Deep sequencing reveals mixed infection with 2009 pandemic influenza A (H1N1) virus strains and the emergence of oseltamivir resistance

Abstract

Mixed infections with seasonal influenza A virus strains are a common occurrence and an important source of genetic diversity. Prolonged viral shedding, as observed in immunocompromised individuals, can lead to mutational accumulation over extended periods. Recently, drug resistance was reported in immunosuppressed patients infected with the 2009 pandemic influenza A (H1N1) virus within a few days after oseltamivir treatment was initiated. To better understand the evolution and emergence of drug resistance in these circumstances, we used a deep sequencing approach to survey the viral population from an immunosuppressed patient infected with H1N1/2009 influenza and treated with neuraminidase inhibitors. This patient harbored 3 genetic variants from 2 phylogenetically distinct viral clades of pandemic H1N1/2009, strongly suggestive of mixed infection. Strikingly, one of these variants also developed drug resistance de novo in response to oseltamivir treatment. Immunocompromised individuals may, therefore, constitute an important source of genetic and phenotypic diversity, both through mixed infection and de novo mutation.

Figure 1.

A, Maximum likelihood tree of 1137 NA sequences of pandemic H1N1/2009 virus strains collected during April–July 2009 and the partially reconstructed variants in this patient. The variant 1 consensus sequences assembled from the intrahost sequences—both wild-type (WT) oseltamivir susceptible and oseltamivir resistant (H275Y; variant 1R)—are marked by red boxes, and the assemblies for variants 2 and 3 are denoted by blue boxes. B, Magnification of the section of the maximum likelihood tree containing the variant 1 assemblies. Of note, both Sanger consensus sequences (A/New York/4095/2009 and A/New York/4438/2009) cluster closely with the variant 1 assemblies. Approximate likelihood ratio test results are shown the relevant nodes, and all branch lengths are drawn to a scale of nucleotide substitutions per site.