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. 2022 Jan 8;8(1):veac001.
doi: 10.1093/ve/veac001. eCollection 2022.

Intra- and inter-host evolution of H9N2 influenza A virus in Japanese quail

Lucas M Ferreri  1 , Ginger Geiger  1 , Brittany Seibert  1 , Adebimpe Obadan  2 , Daniela Rajao  1 , Anice C Lowen  3 , Daniel R Perez  1
Affiliations

Intra- and inter-host evolution of H9N2 influenza A virus in Japanese quail

Lucas M Ferreri et al. Virus Evol. .

Abstract

Influenza A viruses (IAVs) are constantly evolving. Crucial steps in the infection cycle, such as sialic acid (SA) receptor binding on the host cell surface, can either promote or hamper the emergence of new variants. We previously assessed the relative fitness in Japanese quail of H9N2 variant viruses differing at a single amino acid position, residue 216 in the hemagglutinin (HA) viral surface protein. This site is known to modulate SA recognition. Our prior study generated a valuable set of longitudinal samples from quail transmission groups where the inoculum comprised different mixed populations of HA 216 variant viruses. Here, we leveraged these samples to examine the evolutionary dynamics of viral populations within and between inoculated and naïve contact quails. We found that positive selection dominated HA gene evolution, but fixation of the fittest variant depended on the competition mixture. Analysis of the whole genome revealed further evidence of positive selection acting both within and between hosts. Positive selection drove fixation of variants in non-HA segments within inoculated and contact quails. Importantly, transmission bottlenecks were modulated by the molecular signature at HA 216, revealing viral receptor usage as a determinant of transmitted diversity. Overall, we show that selection strongly shaped the evolutionary dynamics within and between quails. These findings support the notion that selective processes act effectively on IAV populations in poultry hosts, facilitating rapid viral evolution in this ecological niche.

Keywords: clonalinterference; influenza; positive selection; poultry; transmission bottleneck; virus population.

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Figures

Figure 1.
Figure 1.
Time series analysis of position HA 216 show that L216 is rapidly selected, but the dynamics depends on the competitors present in the inoculum mixture. In the top panel, the bars show the proportions of HA216 variants present in the inoculum. Below, the stacked area plots represent frequency of amino acids present in tracheal swab samples of inoculated and contact quails. Groups are designated as varΔLQ for mix of variant viruses in absence of L and Q at 216, var + L mix of variants viruses including L216, var + Q mix of viruses including Q216 and var + LQ mix of viruses including L216 and Q216. The specimens collected at 1, 3, 5, and 7 dpi from inoculated quails and 1, 3, 5, 7, and 8 dpc (x-axis) from contact quails were sequenced by NGS, and the amino acid frequency (y-axis) was calculated. Each plot represents a single quail (Q#) and each color represents a variant at position HA 216. Dotted grey lines show timepoints previously analyzed (Obadan et al. 2019). Quails that were euthanize for tissue collection are marked with asterixis. † = virus was below limit of detection in the last days of experiment or data did not meet quality cut off.
Figure 2.
Figure 2.
Intra-group presence of L216 at high frequencies relaxes transmission bottleneck. Shannon entropy was calculated (y-axis) as a measure of diversity for position HA 216. Each dot shows the mean Shannon entropy calculated using 1 dpi and 3 dpi for the inoculated quail. For the contact quails, dots represent the Shannon entropy calculation at 1 dpc. Boxplots delimit lower and upper quartile with central line showing the median.
Figure 3.
Figure 3.
Variants distribution across influenza A genome. Variants representing different type of mutations were found to increase as the infection progressed. Days 1 and 7 are shown for inoculated and contact groups. Days post-infection are shown in the right y-axis. Competition groups are color coded as varΔLQ in red, var + Q in blue, var + LQ in green, and var + L in yellow. The types of mutation are represented by shapes: synonymous (S) as circles, nonsynonymous (N) as tringles, variants in the untranslated regions (U) as diamonds, and stop codons (X) as squares. In the left y-axis the frequency is represented in log scale from 0 to 1. The segments are shown as concatenated in the x-axis. Dashed grey line shows consensus cut off at 0.5 of frequency whereas dashed black line marks 1 of frequency.
Figure 4.
Figure 4.
Common variants across competition and exposure groups show similar dynamics. (A) Variants were detected across quails from different competition and exposure groups. Numbers point to variants that were shared by inoculated and contact animals. Number of quails in which the variant was detected is represented in the heatmap. (B) The frequency dynamics show that some variants are kept at low frequency throughout the infection (PB2 g2327t), can be rapidly fixed (PA a100g (K26E)) or be kept from mid frequency to near fixation (NP c1550t). Direct inoculated quails are shown as dots in solid lines whereas contacts are shown as tringles in dotted lines. Days in the x-axis are shown as days post-infection (dpi). Each box represents the different competition groups.
Figure 5.
Figure 5.
Inclusion of HA L216 allows for a greater diversification at the whole genome level. To assess diversity at the genome level, π was calculated. Each dot shows the mean π calculated using 1 dpi and 3 dpi for the inoculated quails representing the time point at which the naïve quails were introduced—2dpi. For the contact quails, dots represent the π calculation at 1 dpc. Central line in box plots shown the median.
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