ESCMID Online Lecture Library © by Author

LONDON April, 2012

Are viruses the fourth domain of life or “Trucs?” or 1-2-3-4 © by author Didier Raoult Marseille - France ESCMID Online Lecture Library U [email protected] R 1 ‐Microbes (Sedillot,1878) Pasteur! ‐ Pasteurella multocida visible at the microscope

The agent of cholera fowlESCMID Online Lecture Library 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.

ESCMIDJA. Fuerst. Intracellular Online LectureCompartmentation Library in Planctomycetes. Ann. Rev Microbiol. 2005;59:299-328.

JA. Fuerst. Intracellular Compartmentation in Planctomycetes.ESCMID Ann. Online Rev Microbiol. Lecture 2005;59:299-328. Library Photographic credit: Gilbert Greub

Ultrastructure of Criblamydia sequanensis (A), Parachlamydia acanthamoebae (B-C) and Waddlia chondrophila (D-F). We miss © by author nuclei on our plates!

ESCMID Online Lecture Library Greub G, Bertelli C, Croxatto A, Raoult D. New Chlamydiales species question the origin of the nucleus. Confocal microscopy; 600x magnification. Elementary bodies of Parachlamydia acanthamoebae

ESCMID Online Lecture Library U Photographic credit: Gilbert Greub R 3 Archae C.Woese 1975 creates the ribosome based 3 domains

NATURE|Vol 441|18 May 2006 ESSAY Time for a change © by author 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.ESCMID Online Lecture Library Norman R. Pace 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 © by author

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 ESCMID Onlinepartir d’uneLecture selle). Library M.Drancourt There are new human species not found by 16S rDNA

ESCMID Online Lecture Library ? Virus definition

An organism OR a biomolecule ?

Lwoff – 1957 (Stanley vs Burnet) • One dimension < 200 nm Small specialized and • One type of nucleic acid mainly allotropic • Lack of enzymes generating energy • Strict intracellular character • Inability to undergo binary© fissionby author

Lwoff A. The concept of virus. J Gen Microbiol. 1957;17:239-253.

U ESCMID Online Lecture Library R Exceptions

 RNA or DNA Intracellular bacteria

 Binary fission  Giant viruses  Virophages  Organelles  “nanobacteria”and© byprion author  Selfish DNA

U ESCMID Online Lecture Library R What is a Rickettsia ?

 Gram negative bacterium  Strictly intracellular (?)  a1 subgroup of Proteobacteria by 16S rRNA sequence analysis  Transmitted by arthropods:© by author ticks, fleas, lice, mites

U ESCMID Online Lecture Library R 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 dense- cored 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.

Jere W. McBride. Identification of a Glycosylated Ehrlichia canis 19-Kilodalton Major Immunoreactive Protein with a ESCMIDSpecies-Specific Serine-Rich Online Glycopeptide Lecture Epitope. Infect Immun. Library 2007; 75(1): 74–82 Viral factory looking like Morula

U ESCMID Online Lecture Library R

U ESCMIDBABL: intracellularOnline Lecture bacteria Library R

H18 ESCMID Online Lecture Library

Amoeba for pathogen isolation © by author Legionella and Mimivirus

Raoult D, et al. The 1.2-megabase genome sequence of Mimivirus. Science. 2004;306:1344-50. U La ScolaESCMID B, et al. A giant virusOnline in amoebae. Lecture Science;299:2033. Library R 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 © by author  The LLAP collection was bring in Marseilles-France by Richard Birtles an English post-doc. U ESCMID Online Lecture Library R A GIANT VIRUS IN AMOEBAE Bernard La Scola,1 Stephane Audic,2 Catherine Robert,1Liang Jungang,1 Xavier de Lamballerie,3 Michel Drancourt, 1Richard Birtles,1 Jean-Michel Claverie,2* Didier Raoult1*SCIENCE VOL 299 28 MARCH 2003

ESCMID Online Lecture Library U R Mimivirus

– We show that it have a cycle in amoebae with a typical viral eclipse phase:

 ingestion  disappearance © by author  rapid increase in particles DNA + RNA! ESCMID Online Lecture Library U R Mimivirus

It is as big as several bacteria and visible on gram staining

Mimivirus and Mimivirus and Mimivirus and Coxiella burnetiiESCMIDRickettsia Online conorii LectureTropheryma Library whipplei U R The 1.2-megabase genome sequence of Mimivirus Didier Raoult et al. Science. 2004 Nov 19;306(5700):1344-50.

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; © by author 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). ESCMID Online Lecture Library U R 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) Surface- shaded 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 ofby the nucleocapsidauthor 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 inESCMID all panels Online represent 1,000Lecture A. Library (reproduced from [5]) 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

Virology: Gulliver among the Lilliputians. Koonin EV. Raoult D., et al. Science, 2004 Curr Biol 2005; 15(5): R167-R169. Raoult D., et al., Genome Res., 2003 Ogata H., et al., Science, 2001 © by author

ESCMID Online Lecture Library Patients

Respiratory Tested Positive infection Bronchial 110 2 (26) aspiration

BAL 36 6 Others 50 0 Stools 846 0

1) M/47 / diabetic, pneumonia (fever, cough, dysprea) sampled on day 6, low WBC count© (2.300) by author 2) F/72 / diabetic, pneumonia (fever, cough, dysprea) sampled day 3, high risk (18.400) Both cured receiving cipofloxacine

ESCMID OnlineFrom B.La Lecture Scola Library Clinical records Number LBA111 PathoMim Service EXT PN samples LB LB Age 47 72 Sex M F Habits Tabacco (30PA) Tabacco (15PA) Antecedents Diabetes II without dieting Respiratoiry infections, Diabetes II, HTA, Dyslipidemia, coronary Insufficiency

Onset of the 6j 3j disease Functional signs Productive cough Productive cough Dyspnea Dyspnea Fever Fever physical Signs θ°= 38.7 θ° = 39,5 Asculai roaring, crackling right base tachypnea(26 cycles/min) Rx Systematized alveolar opacities of LID Biology GB : 2300 Asculai roaring, CRP : 330 Wheeze BK- © byGB : 18400author CRP : 283 VS : 57 BK-

Treatment Levofloxacine 1fl/j (10j) Levofloxine (15j)

O2 therapy EvolutionESCMID Improved clinical, biological Online and ImprovedLecture clinical, biological Library and radiological radiological PathoMim : negative staining

(ø = 624 nm)

ESCMID Online Lecture Library PathoMim : Ruthunium red staining

ESCMID Online Lecture Library THE VIROPHAGE AS A UNIQUE PARASITE OF THE GIANT MIMIVIRUS

ESCMIDBL Scola et Online al. Nature 000, 1-5 (2008)Lecture doi:10.1038/nature07218 Library Different morphological aspects of mamavirus and Sputnik. 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 © by authorB. 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

ESCMID Online Lecture Library

ESCMID Online Lecture Library FIGURE 4 The four domains of life and their natural viruses. 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

Aftercare Obstetrical Back from Hospital Laos aftercare Admission

August 2009 22/01/2010 29/01/2010 31/03/2010 03/05/2010 Woman IgG NEG 100 200 100 50 IgM NEG 100© by 100author 0 0 IgANEG02500

Man 29/01/2010 31/03/2010 03/05/2010 IgG 400 100 50 ESCMIDIgM Online Lecture 100 0 Library 0 IgA 0 0 0 Two dimensional gel electrophoresis, silver staining and Western blotting

2-D Western blotting Silver staining

woman : positive serum man : positive serum woman : négative serum

Sputnik ORF21

ORF14

Mamavirus

ESCMID Online Lecture Library 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. 2009 Dec 22;106(51):21848-53.

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, by author 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.

ESCMID Online Lecture Library CHIMERIC GENOME

b e

d ESCMID Online Lecture Libraryc Only of viral origin Sympatric virus

ESCMID Online Lecture Library 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. 2011 Jun;13(6):1454‐66.

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.by author 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 theESCMID evolution of these Online large amoebae Lecture‐infecting viruses Library

ESCMID Online Lecture Library

Library 1 : 20 donors Library 2 : 15 donors ESCMID Online Lecture(from C.Desnues) Library 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© ofby nucleic author acids Sequencing (454 pyrosequencing)

Bio-informatic analyses ESCMID Online Lecture Library Purified virus-like particles observed under epifluorescent microscopy

Pooled serum of 10 donors (100 ml)

A B

Concentration & purification

ESCMID Online Lecture Library 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<1e‐05) Blastx / nr (e‐value<1e‐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* 11062 (54.66%) TTV‐like 1566 (7.73%) © by authorSENV** 578 (2.86%) Marseillevirus (MarV) 333 (1.64%) TTV midi 12 (0.06%) TTV‐like mini 9 (0.04%) Anellovirus 2 0.01%) ESCMID Online Lecture* Torque teno virus,Library ** SEN virus Two MarV-like large regions (13,649 bp and 10,173 bp) are recovered after assembly

Marseillevirus

Blood virus © by author

Marseillevirus regions covered by metagenomic reads (A) and genetic map of the two Marseillevirus homologous regions resultingESCMID from metagenomic reads Online assembly (B). Lecture Library Immunofluorescence using Marseillevirus antibodies on viral particles purified from serum 27725

Cytoblock Gelose inclusion of purified viral particles

Paraffin inclusion Thin slice section

© by author Green : MarV polyclonal Ab Red : DAPI 100X ESCMID Online Lecture Library 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):344-53.

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. 33 4 91 32 43 75 Fax. 33 4 91 38 77 72 E-mail. [email protected]

Abstract OBJECTIVE: Metagenomic studies have revealed that Acanthamoeba polyphaga Mimivirus relatives are common in the environment; however, only three Acanthamoeba-gro wing 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 Mimiviru© bys. Results author 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.ESCMID Online Lecture Library

ESCMID Online Lecture Library Fig. 5

Megavirales (other viruses have a size lower than that of the filter’s pores)

Filter Direction of filtration © by author Because of the virus definition we miss viruses!

ESCMID Online Lecture Library Exceptions

 RNA and DNA Intracellular bacteria

 Binary fission  Giant viruses  Virophages  Organelles  “nanobacteria”and© byprion author  Selfish DNA

U ESCMID Online Lecture Library R 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© fissionby author

Lwoff A. The concept of virus. J Gen Microbiol. 1957;17:239-253.

U ESCMID Online Lecture Library R Redefining viruses: lessons from Mimivirus. Didier Raoult & Patrick Forterre Nature Reviews Microbiology 6, 315-319 (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© whichby author homeostatic, metabolizing organisms evolve” — clearly include viruses. We can also paraphrase Engels32 and define life as “the mode of existence of livingESCMID organisms”, Online Lecture Library 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.© by The author definition from Chambers Reference Online, however, includes viruses and nucleic acids, as it does not retain the word cell. ESCMID Online Lecture Library PERSPECTIVES TREE OF LIFE Nature Reviews Microbiology 7, 306-311 (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 by toauthor 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. ESCMID Online Lecture Library METAGENOMIC

 10‐15% organisms have ribosomes  15‐30% viruses are known  55‐70% ??? © by author Sequences DNA of living at the 21st century ESCMID Online Lecture Library Science. 2011 Mar 25;331(6024):1513

Expanding universe. The tally of known viruses is exploding, and this graph doesn’t ESCMIDeven include the Onlineincredible number Lecture that prey Library on bacteria. CORRESPONDENCE

Nature Reviews Microbiology 7, 615 (August 2009) There is no such thing as a tree of life (and of course viruses are out!) Didier Raoult 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© databy challengingauthor 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 siESCMID muove”, meaning Onlinethat facts Lecture will resist Library theories. TRYING TO RECLASSIFY

.Eukarya Raoult D, Forterre P. .Bacteria Redefining viruses: lessons .Archae from Mimivirus. Nat Rev Microbiol. 2008; 6(4):315-9.

.Plasmids .Transposon U U R .RetroposonsESCMID Online Lecture Library R RIBOSOME ENCODING Archae, Bacteria, Eukarya

U ESCMID Online Lecture Library R COMPARING MIMIVIRUS AND INTRACELLULAR BACTERIA, EUKARYA, ARCHAE

If you classify domains on ribosome => no virus ESCMID Online Lecture Library U No ribosome little reduction in viruses R WHAT IS SPECIFIC TO SOME VIRUSES? Capsids

Raoult D, Forterre P. Redefining viruses: lessons from Mimivirus.ESCMID Nat RevOnline Microbiol. Lecture 2008; Library 6(4):315-9

Raoult D, Forterre P. Redefining viruses: lessons from

U Mimivirus.ESCMID Nat OnlineRev Microbiol. Lecture 2008; 6(4):315-9.Library R Other “living things”

Nucleic acid

 Plasmid  Transposons

 Retroposons  Viroids

Prions, Nanons U ESCMID Online Lecture Library R 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 by could author 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 bothESCMID computers and USB Online flash drive Lecture (or Library other external computer devices). 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. 2010 Dec 2;5(12):e15530 Abstract: The discovery of Mimivirus, with its very large genome content, made4? 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© byin the author 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. ESCMIDLooking Onlinefor other genes Lecture then ribosome Library The first frame from the top right corresponds to the dNTP biosynthesis catalyzed by RNR and ThyA from RNA => DNA

ESCMID Online Lecture Library Four enzymes involved in DNA replication: DNAP B, TopoIIA, PCNA and FEN

ESCMID Online Lecture Library

PhylogeneticESCMID tree of the RNA Online polymerase Lecture II beta subunit Library

ESCMID Online Lecture Library Phylogenetic tree of the Transcription factor II B (TFIIB). Gene content analysis of informational genes

HierarchicalESCMID clustering of EukaryaOnline(blue), Lecture Bacteria Library(purple), Archaea (green) and NCLDVs (red) by phyletic pattern

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

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 © by author 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.ESCMID Online Lecture Library Gwyneth Dickey Zakaib Phylogenetic trees try to show how species are related based on similarities in a Family trees 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.

The challenge of microbial diversity: Out on a limb. GwynethESCMID Dickey Zakaib. Online Lecture Library Nature 476, 20-21

The challenge of microbial diversity: Out on a limb. GwynethESCMID Dickey Zakaib. Online Lecture Library Nature 476, 20-21 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.

ALL FOLLOWING SINGLE DIFFERENT COLLECTIONS OF PROPERTIES AMONG CHARACTERISTICS ARE REQUIRED FOR THOSE LISTED ABOVE ARE REQUIRED FOR MEMBERSHIP IN THE ORDER MEGAVIRALES MEMBERSHIP IN THE ORDER

Large size for the viral particle and the • Presence of both DNA and RNA genome: capsid diameter is >150 nm, and genome size >130 kB • Presence of proteins involved in the translation apparatus Jelly-roll capsid • Substantial proportions of duplicated genes and of Presence within the gene repertoire of core genes involved in HGT within the genome genes, including all nine class I core genes *, found in all NCLDV, and all five NCVOG • Substantial proportions of ORFans and metaORFans found in all NCLDV among the repertoire of genes

Common ancestral origin and membership • Presence of a viral factory in the fourth domain of Life • Possible infection by a virophage © by author The reconstructed core gene set of the common ancestor of the NCLDVs (47 NCVOGs) Adapted from Core genes [13,14] ESCMID Online Lecture Library

ESCMID Online Lecture Library Fig. 2 R.E.O.C.E.O. Ribosome Encoding Capsid Encoding Organisms Organisms EUKARYOTA MEGAVIRALES Animalia Asfarviridae

Plantae Iridoviridae

Chromista Ascoviridae Poxviridae Protozoa Phycodnaviridae

Infects ESCMID OnlineDomains of Life Lecture Library Infects or is grazed VIROPHAGES QUESTION THE EXISTENCE OF SATELLITES Desnues C, Raoult D. Nat Rev Microbiol. 2012 Feb 16;10(3):234

ESCMID Online Lecture Library PERSPECTIVES TREE OF LIFE Nature Reviews Microbiology 7, 306-311 (April 2009) OPINION Ten reasons to exclude viruses from the tree of life

David Moreira and Purificación López-García

ESCMID Online Lecture Library It is a biblical concept 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!

ESCMID Online Lecture Library THE TREE OF LIFE IN FACT IS OPPOSITE OF WHAT WE KNOW THE GENEALOGIC TREE

ESCMID Online Lecture Library The rhizome of life: the sympatric Rickettsia felis paradigm demonstrates the random transfer of Genealogy of R. felis genes DNA sequences. Merhej V, Notredame C, Royer- Carenzi M, Pontarotti P, Raoult D. Mol Biol Evol. 2011 Nov;28(11):3213-23

ESCMID Online Lecture Library RHIZOME (OR MYCELIUM) OF LIFE

D.Raoult.The post-Darwinist rhizome of life Lancet. 2010 Jan 9;375(9709):104-5.

U ESCMID Online Lecture Library R According to K.Popper « Never fight for a world » and « new tools create new theories ». The logic of scientific discovery

The STRUCTURE Of SCIENTIFIC According to T.Kuhn « when theories REVOLUTIONS are unstable we need a change in paradigm ».

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

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