Influenza Classification from Short Reads with VAPOR Facilitates Robust Mapping Pipelines and Zoonotic Strain Detection for Routine Surveillance Applications

BackgroundInfluenza viruses are associated with a significant global public health burden. The segmented RNA genome of influenza changes continually due to mutation, and the accumulation of these changes within the antigenic recognition sites of haemagglutinin (HA) and neuraminidase (NA) in turn leads to annual epidemics. Influenza A is also zoonotic, allowing for exchange of segments between human and non-human viruses, resulting in new strains with pandemic potential. These processes necessitate a global surveillance system for influenza monitoring. To this end, whole-genome sequencing (WGS) has begun to emerge as a useful tool. However, due to the diversity and mutability of the influenza genome, and noise in short-read data, bioinformatics processing can present challenges.ResultsConventional mapping approaches can be insufficient when a sub-optimal reference strain is chosen. For short-read datasets simulated from influenza H1N1 HA sequences, read recovery after single-reference mapping was routinely as low as 90% for human-origin influenza sequences, and often lower than 10% for those from avian hosts. To this end, we developed a de Bruijn Graph (DBG)-based classifier of influenza WGS datasets: VAPOR. In real data benchmarking using 257 WGS read sets with corresponding de novo assemblies, VAPOR provided classifications for all samples with a mean of >99.8% identity to assembled contigs. This resulted in an increase in the number of mapped reads by 6.8% on average, up to a maximum of 13.3%. Additionally, using simulations, we demonstrate that classification from reads may be applied to detection of reassorted strains.ConclusionsVAPOR has potential to simplify bioinformatics pipelines for surveillance, providing a novel method for detection of influenza strains of human and non-human origin directly from reads, minimization of potential data loss and bias associated with conventional mapping, and allowing visualization of alignments that would otherwise require slow de novo assembly. Whilst with expertise and time these pitfalls can largely be avoided, with pre-classification they are remedied in a single step. Furthermore, our algorithm could be adapted in future to surveillance of other RNA viruses. VAPOR is available at https://github.com/connor-lab/vapor.

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Influenza classification from short reads with VAPOR facilitates robust mapping pipelines and zoonotic strain detection for routine surveillance applications

Bioinformatics ◽

10.1093/bioinformatics/btz814 ◽

2019 ◽

Vol 36 (6) ◽

pp. 1681-1688

Author(s):

Joel A Southgate ◽

Matthew J Bull ◽

Clare M Brown ◽

Joanne Watkins ◽

Sally Corden ◽

...

Keyword(s):

De Novo ◽

Influenza Viruses ◽

Supplementary Information ◽

Global Public Health ◽

Human Origin ◽

Short Read ◽

Public Health Burden ◽

Routine Surveillance ◽

Surveillance Applications ◽

Strain Detection

Abstract Motivation Influenza viruses represent a global public health burden due to annual epidemics and pandemic potential. Due to a rapidly evolving RNA genome, inter-species transmission, intra-host variation, and noise in short-read data, reads can be lost during mapping, and de novo assembly can be time consuming and result in misassembly. We assessed read loss during mapping and designed a graph-based classifier, VAPOR, for selecting mapping references, assembly validation and detection of strains of non-human origin. Results Standard human reference viruses were insufficient for mapping diverse influenza samples in simulation. VAPOR retrieved references for 257 real whole-genome sequencing samples with a mean of >99.8% identity to assemblies, and increased the proportion of mapped reads by up to 13.3% compared to standard references. VAPOR has the potential to improve the robustness of bioinformatics pipelines for surveillance and could be adapted to other RNA viruses. Availability and implementation VAPOR is available at https://github.com/connor-lab/vapor. Supplementary information Supplementary data are available at Bioinformatics online.

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The Dynamics of Influenza A H3N2 Defective Viral Genomes from a Human Challenge Study

10.1101/814673 ◽

2019 ◽

Cited By ~ 1

Author(s):

Michael A. Martin ◽

Drishti Kaul ◽

Gene S. Tan ◽

Christopher W. Woods ◽

Katia Koelle

Keyword(s):

Genetic Drift ◽

Influenza A ◽

Evolutionary Dynamics ◽

De Novo ◽

Gene Segment ◽

Influenza Viruses ◽

Genomic Variation ◽

Wild Type Virus ◽

Single Nucleotide Variants ◽

Viral Genomes

AbstractThe rapid evolution of influenza is an important contributing factor to its high worldwide incidence. The emergence and spread of genetic point mutations has been thoroughly studied both within populations and within individual hosts. In addition, influenza viruses are also known to generate genomic variation during their replication in the form of defective viral genomes (DVGs). These DVGs are formed by internal deletions in at least one gene segment that render them incapable of replication without the presence of wild-type virus. DVGs have previously been identified in natural human infections and may be associated with less severe clinical outcomes. These studies have not been able to address how DVG populations evolve in vivo in individual infections due to their cross-sectional design. Here we present an analysis of DVGs present in samples from two longitudinal influenza A H3N2 human challenge studies. We observe the generation of DVGs in almost all subjects. Although the genetic composition of DVG populations was highly variable, identical DVGs were observed both between multiple samples within single hosts as well as between hosts. Most likely due to stochastic effects, we did not observe clear instances of selection for specific DVGs or for shorter DVGs over the course of infection. Furthermore, DVG presence was not found to be associated with peak viral titer or peak symptom scores. Our analyses highlight the diversity of DVG populations within a host over the course of infection and the apparent role that genetic drift plays in their population dynamics.ImportanceThe evolution of influenza virus, in terms of single nucleotide variants and the reassortment of gene segments, has been studied in detail. However, influenza is known to generate defective viral genomes (DVGs) during replication, and little is known about how these genomes evolve both within hosts and at the population level. Studies in animal models have indicated that prophylactically or therapeutically administered DVGs can impact patterns of disease progression. However, the formation of naturally-occurring DVGs, their evolutionary dynamics, and their contribution to disease severity in human hosts is not well understood. Here, we identify the formation of de novo DVGs in samples from human challenge studies throughout the course of infection. We analyze their evolutionary trajectories, revealing the important role of genetic drift in shaping DVG populations during acute infections with well-adapted viral strains.

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An R195K Mutation in the PA-X Protein Increases the Virulence and Transmission of Influenza A Virus in Mammalian Hosts

Journal of Virology ◽

10.1128/jvi.01817-19 ◽

2020 ◽

Vol 94 (11) ◽

Cited By ~ 6

Author(s):

Yipeng Sun ◽

Zhe Hu ◽

Xuxiao Zhang ◽

Mingyue Chen ◽

Zhen Wang ◽

...

Keyword(s):

Avian Influenza ◽

Influenza A ◽

Reverse Genetics ◽

Inflammatory Responses ◽

Gene Segment ◽

Genetic Alterations ◽

Influenza Viruses ◽

Interspecies Transmission ◽

Adaptive Mutation ◽

Human Origin

ABSTRACT In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses. IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

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Divergent Human-Origin Influenza Viruses Detected in Australian Swine Populations

Journal of Virology ◽

10.1128/jvi.00316-18 ◽

2018 ◽

Vol 92 (16) ◽

Cited By ~ 6

Author(s):

Frank Y. K. Wong ◽

Celeste Donato ◽

Yi-Mo Deng ◽

Don Teng ◽

Naomi Komadina ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Swine Influenza ◽

Influenza Viruses ◽

Antigenic Drift ◽

Human Origin ◽

Human Influenza ◽

Influenza A Viruses ◽

Data Set ◽

Antigenic Properties

ABSTRACTGlobal swine populations infected with influenza A viruses pose a persistent pandemic risk. With the exception of a few countries, our understanding of the genetic diversity of swine influenza viruses is limited, hampering control measures and pandemic risk assessment. Here we report the genomic characteristics and evolutionary history of influenza A viruses isolated in Australia from 2012 to 2016 from two geographically isolated swine populations in the states of Queensland and Western Australia. Phylogenetic analysis with an expansive human and swine influenza virus data set comprising >40,000 sequences sampled globally revealed evidence of the pervasive introduction and long-term establishment of gene segments derived from several human influenza viruses of past seasons, including the H1N1/1977, H1N1/1995, H3N2/1968, and H3N2/2003, and the H1N1 2009 pandemic (H1N1pdm09) influenza A viruses, and a genotype that contained gene segments derived from the past three pandemics (1968, reemerged 1977, and 2009). Of the six human-derived gene lineages, only one, comprising two viruses isolated in Queensland during 2012, was closely related to swine viruses detected from other regions, indicating a previously undetected circulation of Australian swine lineages for approximately 3 to 44 years. Although the date of introduction of these lineages into Australian swine populations could not be accurately ascertained, we found evidence of sustained transmission of two lineages in swine from 2012 to 2016. The continued detection of human-origin influenza virus lineages in swine over several decades with little or unpredictable antigenic drift indicates that isolated swine populations can act as antigenic archives of human influenza viruses, raising the risk of reemergence in humans when sufficient susceptible populations arise.IMPORTANCEWe describe the evolutionary origins and antigenic properties of influenza A viruses isolated from two separate Australian swine populations from 2012 to 2016, showing that these viruses are distinct from each other and from those isolated from swine globally. Whole-genome sequencing of virus isolates revealed a high genotypic diversity that had been generated exclusively through the introduction and establishment of human influenza viruses that circulated in past seasons. We detected six reassortants with gene segments derived from human H1N1/H1N1pdm09 and various human H3N2 viruses that circulated during various periods since 1968. We also found that these swine viruses were not related to swine viruses collected elsewhere, indicating independent circulation. The detection of unique lineages and genotypes in Australia suggests that isolated swine populations that are sufficiently large can sustain influenza virus for extensive periods; we show direct evidence of a sustained transmission for at least 4 years between 2012 and 2016.

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Time series computational prediction of vaccines for influenza A H3N2 with recurrent neural networks

Journal of Bioinformatics and Computational Biology ◽

10.1142/s0219720020400028 ◽

2020 ◽

Vol 18 (01) ◽

pp. 2040002 ◽

Cited By ~ 3

Author(s):

Rui Yin ◽

Yu Zhang ◽

Xinrui Zhou ◽

Chee Keong Kwoh

Keyword(s):

Neural Networks ◽

Time Series ◽

Recurrent Neural Networks ◽

Influenza A ◽

Viral Evolution ◽

Computational Prediction ◽

Single Step ◽

Influenza Viruses ◽

Computational Method ◽

Influenza Vaccines

Influenza viruses are persistently threatening public health, causing annual epidemics and sporadic pandemics due to rapid viral evolution. Vaccines are used to prevent influenza infections but the composition of the influenza vaccines have to be updated regularly to ensure its efficacy. Computational tools and analyses have become increasingly important in guiding the process of vaccine selection. By constructing time-series training samples with splittings and embeddings, we develop a computational method for predicting suitable strains as the recommendation of the influenza vaccines using recurrent neural networks (RNNs). The Encoder-decoder architecture of RNN model enables us to perform sequence-to-sequence prediction. We employ this model to predict the prevalent sequence of the H3N2 viruses sampled from 2006 to 2017. The identity between our predicted sequence and recommended vaccines is greater than 98% and the [Formula: see text] indicates their antigenic similarity. The multi-step vaccine prediction further demonstrates the robustness of our method which achieves comparable results in contrast to single step prediction. The results show significant matches of the recommended vaccine strains to the circulating strains. We believe it would facilitate the process of vaccine selection and surveillance of seasonal influenza epidemics.

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Within-host evolutionary dynamics of seasonal and pandemic human influenza A viruses in young children

eLife ◽

10.7554/elife.68917 ◽

2021 ◽

Vol 10 ◽

Author(s):

Alvin X Han ◽

Zandra C Felix Garza ◽

Matthijs RA Welkers ◽

René M Vigeveno ◽

Nhu Duong Tran ◽

...

Keyword(s):

Young Children ◽

Influenza A ◽

Evolutionary Dynamics ◽

De Novo ◽

Purifying Selection ◽

Influenza Viruses ◽

Human Influenza ◽

Influenza A Viruses ◽

Virus Diversity ◽

Host Evolution

The evolution of influenza viruses is fundamentally shaped by within-host processes. However, the within-host evolutionary dynamics of influenza viruses remain incompletely understood, in part because most studies have focused on infections in healthy adults based on single timepoint data. Here, we analysed the within-host evolution of 82 longitudinally-sampled individuals, mostly young children, infected with A/H1N1pdm09 or A/H3N2 viruses between 2007 and 2009. For A/H1N1pdm09 infections during the 2009 pandemic, nonsynonymous minority variants were more prevalent than synonymous ones. For A/H3N2 viruses in young children, early infection was dominated by purifying selection. As these infections progressed, nonsynonymous variants typically increased in frequency even when within-host virus titres decreased. Unlike the short-lived infections of adults where de novo within-host variants are rare, longer infections in young children allow for the maintenance of virus diversity via mutation-selection balance creating potentially important opportunities for within-host virus evolution.

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Novel Approaches for The Development of Live Attenuated Influenza Vaccines

Viruses ◽

10.3390/v11020190 ◽

2019 ◽

Vol 11 (2) ◽

pp. 190 ◽

Cited By ~ 17

Author(s):

Pilar Blanco-Lobo ◽

Aitor Nogales ◽

Laura Rodríguez ◽

Luis Martínez-Sobrido

Keyword(s):

Influenza A ◽

Reverse Genetics ◽

Seasonal Influenza ◽

Influenza Viruses ◽

Antigenic Drift ◽

Influenza Vaccines ◽

Global Public Health ◽

Protection Efficacy ◽

Seasonal Epidemics ◽

Rapid Generation

Influenza virus still represents a considerable threat to global public health, despite the advances in the development and wide use of influenza vaccines. Vaccination with traditional inactivate influenza vaccines (IIV) or live-attenuated influenza vaccines (LAIV) remains the main strategy in the control of annual seasonal epidemics, but it does not offer protection against new influenza viruses with pandemic potential, those that have shifted. Moreover, the continual antigenic drift of seasonal circulating influenza viruses, causing an antigenic mismatch that requires yearly reformulation of seasonal influenza vaccines, seriously compromises vaccine efficacy. Therefore, the quick optimization of vaccine production for seasonal influenza and the development of new vaccine approaches for pandemic viruses is still a challenge for the prevention of influenza infections. Moreover, recent reports have questioned the effectiveness of the current LAIV because of limited protection, mainly against the influenza A virus (IAV) component of the vaccine. Although the reasons for the poor protection efficacy of the LAIV have not yet been elucidated, researchers are encouraged to develop new vaccination approaches that overcome the limitations that are associated with the current LAIV. The discovery and implementation of plasmid-based reverse genetics has been a key advance in the rapid generation of recombinant attenuated influenza viruses that can be used for the development of new and most effective LAIV. In this review, we provide an update regarding the progress that has been made during the last five years in the development of new LAIV and the innovative ways that are being explored as alternatives to the currently licensed LAIV. The safety, immunogenicity, and protection efficacy profile of these new LAIVs reveal their possible implementation in combating influenza infections. However, efforts by vaccine companies and government agencies will be needed for controlled testing and approving, respectively, these new vaccine methodologies for the control of influenza infections.

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Defective X-gating caused by de novo gain-of-function mutations in KCNK3 underlies a developmental disorder with sleep apnea

10.1101/2021.08.05.21261490 ◽

2021 ◽

Author(s):

Janina Soermann ◽

Marcus Schewe ◽

Peter Proks ◽

Thibault Jouen-Tachoire ◽

Shanlin Rao ◽

...

Keyword(s):

Sleep Apnea ◽

Developmental Disorder ◽

De Novo ◽

Therapeutic Strategies ◽

K Channel ◽

Global Public Health ◽

Gain Of Function ◽

Public Health Burden ◽

Channel Opening ◽

G Protein Coupled

Sleep apnea is a common disorder that represents a global public health burden. KCNK3 encodes TASK-1, a K+ channel implicated in the control of breathing, but its reported link with sleep apnea remains poorly understood. Here we describe a novel developmental disorder with sleep apnea caused by rare de novo gain-of-function mutations in KCNK3. The mutations cluster around the X-gate, a gating motif which controls channel opening, and produce overactive channels that no longer respond to inhibition by G-protein coupled receptor pathways but which can be inhibited by several clinically relevant drugs. These findings demonstrate a clear role for TASK-1 in sleep apnea and identify possible therapeutic strategies.

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Detection and Characterization of Swine Origin Influenza A(H1N1) Pandemic 2009 Viruses in Humans following Zoonotic Transmission

Journal of Virology ◽

10.1128/jvi.01066-20 ◽

2020 ◽

Vol 95 (2) ◽

pp. e01066-20

Author(s):

Peter W. Cook ◽

Thomas Stark ◽

Joyce Jones ◽

Rebecca Kondor ◽

Natosha Zanders ◽

...

Keyword(s):

Sequence Comparison ◽

Influenza A ◽

Phylogenetic Analyses ◽

Influenza Viruses ◽

Full Genome Sequence ◽

H1n1 Pandemic ◽

Human Origin ◽

First Case ◽

Influenza A H1n1 ◽

Wide Range

ABSTRACTHuman-to-swine transmission of seasonal influenza viruses has led to sustained human-like influenza viruses circulating in the U.S. swine population. While some reverse zoonotic-origin viruses adapt and become enzootic in swine, nascent reverse zoonoses may result in virus detections that are difficult to classify as “swine-origin” or “human-origin” due to the genetic similarity of circulating viruses. This is the case for human-origin influenza A(H1N1) pandemic 2009 (pdm09) viruses detected in pigs following numerous reverse zoonosis events since the 2009 pandemic. We report the identification of two human infections with A(H1N1)pdm09 viruses originating from swine hosts and classify them as “swine-origin” variant influenza viruses based on phylogenetic analysis and sequence comparison methods. Phylogenetic analyses of viral genomes from two cases revealed these viruses were reassortants containing A(H1N1)pdm09 hemagglutinin (HA) and neuraminidase (NA) genes with genetic combinations derived from the triple reassortant internal gene cassette. Follow-up investigations determined that one individual had direct exposure to swine in the week preceding illness onset, while another did not report swine exposure. The swine-origin A(H1N1) variant cases were resolved by full genome sequence comparison of the variant viruses to swine influenza genomes. However, if reassortment does not result in the acquisition of swine-associated genes and swine virus genomic sequences are not available from the exposure source, future cases may not be discernible. We have developed a pipeline that performs maximum likelihood analyses, a k-mer-based set difference algorithm, and random forest algorithms to identify swine-associated sequences in the hemagglutinin gene to differentiate between human-origin and swine-origin A(H1N1)pdm09 viruses.IMPORTANCE Influenza virus infects a wide range of hosts, resulting in illnesses that vary from asymptomatic cases to severe pneumonia and death. Viral transfer can occur between human and nonhuman hosts, resulting in human and nonhuman origin viruses circulating in novel hosts. In this work, we have identified the first case of a swine-origin influenza A(H1N1)pdm09 virus resulting in a human infection. This shows that these viruses not only circulate in swine hosts, but are continuing to evolve and distinguish themselves from previously circulating human-origin influenza viruses. The development of techniques for distinguishing human-origin and swine-origin viruses are necessary for the continued surveillance of influenza viruses. We show that unique genetic signatures can differentiate circulating swine-associated strains from circulating human-associated strains of influenza A(H1N1)pdm09, and these signatures can be used to enhance surveillance of swine-origin influenza.

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Nonreplicating Influenza A Virus Vaccines Confer Broad Protection against Lethal Challenge

mBio ◽

10.1128/mbio.01487-15 ◽

2015 ◽

Vol 6 (5) ◽

Cited By ~ 28

Author(s):

Mariana Baz ◽

Kobporn Boonnak ◽

Myeisha Paskel ◽

Celia Santos ◽

Timothy Powell ◽

...

Keyword(s):

Influenza Virus ◽

Influenza A ◽

Signal Sequence ◽

Influenza Viruses ◽

Global Public Health ◽

Lethal Challenge ◽

Challenge Virus ◽

Cell Responses ◽

Hemagglutinin Protein ◽

Live Attenuated Influenza Virus

ABSTRACT New vaccine technologies are being investigated for their ability to elicit broadly cross-protective immunity against a range of influenza viruses. We compared the efficacies of two intranasally delivered nonreplicating influenza virus vaccines (H1 and H5 S-FLU) that are based on the suppression of the hemagglutinin signal sequence, with the corresponding H1N1 and H5N1 cold-adapted (ca) live attenuated influenza virus vaccines in mice and ferrets. Administration of two doses of H1 or H5 S-FLU vaccines protected mice and ferrets from lethal challenge with homologous, heterologous, and heterosubtypic influenza viruses, and two doses of S-FLU and ca vaccines yielded comparable effects. Importantly, when ferrets immunized with one dose of H1 S-FLU or ca vaccine were challenged with the homologous H1N1 virus, the challenge virus failed to transmit to naive ferrets by the airborne route. S-FLU technology can be rapidly applied to any emerging influenza virus, and the promising preclinical data support further evaluation in humans. IMPORTANCE Influenza viruses continue to represent a global public health threat, and cross-protective vaccines are needed to prevent seasonal and pandemic influenza. Currently licensed influenza vaccines are based on immunity to the hemagglutinin protein that is highly variable. However, T cell responses directed against highly conserved viral proteins contribute to clearance of the virus and confer broadly cross-reactive and protective immune responses against a range of influenza viruses. In this study, two nonreplicating pseudotyped influenza virus vaccines were compared with their corresponding live attenuated influenza virus vaccines, and both elicited robust protection against homologous and heterosubtypic challenge in mice and ferrets, making them promising candidates for further evaluation in humans.

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