Abstract
ABSTRACTKnowledge of influenza virus evolution at the point of transmission and at the intrahost level remains limited, particularly for human hosts. Here, we analyze a unique viral data set of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with cell- and egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects' viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects' nasal wash samples, the viral stock, and the influenza virus A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that (i) the viral stock contained genetic variants that originated and likely were selected for during the passaging process, (ii) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein, and (iii) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation.IMPORTANCE Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza virus sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects' viral populations differ genetically from the viral stock, with subjects' viral populations having lower representation of the amino-acid-changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid-changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections.
Highlights
Influenza viruses circulating among humans are known to rapidly evolve over time
Three key observations can be made from these plots. (i) The large genetic distance between the viral stock and the reference strain [A/Wisconsin/67/2005 (H3N2)], in the PA, HA, and NP gene segments, indicates that viral evolution occurred during generation of the viral stock. (ii) The genetic distances between virus isolated from the subjects’ first nasal wash samples and the viral stock can be appreciable, suggesting a narrow transmission bottleneck
Virus isolated from subjects’ first nasal wash samples, for the HA, are often genetically more similar to one another than they are to the viral stock, putatively indicating that the transmission bottleneck is a selective one. (iii) The genetic distances between virus isolated from a single subject at different time points are sometimes substantial, e.g., the distances between S12:D2, S12: D3, and S12:D6
Summary
Influenza viruses circulating among humans are known to rapidly evolve over time. little is known about how influenza virus evolves across single transmission events and over the course of a single infection. These studies have generally shown that viral populations in recipient hosts differ from the ones in donor hosts, they again can contain variants that are present at low frequencies in donor hosts [18] This finding suggests that the between-host transmission dynamics of less well adapted IAVs are instead better characterized by narrow, selective bottlenecks [17, 18], the size and type of the bottleneck will be affected by the degree of viral maladaptation to the host [17]. We find evidence for continued purifying selection and only limited evolution via de novo mutation, more similar to what was found in studies with well-adapted IAVs
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