Philippe Colson

Sarah Aherfi

La Bernard

Didier Scola

Raoult

Bernard La Scola

Didier Raoult
email: didier.raoult@gmail.com

Full-length title: The role of giant viruses of amoebas in humans

Keywords: Giant virus, human, Megavirales, pathogenicity, amoeba, nucleocytoplasmic large DNA virus, TRUC, pneumonia

The role of giant viruses of amoebas in humans.

TEXT (2,625 words) The emergence of giant viruses of amoebas

The story of giant viruses that infect amoebas (GVA) began with the isolation of the Mimivirus in 1992 [1;2]. This was made possible by using a strategy that consisted of inoculating samples on an axenic culture of Acanthamoeba spp. and was implemented to isolate amoeba-resisting microorganisms such as Legionella spp. [START_REF] Raoult | The discovery and characterization of Mimivirus, the largest known virus and putative pneumonia agent[END_REF]. The first mimivirus isolate was obtained from cooling tower water while investigating a pneumonia outbreak in England. It took a decade to identify that one of the amoeba-resistant microbes was a giant virus, which was visible on light microscopy and looked like a Gram-positive coccus. This was eventually revealed in 2003 in Marseille by using electron microscopy [1;2]. Thus, the investigation triggered in 1992 by pneumonia cases serendipitously led to discovery of the largest viruses known so far, which strongly challenge the concept and definition of viruses [1;3;4]. Moreover, it suggested the link between these GVA and humans and their possible pathogenicity. Dozens of additional mimiviruses, which were classified in the family Mimiviridae, were isolated in amoebas from environmental water samples collected in various geographical areas worldwide [5;6]. In addition, these studies led to the discovery of the first viruses of viruses, named 'virophages', which replicate in the viral factories of mimiviral hosts and can impair their replicative cycle and morphogenesis [7;8]. Moreover, other GVA have been discovered since 2008 [4;9]. Some were classified in the family Marseilleviridae and others include pandoraviruses [10;11], Pithovirus sibericum [START_REF] Legendre | Thirtythousand-year-old distant relative of giant icosahedral DNA viruses with a pandoravirus morphology[END_REF], faustoviruses [START_REF] Reteno | Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae[END_REF] and Mollivirus sibericum [START_REF] Legendre | In-depth study of Mollivirus sibericum, a new 30,000-y-old giant virus infecting Acanthamoeba[END_REF], which represent new putative virus families [START_REF] Aherfi | Giant viruses of amoebas: an update[END_REF]. All these GVA cultured in amoebas display many unique characteristics that put them on the edge of the virus definition, and warrant proposing their reclassification as representatives of a fourth 'TRUC' (an acronym for Things Resisting Uncompleted Classifications) of microbes [START_REF] Raoult | TRUC or the Need for a New Microbial Classification[END_REF] (reviewed by V.

Sharma et al. [4]). They have been proposed for classification in a new viral order,

Megavirales, alongside other double-stranded DNA viruses [START_REF] Iyer | Common origin of four diverse families of large eukaryotic DNA viruses[END_REF]. GVA appear to be common in our biosphere; they have been isolated from marine water, freshwater and soil samples collected in several countries worldwide (https://www.google.com/maps/d/edit?mid=zA3X4ljlz-uM.kFSrbnCtoBLc) [5;17;18]. This has been corroborated by metagenomic studies that detected sequences matching these viruses in similar environmental samples collected in highly diverse geographical areas [19;20] (reviewed by S. Halary et al. [START_REF] Halary | Viral metagenomics: are we missing the giants?[END_REF]). In addition, their hosts, Acanthamoeba spp. (for most of these viruses) or Vermamoeba vermiformis (for faustoviruses) are ubiquitous organisms that are common in human environments, very resistant and described as 'Trojan horses' for their parasitic pathogens [22;23]. Moreover, GVA prevalence was probably underestimated because 'viral' fractions analyzed were most often obtained by filtration through a 0.2 µmlarge pore size, which neglects gigantic virions [START_REF] Colson | Evidence of the megavirome in humans[END_REF]. Taken together, these findings strongly suggest that humans are exposed to GVA. Noteworthy, 12% of 242 samples collected from inanimate surfaces in a Brazilian hospital were positive for Mimivirus DNA by PCR, the incidence being significantly greater in respiratory isolation facilities, and amoebal lysis was obtained from 83% of these samples [START_REF] Dos | High positivity of mimivirus in inanimate surfaces of a hospital respiratory-isolation facility, Brazil[END_REF].

Other studies have reported the isolation of mimiviruses from oysters [START_REF] Andrade | Oysters as hot spots for mimivirus isolation[END_REF] and a leech [START_REF] Boughalmi | First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis[END_REF], and their detection by PCR in monkeys and cattle [START_REF] Dornas | Mimivirus circulation among wild and domestic mammals, Amazon Region, Brazil[END_REF]. In addition, a Marseillevirus was isolated from a diptera [START_REF] Boughalmi | First Isolation of a Giant Virus from Wild Hirudo medicinalis Leech: Mimiviridae isolation in Hirudo medicinalis[END_REF] and a faustovirus was cultured from culicoides [START_REF] Temmam | Faustovirus-like asfarvirus in hematophagous biting midges and their vertebrate hosts[END_REF]. Moreover, mimivirus-like sequences were identified in metagenomes generated from bats, rodents, dromedaries and culicoides, and faustovirus-and pandoravirus-like sequences were detected in metagenomes generated from culicoides [20;21;28] (reviewed S. Halary et al. [START_REF] Halary | Viral metagenomics: are we missing the giants?[END_REF]).

Evidence for a causative role of giant viruses of amoebas in pathogenicity

Causality criteria

An increasing body of data supports the presence of GVA in humans, and in addition, the question of the putative pathogenic role of these viruses has been addressed and documented, mainly for mimiviruses, and more recently for marseilleviruses. Establishing a causative role of viruses in diseases has been a long journey. Criteria developed since 1840 by Henle, Loeffler and Koch to prove the etiologic association between an infectious agent and a specific disease have been deemed less and less appropriate over time [START_REF] Koch | Uber bakteriologische forschung verhandlung des X internationalen medichinischen congresses[END_REF]. Other criteria for causative relations were proposed [START_REF] Hill | The environment and disease: association or causation?[END_REF], including some specifically applied to viruses in 1937, 1957 and 1976 (Box 1) [START_REF] Rivers | Viruses and Koch's Postulates[END_REF][START_REF] Huebner | Criteria for etiologic association of prevalent viruses with prevalent diseases; the virologist's dilemma[END_REF][START_REF] Evans | Causation and disease: the Henle-Koch postulates revisited[END_REF]. However, newly discovered viruses challenge existing postulates, as, for instance, with viruses determining chronic or latent infections. Thus, with the advent of new technologies and improved knowledge in microbiology and virology, criteria considered for suspecting or establishing a causality link have drifted considerably. Notably, sequence-based criteria were introduced in 1996, and metagenomic Koch's postulates were finally proposed in 2012 [34;35]. Since 2003, the presence and possible pathogenic role of GVA has been serendipitously observed or investigated using a broad range of technological approaches including culture, electron microscopy, serology and various techniques based on molecular biology, including metagenomics (Table 1). The findings fulfill several of the criteria considered as proofs of viral disease causation.

Host cells other than phagocytic amoebas for giant viruses of amoebas

All GVA have been isolated on cultures of A. castellani, A. polyphaga, or V. vermiformis [13;36]. Numerous cell lines have been tested for their permissivity to mimiviruses or marseilleviruses. In experimental inoculation tests, Mimivirus was capable of entering professional phagocytes, among which various human myeloid cells including circulating monocytes, monocyte-derived macrophages and myelomonocytic cells, and also mouse myeloid cells [START_REF] Ghigo | Ameobal pathogen mimivirus infects macrophages through phagocytosis[END_REF]. Further experiments conducted with mouse macrophages showed a significant increase in Mimiviral DNA load during a 30-hour period of incubation; in addition, only approximately one quarter of the macrophages were viable after 30 hours, and macrophage extracts led to Mimivirus replication within amoebae and to amoebal lysis. These findings indicated productive infection of macrophage by Mimivirus post-internalization. In addition, Mimivirus was demonstrated to replicate in total human peripheral blood mononuclear cells (PBMC), as measured by the tissue culture infective dose method [START_REF] Silva | Demonstration that Mimivirus can replicate in human peripheral blood mononuclear cells and interact with the human type I Interferon system[END_REF]. Although GBM was not propagated, these results indicated productive infection of these cells.

It should be considered that the host barrier may be far more limited for GVA than for other viruses, because GVA infect their hosts by phagocytosis [START_REF] Ghigo | Ameobal pathogen mimivirus infects macrophages through phagocytosis[END_REF]. This was exemplified by the capability of Mimivirus to enter human macrophages through phagocytosis, and this closely resembled Mimivirus entry in amoebas [START_REF] Ghigo | Ameobal pathogen mimivirus infects macrophages through phagocytosis[END_REF]. In addition, mimiviruses, marseilleviruses or faustoviruses have been isolated from different phagocytic protists, including amoebozoa and chromalveolata, and also mammals, including humans, and also insects [26;48;49].

Mimivirus

Serological-only evidence

Concomitantly with the initial attempts to identify the giant Mimivirus, serological testing of sera from patients with unexplained pneumonia showed that the strongest reactivities were against this amoeba-resisting microbe [40]. Subsequently, the prevalence of antibodies to Mimivirus was assessed using microimmunofluorescence in several studies, in most cases in pneumonia patients hospitalized in intensive care units (ICU) (Table 1). IgG prevalence was most often »10-20% in pneumonia patients, ranging from 0% to 25% [START_REF] Scola | antibodies to Mimivirus in patients with pneumonia[END_REF][START_REF] Berger | Amebaassociated microorganisms and diagnosis of nosocomial pneumonia[END_REF][START_REF] Vanspauwen | Infections with mimivirus in patients with chronic obstructive pulmonary disease[END_REF][START_REF] Bousbia | Serologic prevalence of amoeba-associated microorganisms in intensive care unit pneumonia patients[END_REF].

In contrast, it was 0% and 2.3% in intubated control patients without pneumonia and healthy controls, respectively [START_REF] Scola | antibodies to Mimivirus in patients with pneumonia[END_REF]. Moreover, IgG and IgM elevations or seroconversions were observed in patients with hospital-acquired pneumonia [START_REF] Bousbia | Serologic prevalence of amoeba-associated microorganisms in intensive care unit pneumonia patients[END_REF]. The first strong evidence of infection with a GVA was in a laboratory technician who handled large amounts of Mimivirus and developed unexplained pneumonia [START_REF] Raoult | Strong serological evidence of a link between unexplained pneumonia and exposure to Mimivirus[END_REF]. He exhibited seroconversion to 23 Mimivirus proteins, as assessed by 2-dimensional gel electrophoresis (2DGE) and Western blotting, among which 4 proteins were unique to this virus. Interestingly, this story is very similar to the one that linked Epstein-Barr virus (EBV) to infectious mononucleosis. In 1968, a laboratory technician who worked with EBV developed infectious mononucleosis and concurrently exhibited seroconversion to this virus [START_REF] Golden | Leukocytetransforming agent: prolonged excretion by patients with mononucleosis and excretion by normal individuals[END_REF]. Positive serology to the Sputnik virophage was also observed in two patients of Laotian origin who exhibited fever while returning from Laos [START_REF] Parola | Acanthamoeba polyphaga Mimivirus virophage seroconversion in patients returning from Laos[END_REF]. Serological reactivities were obtained by Western blot, 2DGE and mass spectrometry and targeted two virophage proteins. In addition, one seroconversion could be shown. The serological detection of Sputnik in humans suggests the exposure of humans to this virophage, and the concurrent exposure to mimiviruses, which are the Sputnik hosts [7;8]. Thus, in this study, serological reactivities were also observed to Mamavirus and Acanthamoeba. No virus was isolated. In addition, a significant association was reported between antibodies to Mimivirus L71 protein, which harbors collagen-like motifs, and rheumatoid arthritis in patients [START_REF] Shah | Exposure to mimivirus collagen promotes arthritis[END_REF].

PCR

Detection by PCR of GVA in humans has only been conducted to date in clinical specimens evaluated for mimiviruses and marseilleviruses [START_REF] Ngounga | Real-time PCR systems targeting giant viruses of amoebae and their virophages[END_REF]. Mimivirus DNA was screened for in respiratory samples and was first found in 1 of 32 patients with ICU-acquired pneumonia (Table 1) [START_REF] Scola | antibodies to Mimivirus in patients with pneumonia[END_REF]. Then, mimiviruses were detected by conventional PCR in a Tunisian patient presenting unexplained pneumonia, concurrently with mimivirus isolation [START_REF] Saadi | First isolation of Mimivirus in a patient with pneumonia[END_REF]. Other studies have reported negative PCR testing in human respiratory samples [43;51], which may mean that mimiviruses are uncommon in this setting, or present at a low titer, but the main reason may be the substantial genetic diversity within the family Mimiviridae, which prevents implementation of universal PCR assays [START_REF] Ngounga | Real-time PCR systems targeting giant viruses of amoebae and their virophages[END_REF].

Culture isolation

Two mimiviruses have been isolated to date from clinical samples, in Tunisian patients with unexplained pneumonia (Table 1). In the first case, LBA111 virus was cultured from the bronchoalveolar fluid of a 72-year-old woman [START_REF] Saadi | First isolation of Mimivirus in a patient with pneumonia[END_REF]. The patient was admitted to the hospital for a 3-day fever with cough, dyspnea and hemoptysis; chest X-ray revealed right lower lobe consolidation and the white blood cell count was elevated. Concurrently, antibodies to 9 LBA111 virus proteins were detected by 2D Western blotting. The second case was a 17-year-old girl admitted for fever (40°C) and cough for 15 days, with lower left lung opacity, diarrhea, and leukocytosis [START_REF] Saadi | Shan virus: a new mimivirus isolated from the stool of a Tunisian patient with pneumonia[END_REF]. In this case, Shan virus was isolated from the stool; no respiratory sample was available. In addition, another mimivirus, named Lentillevirus, was isolated from the contact lens storage liquid of a keratitis patient [START_REF] Cohen | Ameba-associated Keratitis, France[END_REF].

Interestingly, its Acanthamoeba host was isolated and revealed to be infected with two amoeba-resisting bacteria and a virophage, Sputnik2.

Experimental evidence

Histopathological features of pneumonia, including thickened alveolar walls, inflammatory infiltrates and diffuse alveolar damage were observed in an experimental mouse model following intracardiac Mimivirus inoculation (Table 1) [START_REF] Khan | Pneumonia in mice inoculated experimentally with Acanthamoeba polyphaga mimivirus[END_REF]. No other experiment model of inoculation to animal has been conducted to date for another GVA. Such approach is of strong interest but questions on the most appropriate inoculum and inoculation route.

Marseilleviruses

The first hint of the presence of a marseillevirus in humans was serendipitously obtained during a metagenomic study that targeted bacterial sequences generated from the stools of a healthy Senegalese young man, and consisted of sequences best matching Marseillevirus among trashed metagenomic reads (Table 1) [20;55]. Subsequently, a close relative to Marseillevirus was isolated from this sample in Acanthamoeba and named Senegalvirus. Another metagenomic study identified reads matching the Marseillevirus genome in the blood of healthy blood donors [39]. This was confirmed by positive serology to Marseillevirus using immunofluorescence and Western blotting, and positive fluorescence in situ hybridization (FISH) and PCR on the blood and infected human lymphocytes. The presence of Marseillevirus was further detected by serology and PCR in other blood donors in France (IgG prevalence, 13-15%; DNA prevalence, 4-10%) [39;56], in Switzerland (IgG, 1.7-2.5%) [START_REF] Mueller | Lausannevirus seroprevalence among asymptomatic young adults[END_REF], and in polytransfused thalassemic patients in France (IgG, 23%; DNA, 9%) [START_REF] Popgeorgiev | Marseillevirus prevalence in multitransfused patients suggests blood transmission[END_REF].

The detection of Marseillevirus DNA in blood donors and recipients has been a controversial issue, as it has not been observed in other studies [START_REF] Sauvage | No Evidence of Marseillevirus-like Virus Presence in Blood Donors and Recipients of Multiple Blood Transfusions[END_REF][START_REF] Desnues | Reply to[END_REF][START_REF] Goodman | Marseillevirus, blood safety, and the human virome[END_REF][START_REF] Phan | Absence of giant blood Marseille-like virus DNA detection by polymerase chain reaction in plasma from healthy US blood donors and serum from multiply transfused patients from Cameroon[END_REF]. However, the body of data supporting the presence of Marseillevirus in humans has continued to grow. In 2013, an 11month-old child was found to exhibit a very high level of IgG to Marseillevirus [START_REF] Popgeorgiev | Marseillevirus adenitis in an 11-month-old child[END_REF]. He presented an unexplained adenitis, and Marseillevirus DNA was detected in his blood, while the virus was visualized in the lymph node by immunohistochemistry and FISH.

Other giant viruses of amoebas

GVA other than mimiviruses and marseilleviruses, including pandoraviruses, faustoviruses and P. sibericum and M. sibericum, have been discovered during the past three years, which has prevented extensive investigation of their presence in humans until now [13;17]. However, Pandoravirus inopinatum was isolated from the contact lens storage liquid of a keratitis patient [START_REF] Antwerpen | Whole-Genome Sequencing of a Pandoravirus Isolated from Keratitis-Inducing Acanthamoeba[END_REF] and sequences related to faustoviruses have been detected in metagenomes generated from human serum [START_REF] Reteno | Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae[END_REF].

Metagenomic data

Metagenomics has emerged during the same period as GVA, representing a new technological approach and powerful tool, although it may lack sensitivity and may allow only detecting sequences best matching with GVA [START_REF] Colson | Evidence of the megavirome in humans[END_REF]. Nevertheless, causing diseases Detection in human metagenomes of sequences related to GVA tends to be correlated with the number of available genomes and time to their release. Mimivirus-like sequences have been detected in metagenomes generated from human coprolites, stools of diarrheal patients and healthy people, nasopharyngeal aspirates from patients with respiratory tract infections, buccal mucosa, saliva and retroauricular crease from healthy people, vagina from healthy women, and blood samples from healthy people or patients with liver diseases of various etiologies (Table 1) [20;21;63]. Notably, it has been recently reported that Mimiviridae representatives dominated, together with Poxviridae representatives, the human gut eukaryotic virome in metagenomic samples of the Human Microbiome Project [START_REF] Rampelli | ViromeScan: a new tool for metagenomic viral community profiling[END_REF].

Virophage-like sequences have also been found in the human gut [START_REF] Zhou | Diversity of virophages in metagenomic data sets[END_REF]. In addition, Marseillevirus-like sequences have been detected in the buccal mucosa, retroauricular crease, vagina and stools from healthy people (Table 1) [20;63]. Recently, metagenome sequences best matching with pandoraviruses, Pithovirus sibericum, faustoviruses or virophages have also been detected in human plasma samples from patients with liver diseases [START_REF] Verneau | MG-Digger: an automated pipeline to search for giant virus-related sequences in metagenomes[END_REF].

Conclusion

The presence and impact of GVA and virophages in humans undoubtedly represent an important field that deserves further investigation in medicine. Such investigations are difficult. However, it has been increasingly demonstrated that GVA can be present in humans.

Evidence is particularly strong for mimiviruses and marseilleviruses, which were isolated from human feces, bronchoalveolar fluid and blood. Regarding the potential pathogenic role of these viruses in humans, the link between amoebal mimiviruses and pneumonia has been the most documented, whereas marseilleviruses have mostly been described in asymptomatic persons, and in an adenitis patient. Furthermore, for all these GVA, one must consider that their tremendous gene repertoires confer on them a strong potential for interaction with other organisms. It is also noteworthy that the closest relatives to faustoviruses are asfarviruses, which cause a common and severe disease in pigs [START_REF] Reteno | Faustovirus, an asfarvirus-related new lineage of giant viruses infecting amoebae[END_REF]. Regarding other megaviruses, they include poxviruses, which are pathogenic in insects and mammals, including humans [START_REF] Colson | Reclassification of giant viruses composing a fourth domain of life in the new order Megavirales[END_REF],

and Acanthocystis turfacea chlorella virus, a phycodnavirus that was found in human pharyngeal samples and tentatively associated with cognitive disorders [START_REF] Yolken | Chlorovirus ATCV-1 is part of the human oropharyngeal virome and is associated with changes in cognitive functions in humans and mice[END_REF]. Until recently, the belief that all viruses are small entities probably limited the detection of GVA in humans.

As this paradigm has been crumbling for a decade, future research should clarify the prevalence and consequence of their presence in humans. It appears particularly relevant to continue searching for mimiviruses in respiratory samples and stools, and for marseilleviruses in blood and in lymph nodes. Nevertheless, a broader panel of human samples from healthy and sick people should be tested; for instance, urine samples might be studied. In addition, investigations should involve a broad range of technological approaches, including serology, immunohistochemistry, immunofluorescence, FISH, targeted and random nucleic acid amplification, Sanger and next-generation sequencing, cytometry, microscopy, and high throughput culture isolation. Particularly, metagenomes currently extensively generated from human samples should be more exhaustively, thoroughly and recurrently screened for the presence of sequences best matching these GVA. Finally, experimental models on cells or animals would be helpful to gain a better understanding of the consequences of GVA presence in humans. Metagenomics: Detection in metagenomes generated from human coprolites, stools of diarrheal patients and healthy people, nasopharyngeal aspirates from patients with respiratory tract infections, buccal mucosa, saliva and retroauricular crease from healthy people, vagina from healthy women, and blood samples from healthy people or patients with liver diseases of various etiologies; detection of virophage-like sequences in the human gut Metagenomics: Marseillevirus-like sequences detection in the buccal mucosa, retroauricular crease, vagina and stools from healthy people

Culture isolation

Isolation from a broncholalveolar fluid and a faeces sample from two pneumonia patients

Association of Mimivirus with unexplained pneumonia

Experimental models Cells: Entry in various human myeloid cells including circulating monocytes, monocyte-derived macrophages and myelomonocytic cells; entry in of mouse myeloid cells; productive infection of macrophage by Mimivirus postinternalization; replication in total human peripheral blood mononuclear cells; interaction with type I IFN production in these cells 1. The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy animals.

Cells

2. The microorganism must be isolated from a diseased organism and grown in pure culture.

3. The cultured microorganism should cause disease when introduced into a healthy organism.

4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Rivers' criteria for proof of viral disease causation (1937) [31]

1. A specific virus must be found associated with a disease with a degree of regularity.

2. The virus must be shown to occur in the sick individual not as an incidental or accidental finding but as the cause of the disease under investigation.

3. Information concerning the presence of antibodies against the agent and the time of their appearance in the serum of patients is equally important as evidence of etiological significance of the virus.

Huebner's prescription for the virologist's dilemma: conditions necessary for establishing a virus as cause of a specific human disease (1957) [START_REF] Huebner | Criteria for etiologic association of prevalent viruses with prevalent diseases; the virologist's dilemma[END_REF] 1. Virus must be a "real" entity: A new virus must be well established by passage in the laboratory in animal or tissue cultures.

2. Origin of virus: the virus must be repeatedly isolated from human specimens and shown not to be a viral contaminant of the experimental animals, cells, or media employed to grow it.

3. Antibody response: An increase in neutralizing or other serologically demonstrable antibodies should regularly result from active infection.

Characterization and comparison with known agents:

A new virus should be fully characterized and compared with other agents including host and host-cell ranges, pathologic lesions, types of cytopathogenic effects, size, susceptibility to physical agents, etc.

5. Constant association with specific illness: The virus must be constantly associated with any well-defined clinical entity and isolated from diseased tissue, if available.

6. Studies with human volunteers: Human beings inoculated with a newly recognized agent in "double blind" studies should reproduce the clinical syndrome.

7. Epidemiologic studies: Both "cross-sectional" and "longitudinal" studies of community or institutional groups to identify patterns of infection and disease.

8. Prevention by a specific vaccine: One of the best ways to establish an agent as the cause.

9. Financial support: A consideration so absolutely necessary that it deserves to be called a postulate.

Evans's criteria for proof of disease causation: a unified concept appropriate for viruses as causative agents of disease based on the Henle-Koch postulates (1976) [START_REF] Evans | Causation and disease: the Henle-Koch postulates revisited[END_REF] 1. Prevalence of the disease is significantly higher in subjects exposed to the putative virus than in those not so exposed.

2. Incidence of the disease is significantly higher in subjects exposed to the putative virus than in those not so exposed (prospective studies).

3. Evidence of exposure to the putative virus is present more commonly in subjects with the disease than in those without the disease.

4. Temporally, the onset of disease follows exposure to the putative virus, always following an incubation period.

5.

A regular pattern of clinical signs follows exposure to the putative virus, presenting a graded response, often from mild to severe.

6. A measurable host immune response, such as an antibody response and/or a cell-mediated response, follows exposure to the putative virus. In those individuals lacking prior experience, the response appears regularly, and in those individuals with prior experience, the response is anamnestic.

7. Experimental reproduction of the disease follows deliberate exposure of animals to the putative virus, but nonexposed control animals remain disease free. Deliberate exposure may be in the laboratory or in the field, as with sentinel animals.

8. Elimination of the putative virus and/or its vector decreases the incidence of the disease. 9. Prevention or modification of infection, via immunization or drugs, decreases the incidence of the disease.

10. The whole thing should make biologic and epidemiologic sense.

Fredricks and Relman's molecular guidelines for establishing microbial disease causation (1996) [START_REF] Fredricks | Sequence-based identification of microbial pathogens: a reconsideration of Koch's postulates[END_REF] 1. A nucleic acid sequence belonging to a putative pathogen should be present in most cases of an infectious disease. Microbial nucleic acids should be found preferentially in those organs or gross anatomic sites known to be diseased (i.e., with anatomic, histologic, chemical, or clinical evidence of pathology) and not in those organs that lack pathology.

2. Fewer, or no, copy numbers of pathogen associated nucleic acid sequences should occur in hosts or tissues without disease.

3. With resolution of disease (for example, with clinically effective treatment), the copy number of pathogen-associated nucleic acid sequences should decrease or become undetectable. With clinical relapse, the opposite should occur.

4. When sequence detection predates disease, or sequence copy number correlates with severity of disease or pathology, the sequence-disease association is more likely to be a causal relationship.

5. The nature of the microorganism inferred from the available sequence should be consistent with the known biological characteristics of that group of organisms. When phenotypes (e.g., pathology, microbial morphology, and clinical features) are predicted by sequence-based phylogenetic relationships, the meaningfulness of the sequence is enhanced.

6. Tissue-sequence correlates should be sought at the cellular level: efforts should be made to demonstrate specific in situ hybridization of microbial sequence to areas of tissue pathology and to visible microorganisms or to areas where microorganisms are presumed to be located.

7. These sequence-based forms of evidence for microbial causation should be reproducible. [START_REF] Mokili | Metagenomics and future perspectives in virus discovery[END_REF] Comparison between a diseased and healthy control animal shows a significant difference between the metagenomic libraries (depicted by the histograms of relative abundance reads).

Metagenomic Koch's postulates (2012)

In order to fulfill the metagenomic Koch's postulates:

1. The metagenomic traits in diseased subject must be significantly different from healthy subject.

2. Inoculation of samples from the disease animal into the healthy control must lead to the induction of the disease state. Comparison of the metagenomes before and after inoculation should suggest the acquisition or increase of new metagenomic traits. New traits can be purified by methods such as serial dilution or time-point sampling of specimens from a disease animal.

3. Inoculation of the suspected purified traits into a healthy animal will induce disease if the traits form the etiology of the disease.

Acanthamoeba polyphaga mimivirus

First giant virus of amoeba

Pandoravirus salinus

First pandoravirus

Pithovirus sibericum

First pithovirus

Mollivirus sibericum

First mollivirus

LBA111 virus and Shan virus

Two first mimiviruses isolated from humans (pneumonia patients)

Marseillevirus

First Marseillevirus

Senegalvirus

First giant virus isolated from a human

Insectomime virus

First giant virus isolated from an insect

Sputnik virophage

First virus of virus

Hirudovirus

Mimivirus isolated from a leech

Giant blood Marseillevirus detected:

-

Furthermore,

  Mimivirus was revealed to induce type I IFN production in infected human PBMC and to inhibit interferon stimulated genes expression in these cells. These findings question if amoebae are the exclusive hosts for the giant Mimivirus. Moreover, inoculation of Jurkat cells, which are immortalized human T lymphocyte cells, with a serum sample positive for Giant blood Marseillevirus (GBM) DNA led to detection of this virus by PCR in the culture supernatant, and viral DNA and virions were detected within Jurkat cells 21 days postinfection by PCR, fluorescence in situ hybridization, or transmission electron microcopy [39].

Figure 1 .

 1 Figure 1. Schematic of the chronology of major findings that support the presence and

Figure 2 .

 2 Figure 2. Schematic of findings that support the presence and possible pathogenic role in

:Box 1 .

 1 Inoculation of immortalized human T lymphocyte cells with a serum sample positive for Giant blood Marseillevirus (GBM) DNA led to virus DNA detection in the culture supernatant, and viral DNA and virions detection within these cells 21 days post-infection by PCR, fluorescence in situ hybridization, or transmission electron microcopy Animal: Pneumonia induction in mice inoculated intracardiacally Mimivirus causes pneumonia in miceReferences for quoted studies are included in the text Evolving criteria for proof of disease causation that can be applied to viruses Henle, Loeffler and Koch's postulate (1884-1890)[START_REF] Koch | Uber bakteriologische forschung verhandlung des X internationalen medichinischen congresses[END_REF]

Fig. 1 Faustovirus

 1 Fig.1

  a virophage isolated from contact lens rinsing fluid of a keratitis patient Isolation of an Acanthamoeba parasite, further revealed to be a pandoravirus, from contact lens rinsing fluid of a keratitis patient

  Fig. 2

TABLETable 1 .

 1 Summary of evidence of associations of mimiviruses or marseilleviruses with humans and of a possible pathogenic role

	Technical approaches	Evidence for mimiviruses	Evidence for marseilleviruses	Elements to consider for causality
	Serology	Presence of specific IgG and IgM antibodies to Mimivirus in	IgG detection in blood donors, young health adults,	Recurrent evidence of serological
		pneumonia patients	multitransfused thalassemia patients, and a lymphadenitis	reactivities, including in association with
		Greater seroprevalence in pneumonia patients than controls Mimivirus seroconversion in pneumonia patients, including one individual who manipulated the virus (reactivity to 23 Mimivirus proteins)	patient	Mimivirus isolation in one case; seroconversion to the Mimivirus and the Sputnik virophage in patients; association with Mimivirus handling in a patient with unexplained pneumonia, and with
		Serological reactivities to the Sputnik virophage in two patients		hospital-acquired pneumonia
		(reactivity to 2 virophages proteins); seroconversion in one case		Detection of antibodies to Marseillevirus in association with Marseillevirus
				antigen/DNA detection in a single case-
				patient
	Immunodetection		Detection of Marseillevirus antigens by immunofluorescence	Association of Marseillevirus with
			and immunochemistry in a lymph node adenitis	lymphadenitis
	Molecular detection	Conventional PCR: Mimivirus DNA found in a	Conventional PCR: Marseillevirus DNA detection in the	Association of Mimivirus with unexplained
		broncholalveolar fluid and a serum sample from two	serum from blood donors, multitransfused thalassemia	pneumonia and of Marseillevirus with
		pneumonia patients	patients, and a lymphadenitis patient	lymphadenitis
			Detection of Marseillevirus DNA by fluorescence in situ
			hybridization in a lymph node adenitis

  in the blood from a blood donor by metagenomics, PCR and FISH -in the blood from multitransfused thalassemia patients by PCR -in a lymph node adenitis by FISH and immunochemistry

	First evidence of Mimivirus
	seropositivity in pneumonia
	patients
	First Mimivirus DNA-positivity of a clinical
	sample (a bronchoalveolar fluid from a
	patient with intensive care unit-acquired pneumonia)	Seropositivity to Marseillevirus in: -blood donors
		-multitransfused thalassemia patients
		-a chiild with adenitis
	Unexplained pneumonia in a laboratory
	technician who handled Mimivirus and
	presented seroconversion to this virus	Seropositivity to Lausannevirus (a
		marseillevirus) in healthy persons
		Seropositivity to Sputnik and
		Mamavirus (a Mimivirus isolate)
		in two febrile patients