Intrahost dynamics of antiviral resistance in influenza A virus reflect complex patterns of segment linkage, reassortment, and natural selection

Abstract

Resistance following antiviral therapy is commonly observed in human influenza viruses. 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. In addition, the short duration of human influenza virus infections limits the available time window in which to examine intrahost evolution. 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 this unique natural experiment, we were able to document the complex dynamics underlying the evolution of antiviral resistance. 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. 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. 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.

Importance: Understanding the evolutionary forces that shape the genetic diversity of influenza virus is crucial for predicting the emergence of drug-resistant strains but remains challenging because multiple processes occur concurrently. We characterized the evolution of antiviral resistance in a single persistent influenza virus infection, representing the first case in which reassortment and the complex patterns of drug resistance emergence and evolution have been determined within an individual host. Deep-sequence data from multiple time points revealed that the evolution of antiviral resistance reflects a combination of frequent mutation, natural selection, and a complex pattern of segment linkage and reassortment. In sum, these data show how immunocompromised hosts may help reveal the drivers of strain emergence.

FIG 1

Timeline of sampling, drug treatment, and genetic diversity. (a) Sampling and drug treatment. White circles correspond to dates at which samples were collected from the patient. Letters correspond to sampling month, starting with April. A, amantadine treatment; O, oseltamivir treatment; Z, zanamivir treatment. Black lines track the length of time for which the patient was treated with each drug. Days are numbered based on time since first sample. (b) Total genetic diversity based on SNPs present within each sample (identified by LoFreq) and average sequence coverage. nps, nasopharyngeal swabs; clt, culture isolates.

FIG 2

Plots of drug resistance frequency. Histograms representing the frequency of ion channel blocker mutations (a) and neuraminidase inhibitor mutations (b) on Ion Torrent sequence reads. A, amantadine treatment; O, oseltamivir treatment; Z, zanamivir treatment. Black lines track the length of time for which the patient was treated with each drug. nps, nasopharyngeal swabs; clt, culture isolates.

FIG 3

Phylogenetic trees of consensus assemblies for all eight segments. All trees are shown as unrooted. Branch lengths correspond to the number of nucleotide substitutions per site (same scale for each tree), and bootstrap values of >60% are shown. The three clades, defined on the basis of the HA tree topology, are colored red, blue, and black, respectively. M tree symbols: *, S31N. NA tree symbols: *, E119V; **, E119V/I222V; ^, del245–248. Segments and the proteins that they encode: HA, hemagglutinin; M, matrix (M1) and ion channel (M2); NA, neuraminidase; NP, nucleoprotein; NS, nonstructural proteins 1 and 2 (NEP and NS2, respectively); PB2, PB1, and PA, polymerases (PB2 = 2,280 nt, PB1 = 2,274 nt, PA = 2,151 nt, HA = 1,701 nt, NP = 1,497 nt, NA = 1,410 nt, M = 982 nt, NS1 = 863 nt). nps, nasopharyngeal swabs; clt, culture isolates.

FIG 4

Schematic representation of lineage dynamics for segments HA, M, NA, and NP. Consensus (a) and haplotype (b) frequencies across time points (see Table S4 and Fig. S2 in the supplemental material). M segment symbols: white asterisk, S31N; white cross, V27A. NA segment symbols: black asterisk, E119V; white asterisk, I222V; white caret, del245–248. Gray arrowheads point to the suspected reassortment event. Note that time points 196 and 225 show the data from the primary swabs and not from the culture isolates. Numbers above each panel are days since first sample was collected; numbers to the left of each graph in panel b are percents.