Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Hoecke, Silvie Van den; Verhelst, Judith; Vuylsteke, Marnik; Saelens, Xavier

doi:10.1186/s12864-015-1284-z

Cited by 84 publications

(106 citation statements)

References 79 publications

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Section: Discussionsupporting

confidence: 76%

“…Higher error rates have been observed with the Ion Torrent PGM than with the Illumina MiSeq, highlighting the importance of the error model in distin- guishing variants from error (38). Similar to other studies, we found that sequencing errors were more often insertions and deletions and primarily located in homopolymer regions (38,39). While the use of long-range, inverse PCR specifically enriched samples for virus with intact genomes, fragmented genomes would not have amplified and, ultimately, were not evaluated.…”

Section: Discussionmentioning

confidence: 98%

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Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

et al. 2015

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f BK virus (BKV) infection causing end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep-sequencing methodology and bioinformatics pipeline that identify BKV variants across the genome and at BKV-specific HLA-A2-, HLA-B0702-, and HLA-B08-restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets, and fragmentation libraries were sequenced on the Ion Torrent Personal Genome Machine (PGM). An error model and variant-calling algorithm were developed to accurately identify rare variants. A total of 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range, 2 to 37; interquartile range, 10), with the majority of variants (77%) detected at a frequency of <5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for the BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies.

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Section: Discussionsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 98%

Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

et al. 2015

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“…Based on the known error rate of the Illumina sequencing platform (36, 37) and the stringency of these variant call criteria, we used a minimum allele frequency cutoff of 1% to identify variants. Each of the identified variants was visually inspected and excluded if the variant was supported by <10 reads (38) or if the average read position of the variant occurred outside the middle 50% of the short read (35). We made one exception to these variant processing protocols.…”

Section: Methodsmentioning

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

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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“…[34–36] Furthermore, the low error rate for Illumina minimizes base call errors in the primer tags that could lead to the creation of artificial variants and distort quasispecies distributions. [33, 48] Thus, SVS allowed us to determine viral variants and quasispecies population with high accuracy and sensitivity. We note that errors from first-strand DNA synthesis during reverse transcription could not be corrected using SVS.…”

Section: Discussionmentioning

confidence: 99%

CD4+ T-cell recovery with suppressive ART-induced rapid sequence evolution in hepatitis C virus envelope but not NS3

Liu

Nardo²,

Li³

et al. 2016

Aids

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Objectives CD4+ T-cell depletion from human immunodeficiency virus (HIV) infection leads to a global decline in anti-hepatitis C virus (HCV) envelope neutralizing antibody (nAb) response, which may play a role in accelerating liver fibrosis. An increase in anti-HCV nAb titers has been reported during antiretroviral therapy (ART) but its impact on HCV remains poorly understood. The objective of this study is to determine the effects of ART on long-term HCV evolution. Design/methods We examined HCV quasispecies structure and long-term evolution in HIV/HCV co-infected subjects with ART-induced CD4+ T-cell recovery, and compared to subjects with CD4+ T-cell depletion from delayed ART. We applied a single-variant sequencing (SVS) method to construct authentic viral quasispecies and compared sequence evolution in HCV envelope, the primary target for humoral immune responses, and NS3, a target for cellular immunity, between the two cohorts. Results The SVS method corrected biases known to skew the proportions of viral variants, revealing authentic HCV quasispeices structures. We observed higher rates of HCV envelope sequence evolution in subjects with ART-induced CD4+ T-cell recovery, compared to subjects with CD4+ T-cell depletion from delayed ART (P=0.03). Evolutionary rates for NS3 were considerably lower than the rates for envelope (P<0.01), with no significant difference observed between the two groups. Conclusions ART-induced CD4+ T-cell recovery results in rapid sequence evolution in HCV envelope, but not in NS3. These results suggest that suppressive ART disproportionally enhances HCV-specific humoral responses more than cellular responses, resulting in rapid sequence evolution in HCV envelope but not NS3.

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Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Cited by 84 publications

References 79 publications

Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

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

CD4+ T-cell recovery with suppressive ART-induced rapid sequence evolution in hepatitis C virus envelope but not NS3

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