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 days 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.

Keywords: influenza virus; next-generation sequencing; within-host evolution.

Figure 1.

Intrahost diversity in the HA and NA proteins. Black vertical lines in panels (A) and (B) represent variable amino acid positions. White vertical bars indicate primer regions. (A) Overview of variable amino acid positions in HA1 and HA2. Red vertical lines indicate amino acid variations in antigenic sites (Wiley et al. 1981; Wilson and Cox 1990). All HA positions are based on H3 numbering without signal peptide. (B) Overview of variable amino acid positions in NA. Yellow vertical lines indicate substitutions associated with reduced inhibition by oseltamivir, green vertical lines indicate variable positions associated with highly reduced inhibition by oseltamivir (Lina et al. 2018). Asterisks indicate positions varying in multiple patients; HA: 119 (two patients), 453 (two), and 498 (two); NA: 264 (two), 292 (two), 307 (two), 329 (two), and 442 (two). (C) Maximum proportions reached by HA1 and HA2 variants. (D) Maximum proportions reached by NA variants. Color coding as in panels (A) and (B). Arrows indicate HA D53N and NA R292K from patient 1707 that co-emerge (see main text).

Figure 2.

Variant proportion changes over time. Orange, green, and brown plots show evolutionary dynamics of antigenic site variants, reduced inhibition variants, and highly reduced inhibition variants, respectively. Patient ID, protein, and amino acid position are specified above each plot. Amino acid changes are indicated in black and white capital letters, where white letters indicate the minority variant enrollment. A vertical dashed line in the NA plots specifies the last day of oseltamivir treatment. Supplementary Fig. S6 shows evolutionary dynamics of all HA and NA variants.

Figure 3.

Serologic analysis of day 0 and day 10 plasma samples. The dashed horizontal line indicates the detection limit of the HI assay at the starting dilution of 1/40 used here. Arrows in the day 0 column indicate plasma samples for which HI titers dropped below detection at day 10. The arrow in the day 10 column indicates a plasma sample for which there was no titer above the detection limit at day 0. Orange filled symbols are the log2 HI titers for the patient with antigenic site variant HA S45N.

Figure 4.

Initial day of variant detection. Diamond shapes specify the first observation in the virus population. Circles indicate the days of sample collection. Open circles indicate days where the variant proportion did not reach the minimum sample inclusion threshold of 1 per cent. Columns on the left show patient ID and amino acid variant. (A) Antigenic site variants (orange). (B) Reduced inhibition variants (green) and highly reduced inhibition variants (brown). Open triangles indicate days of oseltamivir treatment prior to enrollment.