The emergence of oseltamivir resistance in pandemic H1N1/2009 remains rare in the immunocompetent population [1
], and only ~1% of NA sequences available in GenBank possess the H275Y mutation. In the case of immunosuppressed patients who experience prolonged viral shedding, oseltamivir treatment has apparently led to the selection of the 275Y drug resistance mutation on multiple occasions [1
]. Here, we showed that a patient was infected with 3 variants of H1N1/2009 and that the major variant also contained a subpopulation of oseltamivir-resistant virus strains (variant 1R). This subpopulation was not strongly evident on conventional Sanger consensus sequencing, and pyrosequencing assays have been shown to be unreliable for the detection of drug-resistant mutant populations comprising <10% of the virus population [31
]. Therefore, we suggest that the resistance-determining H275Y mutation may be more frequent than is usually supposed but is present as a minority population in infected hosts. Indeed, in this case, H275Y was only detected by careful scrutiny of the Sanger sequencing chromatogram, confirmation by deep sequencing, and the fortuitous overgrowth in culture by the mutant.
Because the H275Y-bearing virus strains identified here were most closely related to oseltamivir-susceptible strains in the global population and because H275Y is well known to have a detrimental effect on viral fitness in the absence of drug [33
], it seems likely that the H275Y mutation appeared de novo in this patient and, thus, is directly linked to the use of oseltamivir. When the primary samples were inoculated into culture, the resistant mutant outgrew the wild-type (and formerly dominant) variant. Similar observations have been made previously during Madin-Darby canine kidney cell culture [12
]. Although the H275Y mutation in seasonal H1N1 influenza A spread rapidly in vivo in the absence of drug pressure, transmission of the equivalent mutant in pandemic H1N1/2009 virus strains has fortunately remained limited thus far [13
In addition to harboring a subpopulation of oseltamivir-resistant virus strains, the patient studied here was noteworthy for the occurrence of a mixed infection: 3 variants were detected that were in 2 phylogenetically distinct clades of the pandemic H1N1/2009 virus (although it is uncertain whether variants 2 and 3 evolved de novo from one another). The occurrence of mixed infection in H1N1/2009 is clearly the precursor to intraserotype reassortment, which is commonplace in seasonal influenza A virus [34
], although distinguishing bona fide reassortment from mixed infection remains an analytical challenge. In this context, it is particularly intriguing that both variants 2 and 3 belong to clade 2, which had not been previously found in samples beyond May 2009. Therefore, clade 2 either continued to circulate undetected in New York through late June or this patient was infected earlier and remained asymptomatic. Indeed, there are examples in the literature of immunosuppressed individuals with prolonged virus shedding [4
]. In addition, it is unclear whether variants 2 and 3 were present in the initial infection along with variant 1 or occurred sequentially. Normally, individuals would be protected from a superinfection with antigenically related virus strains, as appears to be the case for pandemic H1N1/2009 virus strains [36
]. However, in the case of immunosuppressed patients, it is possible that an inadequately protective immune response may be generated against the first infecting virus strain.
Finally, of note, the dominant virus variant in this patient was characterized by a number of unique mutations, including NA D199N, which has not previously been identified in pandemic H1N1/2009 influenza virus strains. Of interest, a D199E mutation in seasonal H1N1 has been associated with reduced susceptibility to oseltamivir [29
], a D198G (universal numbering; equivalent to site 199) mutation in H5N1 is associated with reduced susceptibility to oseltamivir and zanamivir [37
], and a D198N mutation in influenza B virus is associated with high oseltamivir resistance [5
]. In light of newly characterized permissive secondary mutations that enable oseltamivir resistance in seasonal H1N1 virus strains to spread among untreated patients worldwide [36
], we are currently assessing the functional significance of the D199N mutation.