# The impact of viral and host factors on the influenza A virus transmission bottleneck

## TL;DR

This study finds that the influenza A virus has a tight transmission bottleneck that is not affected by host or viral factors.

## Contribution

The study provides the largest set of IAV transmission pairs analyzed to date and confirms a consistent tight transmission bottleneck using two validated models.

## Key findings

- Influenza A virus transmission is restricted to a bottleneck of 1 virion regardless of season, subtype, or host factors.
- Host factors like age, sex, and vaccination status do not alter the transmission bottleneck size.
- Two validated models produced consistent bottleneck estimates, reinforcing the intrinsic tightness of the bottleneck.

## Abstract

Transmission bottlenecks are defined by the number of unique virions or genotypes that establish an infection, and they restrict viral diversity that passes from one infected host to another. Previous work identified a tight transmission bottleneck for seasonal influenza A virus (IAV) based on analysis of 43 household pairs, largely from a single A(H3N2) predominant season. While many viral and host factors are known to influence IAV transmission in households, their impact on the transmission bottleneck is not clear. Nasal swabs were collected daily from IAV infected individuals enrolled in two case-ascertained U.S. household transmission studies, FluTES (2017/2018–2019/2020 seasons) and RVTN (2021/2022 season). Viruses were sequenced in duplicate, and intrahost single nucleotide variants (iSNV) were identified at a 0·5% frequency threshold using a benchmarked pipeline with >99·99% specificity for mutations present in both replicates. Transmission pairs were defined based on co-residence, test date, and genetic distance. For each possible transmission pair, the bottleneck was estimated using a beta binomial and a clonal mutation model. We sequenced 567 samples from 319 individuals and 102 households in duplicate. Based on epidemiologic linkage and a sequence-based cut-off, we defined 59 transmission pairs for the beta binomial model and 56 transmission pairs for the clonal mutation model. Across all pairs, we identified a transmission bottleneck of 1 both using the beta-binomial model (CI 1, 1) and the clonal mutation model (CI: 1.00, 1.22). In our cohort, influenza season, subtype, and host factors (influenza vaccination status, sex, and age) did not alter the transmission bottleneck. IAV is subject to a tight genetic bottleneck during transmission, which limits onward propagation of newly arising nucleotide variants. Tight bottlenecks appear to be intrinsic to the transmission process, as host and viral factors within households do not affect its size.

Previous studies suggest a tight transmission bottleneck for IAV. Host factors such as sex at birth, age, and immune status are well documented modulators of IAV shedding, suggesting they may also play a role in IAV transmission bottleneck size. Year to year variation in circulating strains also suggests that there may be viral factors that are important. However, the impact of viral and host factors on the bottleneck size is presently unclear. In this study, we define and characterize 60 new IAV transmission pairs and find a tight transmission bottleneck regardless of season, subtype, and host factors (sex at birth, age, and immune status). To our knowledge, this is the largest set of IAV transmission pairs analyzed to date and provides additional estimates of the IAV transmission bottleneck. We also use two validated and orthogonal models for inferring the size of the transmission bottleneck and find consistent estimates regardless of method used. Together with prior work, this study demonstrates that the IAV transmission bottleneck is intrinsically tight and not impacted by host or viral factors. Given well-established patterns of IAV evolution, these findings suggests that there is a complex interplay between within-host and population-scale processes that warrant further investigation.