Microbiomes are vast communities of microorganisms and viruses that populate all natural ecosystems. Viruses have been considered to be the most variable component of microbiomes, as supported by virome surveys and examples of high genomic mosaicism. However, recent evidence suggests that the human gut virome is remarkably stable compared with that of other environments. Here, we investigate the origin, evolution and epidemiology of crAssphage, a widespread human gut virus. Through a global collaboration, we obtained DNA sequences of crAssphage from more than one-third of the world's countries and showed that the phylogeography of crAssphage is locally clustered within countries, cities and individuals. We also found fully colinear crAssphage-like genomes in both Old-World and New-World primates, suggesting that the association of crAssphage with primates may be millions of years old. Finally, by exploiting a large cohort of more than 1,000 individuals, we tested whether crAssphage is associated with bacterial taxonomic groups of the gut microbiome, diverse human health parameters and a wide range of dietary factors. We identified strong correlations with different clades of bacteria that are related to Bacteroidetes and weak associations with several diet categories, but no significant association with health or disease. We conclude that crAssphage is a benign cosmopolitan virus that may have coevolved with the human lineage and is an integral part of the normal human gut virome.

APC Microbiome Institute University College Cork Cork Ireland

Biological and Medical Informatics Program San Diego State University San Diego CA USA

Carl R Woese Institute of Genomic Biology University of Illinois at Urbana Champaign Urbana IL USA

CEHA Kampala Uganda

Center for Genomics and Systems Biology and Department of Biology New York University New York NY USA

Centre for Molecular and Biomolecular Informatics Radboud Institute for Molecular Life Sciences Radboud University Medical Centre Nijmegen The Netherlands

Centre of Epidemiology and Microbiology National Institute of Public Health Prague Czech Republic

Centro de Ciencias Genómicas Universidad Nacional Autónoma de México Cuernavaca Mexico

CERELA Tucumán Argentina

Civil and Environmental Engineering and Earth Sciences University of Notre Dame Notre Dame IN USA

Clinical Microbiology and Immunology Sackler school of Medicine Tel Aviv University Tel Aviv Israel

College of Natural and Computational Sciences Hawai'i Pacific University Kaneohe HI USA

Computational Sciences Research Center San Diego State University San Diego CA USA

Computer Science and Engineering University of California San Diego La Jolla CA USA

COVAB Makerere University Kampala Uganda

Departament de Genètica i de Microbiologia Universitat Autònoma De Barcelona Barcelona Spain

Departamento de Genética Molecular y Microbiología Pontificia Universidad Católica de Chile Santiago Chile

Departamento de Química Biológica Facultad de Ciencias Exactas y Naturales Universidad de Buenos Aires Buenos Aires Argentina

Department Estacion Biologica Corrientes Institution Museo Arg Cs Naturales CONICET Corrientes Argentina

Department of Animal Health Columbus Zoo and Aquarium Powell OH USA

Department of Anthropology Dartmouth College Hanover NH USA

Department of Anthropology Northern Illinois University DeKalb IL USA

Department of Anthropology University of Colorado Boulder CO USA

Department of Bacterial Genetics Institute of Microbiology Faculty of Biology University of Warsaw Warsaw Poland

Department of Biology San Diego State University San Diego CA USA

Department of Biology University of Padova Padova Italy

Department of Bionanoscience Kavli Institute of Nanoscience Delft University of Technology Delft The Netherlands

Department of Biosystems KU Leuven Leuven Belgium

Department of Child Health Norwegian Institute of Public Health Oslo Norway

Department of Civil and Environmental Engineering Virginia Tech Blacksburg VA USA

Department of Clinical Microbiology and Immunology Sackler School of Medicine Tel Aviv University Tel Aviv Israel

Department of Computer Science San Diego State University San Diego CA USA

Department of Genetics Microbiology and Statistics Universitat de Barcelona Barcelona Spain

Department of Genetics University Medical Center Groningen Groningen The Netherlands

Department of Informational Technologies ITMO University Saint Petersburg Russia

Department of Medicine University of Chicago Chicago IL USA

Department of Microbiology and Biotechnology Max Rubner Institut Federal Research Institute of Nutrition and Food Kiel Germany

Department of Microbiology and Immunology Faculty of Pharmacy Cairo University Cairo Egypt

Department of Microbiology and Immunology McGill University Montreal Quebec Canada

Department of Microbiology and Immunology University of Otago Dunedin New Zealand

Department of Microbiology and Molecular Genetics University of California Davis Davis CA USA

Department of Molecular Biology and Biochemistry University of California Irvine Irvine CA USA

Department of Molecular Biology and Genetics Federal Research and Clinical Center of Physical Chemical Medicine Moscow Russia

Department of Molecular Biosciences Stockholm University Stockholm Sweden

Department of Pediatrics 2nd Faculty of Medicine Charles University Prague Prague Czech Republic

Department of Pediatrics and Child Health Faculty of Medicine University of Khartoum Khartoum Sudan

Department of Pediatrics Federal Teaching Hospital Abakaliki Ebonyi State University Abakaliki Nigeria

Department of Pediatrics School of Medicine University of Jordan Amman Jordan

Department of Pediatrics University Medical Center Groningen Groningen The Netherlands

Department of Physiology Genetics and Microbiology University of Alicante Alicante Spain

Department of Research and Development Lytech Ltd Moscow Russia

Department of Virology School of Medicine University of Tampere Tampere Finland

Endocrine Centre Baku Baku Azerbaijan

EPHM Lab Civil Engineering Department Monash University Clayton Victoria Australia

Escuela de Tecnología Médica Universidad Andres Bello Santiago Chile

GEMA Center for Genomics Ecology and Environment Universidad Mayor Huechuraba Chile

Institute of Infection and Global Health University of Liverpool Liverpool UK

Laboratorio de Analises Instituto Superior Tecnico Universidade Lisboa Lisboa Portugal

Laboratory of Bioinformatics Federal Research and Clinical Center of Physical Chemical Medicine Moscow Russia

Max Planck Tandem Group in Computational Biology Departamento de Ciencias Biológicas Universidad de los Andes Bogotá Colombia

Mediterranean Institute of Oceanography Aix Marseille Université Université de Toulon CNRS IRD UM 110 Marseille France

MEPHI Aix Marseille Université IRD AP HM CNRS IHU Méditerranée Infection Marseille France

Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering Fudan University Shanghai China

Molecular Genetics Corporación Corpogen Bogotá Colombia

National Food Institute Research Group for Genomic Epidemiology Technical University of Denmark Kongens Lyngby Denmark

Next Generation Sequencing and Microarray Core Facility The Scripps Research Institute La Jolla CA USA

School of Biological Sciences Monash University Clayton Victoria Australia

School of Microbiology University College Cork Cork Ireland

School of Science and Health Western Sydney University Penrith New South Wales Australia

Swanson School of Engineering University of Pittsburgh Pittsburgh PA USA

The Bioinformatics Centre Department of Biology University of Copenhagen Copenhagen Denmark

The Viral Information Institute San Diego State University San Diego CA USA

Theoretical Biology and Bioinformatics Science4Life Utrecht University Utrecht The Netherlands

UWA School of Agriculture and Environment University of Western Australia Perth Western Australia Australia

Wildlife Health Center University of California Davis Davis CA USA

Zobrazit více v PubMed

Sender R, Fuchs S & Milo R Are we really vastly outnumbered? Revisiting the ratio of bacterial to host cells in humans. Cell 164, 337–340 (2016). PubMed

Nguyen S et al. Bacteriophage transcytosis provides a mechanism to cross epithelial cell layers. mBio 8, e01874–17 (2017). PubMed PMC

Reyes A et al. Viruses in the faecal microbiota of monozygotic twins and their mothers. Nature 466, 334–338 (2010). PubMed PMC

Minot S et al. The human gut virome: inter-individual variation and dynamic response to diet. Genome Res. 21, 1616–1625 (2011). PubMed PMC

Reyes A, Semenkovich NP, Whiteson K, Rohwer F & Gordon JI Going viral: next-generation sequencing applied to phage populations in the human gut. Nat. Rev. Microbiol 10, 607–617 (2012). PubMed PMC

Paterson S et al. Antagonistic coevolution accelerates molecular evolution. Nature 464, 275–278 (2010). PubMed PMC

Pedulla ML et al. Origins of highly mosaic mycobacteriophage genomes. Cell 113, 171–182 (2003). PubMed

Heldal M & Bratbak G Production and decay of viruses in aquatic environments. Mar. Ecol. Prog. Ser 72, 205–212 (1991).

Breitbart M, Wegley L, Leeds S, Schoenfeld T & Rohwer F Phage community dynamics in hot springs. Appl. Environ. Microbiol 70, 1633–1640 (2004). PubMed PMC

Steward GF, Smith DC & Azam F Abundance and production of bacteria and viruses in the Bering and Chukchi Seas. Mar. Ecol. Prog. Ser 131, 287–300 (1996).

Minot S et al. Rapid evolution of the human gut virome. Proc. Natl Acad. Sci. USA 110, 12450–12455 (2013). PubMed PMC

Dutilh BE et al. A highly abundant bacteriophage discovered in the unknown sequences of human faecal metagenomes. Nat. Commun 5, 4498 (2014). PubMed PMC

Yutin N et al. Discovery of an expansive bacteriophage family that includes the most abundant viruses from the human gut. Nat. Microbiol 3, 38–46 (2018). PubMed PMC

Shkoporov A et al. ΦCrAss001, a member of the most abundant bacteriophage family in the human gut, infects Bacteroides. Preprint at 10.1101/354837 (2018). PubMed DOI PMC

Barylski J et al. Analysis of spounaviruses as a case study for the overdue reclassification of tailed bacteriophages. Preprint at 10.1101/220434 (2018). PubMed DOI PMC

Adriaenssens E & Brister JR How to name and classify your phage: an informal guide. Viruses 9, 70 (2017). PubMed PMC

Callahan BJ, McMurdie PJ & Holmes SP Exact sequence variants should replace operational taxonomic units in marker-gene data analysis. ISME J. 11, 2639–2643 (2017). PubMed PMC

NCBI Resource Coordinators Database resources of the National Center for Biotechnology Information. Nucleic Acids Res. 44, D7–D19 (2016). PubMed PMC

Nicholls SM et al. Probabilistic recovery of cryptic haplotypes from metagenomic data. Preprint at 10.1101/117838 (2017). DOI

Lim ES et al. Early life dynamics of the human gut virome and bacterial microbiome in infants. Nat. Med 21, 1228–1234 (2015). PubMed PMC

Liang YY, Zhang W, Tong YG & Chen SP crAssphage is not associated with diarrhoea and has high genetic diversity. Epidemiol. Infect 144, 3549–3553 (2016). PubMed PMC

Piper HG et al. Severe gut microbiota dysbiosis is associated with poor growth in patients with short bowel syndrome. JPEN J. Parenter. Enter. Nutr 41, 1202–1212 (2017). PubMed

Vatanen T et al. Variation in microbiome LPS immunogenicity contributes to autoimmunity in humans. Cell 165, 842–853 (2016). PubMed PMC

Huerta-Cepas J, Serra F & Bork P ETE 3: reconstruction, analysis, and visualization of phylogenomic data. Mol. Biol. Evol 33, 1635–1638 (2016). PubMed PMC

Stachler E et al. Quantitative crAssphage PCR assays for human fecal pollution measurement. Environ. Sci. Technol 51, 9146–9154 (2017). PubMed PMC

Stachler E & Bibby K Metagenomic evaluation of the highly abundant human gut bacteriophage crAssphage for source tracking of human fecal pollution. Environ. Sci. Technol. Lett 1, 405–409 (2014).

García-Aljaro C, Ballesté E, Muniesa M & Jofre J Determination of crAssphage in water samples and applicability for tracking human faecal pollution. Microb. Biotechnol 10, 1775–1780 (2017). PubMed PMC

Ahmed W et al. Evaluation of the novel crAssphage marker for sewage pollution tracking in storm drain outfalls in Tampa, Florida. Water Res. 131, 142–150 (2017). PubMed

Yatsunenko T et al. Human gut microbiome viewed across age and geography. Nature 486, 222–227 (2012). PubMed PMC

Santiago-Rodriguez TM et al. Natural mummification of the human gut preserves bacteriophage DNA. FEMS Microbiol. Lett 363, fnv219 (2016). PubMed PMC

Maixner F et al. The 5300-year-old Helicobacter pylori genome of the Iceman. Science 351, 162–165 (2016). PubMed PMC

Altschul SF, Gish W, Miller W, Myers EW & Lipman DJ Basic local alignment search tool. J. Mol. Biol 215, 403–410 (1990). PubMed

Guerin E et al. Biology and taxonomy of crAss-like bacteriophages, the most abundant virus in the human gut. Cell Host Microbe 24, 653–664 (2018). PubMed

Raymond F et al. The initial state of the human gut microbiome determines its reshaping by antibiotics. ISME J. 10, 707–720 (2016). PubMed PMC

Moeller AH et al. Rapid changes in the gut microbiome during human evolution. Proc. Natl Acad. Sci. USA 111, 16431–16435 (2014). PubMed PMC

Moeller AH et al. Cospeciation of gut microbiota with hominids. Science 353, 380–382 (2016). PubMed PMC

Zhernakova A et al. Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity. Science 352, 565–569 (2016). PubMed PMC

Tigchelaar EF et al. Cohort profile: LifeLines DEEP, a prospective, general population cohort study in the northern Netherlands: study design and baseline characteristics. BMJ Open 5, e006772 (2015). PubMed PMC

David LA et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature 505, 559–563 (2014). PubMed PMC

Turnbaugh PJ et al. The effect of diet on the human gut microbiome: a metagenomic analysis in humanized gnotobiotic mice. Sci. Transl. Med 1, 6ra14 (2009). PubMed PMC

Singh RK et al. Influence of diet on the gut microbiome and implications for human health. J. Transl. Med 15, 73 (2017). PubMed PMC

De Filippo C et al. Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc. Natl Acad. Sci. USA 107, 14691–14696 (2010). PubMed PMC

Kovatcheva-Datchary P et al. Dietary fiber-induced improvement in glucose metabolism is associated with increased abundance of Prevotella. Cell Metab. 22, 971–982 (2015). PubMed

Edwards RA, McNair K, Faust K, Raes J & Dutilh BE Computational approaches to predict bacteriophage-host relationships. FEMS Microbiol. Rev 40, 258–272 (2016). PubMed PMC

Manrique P et al. Healthy human gut phageome. Proc. Natl Acad. Sci. USA 113, 10400–10405 (2016). PubMed PMC

Kupczok A et al. Rates of mutation and recombination in Siphoviridae phage genome evolution over three decades. Mol. Biol. Evol 35, 1147–1159 (2018). PubMed PMC

Schrago CG & Russo CAM Timing the origin of New World monkeys. Mol. Biol. Evol 20, 1620–1625 (2003). PubMed

Bankevich A et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol 19, 455–477 (2012). PubMed PMC

Hyatt D et al. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinform. 11, 119 (2010). PubMed PMC

Sievers F et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol 7, 539 (2011). PubMed PMC

Nguyen L-T, Schmidt HA, von Haeseler A & Minh BQ IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol 32, 268–274 (2015). PubMed PMC

Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A & Jermiin LS ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14, 587–589 (2017). PubMed PMC

Zhou X, Shen X, Hittinger CT & Rokas A Evaluating fast maximum likelihood-based phylogenetic programs using empirical phylogenomic data sets. Mol. Biol. Evol 35, 486–503 (2017). PubMed PMC

Dutilh BE et al. Assessment of phylogenomic and orthology approaches for phylogenetic inference. Bioinformatics 23, 815–824 (2007). PubMed

Cinek O et al. Quantitative crAssphage real-time PCR assay derived from data of multiple geographically distant populations. J. Med. Virol 90, 767–771 (2018). PubMed

Liang Y, Jin X, Huang Y & Chen S Development and application of a real-time polymerase chain reaction assay for detection of a novel gut bacteriophage (crAssphage). J. Med. Virol 90, 464–468 (2018). PubMed

Langmead B & Salzberg SL Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012). PubMed PMC

Li H et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009). PubMed PMC

Ewing B & Green P Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8, 186–194 1998). PubMed

Ewing B, Hillier L, Wendl MC & Green P Base-calling of automated sequencer traces using phred. I. Accuracy assessment. Genome Res. 8, 175–185 (1998). PubMed

Rice P, Longden I & Bleasby A EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000). PubMed

Knudsen BE, Bergmark L & Pamp SJ SOP—DNA isolation QIAamp Fast DNA Stool modified. Figshare 10.6084/m9.figshare.3475406.v4 (2016). DOI

National Center for Biotechnology Information SRA Handbook (National Center for Biotechnology Information, 2009).

Stewart CA et al. Jetstream: a self-provisioned, scalable science and engineering cloud environment. In Proc. 2015 XSEDE Conference Scientific Advancements Enabled by Enhanced Cyberinfrastructure 29 (ACM, 2015).

Towns J et al. XSEDE: accelerating scientific discovery. Comput. Sci. Eng 16, 62–74 (2014).

Edwards R SearchSRA (2017); 10.5281/zenodo.1043562 DOI

Torres PJ, Edwards RA & McNair K PARTIE: a partition engine to separate metagenomics and amplicon projects in the Sequence Read Archive. Bioinformatics 33, 2389–2391 (2017). PubMed PMC

Schmieder R & Edwards R Quality control and preprocessing of metagenomic datasets. Bioinformatics 27, 863–864 (2011). PubMed PMC

Cantu VA, Sadural J & Edwards R PRINSEQ++, a multi-threaded tool for fast and efficient quality control and preprocessing of sequencing datasets. Preprint at 10.7287/peerj.preprints.27553v1 (2019). DOI

Levi K, Rynge M, Eroma A & Edwards RA Searching the Sequence Read Archive using Jetstream and Wrangler. In Proc. Practice and Experience on Advanced Research Computing (2018).

Stallman RM, McGrath R & Smith PD GNU Make: A Program for Directing Recompilation, for version 3.81 (Free Software Foundation, 2004).

Edgar RC MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004). PubMed PMC

Letunic I & Bork P Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 44, W242–W245 (2016). PubMed PMC

Berke L & Snel B The histone modification H3K27me3 is retained after gene duplication and correlates with conserved noncoding sequences in Arabidopsis. Genome Biol. Evol 6, 572–579 (2014). PubMed PMC

Meyer F et al. The metagenomics RAST server—a public resource for the automatic phylogenetic and functional analysis of metagenomes. BMC Bioinform. 9, 386 (2008). PubMed PMC

Zhao Y, Tang H & Ye Y RAPSearch2: a fast and memory-efficient protein similarity search tool for next-generation sequencing data. Bioinformatics 28, 125–126 (2012). PubMed PMC

Vlčková K et al. Impact of stress on the gut microbiome of free-ranging western lowland gorillas. Microbiology 164, 40–44 (2018). PubMed

McNair K, Zhou C, Dinsdale EA, Souza B & Edwards RA PHANOTATE: a novel approach to gene identification in phage genomes. Bioinformatics 10.1093/bioinformatics/btz265 (2019). PubMed DOI PMC

Katoh K & Standley DM MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol 30, 772–780 (2013). PubMed PMC

Benjamini Y & Hochberg Y Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol 57, 289–300 (1995).

Dutilh Bas E., and Edwards Robert A. crAssphage Data Repository on GitHub (Github, 2018); 10.5281/zenodo.1230436 DOI

Convergence of gut phage communities but not bacterial communities following wild mouse bacteriophage transplantation into captive house mice