Defensive symbiosis against giant viruses in amoebae.

Protists are important regulators of microbial communities and key components in food webs with impact on nutrient cycling and ecosystem functioning. In turn, their activity is shaped by diverse intracellular parasites, including bacterial symbionts and viruses. Yet, bacteria-virus interactions within protists are poorly understood. Here, we studied the role of bacterial symbionts of free-living amoebae in the establishment of infections with nucleocytoplasmic large DNA viruses (Nucleocytoviricota). To investigate these interactions in a system that would also be relevant in nature, we first isolated and characterized a giant virus (Viennavirus, family Marseilleviridae) and a sympatric potential Acanthamoeba host infected with bacterial symbionts. Subsequently, coinfection experiments were carried out, using the fresh environmental isolates as well as additional amoeba laboratory strains. Employing fluorescence in situ hybridization and qPCR, we show that the bacterial symbiont, identified as Parachlamydia acanthamoebae, represses the replication of the sympatric Viennavirus in both recent environmental isolates as well as Acanthamoeba laboratory strains. In the presence of the symbiont, virions are still taken up, but viral factory maturation is inhibited, leading to survival of the amoeba host. The symbiont also suppressed the replication of the more complex Acanthamoeba polyphaga mimivirus and Tupanvirus deep ocean (Mimiviridae). Our work provides an example of an intracellular bacterial symbiont protecting a protist host against virus infections. The impact of virus-symbiont interactions on microbial population dynamics and eventually ecosystem processes requires further attention.

Virophages: association with human diseases and their predicted role as virus killers.

The fascinating discovery of the first giant virus, Acanthamoeba polyphaga mimivirus (APMV), belonging to the family Mimiviridae in 2008, and its associated virophage, Sputnik, have left the world of microbiology awestruck. To date, about 18 virophages have been isolated from different environmental sources. With their unique feature of resisting host cell infection and lysis by giant viruses, analogous to bacteriophage, they have been assigned under the family Lavidaviridae. Genome of T-27, icosahedral-shaped, non-enveloped virophages, consist of dsDNA encoding four proteins, namely, major capsid protein, minor capsid protein, ATPase and cysteine protease, which are essential in the formation and assembly of new virophage particles during replication. A few virophage genomes have been observed to contain additional sequences like PolB, ZnR and S3H. Another interesting characteristic of virophage is that Mimivirus lineage A is immune to infection by the Zamilon virophage through a phenomenon termed MIMIVIRE, resembling the CRISPR-Cas mechanism in bacteria. Based on the fact that giant viruses have been found in clinical samples of hospital-acquired pneumonia and rheumatoid arthritis patients, virophages have opened a novel era in the search for cures of various diseases. This article aims to study the prospective role of virophages in the future of human therapeutics.

Translating the language of giants: translation-related genes as a major contribution of giant viruses to the virosphere.

Giant viruses of amoebas are a remarkable group of viruses. In addition to their large size and peculiar structures, the genetic content of these viruses is also special. Among the genetic features of these viruses that stand out is the presence of coding regions for elements involved in translation, a complex biological process that occurs in cellular organisms. No viral genome described so far has such a complex genetic arsenal as those of giant viruses, which code for several of these elements. Currently, tupanviruses have the most complete set of translation genes in the known virosphere. In this review, we have condensed what is currently known about translation genes in different groups of giant viruses and theorize about their biological importance, origin, and evolution, and what might possibly be found in the coming years.

Co-Isolation and Characterization of Two Pandoraviruses and a Mimivirus from a Riverbank in Japan.

Giant viruses, like pandoraviruses and mimiviruses, have been discovered from diverse environments, and their broad global distribution has been established. Here, we report two new isolates of Pandoravirus spp. and one Mimivirus sp., named Pandoravirus hades, Pandoravirus persephone, and Mimivirus sp. isolate styx, co-isolated from riverbank soil in Japan. We obtained nearly complete sequences of the family B DNA polymerase gene (polB) of P. hades and P. persephone; the former carried two known intein regions, while the latter had only one. Phylogenetic analysis revealed that the two new pandoravirus isolates are closely related to Pandoravirus dulcis. Furthermore, random amplified polymorphic DNA analysis revealed that P. hades and P. persephone might harbor different genome structures. Based on phylogenetic analysis of the partial polB sequence, Mimivirus sp. isolate styx belongs to mimivirus lineage A. DNA staining suggested that the Pandoravirus spp. asynchronously replicates in amoeba cells while Mimivirus sp. replicates synchronously. We also observed that P. persephone- or Mimivirus sp. isolate styx-infected amoeba cytoplasm is extruded by the cells. To the best of our knowledge, we are the first to report the isolation of pandoraviruses in Asia. In addition, our results emphasize the importance of virus isolation from soil to reveal the ecology of giant viruses.

Giant virus vs amoeba: fight for supremacy.

Since the discovery of mimivirus, numerous giant viruses associated with free-living amoebae have been described. The genome of giant viruses can be more than 2.5 megabases, and virus particles can exceed the size of many bacteria. The unexpected characteristics of these viruses have made them intriguing research targets and, as a result, studies focusing on their interactions with their amoeba host have gained increased attention. Studies have shown that giant viruses can establish host-pathogen interactions, which have not been previously demonstrated, including the unprecedented interaction with a new group of small viruses, called virophages, that parasitize their viral factories. In this brief review, we present recent advances in virophage-giant virus-host interactions and highlight selected studies involving interactions between giant viruses and amoebae. These unprecedented interactions involve the giant viruses mimivirus, marseillevirus, tupanviruses and faustovirus, all of which modulate the amoeba environment, affecting both their replication and their spread to new hosts.

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.

The Complex Nature of Tupanviruses.

The discovery of giant viruses revealed a new level of complexity in the virosphere, raising important questions about the diversity, ecology, and evolution of these viruses. The family Mimiviridae was the first group of amoebal giant viruses to be discovered (by Bernard La Scola and Didier Raoult team), containing viruses with structural and genetic features that challenged many concepts of classic virology. The tupanviruses are among the newest members of this family and exhibit structural, biological, and genetic features never previously observed in other giant viruses. The complexity of these viruses has put us one step forward toward the comprehension of giant virus biology and evolution, but also has raised important questions that still need to be addressed. In this chapter, we tell the history behind the discovery of one of the most complex viruses isolated to date, highlighting the unique features exhibited by tupanviruses, and discuss how these giant viruses have contributed to redefining limits for the virosphere.

"Tupanvirus", a new genus in the family Mimiviridae.

The genus "Tupanvirus" is a new proposed taxon to be included in the family Mimiviridae. The two known tupanvirus isolates were isolated from soda lake and oceanic sediments samples collected in Brazil and were named "tupanvirus soda lake" and "tupanvirus deep ocean", respectively. These viruses exhibit similarities to amoeba-infecting mimiviruses, but there are also several differences that place them in a separate group within the family Mimiviridae. Their virions have a mean size of 1.2 µm, which include a mimivirus-like capsid and a large cylindrical tail, both covered by fibrils. The linear double-stranded DNA genomes of up to 1,516,267 base pairs encode over 1,200 genes, among which ~ 30% have no homologs in any database, including in other mimivirus genomes. Compared to other mimiviruses, tupanviruses exhibit a broader host range and cause a cytotoxic effect in host and non-host organisms, a phenotype that is not observed for other mimiviruses. Remarkably, these viruses possess the most complete gene set related to the protein synthesis process, including 20 aminoacyl-tRNA synthetases, 67-70 tRNAs, many translation factors, and genes involved in maturation and modification of tRNA and mRNA, among others. Moreover, diverse phylogenomic analyses put tupanviruses in a distinct group within the family Mimiviridae. In light of the set of different features observed for these giant viruses, we propose establishment of a new genus to allow proper classification of two known tupanviruses and possibly many more similar viruses yet to be characterized.

Persistence of infectious Enterovirus within free-living amoebae - A novel waterborne risk pathway?

Free-living amoebae (FLA) are phagocytic protozoa found in natural and engineered water systems. They can form disinfectant-resistant cysts, which can harbor various human pathogenic bacteria, therefore providing them with a means of environmental persistence and dispersion through water distribution and other engineered water systems. The association of FLA with human viruses has been raised, but the limited data on the persistence of infectious virions within amoebae leaves this aspect unresolved. Enteroviruses can cause a wide range of illness and replicate in human respiratory and gastrointestinal tracts, both of which could be exposed through contact with contaminated waters if virus detection and removal are compromised by virion internalization in free-living protozoa. This is especially problematic for high-risk contaminants, such as coxsackieviruses, representative members of the Enterovirus genus that are likely infectious at low doses and cause a variety of symptoms to a vulnerable portion of the population (particularly infants). To investigate Enterovirus persistence within free-living amoebae we co-cultured an infectious clinical coxsackievirus B5 (CVB5) isolate, with the commonly reported tap water amoeba Vermamoeba vermiformis, after which we tracked virus localization and persistence in co-culture over time through a combination of advanced imaging, molecular and cell culture assays. Our results clearly demonstrate that infectious CVB5 can persist in all life stages of the amoebae without causing any visible injury to them. We also demonstrated that the amoeba generated vesicles containing virions that were expelled into the bulk liquid surroundings, a finding previously described for FLA-bacteria interactions, but not for FLA and human pathogenic viruses. Therefore, our findings suggest that the ability of CVB5 to persist in V. vermiformis could be a novel waterborne risk pathway for the persistence and dispersion of infectious human enteric viruses through water systems.

Tailed giant Tupanvirus possesses the most complete translational apparatus of the known virosphere.

Here we report the discovery of two Tupanvirus strains, the longest tailed Mimiviridae members isolated in amoebae. Their genomes are 1.44-1.51 Mb linear double-strand DNA coding for 1276-1425 predicted proteins. Tupanviruses share the same ancestors with mimivirus lineages and these giant viruses present the largest translational apparatus within the known virosphere, with up to 70 tRNA, 20 aaRS, 11 factors for all translation steps, and factors related to tRNA/mRNA maturation and ribosome protein modification. Moreover, two sequences with significant similarity to intronic regions of 18 S rRNA genes are encoded by the tupanviruses and highly expressed. In this translation-associated gene set, only the ribosome is lacking. At high multiplicity of infections, tupanvirus is also cytotoxic and causes a severe shutdown of ribosomal RNA and a progressive degradation of the nucleus in host and non-host cells. The analysis of tupanviruses constitutes a new step toward understanding the evolution of giant viruses.

Giant Viruses of Amoebae: A Journey Through Innovative Research and Paradigm Changes.

Giant viruses of amoebae were discovered serendipitously in 2003; they are visible via optical microscopy, making them bona fide microbes. Their lifestyle, structure, and genomes break the mold of classical viruses. Giant viruses of amoebae are complex microorganisms. Their genomes harbor between 444 and 2,544 genes, including many that are unique to viruses, and encode translation components; their virions contain >100 proteins as well as mRNAs. Mimiviruses have a specific mobilome, including virophages, provirophages, and transpovirons, and can resist virophages through a system known as MIMIVIRE (mimivirus virophage resistance element). Giant viruses of amoebae bring upheaval to the definition of viruses and tend to separate the current virosphere into two categories: very simple viruses and viruses with complexity similar to that of other microbes. This new paradigm is propitious for enhanced detection and characterization of giant viruses of amoebae, and a particular focus on their role in humans is warranted.

Mimivirus: leading the way in the discovery of giant viruses of amoebae.

The accidental discovery of the giant virus of amoeba - Acanthamoeba polyphaga mimivirus (APMV; more commonly known as mimivirus) - in 2003 changed the field of virology. Viruses were previously defined by their submicroscopic size, which probably prevented the search for giant viruses, which are visible by light microscopy. Extended studies of giant viruses of amoebae revealed that they have genetic, proteomic and structural complexities that were not thought to exist among viruses and that are comparable to those of bacteria, archaea and small eukaryotes. The giant virus particles contain mRNA and more than 100 proteins, they have gene repertoires that are broader than those of other viruses and, notably, some encode translation components. The infection cycles of giant viruses of amoebae involve virus entry by amoebal phagocytosis and replication in viral factories. In addition, mimiviruses are infected by virophages, defend against them through the mimivirus virophage resistance element (MIMIVIRE) system and have a unique mobilome. Overall, giant viruses of amoebae, including mimiviruses, marseilleviruses, pandoraviruses, pithoviruses, faustoviruses and molliviruses, challenge the definition and classification of viruses, and have increasingly been detected in humans.

Amoebae, Giant Viruses, and Virophages Make Up a Complex, Multilayered Threesome.

Viral infection had not been observed for amoebae, until the Acanthamoeba polyphaga mimivirus (APMV) was discovered in 2003. APMV belongs to the nucleocytoplasmatic large DNA virus (NCLDV) family and infects not only A. polyphaga, but also other professional phagocytes. Here, we review the Megavirales to give an overview of the current members of the Mimi- and Marseilleviridae families and their structural features during amoebal infection. We summarize the different steps of their infection cycle in A. polyphaga and Acanthamoeba castellani. Furthermore, we dive into the emerging field of virophages, which parasitize upon viral factories of the Megavirales family. The discovery of virophages in 2008 and research in recent years revealed an increasingly complex network of interactions between cell, giant virus, and virophage. Virophages seem to be highly abundant in the environment and occupy the same niches as the Mimiviridae and their hosts. Establishment of metagenomic and co-culture approaches rapidly increased the number of detected virophages over the recent years. Genetic interaction of cell and virophage might constitute a potent defense machinery against giant viruses and seems to be important for survival of the infected cell during mimivirus infections. Nonetheless, the molecular events during co-infection and the interactions of cell, giant virus, and virophage have not been elucidated, yet. However, the genetic interactions of these three, suggest an intricate, multilayered network during amoebal (co-)infections. Understanding these interactions could elucidate molecular events essential for proper viral factory activity and could implicate new ways of treating viruses that form viral factories.

Viruses in close associations with free-living amoebae.

As both groups of organisms, free-living amoebae (FLA) and viruses, can be found in aquatic environments side by side, it appears obvious that there are multiple interactions with respect to host-endocytobiont relationships. Several relationships between viruses and protozoan hosts are described and it was the discovery of the so called "giant viruses," associated with amoebae, which gave another dimension to these interactions. Mimiviruses, Pandoraviruses and Pithoviruses are examples for interesting viral endocytobionts within FLA. In the Mimivirus viral factories, viral DNA undergoes replication and transcription, and the DNA is prepared to be packed in procapsids. Theses Mimivirus factories can be considered as efficient "production lines" where, at any given moment, all stages of viral generation including membrane biogenesis, capsid assembly and genome encapsidation, are occurring concomitantly. There are some hints that similar replication factories are involved as well during the Pandoravirus development. Some scientists favour the assumption that the giant viruses have received many of their genes from their hosts or from sympatric occurring endocytobionts via lateral gene transfer. This hypothesis would mean that this type of transfer has been an important process in the evolution of genomes in the context of the intracellular parasitic or endocytobiotic lifestyle. In turn, that would migitate against hypothesizing development of a new branch in the tree of life. Based on the described scenarios to explain the presence of genes related to translation, it is also possible that earlier ancestors of today's DNA viruses were involved in the origin of eukaryotes. That possibly could in turn support the idea that cellular organisms could have evolved from viruses with growing autarkic properties. In future we expect the discovery of further (giant) viruses within free-living amoebae and other protozoa through genomic, transcriptomic and proteomic analyses.

Looking at protists as a source of pathogenic viruses.

In the environment, protozoa are predators of bacteria and feed on them. The possibility that some protozoa could be a source of human pathogens is consistent with the discovery that free-living amoebae were the reservoir of Legionella pneumophila, the agent of Legionnaires' disease. Later, while searching for Legionella in the environment using amoeba co-culture, the first giant virus, Acanthamoeba polyphaga mimivirus, was discovered. Since then, many other giant viruses have been isolated, including Marseilleviridae, Pithovirus sibericum, Cafeteria roenbergensis virus and Pandoravirus spp. The methods used to isolate all of these viruses are herein reviewed. By analogy to Legionella, it was originally suspected that these viruses could be human pathogens. After showing by indirect evidence, such as sero-epidemiologic studies, that it was possible for these viruses to be human pathogens, the recent isolation of some of these viruses (belonging to the Mimiviridae and Marseilleviridae families) in humans in the context of pathologic conditions shows that they are opportunistic human pathogens in some instances.

Infection cycles of large DNA viruses: emerging themes and underlying questions.

The discovery of giant DNA viruses and the recent realization that such viruses are diverse and abundant blurred the distinction between viruses and cells. These findings elicited lively debates on the nature and origin of viruses as well as on their potential roles in the evolution of cells. The following essay is, however, concerned with new insights into fundamental structural and physical aspects of viral replication that were derived from studies conducted on large DNA viruses. Specifically, the entirely cytoplasmic replication cycles of Mimivirus and Vaccinia are discussed in light of the highly limited trafficking of large macromolecules in the crowded cytoplasm of cells. The extensive spatiotemporal order revealed by cytoplasmic viral factories is described and contended to play an important role in promoting the efficiency of these 'nuclear-like' organelles. Generation of single-layered internal membrane sheets in Mimivirus and Vaccinia, which proceeds through a novel membrane biogenesis mechanism that enables continuous supply of lipids, is highlighted as an intriguing case study of self-assembly. Mimivirus genome encapsidation was shown to occur through a portal different from the 'stargate' portal that is used for genome release. Such a 'division of labor' is proposed to enhance the efficacy of translocation processes of very large viral genomes. Finally, open questions concerning the infection cycles of giant viruses to which future studies are likely to provide novel and exciting answers are discussed.

Growing a giant: evaluation of the virological parameters for mimivirus production.

Acanthamoeba polyphaga mimivirus (APMV) was described in 2003, and due to its unique structural and genetic complexity, the viral family Mimiviridae was created. APMV prompted the creation of an open field of study on the function of hundreds of never-before-seen open reading frames (ORFs) and their roles in virus-host interactions. In recent years, several giant viruses have been isolated from different environments and specimens. Although the scientific community has experienced a remarkable advancement in the comprehension of the mimivirus replication cycle in the last years, few studies have been devoted to the investigation of the methodological features and conditions for mimivirus cultivation. In this work, conditions for the cultivation of mimivirus isolates were investigated to obtain relevant information about the production of infectious particles, total viral particles and viral DNA. The results suggest that low viral doses are more efficient for the production of infectious particles, yielding up to 5000 TCID50 for each inoculated TCID50. Besides methodological information, these data also reveal, for the first time, the ratio between total and infectious particles (in TCID50) that are produced during mimivirus cultivation in laboratory conditions. All of this information can be used as a worldwide guide for the production of mimiviruses and can help prompt mimivirological studies in different fields.

Giant viruses of amoebae as potential human pathogens.

Giant viruses infecting phagocytic protists are composed of mimiviruses, the record holders of particle and genome size amongst viruses, and marseilleviruses. Since the discovery in 2003 at our laboratory of the first of these giant viruses, the Mimivirus, a growing body of data has revealed that they are common inhabitants of our biosphere. Moreover, from the outset, the story of Mimivirus has been linked to that of patients exhibiting pneumonia and it was shown that patients developed antibodies to this amoebal pathogen. Since then, there have been several proven cases of human infection or colonization with giant viruses of amoebae, which are known to host several bacteria that are human pathogens. Mimiviruses and marseilleviruses represent a major challenge in human pathology, as virological procedures implemented to date have not used appropriate media to allow their culture, and molecular techniques have used filtration steps that likely prevented their detection. Nevertheless, there is an increasing body of evidence that mimiviruses might cause pneumonia and that humans carry marseilleviruses, and re-analyses of metagenomic databases have provided evidence that these giant viruses can be common in human samples. The proportion of human infections related to these giant mimiviruses and marseilleviruses and the precise short- and long-term consequences of these infections have been scarcely investigated so far and should be the subject of future works.

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.