Influenza A Virus Population Genetics Within and Between Hosts Open Access
Holmes, Katie (Fall 2025)
Abstract
Seasonal influenza virus evolution at large geographic and temporal scales is characterized by a clear pattern of positive selection. In contrast, the within-host evolutionary dynamics of influenza viruses show strong genetic drift and purifying selection. Viral range expansion, both between hosts during transmission and spread within a host during infection, is subject to bottleneck events that shape viral diversity. Resulting populations after a bottleneck event may be genetically dissimilar to each other and to the original population. Since genetic diversity represents adaptive potential, such losses of diversity are thought to limit the opportunity for viral populations to undergo antigenic change and other adaptive processes. Thus, a detailed picture of evolutionary dynamics is critical to understanding the forces driving viral evolution at an epidemiologic scale. To advance this understanding, we used a barcoded virus library and a guinea pig model of transmission to decipher where in the transmission process influenza A virus populations lose diversity. In inoculated guinea pigs, we show that a high level of viral barcode diversity is maintained. Within-host continuity in the barcodes detected across time furthermore indicates that stochastic effects are not pronounced within the inoculated hosts. Importantly, in both aerosol-exposed and direct contact animals, we observed many barcodes at the earliest time point(s) positive for infectious virus, indicating robust transfer of diversity through the environment. This high viral diversity is short-lived, however, with a sharp decline seen 1-2 days after initiation of infection. Although major losses of diversity at transmission are well described for influenza A virus, our data indicate that events that occur following viral transfer and during the earliest stages of natural infection have a central role in this process. We used the same barcoded virus library in a cell culture model system to investigate genetic drift in a spatially expanding viral population. We show that increased spatial constraint suppresses viral loads further from the source population. We also demonstrate that genetic bottlenecks in expanding viral populations are present even on simple landscapes of infection and over relatively short distances. Our data indicate that spatial structure impacts viral propagation and the genetic composition of dispersed populations, suggesting an interplay between spatial heterogeneity and genetic drift that may be central to our understanding of within-host dynamics of viral evolution. Taken together, these findings offer insights towards a more holistic, multiscale population genetics framework for influenza viruses, the dynamics of which are shaped not just by mutation and selection but also by stochasticity.
Table of Contents
Table of Contents
Chapter 1: Introduction…………………………………………………………………………1
Introduction………………………………………………………………………………1
Influenza Virus Species……………………………………………………………….....1
Influenza in Humans………………………………………………………………….....2
Influenza in Non-Human Hosts…………………………………………………………3
Zoonoses, Reverse Zoonoses, and Pandemics/Panzootics……………………………..5
The Influenza Viral Life Cycle……………………………………………………….....6
Influenza Infection Across Scales………………………………………………….........7
i. Infection Landscape Within Hosts……………………………………...7
ii. Transmission Bottlenecks………………………………………………..8
Fig 1. Stages of viral transmission between hosts……….…………………..8
Influenza Viral Evolution………………………………………………………………..9
Fig 2. The stochastic population bottleneck acting during transmission impacts viral genetic diversity in the recipient host…………………………………………………………..10
Dissertation Aims…………………………………………………………………….....12
References Cited……………………………………………..………………………….14
Chapter 2: Viral expansion after transfer is a primary driver of influenza A virus transmission bottlenecks………………………..………………………………….…………..23
Abstract………………………………………………………………………………….23
Introduction……………………………………………………………………………..24
Results…………………………………………………………………………………...27
Fig 1. NA barcode diversity is maintained in both plasmid and virus stocks, and the barcode does not affect overall diversity………………………………………………………28
Fig 2. Population diversity declines between inoculated and exposed guinea pigs…..….31
Fig 3. Initial high barcode diversity in exposed animals plummets after the first 1-2 days of viral positivity ……………………………………………………………...…………….……33
Fig 4. Infectious viruses isolated early after transmission carry diverse barcodes……...36
Discussion……………………………………………………………………………….37
Materials & Methods……………..…………………………………………………….42
Supplemental Information……………………………………………………………..54
S1 Fig. Pan/99 NA-BC infects inoculated animals and transmits to exposed animals…..54
S2 Fig. Population diversity declines between inoculated and exposed guinea pigs…….55
S3 Fig. Changes in richness and evenness both contribute to alterations in diversity…..58
S4 Fig. Targeted amplicon sequencing of the barcode region is reliable and reproducible……………………………………………………………………………………….60
S5 Fig. Growth-induced bottlenecks are not detected in inoculated animals, even at low inoculation doses………………………………………………………………………………….62
References Cited……………………………………………………………………..….63
Chapter 3: Stochasticity drives viral population dynamics during spatially structured dispersal…………………………………………………………………………………….…...70
Abstract……………………………………………………………………………….…70
Introduction……………………………………………………………………………..71
Results…………………………………………………………………………………...74
Fig 1. A cell culture model of spatially structured viral spread allows for harvesting of cells from pre-defined locations…………..…………………………………………………….75
Fig 2. Spatial constraint, conferred by modifying viscosity of culture medium, increases heterogeneity in viral population density ……………………………………………….…….77
Fig 3. Genetic heterogeneity develops during spatially structured viral spread…………79
Discussion……………………………………………………………………………….81
Materials & Methods ……………………………….………………………………….85
Supplemental Information…………………………………………………………......91
S1 Fig. Genetic bottlenecks occur even at relatively short distances from the source population.……………………………………………………………………………...…….……91
S2 Fig. Viral spread yields population heterogeneity………………………………………..93
S3 Fig. Spatial networks demonstrate long-distance viral dispersal from the source population, as well as local spread after long-distance dispersal………………………….95
S4 Fig. Spatial constraint at all levels leads to genetic bottlenecks during viral dispersal, even close to the source population………………………………………………………….....97
References Cited..…………………………………………………………………..…...99
Chapter 4: Discussion………………………………………………………………………....105
Stochasticity shapes IAV population dynamics………………………………...…...105
Broader implications………………………………………………………………….107
Conclusions………………………………………………………………………….…110
References…………………………………………………………………………..….111
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