Indeed, most of what is known about the evolution of influenza A virus comes from population-level epidemiological studies based on the analysis of viral consensus sequences (reviewed in reference 4). However, despite their obvious importance, the frequencies, patterns, and consequences of mutation, reassortment, and natural selection as they occur within individual hosts are poorly understood. These processes are also central to the host adaptation of newly emerging influenza viruses following cross-species transmission and to the evolution of drug resistance in human populations. Influenza A viruses are characterized by rapidly accumulating genetic diversity caused by a combination of error-prone replication ( 1), frequent reassortment during coinfections ( 2), and strong natural selection ( 3). In sum, we were able to reveal, for the first time, the complex interaction between multiple evolutionary processes as they occur within an individual host. Notably, despite frequent reassortment, phylogenetic analysis also provided evidence for specific patterns of segment linkage, with a strong association between the hemagglutinin (HA)- and matrix (M)-encoding segments that matches that previously observed at the epidemiological scale. Individual resistance mutations appeared weeks before they became dominant, evolved independently on cocirculating lineages, led to a genome-wide reduction in genetic diversity through a selective sweep, and were placed into new combinations by reassortment. In this unique natural experiment, we were able to document the complex dynamics underlying the evolution of antiviral resistance. Using single-molecule sequencing, we mapped, in detail, the mutational spectrum of an H3N2 influenza A virus population sampled from an immunocompromised patient who shed virus over a 21-month period. In addition, the short duration of human influenza virus infections limits the available time window in which to examine intrahost evolution.

Although this evolutionary process is initiated within individual hosts, little is known about the pattern, dynamics, and drivers of antiviral resistance at this scale, including the role played by reassortment. Resistance following antiviral therapy is commonly observed in human influenza viruses.

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