Discovery and Further Studies on Giant Viruses at the IHU Mediterranee Infection That Modified the Perception of the Virosphere.

The history of giant viruses began in 2003 with the identification of Acanthamoeba polyphaga mimivirus. Since then, giant viruses of amoeba enlightened an unknown part of the viral world, and every discovery and characterization of a new giant virus modifies our perception of the virosphere. This notably includes their exceptional virion sizes from 200 nm to 2 µm and their genomic complexity with length, number of genes, and functions such as translational components never seen before. Even more surprising, Mimivirus possesses a unique mobilome composed of virophages, transpovirons, and a defense system against virophages named Mimivirus virophage resistance element (MIMIVIRE). From the discovery and isolation of new giant viruses to their possible roles in humans, this review shows the active contribution of the University Hospital Institute (IHU) Mediterranee Infection to the growing knowledge of the giant viruses' field.

Legionella saoudiensis sp. nov., isolated from a sewage water sample.

A Gram-stain-negative, bacilli-shaped bacterial strain, LS-1T, was isolated from a sewage water sample collected in Jeddah, Saudi Arabia. The taxonomic position of strain LS-1T was investigated using a polyphasic taxonomic approach. Phylogenetic analysis based on 16S rRNA gene sequences and those of four other genes indicated that strain LS-1T belongs to the genus Legionella in the family Legionellaceae. Regarding the 16S rRNA gene, the most closely related species are Legionella rowbothamii LLAP-6T (98.6 %) and Legionella lytica L2T (98.5 %). The mip gene sequence of strain LS-1T showed 94 % sequence similarity with that of L. lytica L2T and 93 % similarity with that of L. rowbothamii LLAP-6T. Strain LS-1T grew optimally at a temperature of 32 °C on a buffered charcoal yeast extract (BCYE) agar plate in a 5 % CO2 atmosphere and had a flagellum. The combined phylogenetic, phenotypic and genomic sequence data suggest that strain LS-1T represents a novel species of the genus Legionella, for which the name Legionella saoudiensis sp. nov. is proposed. The type strain is LS-1T (=DSM 101682T=CSUR P2101T).

Isolation of new Brazilian giant viruses from environmental samples using a panel of protozoa.

The Megavirales are a newly described order capable of infecting different types of eukaryotic hosts. For the most part, the natural host is unknown. Several methods have been used to detect these viruses, with large discrepancies between molecular methods and co-cultures. To isolate giant viruses, we propose the use of different species of amoeba as a cellular support. The aim of this work was to isolate new Brazilian giant viruses by comparing the protozoa Acanthamoeba castellanii, A. polyphaga, A. griffini, and Vermamoeba vermiformis (VV) as a platform for cellular isolation using environmental samples. One hundred samples were collected from 3 different areas in September 2014 in the Pampulha lagoon of Belo Horizonte city, Minas Gerais, Brazil. PCR was used to identify the isolated viruses, along with hemacolor staining, labelling fluorescence and electron microscopy. A total of 69 viruses were isolated. The highest ratio of isolation was found in A. polyphaga (46.38%) and the lowest in VV (0%). Mimiviruses were the most frequently isolated. One Marseillevirus and one Pandoravirus were also isolated. With Brazilian environmental samples, we demonstrated the high rate of lineage A mimiviruses. This work demonstrates how these viruses survive and circulate in nature as well the differences between protozoa as a platform for cellular isolation.

Fetuin is the key for nanon self-propagation.

"Nanobacteria", also known as nanons or calciprotein particles (CPP), are nano-sized protein mineral complexes which have been isolated from numerous biological sources. Nanons possess self-replication properties and contain only serum proteins (e.g. Fetuin-A, Albumin). Herein, we develop a simplified in vitro model of nanons propagation composed of only fetuin-A as a protein. Using this model, we demonstrate that fetuin from nanons possesses a different, non-native conformation. Moreover, we show that nanons induce soluble fetuin-A precipitation which could serve as a template for calcification. This phenomenon explains the observed self-propagating properties that mimic infectious behavior. We also demonstrate that renal calculi are capable of inducing a conformational change in fetuin-A, suggesting that the propagation phenomenon of nanons may occur in vivo.

Zamilon, a novel virophage with Mimiviridae host specificity.

Virophages, which are potentially important ecological regulators, have been discovered in association with members of the order Megavirales. Sputnik virophages target the Mimiviridae, Mavirus was identified with the Cafeteria roenbergensis virus, and virophage genomes reconstructed by metagenomic analyses may be associated with the Phycodnaviridae. Despite the fact that the Sputnik virophages were isolated with viruses belonging to group A of the Mimiviridae, they can grow in amoebae infected by Mimiviridae from groups A, B or C. In this study we describe Zamilon, the first virophage isolated with a member of group C of the Mimiviridae family. By co-culturing amoebae with purified Zamilon, we found that the virophage is able to multiply with members of groups B and C of the Mimiviridae family but not with viruses from group A. Zamilon has a 17,276 bp DNA genome that potentially encodes 20 genes. Most of these genes are closely related to genes from the Sputnik virophage, yet two are more related to Megavirus chiliensis genes, a group B Mimiviridae, and one to Moumouvirus monve transpoviron.

Draft genome sequences of Terra1 and Terra2 viruses, new members of the family Mimiviridae isolated from soil.

Since the discovery of Mimivirus, the founding member of the family Mimiviridae, three lineages, A-C, have been delineated among the mimiviruses of amoebae. To date, all giant viruses with annotated genomes have been isolated from water samples. Here, we describe the genome of two mimiviruses, Terra1 virus and Terra2 virus, which were recovered by co-culturing on Acanthamoeba spp. from soil samples. These genomes are predicted to harbor 1055 and 890 genes, respectively. Comparative genomics and phylogenomics show that Terra1 virus and Terra2 virus are classified within lineages C and A of the amoebae-associated mimiviruses, respectively. The genomic architecture of both viruses show conserved collinear central regions flanked by less conserved areas towards the extremities, when compared with other mimivirus genomes. A cluster of genes that are orthologous to bacterial genes and have no counterpart in other viral genomes except in lineage C mimiviruses was identified in Terra1 virus.

Complete genome sequence of Courdo11 virus, a member of the family Mimiviridae.

Giant viruses of amoebae were discovered 10 years ago and led to the description of two new viral families: Mimiviridae and Marseilleviridae. These viruses exhibit remarkable features, including large capsids and genomes that are similar in size to those of small bacteria and their large genetic repertoires include genes that are unique among viruses. The family Mimiviridae has grown during the past decade since the discovery of its initial member, Mimivirus, and continues to expand. Here, we describe the genome of a new giant virus that infects Acanthamoeba spp., Courdo11 virus, isolated in 2010 by inoculating Acanthamoeba spp. with freshwater collected from a river in southeastern France. The Courdo11 virus genome is a double stranded DNA molecule composed of 1,245,674 nucleotides. The comparative analyses of Courdo11 virus with the genomes of other giant viruses showed that it belongs to lineage C of mimiviruses of amoebae, being most closely related to Megavirus chilensis and LBA 111, the first mimivirus isolated from a human. Major characteristics of the M. chilensis genome were identified in the Courdo11 virus genome, found to encode three more tRNAs. Genomic architecture comparisons mirrored previous findings that showed conservation of collinear regions in the middle part of the genome and diversity towards the extremities. Finally, fourteen ORFans were identified in the Courdo11 virus genome, suggesting that the pan-genome of mimiviruses of amoeba might reach a plateau.

First isolation of a giant virus from wild Hirudo medicinalis leech: Mimiviridae isolation in Hirudo medicinalis.

Giant viruses and amoebae are common in freshwater, where they can coexist with other living multicellular organisms. We screened leeches from the species Hirudo medicinalis for giant viruses. We analyzed five H. medicinalis obtained from Tunisia (3) and France (2). The leeches were decontaminated and then dissected to remove internal parts for co-culture with Acanthamoeba polyphaga. The genomes of isolated viruses were sequenced on a 454 Roche instrument, and a comparative genomics analysis was performed. One Mimivirus was isolated and the strain was named Hirudovirus. The genome assembly generated two scaffolds, which were 1,155,382 and 25,660 base pairs in length. Functional annotations were identified for 47% of the genes, which corresponds to 466 proteins. The presence of Mimividae in the same ecological niche as wild Hirudo may explain the presence of the mimivirus in the digestive tract of the leech, and several studies have already shown that viruses can persist in the digestive tracts of leeches fed contaminated blood. As leeches can be used medically and Mimiviruses have the potential to be an infectious agent in humans, patients treated with leeches should be surveyed to investigate a possible connection.

First isolation of a Marseillevirus in the Diptera Syrphidae Eristalis tenax.

OBJECTIVE: Giant viruses and amoebae are common in freshwater, where they can coexist with various insects. We screened insect larvae to detect giant viruses using a high-throughput method. METHODS: We analyzed 86 Eristalis tenax larvae obtained from stagnant water reservoirs in Tunisia. The larvae were decontaminated and then dissected to remove internal parts for coculture with Acanthamoeba polyphaga. Genome sequencing of isolated viruses was performed on a 454 Roche instrument, and comparative genomics were performed. RESULTS: One Marseillevirus, named Insectomime virus, was isolated. The genome assembly generated two scaffolds, which were 382,776 and 3,855 bp in length. Among the 477 identified predicted proteins, the best hit for 435 of the identified proteins was a Marseillevirus or Lausannevirus protein. Tunisvirus was the most closely related to Insectomime, with 446 orthologs. One Insectomime protein shared with Lausannevirus and Tunisvirus showed the highest similarity with a protein from an aphid. CONCLUSION: The isolation of a Marseillevirus from an insect expands the diversity of environments in which giant viruses have been isolated. The coexistence of larvae and giant viruses in stagnant water may explain the presence of the giant virus in the larva internal structures. This study illustrates the putative role of amoeba in lateral gene transfer not only between the organisms it phagocytoses, but also between organisms living in the same environment. © 2013 S. Karger AG, Basel.

Alcohol disinfection procedure for isolating giant viruses from contaminated samples.

OBJECTIVE: Giant viruses of the Megavirales order have been neglected in the literature because they are removed from samples during viral purification for viral metagenomic studies. Isolation via amoeba coculture has low efficiency and is extremely time-consuming. Thus, our objective was to improve Megavirales detection and recovery by using a new protocol that will eliminate most bacteria present in environmental samples while preserving giant virus viability. METHODS: In this study, we tested the ability of a number of disinfection protocols to kill contaminating bacteria. These treatments were ethanol, UV irradiation, desiccation, glutaraldehyde and thermal shock. RESULTS: Of all the treatments, a brief ethanol treatment did not significantly reduce the titer of viable viral particles of Acanthamoeba polyphaga mimivirus or Marseillevirus, whereas it efficiently killed Escherichia coli. This treatment was applied to environmental samples that previously tested positive for giant viruses and was shown to eliminate contaminating bacteria, whereas it allowed for the isolation of the giant viruses. CONCLUSION: Our results demonstrate that ethanol treatment can be used to evaluate large collections of environmental samples for the presence of giant viruses and to provide insight into understanding their ecology. This study should also facilitate the isolation of giant viruses using other species of protozoa in addition to Acanthamoeba spp.

Shan virus: a new mimivirus isolated from the stool of a Tunisian patient with pneumonia.

OBJECTIVE: Following the isolation of a Marseillevirus from the stool of a healthy young Senegalese and a Mimivirus from a Tunisian patient with pneumonia, we attempted to isolate other giant viruses of amoebae from a large human stool collection. METHODS: During the period 2010-2011, a total of 1,605 stool samples, including 115 from Tunisian patients with pneumonia, were cultured on amoebae. We used a recently developed high-throughput isolation system to detect amoebae plaque lysis on agar plates; this method allows for the testing of 100 samples per plate per week. The giant virus was identified by sequencing of genes conserved in Megavirales. RESULTS: A single giant virus, called Shan, was isolated from the stool of a Tunisian patient with pneumonia who responded poorly to antibiotics. This virus has an icosahedral shape typical of members of the family Mimiviridae and a size of 640 ± 10 nm. Phylogenetic analyses showed that Shan virus was classified as a member of Mimivirus lineage C that infects amoebae. CONCLUSION: Only one isolate was obtained in this study, suggesting that giant viruses of amoebae are rare in human stool. The isolation of Shan virus from a patient with pneumonia brings into question the etiological role of this virus and its subsequent release in stool.

Complete genome sequence of Cannes 8 virus, a new member of the proposed family "Marseilleviridae".

Marseillevirus is a giant virus that was isolated in 2007 by culturing water collected from a cooling tower in Paris, France, on Acanthamoeba polyphaga. Since then, five other marseilleviruses have been detected in environmental or human samples. The genomes of two of the six marseilleviruses have been described in detail. We describe herein the genome of Cannes 8 virus, a new member of the proposed family "Marseilleviridae." Cannes 8 virus was isolated from water collected from a cooling tower in Cannes in southeastern France. Its genome is a circular double-stranded DNA molecule with 374,041 base pairs, larger than the Marseillevirus and Lausannevirus genomes. This genome harbors 484 open reading frames predicted to encode proteins with sizes ranging from 50 to 1,537 amino acids, among which 380 (79%) and 272 (56%) are bona fide orthologs of Marseillevirus and Lausannevirus proteins, respectively. In addition, 407 and 336 predicted proteins have significant hits against Marseillevirus and Lausannevirus proteins, respectively, and 294 proteins are shared by all three marseilleviruses. The Cannes 8 virus genome has a high level of collinearity (for 96% of orthologs) with the Marseillevirus genome. About two-thirds of the Cannes 8 virus gene repertoire is composed of family ORFans. The description and annotation of the genomes of new marseilleviruses that will undoubtedly be recovered from environmental or clinical samples will be helpful to increase our knowledge of the pan-genome of the family "Marseilleviridae."

Open membranes are the precursors for assembly of large DNA viruses.

Nucleo cytoplasmic large DNA viruses (NCLDVs) are a group of double-stranded DNA viruses that replicate their DNA partly or entirely in the cytoplasm in association with viral factories (VFs). They share about 50 genes suggesting that they are derived from a common ancestor. Using transmission electron microscopy (TEM) and electron tomography (ET) we showed that the NCLDV vaccinia virus (VACV) acquires its membrane from open membrane intermediates, derived from the ER. These open membranes contribute to the formation of a single open membrane of the immature virion, shaped into a sphere by the assembly of the viral scaffold protein on its convex side. We now compare VACV with the NCLDV Mimivirus by TEM and ET and show that the latter also acquires its membrane from open membrane intermediates that accumulate at the periphery of the cytoplasmic VF. In analogy to VACV this membrane is shaped by the assembly of a layer on the convexside of its membrane, likely representing the Mimivirus capsid protein. By quantitative ET we show for both viruses that the open membrane intermediates of assembly adopt an 'open-eight' conformation with a characteristic diameter of 90 nm for Mimi- and 50 nm for VACV. We discuss these results with respect to the common ancestry of NCLDVs and propose a hypothesis on the possible origin of this unusual membrane biogenesis.

First isolation of Mimivirus in a patient with pneumonia.

BACKGROUND: Mimiviridae Mimivirus, including the largest known viruses, multiply in amoebae. Mimiviruses have been linked to pneumonia, but they have never been isolated from patients. To further understand the pathogenic role of these viruses, we aimed to isolate them from a patient presenting with pneumonia. METHODS: We cultured, on Acanthamoeba polyphaga amoebae, pulmonary samples from 196 Tunisian patients with community-acquired pneumonia during the period 2009-2010. An improved technique was used for Mimivirus isolation, which used agar plates where the growth of giant viruses is revealed by the formation of lysis plaques. Mimivirus serology was tested by microimmunofluorescence and by bidimensional immunoproteomic analysis using Mimivirus strains, to identify specific immunoreactive proteins. The new Mimivirus strain genome sequencing was performed on Roche 454 GS FLX Titanium, then AB SOLiD instruments. RESULTS: We successfully isolated a Mimivirus (LBA111), the largest virus ever isolated in a human sample, from a 72-year-old woman presenting with pneumonia. Electron microscopy revealed a Mimivirus-like virion with a size of 554 ± 10 nm. The LBA111 genome is 1.23 megabases, and it is closely related to that of Megavirus chilensis. Furthermore, the serum from the patient reacted specifically to the virus compared to controls. CONCLUSIONS: This is the first Mimivirus isolated from a human specimen. The findings presented above together with previous works establish that mimiviruses can be associated with pneumonia. The common occurrence of these viruses in water and soil makes them probable global agents that are worthy of investigation.

Broad spectrum of mimiviridae virophage allows its isolation using a mimivirus reporter.

The giant virus Mimiviridae family includes 3 groups of viruses: group A (includes Acanthamoeba polyphaga Mimivirus), group B (includes Moumouvirus) and group C (includes Megavirus chilensis). Virophages have been isolated with both group A Mimiviridae (the Mamavirus strain) and the related Cafeteria roenbergensis virus, and they have also been described by bioinformatic analysis of the Phycodnavirus. Here, we found that the first two strains of virophages isolated with group A Mimiviridae can multiply easily in groups B and C and play a role in gene transfer among these virus subgroups. To isolate new virophages and their Mimiviridae host in the environment, we used PCR to identify a sample with a virophage and a group C Mimiviridae that failed to grow on amoeba. Moreover, we showed that virophages reduce the pathogenic effect of Mimivirus (plaque formation), establishing its parasitic role on Mimivirus. We therefore developed a co-culture procedure using Acanthamoeba polyphaga and Mimivirus to recover the detected virophage and then sequenced the virophage's genome. We present this technique as a novel approach to isolating virophages. We demonstrated that the newly identified virophages replicate in the viral factories of all three groups of Mimiviridae, suggesting that the spectrum of virophages is not limited to their initial host.

Amoebae as battlefields for bacteria, giant viruses, and virophages.

When amoebae are simultaneously infected with Acanthamoeba polyphaga Mimivirus (APM) and the strictly intracellular BABL1 bacterium, the latter is always lost after serial subculturing. We showed that the virophage Sputnik 1, by reducing APM fitness, preserved BABL1 growth in acute and chronic models. This capability of a virophage to modulate the virulence of mimiviruses highlights the competition that occurs between them during natural host infection.

High-throughput isolation of giant viruses of the Mimiviridae and Marseilleviridae families in the Tunisian environment.

Giant viruses of the Megavirales order have been recently isolated from aquatic environments and have long been neglected because they are removed from samples during viral purification for viral metagenomic studies. Due to bacterial overgrowth and susceptibility to high concentrations of antibiotics, isolation by amoeba co-culture has a low efficiency and is highly time-consuming. Thus, few environments have been exhaustively investigated to date, although the ubiquitous distribution of the Acanthamoeba sp. suggests that these viruses could also be ubiquitous. In this work, we have implemented a high-throughput method to detect amoebae lysis on agar plates that allows the testing of hundreds of samples in a few days. Using this procedure, a total of 11 new Marseilleviridae strains and four new Mimiviridae strains, including a virus infected with a virophage, were isolated from 1000 environmental samples from Tunisia. Of these, four corresponded to new genotypic variants. These isolates are the first African environmental isolates identified from these two families, and several samples were obtained from a hypersaline aquatic environment. These results demonstrate that this technique can be used for the evaluation and characterization of large collections of giant viruses to provide insight into understanding their ecology.

"Marseilleviridae", a new family of giant viruses infecting amoebae.

The family "Marseilleviridae" is a new proposed taxon for giant viruses that infect amoebae. Its first member, Acanthamoeba polyphaga marseillevirus (APMaV), was isolated in 2007 by culturing on amoebae a water sample collected from a cooling tower in Paris, France. APMaV has an icosahedral shape with a diameter of ≈250 nm. Its genome is a double-stranded circular DNA that is 368,454 base pairs (bp) in length. The genome has a GC content of 44.7 % and is predicted to encode 457 proteins. Phylogenetic reconstructions showed that APMaV belongs to a new viral family among nucleocytoplasmic large DNA viruses, a group of viruses that also includes Acanthamoeba polyphaga mimivirus (APMV) and the other members of the family Mimiviridae as well as the members of the families Poxviridae, Phycodnaviridae, Iridoviridae, Ascoviridae, and Asfarviridae. In 2011, Acanthamoeba castellanii lausannevirus (ACLaV), another close relative of APMaV, was isolated from river water in France. The ACLaV genome is 346,754 bp in size and encodes 450 genes, among which 320 have an APMaV protein as the closest homolog. Two other giant viruses closely related to APMaV and ACLaV have been recovered in our laboratory from a freshwater sample and a human stool sample using an amoebal co-culture method. The only currently identified hosts for "marseilleviruses" are Acanthamoeba spp. The prevalence of these viruses in the environment and in animals and humans remains to be determined.

Related giant viruses in distant locations and different habitats: Acanthamoeba polyphaga moumouvirus represents a third lineage of the Mimiviridae that is close to the megavirus lineage.

The 1,021,348 base pair genome sequence of the Acanthamoeba polyphaga moumouvirus, a new member of the Mimiviridae family infecting Acanthamoeba polyphaga, is reported. The moumouvirus represents a third lineage beside mimivirus and megavirus. Thereby, it is a new member of the recently proposed Megavirales order. This giant virus was isolated from a cooling tower water in southeastern France but is most closely related to Megavirus chiliensis, which was isolated from ocean water off the coast of Chile. The moumouvirus is predicted to encode 930 proteins, of which 879 have detectable homologs. Among these predicted proteins, for 702 the closest homolog was detected in Megavirus chiliensis, with the median amino acid sequence identity of 62%. The evolutionary affinity of moumouvirus and megavirus was further supported by phylogenetic tree analysis of conserved genes. The moumouvirus and megavirus genomes share near perfect orthologous gene collinearity in the central part of the genome, with the variations concentrated in the terminal regions. In addition, genomic comparisons of the Mimiviridae reveal substantial gene loss in the moumouvirus lineage. The majority of the remaining moumouvirus proteins are most similar to homologs from other Mimiviridae members, and for 27 genes the closest homolog was found in bacteria. Phylogenetic analysis of these genes supported gene acquisition from diverse bacteria after the separation of the moumouvirus and megavirus lineages. Comparative genome analysis of the three lineages of the Mimiviridae revealed significant mobility of Group I self-splicing introns, with the highest intron content observed in the moumouvirus genome.

Mimivirus shows dramatic genome reduction after intraamoebal culture.

Most phagocytic protist viruses have large particles and genomes as well as many laterally acquired genes that may be associated with a sympatric intracellular life (a community-associated lifestyle with viruses, bacteria, and eukaryotes) and the presence of virophages. By subculturing Mimivirus 150 times in a germ-free amoebal host, we observed the emergence of a bald form of the virus that lacked surface fibers and replicated in a morphologically different type of viral factory. When studying a 0.40-µm filtered cloned particle, we found that its genome size shifted from 1.2 (M1) to 0.993 Mb (M4), mainly due to large deletions occurring at both ends of the genome. Some of the lost genes are encoding enzymes required for posttranslational modification of the structural viral proteins, such as glycosyltransferases and ankyrin repeat proteins. Proteomic analysis allowed identification of three proteins, probably required for the assembly of virus fibers. The genes for two of these were found to be deleted from the M4 virus genome. The proteins associated with fibers are highly antigenic and can be recognized by mouse and human antimimivirus antibodies. In addition, the bald strain (M4) was not able to propagate the sputnik virophage. Overall, the Mimivirus transition from a sympatric to an allopatric lifestyle was associated with a stepwise genome reduction and the production of a predominantly bald virophage resistant strain. The new axenic ecosystem allowed the allopatric Mimivirus to lose unnecessary genes that might be involved in the control of competitors.