Longitudinal sampling is required to maximize detection of intrahost A/H3N2 virus variants

Abstract

Seasonal human influenza viruses continually change antigenically to escape from neutralizing antibodies. It remains unclear how genetic variation in the intrahost virus population and selection at the level of individual hosts translates to the fast-paced evolution observed at the global level because emerging intrahost antigenic variants are rarely detected. We tracked intrahost variants in the hemagglutinin and neuraminidase surface proteins using longitudinally collected samples from 52 patients infected by A/H3N2 influenza virus, mostly young children, who received oseltamivir treatment. We identified emerging putative antigenic variants and oseltamivir resistant variants, most of which remained detectable in samples collected at subsequent days, and identified variants that emerged intrahost immediately prior to increases in global rates. In contrast to most putative antigenic variants, oseltamivir resistant variants rapidly increased to high frequencies in the virus population. Importantly, the majority of putative antigenic variants and oseltamivir resistant variants were first detectable four or more days after onset of symptoms or start of treatment, respectively. Our observations demonstrate that de novo variants emerge, and may be positively selected, during the course of infection. Additionally, based on the 4-7 day post-treatment delay in emergence of oseltamivir resistant variants in six out of the eight individuals with such variants, we find that limiting sample collection for routine surveillance and diagnostic testing to early timepoints after onset of symptoms can potentially preclude detection of emerging, positively selected variants.

Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children

“…The A/H3N2 virus samples were collected from 51 unlinked individuals as part of an oseltamivir dosage trial 20,21 . 48 of the 51 A/H3N2 virus infected individuals were young children (median age=2 years; interquartile range (IQR)=2-3 years) at the time of sampling and most had low or no detectable anti-influenza virus antibody titers on day 0 and 10 post-symptom onset 21 .…”

“…The A/H3N2 virus samples were collected from 51 unlinked individuals as part of an oseltamivir dosage trial 20,21 . 48 of the 51 A/H3N2 virus infected individuals were young children (median age=2 years; interquartile range (IQR)=2-3 years) at the time of sampling and most had low or no detectable anti-influenza virus antibody titers on day 0 and 10 post-symptom onset 21 . Given that young children are substantial contributors to influenza virus transmission 22,23 , the samples analysed here offer a valuable opportunity to investigate the within-host IAV evolutionary dynamics in this key population.…”

“…Aggregating over all samples, most nonsynonymous variants were found in the nucleoprotein (NP) and neuraminidase (NA) gene segments (nonsynonymous to synonymous variant (NS/S) ratios = 1.69 (NP) and 1.32 (NA) whereas NS/S ratios were 1 for all other gene segments; Figure S1 and Table S1). While nonsynonymous NA mutations associated with oseltamivir resistance were positively selected for a subset of individuals in response to the antiviral treatment 21 , nonsynonymous changes to NP were likely mediated by protein stability, T-cell immune response and/or host cellular factors (see next section).…”

“…Despite the dominance of purifying selection in seasonal A/H3N2 intra-host viral populations, we detected several nonsynonymous variants of interest. Amino acid variants emerging in the HA and NA proteins were discussed in a previous work 21 (see Supplementary Materials). In the nucleoprotein, there were two notable nonsynonymous variants, D101N/G and G384R, that appeared in multiple individuals who were sampled independently between 2007 and 2009 (Figure 4A and S5C).…”

Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children

“…Consolidating over all samples, most nonsynonymous variants were found in the nucleoprotein (NP) and neuraminidase (NA) gene segments (nonsynonymous to synonymous variant (NS/S) ratios = 1.69 (NP) and 1.32 (NA) whereas NS/S ratios were ≤ 1 for all other gene segments; Figure 2 figure supplement 1 and Supplemental File 1). While nonsynonymous NA mutations associated with oseltamivir resistance were positively selected for a subset of individuals in response to the antiviral treatment 21 , nonsynonymous changes to NP were likely mediated by protein stability, T-cell immune response and/or host cellular factors (see next section). All rates are stratified by substitution type (synonymousblue; nonsynonymousred; greystop-codon).…”