are a common occurrence in the case of seasonal influenza virus and, thus, an important source of genetic diversity, but are not normally apparent from consensus sequencing [7]. To determine whether this is also true of the pandemic H1N1/2009 virus and what the consequences of this process might be for viral evolution, we investigated the increase of oseltamivir resistance in a longitudinal collection from an immunosuppressed patient infected with pandemic H1N1/2009 virus with use of massively parallel (deep) sequencing.