Quantifying influenza virus diversity and transmission in humans

Poon, Leo L. M.; Song, Timothy; Rosenfeld, Roni; Lin, Xudong; Rogers, Matthew B.; Zhou, Bin; Sebra, Robert; Halpin, Rebecca; Guan, Yi; Twaddle, Alan; DePasse, Jay V.; Stockwell, Timothy B.; Wentworth, David E.; Holmes, Edward C.; Greenbaum, Benjamin; Peiris, J. S. Malik; Cowling, Benjamin J.; Ghedin, Elodie

doi:10.1038/ng.3479

Cited by 179 publications

(254 citation statements)

References 27 publications

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“…This rapid evolution occurs via a selective transmission bottleneck and continuing purifying selection acting on the founding viral population. While a large and phenotypically heterogeneous founding population size may be unique to this experimental study, recent estimates of transmission bottleneck sizes in natural human-to-human infections are much larger than anticipated by many—on the order of 100 to 250 virions (16). This indicates that the fitness differences within a viral population, rather than the initial levels of genetic diversity, may be the primary factor setting the speed limit of IAV adaptation.…”

Section: Discussionmentioning

confidence: 99%

“…These studies include ones that characterize IAV evolution across transmission events in attempts to determine the size and type of transmission bottleneck (9–19). While some of these studies have focused on the evolutionary dynamics of well-adapted viruses already circulating in these or similar host populations (9–16), others have instead probed the evolutionary dynamics of maladapted viruses in new host populations to determine the adaptive potential of IAV following cross-species viral spillover (17–19). …”

Section: Introductionmentioning

confidence: 99%

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Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification

Leonard

McClain²,

Smith

et al. 2016

J Virol

Self Cite

108

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Knowledge 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.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification

Leonard

McClain²,

Smith

et al. 2016

J Virol

Self Cite

108

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“…In the adults, our estimate of an infection bottleneck of 25 virions is lower than the bottleneck sizes of other viruses that cause acute infections. The bottleneck size for Ebolavirus is an average of 100 virions, with a range of around 1–1000 virions (20), and the mean bottlenecks for influenza H1N1/2009 and H3N2 are 90 virions and 144 virions, respectively, with a standard deviation of 55 virions for both (28) (Supplemental Figure 2). …”

Section: Discussionmentioning

confidence: 99%

Deep sequencing of RSV from an adult challenge study and from naturally infected infants reveals heterogeneous diversification dynamics

et al. 2017

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As RNA virus mutation occurs during replication within host cells, we hypothesized that viral evolution during acute infections in healthy hosts reflects host immune pressure. We therefore investigated the within-host diversification of human respiratory syncytial virus (RSV), a highly prevalent cause of acute respiratory infections. We evaluated healthy adults experimentally infected with an identical inoculum and infants hospitalized with naturally acquired infections. In aggregate, viral diversification in adults peaked at day 3, with overrepresentation of diversity in the matrix protein 2 (M2) and non-structural protein 2 (NS2) genes. In one subject, delayed viral clearance was accompanied by a late peak of diversity at day 10 in known and predicted B and T cell epitopes. In contrast, infant infections showed much less viral diversity. Our findings suggest multiple overlapping mechanisms for early control of acute viral infections, which may differ between age groups and host immune responses.

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“…Although the initial benchmarking experiment validated our ability to accurately detect rare variants in influenza virus populations, the experiment was run under relatively artificial conditions. Patient-derived populations are typically less divergent than WSN33 and PR8 (4,5,23), and the number of viral genomes in patient samples is much lower than that found in cell culture. To mimic patientspecific conditions, we generated 20 viral clones, each with a single point mutation in the WSN33 background.…”

Section: Resultsmentioning

confidence: 99%

“…SISPA controls for any errors that may arise during library amplification, including PCR biases. The method has been used in conjunction with statistical algorithms to control for accuracy in studies of intrahost influenza virus diversity (5,22,23). However, the bar-coding reaction used in SISPA can be biased in unpredictable ways, resulting in uneven coverage and sensitivity across the genome (24).…”

mentioning

confidence: 99%

Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling

McCrone

Lauring

2016

J Virol

115

164

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With next-generation sequencing technologies, it is now feasible to efficiently sequence patient-derived virus populations at a depth of coverage sufficient to detect rare variants. However, each sequencing platform has characteristic error profiles, and sample collection, target amplification, and library preparation are additional processes whereby errors are introduced and propagated. Many studies account for these errors by using ad hoc quality thresholds and/or previously published statistical algorithms. Despite common usage, the majority of these approaches have not been validated under conditions that characterize many studies of intrahost diversity. Here, we use defined populations of influenza virus to mimic the diversity and titer typically found in patient-derived samples. We identified single-nucleotide variants using two commonly employed variant callers, Deep-SNV and LoFreq. We found that the accuracy of these variant callers was lower than expected and exquisitely sensitive to the input titer. Small reductions in specificity had a significant impact on the number of minority variants identified and subsequent measures of diversity. We were able to increase the specificity of DeepSNV to >99.95% by applying an empirically validated set of quality thresholds. When applied to a set of influenza virus samples from a household-based cohort study, these changes resulted in a 10-fold reduction in measurements of viral diversity. We have made our sequence data and analysis code available so that others may improve on our work and use our data set to benchmark their own bioinformatics pipelines. Our work demonstrates that inadequate quality control and validation can lead to significant overestimation of intrahost diversity. IMPORTANCEAdvances in sequencing technology have made it feasible to sequence patient-derived viral samples at a level sufficient for detection of rare mutations. These high-throughput, cost-effective methods are revolutionizing the study of within-host viral diversity. However, the techniques are error prone, and the methods commonly used to control for these errors have not been validated under the conditions that characterize patient-derived samples. Here, we show that these conditions affect measurements of viral diversity. We found that the accuracy of previously benchmarked analysis pipelines was greatly reduced under patientderived conditions. By carefully validating our sequencing analysis using known control samples, we were able to identify biases in our method and to improve our accuracy to acceptable levels. Application of our modified pipeline to a set of influenza virus samples from a cohort study provided a realistic picture of intrahost diversity and suggested the need for rigorous quality control in such studies. Many viral pathogens are thought to exist as a cloud of closely related mutants within an infected individual (1). Until recently, our understanding of intrahost viral dynamics and the impact of viral diversity on evolution and pathogenesis has been li...

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Quantifying influenza virus diversity and transmission in humans

Cited by 179 publications

References 27 publications

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification

Deep sequencing of RSV from an adult challenge study and from naturally infected infants reveals heterogeneous diversification dynamics

Measurements of Intrahost Viral Diversity Are Extremely Sensitive to Systematic Errors in Variant Calling

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