Influenza B Viruses Exhibit Lower Within-Host Diversity than Influenza A Viruses in Human Hosts

Abstract

Influenza B virus (IBV) undergoes seasonal antigenic drift more slowly than influenza A virus, but the reasons for this difference are unclear. While the evolutionary dynamics of influenza viruses play out globally, they are fundamentally driven by mutation, reassortment, drift, and selection at the level of individual hosts. These processes have recently been described for influenza A virus, but little is known about the evolutionary dynamics of IBV during individual infections and transmission events. Here, we define the within-host evolutionary dynamics of IBV by sequencing virus populations from naturally infected individuals enrolled in a prospective, community-based cohort over 8,176 person-seasons of observation. Through analysis of high depth-of-coverage sequencing data from samples from 91 individuals with influenza B, we find that IBV accumulates lower genetic diversity than previously observed for influenza A virus during acute infections. Consistent with studies of influenza A viruses, the within-host evolution of IBVs is characterized by purifying selection and the general absence of widespread positive selection of within-host variants. Analysis of shared genetic diversity across 15 sequence-validated transmission pairs suggests that IBV experiences a tight transmission bottleneck similar to that of influenza A virus. These patterns of local-scale evolution are consistent with the lower global evolutionary rate of IBV.IMPORTANCE The evolution of influenza virus is a significant public health problem and necessitates the annual evaluation of influenza vaccine formulation to keep pace with viral escape from herd immunity. Influenza B virus is a serious health concern for children, in particular, yet remains understudied compared to influenza A virus. Influenza B virus evolves more slowly than influenza A virus, but the factors underlying this are not completely understood. We studied how the within-host diversity of influenza B virus relates to its global evolution by sequencing viruses from a community-based cohort. We found that influenza B virus populations have lower within-host genetic diversity than influenza A virus and experience a tight genetic bottleneck during transmission. Our work provides insights into the varying dynamics of influenza viruses in human infection.

Keywords: bottleneck; diversity; evolution; influenza B virus; influenza virus; transmission.

FIG 1

Viral load and sequencing coverage. (A) Boxplot of viral load (genome copies per microliter of swab transport medium, y axis) by day of sampling relative to symptom onset (x axis). The boxes display median and 25th and 75th percentiles, with whiskers extending to the most extreme point within the range of the median ± 1.5 times the interquartile range. (B) Sequencing coverage is plotted with read depth on the y axis and location within a concatenated influenza B virus genome on the x axis. The mean coverage for each sample was calculated over a sliding window of size 200 and a step size of 100. The data are displayed for all samples at each window as a boxplot, showing the median and 25th and 75th percentiles, with whiskers extending to the most extreme point within the range of the median ± 1.5 times the interquartile range; all values outside this range are shown as individual points.

FIG 2

Intrahost minority SNV by day post-symptom onset and viral load. (A) The number of minority iSNV per sample is plotted on the y axis by day post-symptom onset on the x axis. Data are displayed as boxplots representing the median and 25th and 75th percentiles, with whiskers extending to the most extreme point within the range of the median ± 1.5 times the interquartile range. The raw data points are shown in black overlaid on top of the boxplots; points from mixed infection samples are shown in orange. (B) Scatterplot relating the number of minority iSNV per sample on the y axis to the log₁₀ of viral load, in genome copies per microliter, on the x axis. Data points from the mixed infection are shown in orange. (C) Frequency of minority iSNV in samples sequenced in duplicate. Orange dots represent variants identified in samples with a viral load of 10³ to 10⁴ genome copies per microliter, and blue dots represent variants in samples with a viral load of 10⁴ to 10⁵ genome copies per microliter.

FIG 3

Intrahost SNV frequency by genome position and mutation type. All minority (<50%) iSNV from 99 samples are displayed with their frequency on the y axis and their position within a concatenated influenza B virus genome on the x axis. Synonymous mutations are shown in orange, and nonsynonymous mutations are shown in blue.

FIG 4

Intrahost SNV by vaccination status and IBV lineage. (A) Numbers of minority iSNV per sample across all 99 samples are shown (y axis) by current-season vaccination status of the host (x axis). Samples from the mixed infection are shown in orange. (B) Numbers of minority iSNV per sample are shown (y axis) by IBV lineage (x axis). Samples from the mixed infection are shown in orange. (C) Pairwise nucleotide diversity (π, y axis) by influenza virus type (x axis), stratified by iSNV frequency cutoff (top). Medians are shown as red lines. Data for influenza A virus are from 243 samples described by McCrone et al. (18). Data on influenza B virus are from 97 high-quality samples in the present study. Samples from mixed infections in both studies are excluded. (D) Numbers of minority iSNV in 43 of the 99 high-quality samples (y axis), consisting of B/Yamagata from the 2014–2015 season, B/Victoria from the 2015–2016 season, and B/Yamagata from the 2016–2017 season based on alignments to the original references from the 2012–2013 season versus season-matched reference genomes (x axis).

FIG 5

Identification of household transmission pairs. Maximum likelihood phylogenetic tree of all B/Victoria (A) and B/Yamagata (B) samples from this study. Concatenated consensus coding sequences were aligned with MUSCLE, and phylogenetic trees were constructed with RAxML. Tip labels are denoted as enrollee ID, household ID, season, and lineage, separated by underscores; tip labels are colored by season. (C) Histogram of genetic distance, as measured by L1-norm, between household pairs and random community pairs from the same season and lineage. The bar heights for each group are normalized to the maximum for each group for comparison. Community pairs are shown in orange, and household pairs are shown in blue. The dotted red line indicates the 5th percentile of the community pair distribution.

FIG 6

Shared diversity across household transmission pairs with influenza B virus. iSNV for 15 validated transmission pairs using samples closest to the time of transmission (inferred based on day of symptom onset) are shown. Each iSNV is plotted as a point, with its frequency in the recipient (y axis) versus its frequency in the donor (x axis).