1,721,047 research outputs found
Quantifying progeny production from individual influenza virus-infected cells
Get PDFThesis (Ph.D.)--University of Washington, 2022The distribution of progeny virions produced by virus-infected cells is extremely heterogeneous. This trend has been observed in diverse viruses, including bacteriaphage and pathogenic human viruses. To date, it has been difficult to explain why some infected cells generate thousands of progeny virions while others – infected under identical conditions – produce no progeny.Established methods for quantifying progeny from single cells rely on the isolation of individual cells during infection. Such methods are not compatible with contemporary single-cell assays. With limited information gathered about the host and virus processes that occur during infection, the factors that might influence progeny production at the single-cell level have remained largely inaccessible.
I have developed new methods to quantify the amount of progeny produced by single influenza virus-infected cells; these methods do not require single-cell isolation during in- fection. Applying these methods, I have simultaneously measured viral transcription, viral genotype, and progeny virion production in the same influenza-infected cells. The correlation between viral transcription and progeny production is surprisingly poor at an early time of influenza infection. Using the viral gene expression information provided by single-cell RNA sequencing, I learned that cells with extremely high viral transcription often lack the influenza non-structural (NS) gene, precluding them from contributing infectious progeny.
While this system was developed to study influenza virus progeny production in single cells, individually-traceable virions may be useful in several areas of virology. The general approach to generate highly-diverse libraries of barcoded virions could likely be applied to other viruses with established reverse genetics systems. In the course of developing these methods, further opportunities for optimization have become apparent. I have outlined potential future applications that could be facilitated by either the current virus libraries or more diverse libraries that are developed in the future
A lentiviral vector deep mutational scanning system for studying virus evolution and escape from neutralization by antibodies and polyclonal serum
No full textThesis (Ph.D.)--University of Washington, 2023Viral entry proteins are critical for viral replication of enveloped viruses. These proteins allow viruses to bind receptors on cells and accomplish membrane fusion. Since viral entry proteins are located on the viral surface and the surface of infected cells, they are also the targets of host immune responses. For all of these reasons, mutations to viral entry protein can have a wide range of effects. These effects include the ability to better infect new host species, changes to receptor tropism and affinity, and evasion of host immune responses. By studying the effects of mutations to viral entry proteins, we can inform monitoring of emerging viral pathogens as well as the design of vaccines and other therapeutics.Studying the effects of mutations on viral entry proteins is not straightforward. Traditional methods of measuring mutation effects on viral entry proteins have drawbacks. Often, mutations are cloned individually or in a few combinations and tested in viral replication or neutralization assays, which can be time and resource expensive. These methods may rely on using replicative viruses with full-length genomes, raising the biosafety level required to perform these
experiments. Mutations also often have different effects in different strains of a virus, making it difficult to gain a full picture of the mutation’s potential effects unless these already intensive experiments are extended to multiple strains. Some of these challenges have been alleviated by high throughput techniques like phage display, yeast display, deep mutational scanning of full- length replicative viruses, and cell surface display. However, these methods each have drawbacks of their own, such as not displaying the full viral entry proteins, only being able to measure ligand or antibody binding rather than viral entry protein function, not being able to measure effects of combinations of mutations, or being difficult to perform using certain viruses due to technical reasons or biosafety concerns.
We have developed a lentiviral vector-based system for deep mutational scanning of viral entry proteins that overcomes many limitations of previous studies. Non-replicative lentiviral vectors can be pseudotyped by displaying viral entry proteins from viruses of interest on their surface. We developed a method to generate a genotype-phenotype link between a lentivirus genome and a viral entry protein mutant displayed on the surfaces of the virion for large mutant libraries of viral entry proteins. These mutant lentivirus libraries can then be used to measure mutation effects on function and immune escape of full viral entry proteins. By using a nucleotide barcoding method, this system is also able to measure the effects of combinations of mutations in viral entry proteins. Measurements of effects of combinations of mutations allows us to investigate epistasis in mutation effects and map virus escape from immunity targeting multiple epitopes simultaneously.
We used the lentiviral vector system to perform deep mutational scanning of the SARS- CoV-2 spike protein. This allowed us to safely measure the effects of mutations to the full SARS-CoV-2 Spike protein in a biosafety level 2 setting. Using mutant libraries of Omicron BA.1 and Delta strain spike proteins, we mapped escape mutations to neutralizing antibodies targeting various domains of the spike protein. We also compared our measurements of the functional effects of mutations to the spike protein to previous studies and natural sequence data, and found our results were more correlated with enrichment of mutations in natural sequences. This approach can be used to rapidly characterize mutation effects on function and escape from neutralization by sera or therapeutics for emerging pathogens.
We also tailored the lentiviral vector system to map the neutralizing specificity of human anti-HIV sera. We designed mutant libraries of HIV Envelope mutants with combinations of mutations based on previous deep mutational scanning studies and natural sequence data. We used these libraries to map escape from neutralizing antibodies and human sera targeting CD4 binding site of HIV Envelope. Individual mutation effects and antibody epitopes were inferred using a biophysical model to deconvolute mutation effects from the combinations of mutations in the mutant libraries. Most sera mapped had neutralizing specificities similar to individually characterized monoclonal antibodies, but the neutralizing specificity of one serum was best explained by two epitopes within the CD4 binding site. Thus, this approach allows us to map multi-epitope targeting polyclonal immunity and can be used to characterize and evaluate infection or vaccination elicited polyclonal antibody responses to viral entry proteins
Uncovering the dynamics of viral evolution and pathogenesis from high-throughput datasets: a computational perspective
Get PDFThesis (Ph.D.)--University of Washington, 2023High-throughput experiments, including deep sequencing and deep mutational scanning (DMS), provide insight into the genotypic and phenotypic landscapes traversed by an evolving virus. However, interpreting the large amount of data produced by these techniques requires a robust computational strategy. Recognizing this challenge, in my dissertation, I describe how I used a computational approach to tackle three distinct but interconnected aspects of viral evolution. In the first chapter, I characterize the adaptive progression that enables measles to infect the brain. Ordinarily, a Measles infection is acute and self-limiting. However, through unknown mechanisms, Measles can persist after acute infection, remain undetected in the body, migrate to the brain, and become neurotropic. Previous studies of measles infections of the brain have been limited by low genetic resolution and restricted sampling schemes. Using the most comprehensive spatially-sampled neurotropic measles dataset to date, our study offers compelling clues into the evolutionary processes that allowed measles to colonize the brain in a patient who succumbed to this rare disease. In the next chapter, I determine how superspreading influences the transmission of SARS-CoV-2 viral diversity between hosts. Most studies of the impact of transmission on shared viral diversity for respiratory viruses involve household or nosocomial transmission scenarios. In contrast, the dynamics of shared viral diversity in superspreading events are poorly understood, despite playing a significant role in the global spread of viruses. To address this, I investigated the spread of viral diversity during a SARS-CoV-2 superspreading event on a fishing boat to see if circumstances highly conducive to transmission exhibit unique patterns of viral evolution. I found that superspreading imposed a narrow bottleneck on viral diversity between hosts despite the unique transmission scenario. In the final chapter, I describe an interactive visualization tool to help analyze large mutation-function datasets from high-throughput experiments like deep-mutational scanning. The mutation-based data generated by these approaches is often best understood in the context of a protein’s 3D structure. However, current approaches for visualizing mutation data in the context of a protein’s structure are cumbersome and require multiple steps and software. To streamline the visualization of mutation-associated data in the context of a protein structure, I developed a web-based tool called 'dms-viz'. With 'dms-viz', researchers can easily create, analyze, and share customized visualizations of their mutation-based datasets with the broader research community. In my graduate research, I developed and applied computational methods to study viral evolution. First, I explored how virus evolution can occur within an individual host. Then, I characterized the impact of transmission between hosts on viral evolution. And finally, I developed a computational tool to help analyze large mutation-based datasets to help answer a myriad of evolutionary questions
Pseudotyped lentiviral systems for studying viral entry proteins from emerging viruses with pandemic potential
No full textThesis (Ph.D.)--University of Washington, 2021Viral entry proteins facilitate viral entry into host cells through receptor binding and membrane fusion. They play a critical role in the viral life cycle and, as prominent surface proteins, are common targets of the host immune response. Mutations in viral entry proteins can allow viruses to infect new hosts (including humans), better spread between hosts, or evade immune responses and some therapeutics. A better understanding of how viral entry proteins interact with host proteins, elicit host antibody responses, or are affected by mutations is important for being tackling current and emerging viral threats. However, viral entry proteins are also heavily glycosylated, multimeric, metastable proteins that are challenging to work with. Furthermore, studying viral entry proteins in their native viral context is challenging due to inefficient reverse genetics systems or the need to work under high biosafety level containment. As such, many viral entry proteins—especially those from emerging viruses—cannot be studied with many high- throughput experimental techniques, such as mutational scanning experiments. Pseudotyping viral entry proteins on easier- and safer-to-work-with viral particles is one way to facilitate research of viral entry proteins from emerging viruses. When SARS- CoV-2 was first identified in late 2019, there was an urgent need to develop tools to safely and easily study the virus. Working with SARS-CoV-2 itself requires a biosafety level 3 facility, but pseudotyping its entry protein, spike, onto lentiviral particles allows SARS-CoV-2 spike to be studied under commonly available biosafety level 2 conditions. In the following chapters, I describe a protocol for pseudotyping lentiviral particles with SARS-CoV-2 spike to facilitate research into this emerging virus. I also describe the use of spike-pseudotyped lentiviral particles to investigate the neutralizing antibody response to SARS-CoV-2 and the effects of mutations to spike on its function as a viral entry protein. Spike-pseudotyped lentiviral particles are especially useful for measuring SARS-CoV-2-neutralizing antibodies and I provide a detailed protocol for a SARS-CoV-2 neutralization assay using spike-pseudotyped lentiviral particles I then use this assay to investigate the dynamics of the neutralizing antibody response to SARS-CoV-2 in the first several months following infection. Titers of SARS-CoV-2 neutralizing antibodies decline modestly from ≈1 to ≈3 months post symptom onset, which is typical of the neutralizing antibody response to other acute respiratory viruses. The SARS-CoV-2 pandemic has also provided an example of the importance of prospectively characterizing the effects of mutations to viral entry proteins. Pseudotyped lentiviral particles provide a genetically tractable system for assessing the effects of mutations to viral entry proteins from emerging viruses at high-throughput. As such, I have worked to develop a pseudotyped lentivirus-based system for screening the functional and antigenic effects of mutations to viral entry proteins from emerging viruses in high-throughput. This system is still being developed, but I briefly discuss current progress and describe a Python package I helped write to facilitate the analysis of such deep mutational scanning experiments. In summary, the following chapters describe the use of pseudotyped lentiviral particles as a a flexible, rapidly deployable tool for studying viral entry proteins from emerging viruses, such as SARS-CoV-2. Using spike-pseudotyped lentiviral particles, I contributed to some of our earliest understanding of the dynamics of the neutralizing antibody response to SARS-CoV-2 in the first several months following infection. Nonetheless, I think the full potential of using pseudotyped lentiviral particles to study viral entry proteins from emerging viruses has yet to be realized. As such, I also discuss current progress (including computational tools) and future work towards using pseudotyped lentiviral particles to measure the functional and antigenic effects of mutations to viral entry proteins from emerging viruses at high-throughput
Epistasis and pleiotropy in viral protein evolution
Get PDFThesis (Ph.D.)--University of Washington, 2025Viruses evolve under Darwinian selection, and forecasting their evolution requires understanding which mutations confer fitness advantages. Over the past decade, advances in high-throughput experiments have made it possible to measure the phenotypic effects of all mutations to key viral proteins. Yet we continue to fall short when predicting which viral mutations will rise in frequency. The main problem is that the effect of a mutation is not fixed—it depends on the genetic background in which it occurs and on an immune context that is often unknown and dynamic. A mutation that is deleterious in one background may become tolerated in another. A mutation that benefits one phenotype but harms another can create conflicts that constrain selection. These phenomena, known as epistasis and pleiotropy, complicate efforts to make accurate viral forecasts. In chapter 2, we examine how viral mutations combine to escape antibodies in human sera. We introduce a simple biophysical model that explains how the effect of a mutation on antibody escape depends on epistatic interactions with other mutations. We then show how these mutation effects can be inferred directly from deep mutational scanning datasets. In chapter 3, we investigate how pleiotropy constrains viral protein evolution. We use deep mutational scanning to measure the effects of mutations to human influenza virus hemagglutinin on three phenotypes: cell entry, acid stability, and serum antibody neutralization. By quantifying mutation effects across multiple phenotypes, we identify viral mutations that are beneficial in one context but deleterious in another, revealing evolutionary trade-offs. Finally, in chapter 4, we compare the effects of viral mutations to H3, H5, and H7 influenza virus hemagglutinin to explore how mutation effects differ across three sequentially divergent but structurally conserved proteins
Complete mapping of HIV-1 escape from broadly neutralizing antibodies, vaccines, and drugs
No full textThesis (Ph.D.)--University of Washington, 2019The expansive global diversity of HIV-1 Env presents significant hurdles in developing a broadly protective vaccine. This diversity is a result of HIV Env’s exceptional evolutionary capacity, which allows it to evade the extraordinary diversity of the humoral immune system during infection. However, the evolutionary arms race between Env and humoral immunity occasionally drives the development of broadly neutralizing antibodies (bnAbs) capable of neutralizing diverse strains. Mapping the epitope specificity of bnAbs has revealed conserved regions of Env, which are promising targets for structure-based vaccine design. Additionally, bnAbs’ broad activity and potential to direct the killing of infected cells make them promising antiviral immunotherapeutic drugs for HIV prevention, therapy, and cure strategies. Translating bnAbs into vaccines and therapies will require both a detailed understanding of how bnAbs interact with Env as well as assessing their potential for viral escape. While structural studies provide atomic-level views of HIV-antibody interactions, they fail to reveal the functional interactions necessary for neutralization and the viral mutations that disrupt these interactions. Neutralization and binding assays using mutants can provide such information for specific mutations, but even the largest studies employing one-at-a-time mutagenesis can only assay a small fraction of all possible Env mutations. To overcome these shortcomings, we have developed mutational antigenic profiling, a deep mutational scanning approach that completely maps the functional interface between HIV and an antibody in a single massively parallel experiment. This involves generating libraries of HIV that carry all possible amino-acid mutations to Env (12,730 amino-acid mutations), incubating these viral libraries with or without an antibody, infecting T cells, and using deep sequencing to quantify the enrichment of each mutation in the antibody selected versus non-selected libraries. Profiling escape from bnAb PGT151 identified all previously known and revealed numerous additional escape mutations. Benchmarking these data against traditional neutralization assays further validated that we accurately quantified the effect of all amino-acid mutations to Env. Additionally, evaluating the effect of each amino acid at each site elucidated the biochemical mechanisms of escape throughout the epitope, highlighting the previously unappreciated role for charge-charge repulsions. To gain a broad view of HIV antibody escape, we mapped escape from a panel of nine bnAbs targeting the five best-characterized Env epitopes. Importantly, many of these bnAbs are being clinically developed as immunotherapeutics. While prior studies had defined each of these bnAbs’ structural epitope, our unbiased mapping defined their functional epitopes, or the sites at which mutations mediated escape in the context of replication competent viruses, for the first time. For most bnAbs, mutations at only a small fraction of structurally defined contact sites mediated escape, and escape often occurred at sites that are near but do not directly contact the antibody. Further, these data helped to interpret viral mutations observed in immunotherapy clinical trials—in vivo escape occurred in the functional epitope, some of which was previously missed since it was far from the structural epitope. Additionally, this data allowed for an unbiased quantification of the ease of viral escape for each bnAb, which we found is distinct from antibody breadth. We also mapped escape from a pool of two bnAbs; we found that there were no mutations that robustly escaped both antibodies, agreeing with the results of two recently completed clinical trials that administered this combination. Further, we profiled escape from two antibodies across multiple viral strains, providing the first unbiased quantifications of strain-specific differences in antibody escape. Next, we leveraged mutational antigenic profiling to directly refine structure-based vaccine design. We contrasted escape from bnAb VRC34.01 with escape from two murine antibodies that were elicited with immunogens based on the VRC34.01 epitope. This revealed distinct differences in the recognition of natural and vaccine-elicited antibodies, and provide a template to guide the iterative rounds of vaccine design. We then adapted this approach to better delineate the genotypic determinants of resistance to the only clinically approved HIV fusion inhibitor, enfuvirtide. Again, we identified both previously characterized and novel resistance mutations. Many resistance mutations were allosteric to the drug’s binding site, which shed light on diverse mechanisms of resistance. Further, this complete map of resistance may be of use in the clinical monitoring of resistance during therapy and the genotypic prediction of enfuvirtide sensitivity prior to treatment. Few protein-protein interfaces have been as heavily studied as those between bnAbs and Env, as these interactions provide the motivation for many HIV treatment and prevention efforts. Mutational antigenic profiling yields an unprecedented view of these interfaces, redefining out understanding of an antibody’s functional epitope. The complete maps of viral escape detailed in this thesis provide a mutation-level antigenic atlas for understanding viral immune escape and guiding the development of antibody immunotherapies and vaccines
High-throughput analysis of the antigenic effects of mutations to influenza hemagglutinin
No full textThesis (Ph.D.)--University of Washington, 2024Influenza virus rapidly evolves to escape neutralization by polyclonal antibodies. However, we have a limited understanding of how the effects of viral mutations on antibody neutralization vary across the human population, and how this heterogeneity might affect virus evolution. In my dissertation, I address this question by mapping the antigenic effects of influenza mutations against sera from defined age cohorts. First, I describe the development of an improved deep mutational scanning system to measure how mutations in hemagglutinin (HA) affect neutralization by human sera. I engineer a chimeric, barcoded HA construct, which both improves sample throughput while also allowing for absolute quantification of both escape and sensitizing mutations. I show that the resulting barcoded libraries can be used to map the HA epitopes targeted by monoclonal antibodies, antibody cocktails, and polyclonal sera, and that these measurements are consistent with results from traditional neutralization assays. In the remainder of my thesis, I use a barcoded library in the background of the A/Hong Kong/45/2019 H3 HA protein to analyze heterogeneity in serum antibody targeting across individuals and age cohorts. I find that the effects of HA mutations on serum neutralization differ across age groups, and that these differences can be partially rationalized in terms of exposure histories. For instance, mutations that revert to amino acids found in the HAs of older viral strains often increase neutralization sensitivity in older individuals, but not young children. I incorporate data from similar experiments using the earlier, non-barcoded A/Perth/16/2009 H3 HA library, which also found substantial differences between child and adult escape maps. Natural mutations that fixed in influenza variants after 2020 cause the greatest escape from sera from children and teenagers, suggesting that antigenic pressure from younger age groups play a more prominent role in driving viral evolution. Overall, my graduate research demonstrates that influenza faces distinct antigenic selection regimes from different age groups, and that this heterogeneity may have a substantial impact on viral evolution. More rigorous characterization of this immune heterogeneity has the potential to improve both evolutionary forecasting and vaccine effectiveness
Quantifying how the mutational tolerance of HIV's envelope protein shapes its evolution
Get PDFThesis (Ph.D.)--University of Washington, 2017-08HIV's most rapidly evolving proteins is its envelope protein (Env). This rapid evolution is driven by continuous selection to evade immunity within HIV-infected hosts. However, as Env evolves, it is also under functional constraint to perform essential functions in the viral lifecycle, including receptor binding and membrane fusion. Since both of the above forces strongly shape Env's evolution, their effects have been difficult to disentangle from one another. As a result, our understanding of these forces is far from complete. A central goal of my graduate research has been to experimentally measure the functional constraint on Env in the lab in the absence of external immune selection. There are ~10,000 single amino-acid mutations to a protein of Env's length (=19 * ~850). Using a high-throughput technique called deep mutational scanning, I measured the effects of each of these mutations to Env in context of viral replication in cell culture. The results provide an in-depth profile of Env's ability to tolerate each of the 20 amino acids at each site in the protein. Using these data, I examined Env's mutational tolerance variable loops, which rapidly evolve to evade antibodies. It is possible that these loops have a high tolerance for mutations, and that that is one reason they so readily evolve. However, I did not find statistical support that these loops are more tolerant of mutations than other parts of the protein, suggesting that their variability in nature may mainly be due to high levels of diversifying pressure from antibodies. I also examined epitopes of broadly neutralizing antibodies targeting the CD4 binding site. These epitopes that are highly conserved in nature and are targets in vaccine design. A common assumption is that this conservation is due to high functional constraint at these sites. Indeed, I found that they were less tolerant of mutations than other parts of Env, providing rigorous support for a long-standing hypothesis, and suggesting that these epitopes may have a diminished evolutionary capacity to evade antibodies relative to other sites in the protein, which would make them more vulnerable to immune targeting. Another central goal of my thesis has been to compare Env's mutational tolerance among divergent strains. The same mutation (e.g., A12N) can have different effects in two related proteins due to epistasis (e.g., A12N may only be tolerated in one homolog, but not the other). However, the extent that mutational effects to Env differ between divergent HIV strains is largely unknown. To address this knowledge gap, I repeated the deep mutational-scanning experiment of two Env homologs that have 85% amino-acid identity. The results allowed me to compare each homolog's ability to tolerate each of the 20 amino acids at 616 homologous sites. I found that at a small fraction of sites, the amino acids tolerated in one homolog were largely distinct from the amino acids tolerated in the other homolog. However, only a few sites showed such extreme differences; most sites had changes in mutational tolerance that were only small-to-intermediate in effect size. Thus, these results indicate that Env's mutational tolerance is still substantially conserved between homologs. Overall, my graduate research has increased our knowledge of how Env's underlying mutational tolerance shapes the evolution of antibody epitopes, providing experimental support for the assumption that conserved epitopes targeted in vaccine design are indeed less tolerant of mutations than the rest of the protein, and may thus be less likely to evade an immune response. This work also provides a comprehensive measure of differences in mutational effects across Env, finding that mutational effects are largely conserved between divergent homologs. More broadly, this work was also the first time that deep mutational scanning had been used to comprehensively measure mutational effects to an HIV protein in context of viral replication. In the future, this technique could be adapted to study any phenotype that is selectable in the lab (e.g., antibody escape)
Influenza viruses with receptor-binding neuraminidases
No full textThesis (Ph.D.)--University of Washington, 2015University of Washington Abstract Influenza viruses with receptor-binding neuraminidases Kathryn A. Hooper Chair of the Supervisory Committee: Assistant Member Jesse D. Bloom Division of Basic Sciences and Computational Biology, Fred Hutchinson Cancer Research Center For the vast majority of influenza viruses characterized to date, the two main surface proteins have opposing and mutually exclusive functions. Hemagglutinin (HA) is the viral entry protein, mediating attachment of the virus to the host cell via sialic acid-receptors and fusion of the viral and endosomal membranes. In contrast, neuraminidase (NA) is the viral exit protein, and is a sialidase that cleaves receptors from the surface of the producing cell to facilitate viral release. In the last few years, a growing body of evidence has shown that the receptor binding and receptor cleaving activities need not be mutually exclusive, and that single amino-acid mutations can allow NA to act as the receptor-binding protein. These mutations are D151G in N2 subtype NAs and G147R in N1 subtype NAs. Here I describe the characterization of the G147R N1 NA mutation in Chapter II which was serendipitously discovered as a tissue-culture adaptation mutation. In Chapter III, I examine the effect of the G147R mutation in the background of naturally circulating viruses from three different viral lineages. I find that the receptor-binding NA mutation has no effect on the fitness of a pandemic human H1N1 virus in cell culture or in a mouse model of pathogenesis. The presence of the receptor-binding NA also does not affect sensitivity of the virus to neutralization by an anti-HA IgG, although it does slightly protect against neutralization by the Fab domain of the antibody. The physiological relevance of escape from anti-HA Fab, however, remains unclear. Overall, the identification of NA receptor-binding mutations represents a new aspect of influenza virus biology which warrants further investigation. The first NA-binding mutation was discovered when viruses failed to behave as expected in traditional hemagglutination inhibition assays. The acquisition of this D151G mutation by many recent H3N2 strains has remained a problem for the determination of vaccine efficacy in anti-sera hemagglutination inhibition assays. My work extended the finding that receptor-binding mutations can occur in N2 subtype NAs to the N1 subtype, as well. This suggests that the overall spectrum of NA-binding mutations may be much greater than is currently understood. Furthermore, because the N1 binding mutation does not significantly compromise the fitness of a recent human virus, my work also suggests that NA-binding mutations may have the potential to become more widespread in the future
Evolutionary dynamics of influenza virus across spatiotemporal scales
No full textThesis (Ph.D.)--University of Washington, 2019RNA viruses like influenza mutate rapidly to form genetically diverse populations. Recent high-throughput deep sequencing techniques make it possible to track influenza’s evolutionary dynamics at high resolution, showing how viral populations diversify and adapt in just days or weeks. In my dissertation, I examine how influenza viruses evolve across different spatiotemporal scales. First, I characterize a cooperative interaction between two distinct influenza variants that differ by a single nucleotide mutation. In cell culture, a mixture of the two viral variants grows to higher titers than either variant alone, and populations maintain an equal mixture of the two variants through several passages. Next, I show that this mixture of cooperative variants arises primarily in cell culture rather than in clinical samples. This work provides one of the first examples of a specific cooperative interaction between RNA viruses. In the rest of my thesis, I focus on how influenza viruses evolve within infected hosts. First, I characterize influenza’s evolutionary dynamics within chronically infected individuals. In multi-week infections, I observe extensive parallelism in the mutations that arise within and between hosts. The same small set of antigenic variants arises recurrently within an individual, in multiple individuals in our study, and in the global influenza population. Next, I resolve a discrepancy between two recent estimates of how much genetic diversity is present within acute influenza infections and what proportion of this genetic diversity is transmitted. I identify a major technical issue in the raw sequencing data for one study that contributes to that study’s estimate of high genetic diversity and a large transmission bottleneck. Altogether, this work expands our understanding of the evolutionary forces that shape viral populations across multiple spatiotemporal scales
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