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.

A Brief History of Giant Viruses' Studies in Brazilian Biomes.

Almost two decades after the isolation of the first amoebal giant viruses, indubitably the discovery of these entities has deeply affected the current scientific knowledge on the virosphere. Much has been uncovered since then: viruses can now acknowledge complex genomes and huge particle sizes, integrating remarkable evolutionary relationships that date as early as the emergence of life on the planet. This year, a decade has passed since the first studies on giant viruses in the Brazilian territory, and since then biomes of rare beauty and biodiversity (Amazon, Atlantic forest, Pantanal wetlands, Cerrado savannas) have been explored in the search for giant viruses. From those unique biomes, novel viral entities were found, revealing never before seen genomes and virion structures. To celebrate this, here we bring together the context, inspirations, and the major contributions of independent Brazilian research groups to summarize the accumulated knowledge about the diversity and the exceptionality of some of the giant viruses found in Brazil.

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.

Virus-encoded histone doublets are essential and form nucleosome-like structures.

The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of "minimalist" histones in archaea and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones are essential for viral infectivity, localize to cytoplasmic viral factories after virus infection, and ultimately are found in the mature virions. Cryogenic electron microscopy (cryo-EM) structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of "organisms" that require nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses.

Extracellular amoebal-vesicles: potential transmission vehicles for respiratory viruses.

Human respiratory syncytial virus (RSV) is a major cause of acute respiratory tract infections in children and immunocompromised adults worldwide. Here we report that amoebae-release respirable-sized vesicles containing high concentrations of infectious RSV that persisted for the duration of the experiment. Given the ubiquity of amoebae in moist environments, our results suggest that extracellular amoebal-vesicles could contribute to the environmental persistence of respiratory viruses, including potential resistance to disinfection processes and thereby offering novel pathways for viral dissemination and transmission.

Alohomora! What the entry mechanisms tell us about the evolution and diversification of giant viruses and their hosts.

The virosphere is fascinatingly vast and diverse, but as mandatory intracellular parasites, viral particles must reach the intracellular space to guarantee their species' permanence on the planet. While most known viruses that infect animals explore the endocytic pathway to enter the host cell, a diverse group of ancient viruses that make up the phylum Nucleocytoviricota appear to have evolved to explore new access' routes to the cell's cytoplasm. Giant viruses of amoeba take advantage of the phagocytosis process that these organisms exploit a lot, while phycodnavirus must actively break through a algal cellulose cell wall. The mechanisms of entry into the cell and the viruses themselves are diverse, varying in the steps of adhesion, entry, and uncoating. These are clues left by evolution about how these organisms shaped and were shaped by convoluting with eukaryotes.

Amoeba Genome Reveals Dominant Host Contribution to Plastid Endosymbiosis.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function ("dark" genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

Can Neurotropic Free-Living Amoeba Serve as a Model to Study SARS-CoV-2 Pathogenesis?

Of the single-celled eukaryotic microbes, Naegleria fowleri, Balamuthia mandrillaris, and Acanthamoeba spp. are known to cause fatal encephalitis in humans. Being eukaryotes, these cells have been used as a model for studying and understanding complex cellular processes in humans like cell motility, phagocytosis, and metabolism. The ongoing pandemic caused by SARS-CoV-2 that infects multiple organs has emerged as a challenge to unravel its mode of infection and the pathogenicity resulting in eukaryotic cell death. Working with these single-celled eukaryotic microbes provided us the opportunity to plan bioinformatic approaches to look into the likelihood of studying the known and alternative mode of infection of the SARS-CoV-2 in eukaryotic cells. Genome databases of N. fowleri, B. mandrillaris, and Acanthamoeba spp. were used to explore the expression of angiotensin-converting enzyme 2 (ACE2), androgen-regulated serine protease precursor (TMPRSS2), CD4, CD147, and furin that are known to be cardinal for SARS-CoV-2 in recognition and binding to human cells. It was hypothesized that if a receptor-dependent or phagocytosis-assisted SARS-CoV-2 uptake does occur in free-living amoebae (FLA), this model can provide an alternative to human cells to study cellular recognition and binding of SARS-CoV-2 that can help design drugs and treatment modalities in COVID-19. We show that, of the FLA, ACE2 and TMPRSS2 are not expressed in Acanthamoeba spp. and B. mandrillaris, but primitive forms of these cell recognition proteins were seen to be encoded in N. fowleri. Acanthamoeba spp. and N. fowleri encode for human-like furin which is a known SARS-CoV-2 spike protein involved in host cell recognition and binding.

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.

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation.

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

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.

Protozoal giant viruses: agents potentially infectious to humans and animals.

The discovery of giant viruses has revolutionised the knowledge on viruses and transformed the idea of three domains of life. Here, we discuss the known protozoal giant viruses and their potential to infect also humans and animals.

Free-living amoebae and squatters in the wild: ecological and molecular features.

Free-living amoebae are protists frequently found in water and soils. They feed on other microorganisms, mainly bacteria, and digest them through phagocytosis. It is accepted that these amoebae play an important role in the microbial ecology of these environments. There is a renewed interest for the free-living amoebae since the discovery of pathogenic bacteria that can resist phagocytosis and of giant viruses, underlying that amoebae might play a role in the evolution of other microorganisms, including several human pathogens. Recent advances, using molecular methods, allow to bring together new information about free-living amoebae. This review aims to provide a comprehensive overview of the newly gathered insights into (1) the free-living amoeba diversity, assessed with molecular tools, (2) the gene functions described to decipher the biology of the amoebae and (3) their interactions with other microorganisms in the environment.

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 multiple origins of proteins present in tupanvirus particles.

In the last few decades, the isolation of amoebae-infecting giant viruses has challenged established principles related to the definition of virus, their evolution, and their particle structures represented by a variety of shapes and sizes. Tupanviruses are one of the most recently described amoebae-infecting viruses and exhibit a peculiar morphology with a cylindrical tail attached to the capsid. Proteomic analysis of purified viral particles revealed that virions are composed of over one hundred proteins with different functions. The putative origin of these proteins had not yet been investigated. Here, we provide evidences for multiple origins of the proteins present in tupanvirus particles, wherein 20% originate from members of the archaea, bacteria and eukarya.

Tupanvirus-infected amoebas are induced to aggregate with uninfected cells promoting viral dissemination.

The discovery of giant viruses in the last years has fascinated the scientific community due to virus particles size and genome complexity. Among such fantastic discoveries, we have recently described tupanviruses, which particles present a long tail, and has a genome that contains the most complete set of translation-related genes ever reported in the known virosphere. Here we describe a new kind of virus-host interaction involving tupanvirus. We observed that tupanvirus-infected amoebas were induced to aggregate with uninfected cells, promoting viral dissemination and forming giant host cell bunches. Even after mechanical breakdown of bunches, amoebas reaggregated within a few minutes. This remarkable interaction between infected and uninfected cells seems to be promoted by the expression of a mannose receptor gene. Our investigations demonstrate that the pre-treatment of amoebas with free mannose inhibits the formation of bunches, in a concentration-dependent manner, suggesting that amoebal-bunch formation correlates with mannose receptor gene expression. Finally, our data suggest that bunch-forming cells are able to interact with uninfected cells promoting the dissemination and increase of tupanvirus progeny.

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.