ESCMID Online Lecture Library. by author. Are viruses the fourth domain of life or Trucs? or LONDON April, 2012

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1 LONDON April, 2012 Are viruses the fourth domain of life or Trucs? or Didier Raoult Marseille - France didier.raoult@gmail.com U R

2 1 Microbes (Sedillot,1878) Pasteur! Pasteurella multocida visible at the microscope The agent of cholera fowl

3 Prokaryotes Eukaryotes (E.Chatton,1925) dichotomy based on the presence of a nucleus 2 There exist bacteria having a nucleus and histones. The definitions of Eukaryotes, Prokaryotes are false. JA. Fuerst. Intracellular Compartmentation in Planctomycetes. Ann. Rev Microbiol. 2005;59:

4 JA. Fuerst. Intracellular Compartmentation in Planctomycetes. Ann. Rev Microbiol. 2005;59:

5 Photographic credit: Gilbert Greub Ultrastructure of Criblamydia sequanensis (A), Parachlamydia acanthamoebae (B-C) and Waddlia chondrophila (D-F). We miss nuclei on our plates! Greub G, Bertelli C, Croxatto A, Raoult D. New Chlamydiales species question the origin of the nucleus.

6 Confocal microscopy; 600x magnification. Elementary bodies of Parachlamydia acanthamoebae Photographic credit: Gilbert Greub U R

7 3 NATURE Vol May 2006 Archae ESSAY Time for a change Prokaryote: gene-sequence comparisons show the tree of life consists of bacteria, eukarya and archaea. The use of the term prokaryote fails to recognize that an idea about life s origins has been proved wrong. Norman R. Pace C.Woese 1975 creates the ribosome based 3 domains

8 Archae - Archae is inappropriate name as it induces the opinion that this organism an ancestral and are extramophiles. - It produces most of the methane on the earth - In fact there are more archae cells in human body then Eukaryotic cells! - 10 % of the prokaryotes of the gut 500 nm Microscopie Electronique : Forme la plus proche des Methanobrevibacter Gram Contrôle de la culture par microscopie optique: Les méthanogènes sont visualisés par la fluorescence de l'un de leurs co-enzymes uniques, le F420. (Culture de Methanobrevibacter marseillense à partir d une selle). M.Drancourt

9 There are new human species not found by 16S rdna

10 ? Virus definition An organism OR a biomolecule? Lwoff 1957 (Stanley vs Burnet) One dimension < 200 nm One type of nucleic acid Lack of enzymes generating energy Strict intracellular character Inability to undergo binary fission Small specialized and mainly allotropic Lwoff A. The concept of virus. J Gen Microbiol. 1957;17: U R

11 Exceptions RNA or DNA Intracellular bacteria Binary fission Giant viruses Virophages Organelles nanobacteria and prion Selfish DNA U R

12 What is a Rickettsia? Gram negative bacterium Strictly intracellular (?) a1 subgroup of Proteobacteria by 16S rrna sequence analysis Transmitted by arthropods: ticks, fleas, lice, mites U R

13 FIG. 6. Electron photomicrograph of an ultrathin section of E. canis-infected DH82 cells demonstrating E. canis gp19 localization in a morula containing both reticulate and densecored ehrlichiae (A) and a corresponding ultrathin section containing uninfected DH82 cells (negative control) (B). Cells in both panels were reacted with mouse anti-gp19 antibody (1:10,000). Bar = 1 μm. Morula of Ehrlichia Jere W. McBride. Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a Species-Specific Serine-Rich Glycopeptide Epitope. Infect Immun. 2007; 75(1): 74 82

14 Viral factory looking like Morula U R

15 U R

16 BABL: intracellular bacteria U R

17 H18

18 Amoeba for pathogen isolation Legionella and Mimivirus Raoult D, et al. The 1.2-megabase genome sequence of Mimivirus. Science. 2004;306: La Scola B, et al. A giant virus in amoebae. Science;299:2033. U R

19 Mimivirus In 1992 Rowbotham working on relationships between amoebae and Legionella isolated a new Gram positive coccus in a cooling tower after a pneumonia outbreak in Bradford, England, using amoebae as a culture system. This was considered a LLAP (Legionella Like Amoebal Pathogen) with others The LLAP collection was bring in Marseilles-France by Richard Birtles an English post-doc. U R

20 A GIANT VIRUS IN AMOEBAE Bernard La Scola, 1 Stephane Audic, 2 Catherine Robert, 1 Liang Jungang, 1 Xavier de Lamballerie, 3 Michel Drancourt, 1 Richard Birtles, 1 Jean-Michel Claverie, 2* Didier Raoult 1* SCIENCE VOL MARCH 2003 U R

21 We show that it have a cycle in amoebae with a typical viral eclipse phase: ingestion disappearance rapid increase in particles Mimivirus DNA + RNA! U R

22 Mimivirus It is as big as several bacteria and visible on gram staining Mimivirus and Mimivirus and Mimivirus and Coxiella burnetii Rickettsia conorii Tropheryma whipplei U R

23 The 1.2-megabase genome sequence of Mimivirus Didier Raoult et al. Science Nov 19;306(5700): Map of the Mimivirus chromosome. The predicted protein coding sequences are shown on both strands and colored according to the function category of their matching COG. Genes with no COG match are shown in gray. Abbreviations for the COG functional categories are as follows: E, amino acid transport and metabolism; F, nucleotide transport and metabolism; J, translation; K, transcription; L, replication, recombination, and repair; M, cell wall/membrane biogenesis; N, cell motility; O, posttranslational modification, protein turnover, and chaperones; Q, secondary metabolites biosynthesis, transport, and catabolism; R, general function prediction only; S, function unknown. Small red arrows indicate the location and orientation of trnas. The A+C excess profile is shown on the innermost circle, exhibiting a peak around position 380,000 (2)(fig.S1). U R

24 The Three Dimensional Structure of Mimivirus Klose T., Kuznetsov Y.G., Xiao C., Sun S., McPherson A., Rossmann M.G. CryoEM reconstruction of Mimivirus applying only fivefold symmetry averaging. (A C) Surfaceshaded rendering of cryoem reconstruction of Mimivirus. (A) View onto the starfish-shaped feature associated vertex, (B) looking from one side, and(c) looking from the opposite side of the starfish - associated vertex. (D) The starfish - associated vertex was removed to show the internal nucleocapsid with its concave surface facing the special vertex. (E) Central slice of the reconstruction viewed from the side of the particle showing the concave face of the nucleocapsid and the low density space beneath the starfish -associated vertex. A perfectly icosahedral particle is outlined in gray to show the extension of the unique vertex. (F) Central slice of the reconstruction viewed along the fivefold axis from the starfish-shaped feature showing the enveloped nucleocapsid surrounded by a lower density space. The coloring is based on radial distance from the center of the virus. Gray is from 0 to 1,800 A, red from 1,800 to 2,100 A, and rainbow coloring from red to blue between 2,100 and 2,500 A. The scale bars in all panels represent 1,000 A. (reproduced from [5])

25 Mimivirus as organism Mimivirus has a : large genome (larger than many sequenced bacteria) is as big as several bacteria has plenty of RNA many of its protein in the virion Raoult D., et al. Science, 2004 Raoult D., et al., Genome Res., 2003 Ogata H., et al., Science, 2001 Virology: Gulliver among the Lilliputians. Koonin EV. Curr Biol 2005; 15(5): R167-R169.

26 Respiratory infection Bronchial aspiration Patients Tested Positive (26) BAL 36 6 Others 50 0 Stools ) M/47 / diabetic, pneumonia (fever, cough, dysprea) sampled on day 6, low WBC count (2.300) 2) F/72 / diabetic, pneumonia (fever, cough, dysprea) sampled day 3, high risk (18.400) Both cured receiving cipofloxacine From B.La Scola

27 Number LBA111 PathoMim Service EXT PN samples LB LB Age Sex M F Habits Tabacco (30PA) Tabacco (15PA) Antecedents Diabetes II without dieting Respiratoiry infections, Diabetes II, HTA, Dyslipidemia, coronary Insufficiency Onset of the disease Functional signs 6j Productive cough Dyspnea Fever physical Signs θ = 38.7 Asculai roaring, crackling right base Rx Treatment Evolution Systematized alveolar opacities of LID Levofloxacine 1fl/j (10j) O 2 therapy Improved clinical, biological and radiological Clinical records 3j Productive cough Dyspnea Fever θ = 39,5 tachypnea(26 cycles/min) Biology GB : 2300 CRP : 330 BK- Asculai roaring, Wheeze GB : CRP : 283 VS : 57 BK- Levofloxine (15j) Improved clinical, biological and radiological

28 (ø = 624 nm) PathoMim : negative staining

29 PathoMim : Ruthunium red staining

30 THE VIROPHAGE AS A UNIQUE PARASITE OF THE GIANT MIMIVIRUS BL Scola et al. Nature 000, 1-5 (2008) doi: /nature07218 Different morphological aspects of mamavirus and Sputnik.

31 CHAPTER 3 Sputnik, a Virophage Infecting the Viral Domain of Life Christelle Desnues,* Mickaël Boyer,* and Didier Raoult Contents I. The Mimiviridae Family and the History of Sputnik 65 A. Mimivirus, Mamavirus, and Sputnik 65 B. Other Mimi like viruses associated with amoebas and a second virophage 67 C. The marine Cafeteria roenbergensis virus and its virophage Mavirus 68 II. Sputnik Structure: Morphology, Chemical Composition, and Protein Components 69 III. Life Cycle: Host Cells, Entry, Uncoating, DNA Replication, Transcription, Translation, Assembly, Maturation, and Release 71 A. Entry in the amoeba 71 B. Virophage hijacking of the viral factory 72 C. Production and release of progeny virions 72 D. Sputnik coinfection with other viruses 73 IV. Genomics: Gene Content, Specific Genes, Laterally Transferred Genes, ORFans, Gene Expression, and Metagenomics 73 A. Genome organization 73 B. Gene content and sources of Sputnik genes 73 C. Gene expression 77 D. Proteomics 78 * These authors contributed equally. The authors declare no conflict of interest. URMITE, Centre National de la Recherche Scientifique UMR IRD 6236, Faculte deme decine, Aix Marseille Universite, Marseille Cedex 5, France Advances in Virus Research, Volume 82 ISSN , DOI: /B Elsevier Inc. All rights reserved 63

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33 FIGURE 4 The four domains of life and their natural viruses.

34 Human seroconversion to Sputnik virophage Laotian woman 29 year old and her husband 36, back from Laos Day 7: fever, fatigue, myalgia, nausea Hypereosinophilia, Transaminasitis => ttt anti parasitaire Woman Man Obstetrical aftercare Back from Laos Hospital Admission Aftercare August /01/ /01/ /03/ /05/2010 IgG NEG IgM NEG IgA NEG /01/ /03/ /05/2010 IgG IgM IgA 0 0 0

35 Two dimensional gel electrophoresis, silver staining and Western blotting Sputnik Mamavirus A. castellani 2-D Western blotting Silver staining woman : positive serum man : positive serum woman : négative serum ORF21 ORF14

36 Giant Marseillevirus highlights the role of amoebae as a melting pot in emergence of chimeric microorganisms. Boyer M, Yutin N, Pagnier I, Barrassi L, Fournous G, Espinosa L, Robert C, Azza S, Sun S, Rossmann MG, Suzan- Monti M, La Scola B, Koonin EV, Raoult D. Proc Natl Acad Sci U S A Dec 22;106(51): Abstract Giant viruses such as Mimivirus isolated from amoeba found in aquatic habitats show biological sophistication comparable to that of simple cellular life forms and seem to evolve by similar mechanisms, including extensive gene duplication and horizontal gene transfer (HGT), possibly in part through a viral parasite, the virophage. We report here the isolation of "Marseille" virus, a previously uncharacterized giant virus of amoeba. The virions of Marseillevirus encompass a 368-kb genome, a minimum of 49 proteins, and some messenger RNAs. Phylogenetic analysis of core genes indicates that Marseillevirus is the prototype of a family of nucleocytoplasmic large DNA viruses (NCLDV) of eukaryotes. The genome repertoire of the virus is composed of typical NCLDV core genes and genes apparently obtained from eukaryotic hosts and their parasites or symbionts, both bacterial and viral. We propose that amoebae are "melting pots" of microbial evolution where diverse forms emerge, including giant viruses with complex gene repertoires of various origins.

37 e CHIMERIC GENOME d Only of viral origin c a b

38 Sympatric virus

39 Lausannevirus, a giant amoebal virus encoding histone doublets. Thomas V, Bertelli C, Collyn F, Casson N, Telenti A, Goesmann A, Croxatto A, Greub G. Environ Microbiol Jun;13(6): Abstract Large viruses infecting algae or amoebae belong to the NucleoCytoplasmic Large DNA Viruses (NCLDV) and present genotypic and phenotypic characteristics that have raised major interest among microbiologists. Here, we describe a new large virus discovered in Acanthamoeba castellanii co culture of an environmental sample. The virus, referred to as Lausannevirus, has a very limited host range, infecting Acanthamoeba spp. but being unable to infect other amoebae and mammalian cell lines tested. Within A. castellanii, this icosahedral virus of about 200 nm exhibits a development cycle similar to Mimivirus, with an eclipse phase 2 h post infection and a logarithmic growth leading to amoebal lysis in less than 24 h. The 346 kb Lausannevirus genome presents similarities with the recently described Marseillevirus, sharing 89% of genes, and thus belongs to the same family as confirmed by core gene phylogeny. Interestingly, Lausannevirus and Marseillevirus genomes both encode three proteins with predicted histone folds, including two histone doublets, that present similarities to eukaryotic and archaeal histones. The discovery of Lausannevirus and the analysis of its genome provide some insight in the evolution of these large amoebae infecting viruses

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41 Library 1 : 20 donors Library 2 : 15 donors (from C.Desnues)

42 Human blood virome: samples from 10 healthy donors Viral particles are separated from cells by centrifugation (40 ml/patient blood samples) 10 ml supernatants of each samples were pooled (total volume : 100 ml) Filtration through 0.45 µm membrane filter Concentration and purification of virus-like particles (CsCl discontinuous gradient) Epifluorescence microscopy and electron microscopy (check for human contamination) Extraction of nucleic acids Sequencing (454 pyrosequencing) Bio-informatic analyses

43 Purified virus-like particles observed under epifluorescent microscopy A Pooled serum of 10 donors (100 ml) B Concentration & purification SYBR gold stained human serum filtered through 0.45 µm membrane (A) and concentrated (B) under epifluorescent microscopy (100X magnification)

44 Repartition of the sequences after BLAST against NR Taxonomic distribution of the 20,238 reads based on GenBank taxonomic classification of the Best Blast Hit Blastn / nr (e value<1 e 05) Blastx / nr (e value<1 e 05) Viruses 13,708 (67.7%) 12,230 (60.4%) Bacteria 1,932 (9.5%) 4,421 (21.8%) Eukaryota 728 (3.6%) 254 (1.3%) Archaea 0 (0.0%) 4 (0.02%) other sequences 8,89 (4.4%) 197 (1.0%) TOTAL 17,257 (85.3%) 17,106 (84.5%) Classification of viral reads based on significant hits (E value < E 05) from blastn searches against nr Virus No. (%) reads TTV* (54.66%) TTV like 1566 (7.73%) SENV** 578 (2.86%) Marseillevirus (MarV) 333 (1.64%) TTV midi 12 (0.06%) TTV like mini 9 (0.04%) Anellovirus %) * Torque teno virus, ** SEN virus

45 Two MarV-like large regions (13,649 bp and 10,173 bp) are recovered after assembly A Marseillevirus Blood virus Marseillevirus regions covered by metagenomic reads (A) and genetic map of the two Marseillevirus homologous regions resulting from metagenomic reads assembly (B).

46 Immunofluorescence using Marseillevirus antibodies on viral particles purified from serum Cytoblock Gelose inclusion of purified viral particles Paraffin inclusion Thin slice section Green : MarV polyclonal Ab Red : DAPI 100X

47 Tentative characterization of new environmental giant viruses by MALDI-TOF mass spectrometry La Scola B, Campocasso A, N'Dong R, Fournous G, Barrassi L, Flaudrops C, Raoult D. Intervirology. 2010;53(5): Pôle des Maladies Infectieuses, Assistance Publique-Hôpitaux de Marseille and URMITE UMR CNRS-IRD 6236, IFR48, Faculté de Médecine, Université de la Méditerranée, Marseille, France Corresponding authors: Prof.Bernard La Scola, URMITE, Faculté de Médecine, 27 Boulevard Jean Moulin, 13385Marseille cedex 5, France. Tel Fax Bernard.lascola@univmed.fr Abstract OBJECTIVE: Metagenomic studies have revealed that Acanthamoeba polyphaga Mimivirus relatives are common in the environment; however, only three Acanthamoeba-growing giant viruses have been isolated from hundreds of environmental samples. We attempted herein to isolate new Acanthamoeba-growing giant viruses from environmental samples. METHODS: We inoculated 105 environmental samples by our usual procedure but with the addition of selected antibiotics to inhibit bacterial overgrowth. RESULTS: We isolated 19 giant viruses with capsid sizes of 150 to 600 nm, including one associated with a virophage. For the first time some were isolated from saltwater and soil samples. Tentative characterization using the PolB gene sequence was possible for some of these viruses. They were closely related to each other but different from the two previous isolates of Acanthamoeba polyphaga Mimivirus. Results obtained by MALDI-TOF MS analysis of viral particles were congruent with that of PolB sequencing. CONCLUSION: Our data confirm that Acanthamoeba-growing giant viruses are common in the environment. Additionally, MALDI- TOF MS analysis can be used for the initial screening of new viruses to avoid redundant analysis. However, due to their genetic variability, it is likely that the genome sequences of most of these viruses will have to be determined for accurate classification.

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49 Fig. 5 Megavirales (other viruses have a size lower than that of the filter s pores) Filter Direction of filtration Because of the virus definition we miss viruses!

50 Exceptions RNA and DNA Intracellular bacteria Binary fission Giant viruses Virophages Organelles nanobacteria and prion Selfish DNA U R

51 Virus definition An organism OR a biomolecule? Lwoff 1957 (Stanley vs Burnet) One dimension < 200 nm One type of nucleic acid Lack of enzymes generating energy Strict intracellular character Inability to undergo binary fission Lwoff A. The concept of virus. J Gen Microbiol. 1957;17: U R

52 Redefining viruses: lessons from Mimivirus. Didier Raoult & Patrick Forterre Nature Reviews Microbiology 6, (April 2008) Defining living entities The general consensus of what constitutes life can be sampled by consulting a global resource such as Wikipedia (the largest free online encyclopaedia; see Further information), which defines life as a condition that distinguishes organisms from inorganic objects. life is a characteristic of self organizing, self recycling systems consisting of populations of replicators that are capable of mutation, around most of which homeostatic, metabolizing organisms evolve clearly include viruses. We can also paraphrase Engels32 and define life as the mode of existence of living organisms,

53 DEFINING ORGANISMS The definition of an organism is a difficult problem in itself and is subject to controversy. An organism has been defined as An individual living system such as animal, plant, fungus or microorganism by Wikipedia, An individual animal, plant or single-celled life form by the Oxford English Dictionary Online and Any living structure capable of growth and reproduction by Chambers Reference Online (see Further information). The definitions from Wikipedia and the Oxford English Dictionary Online exclude intracellular parasites, symbionts, organelles and viruses. The definition from Chambers Reference Online, however, includes viruses and nucleic acids, as it does not retain the word cell.

54 PERSPECTIVES TREE OF LIFE Nature Reviews Microbiology 7, (April 2009) OPINION Ten reasons to exclude viruses from the tree of life David Moreira and Purificación López-García Abstract When viruses were discovered, they were accepted as missing links between the inert world and living organisms. However, this idea was soon abandoned as information about their molecular parasitic nature accumulated. Recently, the notion that viruses are living organisms that have had a role in the evolution of some essential features of cells has experienced a renaissance owing to the discovery of unusually large and complex viruses that possess typical cellular genes. Here, we contend that there is strong evidence against the notion that viruses are alive and represent ancient lineages of the tree of life.

55 10 15% organisms have ribosomes 15 30% viruses are known 55 70%??? METAGENOMIC Sequences DNA of living at the 21st century

56 Science Mar 25;331(6024):1513 Expanding universe. The tally of known viruses is exploding, and this graph doesn t even include the incredible number that prey on bacteria.

57 CORRESPONDENCE There is no such thing as a tree of life (and of course viruses are out!) Didier Raoult Nature Reviews Microbiology 7, 615 (August 2009) Indeed, viruses are a part of biology (a word that is derived from the Greek biola, meaning the study of life). The controversy about the word life (also quoted as unscientific by K. Popper3) is not my cup of tea. If I had to choose a definition of life, I would prefer that of Shakespeare in Macbeth: it is A tale, told by an idiot, full of sound and fury, signifying nothing. Finally, I am primarily a biologist (a man studying life, etymologically), and I am therefore happy to provide data challenging old concepts and definitions. If this is hurting dogmas I do not really care, and I repeat, as quoted by Galileo in response to church sanctions, eppur si muove, meaning that facts will resist theories.

58 TRYING TO RECLASSIFY Eukarya Bacteria Archae Virus Retrovirus Satellite virus Viroids Plasmids Transposon Retroposons Raoult D, Forterre P. Redefining viruses: lessons from Mimivirus. Nat Rev Microbiol. 2008; 6(4): U U R R

59 RIBOSOME ENCODING Archae, Bacteria, Eukarya U R

60 COMPARING MIMIVIRUS AND INTRACELLULAR BACTERIA, EUKARYA, ARCHAE If you classify domains on ribosome => no virus No ribosome little reduction in viruses U R

61 WHAT IS SPECIFIC TO SOME VIRUSES? Capsids Same structure : same origin? Raoult D, Forterre P. Redefining viruses: lessons from Mimivirus. Nat Rev Microbiol. 2008; 6(4):315-9

62 Raoult D, Forterre P. Redefining viruses: lessons from Mimivirus. Nat Rev Microbiol. 2008; 6(4): U R

63 Other living things Nucleic acid Plasmid Transposons Retroposons Viroids Satellite DNA Proteins? Prions, Nanons U R

64 Amoebae as genitors and reservoirs of giant viruses. Raoult D, Boyer M Intervirology. 2010;53(5):321-9 Information (DNA/RNA) Sterage (cell, virion) Genetic exchanges between ribosome encoding organisms (REO) and capsid encoding organisms (CEO) could be comparable to information transmission in computer network. CEO could exchange genetic information with REO and could be regarded as USB flash drive (or other external computer devices) which stores information and transfers it between computers. Selfish genetic elements could be similar to computer virus able to parasitize both computers and USB flash drive (or other external computer devices).

65 Phylogenetic and Phyletic Studies of Informational Genes in Genomes Highlight Existence of a 4th Domain of Life Including Giant Viruses Mickaël Boyer., Mohammed-Amine Madoui., Gregory Gimenez, Bernard La Scola, Didier Raoult PLoS One Dec 2;5(12):e ? Abstract: The discovery of Mimivirus, with its very large genome content, made it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation, we found in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supports existence of a clade containing NCLDVs clearly distinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life. Looking for other genes then ribosome

66 The first frame from the top right corresponds to the dntp biosynthesis catalyzed by RNR and ThyA from RNA => DNA

67 Four enzymes involved in DNA replication: DNAP B, TopoIIA, PCNA and FEN

68 Phylogenetic tree of the RNA polymerase II beta subunit

69 Phylogenetic tree of the Transcription factor II B (TFIIB).

70 Gene content analysis of informational genes Hierarchical clustering of Eukarya (blue), Bacteria (purple), Archaea (green) and NCLDVs (red) by phyletic pattern

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72 The challenge of microbial diversity: Out on a limb Gwyneth Dickey Zakaib.Nature 476, So Raoult is currently standing alone. GIANT HUNTING Raoult is still trying to work out how his huge viruses evolved. One idea is that they could have come from a more complex ancestor that was later reduced to a parasitic shell. Raoult continues to search for new giant viruses and wants to use their genome sequences to work out when this group of viruses emerged, based on a molecular clock deduced from the viruses mutation rates. And he avoids conversing with members of the virology community who are unwilling to challenge the dogma of their field. I m more excited by discussions with the people trying to put a little bit of disorder in the way we think about biology, he says. Gwyneth Dickey Zakaib

73 Family trees The challenge of microbial diversity: Out on a limb. Gwyneth Dickey Zakaib. Nature 476, Phylogenetic trees try to show how species are related based on similarities in a common gene or protein sequence. In theory, the more similar the sequences, the more closely the organisms are related. But dierent models of the same sequence, such as the protein RNA polymerase II mapped here, can turn up dierent results.

74 The challenge of microbial diversity: Out on a limb. Gwyneth Dickey Zakaib. Nature 476, 20-21

75 ALL FOLLOWING SINGLE CHARACTERISTICS ARE REQUIRED FOR MEMBERSHIP IN THE ORDER Large size for the viral particle and the genome: capsid diameter is >150 nm, and genome size >130 kb Jelly-roll capsid Presence within the gene repertoire of core genes, including all nine class I core genes *, found in all NCLDV, and all five NCVOG found in all NCLDV Common ancestral origin and membership in the fourth domain of Life DEFINING MEGAVIRALES Reclassification of giant viruses composing a fourth domain of life in the new order Megavirales. Colson P, de Lamballerie XN, Fournous G, Raoult D. Intervirology. The reconstructed core gene set of the common ancestor of the NCLDVs (47 NCVOGs) Adapted from [13,14] MEGAVIRALES Core genes DIFFERENT COLLECTIONS OF PROPERTIES AMONG THOSE LISTED ABOVE ARE REQUIRED FOR MEMBERSHIP IN THE ORDER Presence of both DNA and RNA Presence of proteins involved in the translation apparatus Substantial proportions of duplicated genes and of genes involved in HGT within the genome Substantial proportions of ORFans and metaorfans among the repertoire of genes Presence of a viral factory Possible infection by a virophage

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77 Fig. 2 Infects Ribosome Encoding Organisms Infects or is grazed R.E.O.C.E.O. EUKARYOTA Animalia Plantae Chromista Protozoa BACTERIA ARCHAEA Domains of Life Capsid Encoding Organisms MEGAVIRALES Asfarviridae Iridoviridae Ascoviridae Poxviridae Phycodnaviridae Mimiviridae Marseilleviridae Other C.E.O Virophages

78 VIROPHAGES QUESTION THE EXISTENCE OF SATELLITES Desnues C, Raoult D. Nat Rev Microbiol Feb 16;10(3):234

79 PERSPECTIVES TREE OF LIFE Nature Reviews Microbiology 7, (April 2009) OPINION Ten reasons to exclude viruses from the tree of life David Moreira and Purificación López-García Abstract When viruses were discovered, they were accepted as missing links between the inert world and living organisms. However, this idea was soon abandoned as information about their molecular parasitic nature accumulated. Recently, the notion that viruses are living organisms that have had a role in the evolution of some essential features of cells has experienced a renaissance owing to the discovery of unusually large and complex viruses that possess typical cellular genes. Here, we contend that there is strong evidence against the notion that viruses are alive and represent ancient lineages of the tree of life.

80 CORRESPONDENCE There is no such thing as a tree of life (and of course viruses are out!) Didier Raoult Nature Reviews Microbiology 7, 615 (August 2009) Indeed, viruses are a part of biology (a word that is derived from the Greek biola, meaning the study of life). The controversy about the word life (also quoted as unscientific by K. Popper3) is not my cup of tea. If I had to choose a definition of life, I would prefer that of Shakespeare in Macbeth: it is A tale, told by an idiot, full of sound and fury, signifying nothing. Finally, I am primarily a biologist (a man studying life, etymologically), and I am therefore happy to provide data challenging old concepts and definitions. If this is hurting dogmas I do not really care, and I repeat, as quoted by Galileo in response to church sanctions, eppur si muove, meaning that facts will resist theories.

81 WHAT ABOUT THE TREE OF LIFE OF Arbre Darwin DARWIN It is a biblical concept

82 The tree does no reprent life! No even plant life Algae, biofilm, rhizome, mycelium (9.7 km long, 2,200 years old in Oregon) are as representative of life!

83 THE TREE OF LIFE IN FACT IS OPPOSITE OF WHAT WE KNOW THE GENEALOGIC TREE

84 Genealogy of R. felis genes The rhizome of life: the sympatric Rickettsia felis paradigm demonstrates the random transfer of DNA sequences. Merhej V, Notredame C, Royer- Carenzi M, Pontarotti P, Raoult D. Mol Biol Evol Nov;28(11):

85 RHIZOME (OR MYCELIUM) OF LIFE D.Raoult.The post-darwinist rhizome of life Lancet Jan 9;375(9709): U R

86 The logic of scientific discovery The STRUCTURE Of SCIENTIFIC REVOLUTIONS Thomas S. KUHN According to K.Popper «Never fight for a world» and «new tools create new theories». According to T.Kuhn «when theories are unstable we need a change in paradigm». After - Microbes (1) - Eukaryotes (2) - Domains (3)

87 «Postmodern science makes the theory of its own evolution as discontinuous, catastrophic, nonrectifiable, paradoxical.» P.97

88 4 CONCLUSION Finally defining TRUC («thing» in French) with 4 parts, bacteria, eukarya, archae and megavirales. As an acronym of Things Resisting Uncomplete Classification (including Megavirales).

89