Giant Virus Global Proteomics Innovation: Comparative Evaluation of In-Gel and In-Solution Digestion Methods.

With their unusually large genome and particle sizes, giant viruses (GVs) defy the conventional definition of viruses. Although most GVs isolated infect unicellular protozoans, such as amoeba, studies in the last decade have established their much wider prevalence infecting most eukaryotic supergroups and some giant viral families with the potential to be human pathogens. Their complexity, almost autonomous life cycle, and enigmatic evolution necessitate the study of GVs. The accurate assessment of GV proteome is a veritable challenge. We have compared the coverage of global protein identification using different methods for GVs isolated in Mumbai, Mimivirus Bombay (MVB), Powai Lake Megavirus (PLMV), and Kurlavirus (KV), along with two previously studied GVs, Acanthamoeba polyphaga Mimivirus (APMV) and Marseillevirus (MV). Our study shows that the simultaneous use of in-gel and in-solution digestion methods can significantly increase the coverage of protein identification in the global proteome analysis of purified GV particles. Combining the two methods of analyses, we identified an additional 72 proteins in APMV and 114 in MV compared with what have been previously reported. Similarly, proteomes of MVB, PLMV, and KV were analyzed, and a total of 242 proteins in MVB, 287 proteins in PLMV, and 174 proteins in KV were identified. Our results suggest that a combined methodology of in-gel and in-solution methods is more efficient and opens up new avenues for innovation in global proteome analysis of GVs. Future planetary health research on GVs can benefit from consideration of a broader range of proteomics methodologies as illustrated by the present study.

Could giant viruses be considered as a biotechnological tool for preventing and controlling Acanthamoeba infections?

AIM: The aim of the study was to evaluate the efficiency of mimivirus as a potential therapeutic and prophylactic tool against Acanthamoeba castellanii, the etiological agent of Acanthamoeba keratitis, a progressive corneal infection, that is commonly associated with the use of contact lenses and can lead to blindness if not properly treated. METHODS AND RESULTS: Mimivirus particles were tested in different multiplicity of infection, along with commercial multipurpose contact lenses' solutions, aiming to assess their ability to prevent encystment and excystment of A. castellanii. Solutions were evaluated for their amoebicidal potential and cytotoxicity in MDCK cells, as well as their effectiveness in preventing A. castellanii damage in Madin-Darby canine kidney (MDCK) cells. Results indicated that mimivirus was able to inhibit the formation of A. castellanii cysts, even in the presence of Neff encystment solution. Mimivirus also showed greater effectiveness in controlling A. castellanii excystment compared to commercial solutions. Additionally, mimivirus solution was more effective in preventing damage caused by A. castellanii, presented greater amoebicidal activity, and were less cytotoxic to MDCK cells than commercial MPS. CONCLUSIONS: Mimivirus demonstrates a greater ability to inhibit A. castellanii encystment and excystment compared to commercial multipurpose contact lens solutions. Additionally, mimivirus is less toxic to MDCK cells than those commercial solutions. New studies utilizing in vivo models will be crucial for confirming safety and efficacy parameters.

Surface fibrils on the particles of nucleocytoviruses: A review.

The capsid has a central role in viruses' life cycle. Although one of its major functions is to protect the viral genome, the capsid may be composed of elements that, at some point, promote interaction with host cells and trigger infection. Considering the scenario of multiple origins of viruses along the viral evolution, a substantial number of capsid shapes, sizes, and symmetries have been described. In this context, capsids of giant viruses (GV) that infect protists have drawn the attention of the scientific community, especially in the last 20 years, specifically for having bacterial-like dimensions with hundreds of different proteins and exclusive features. For instance, the surface fibrils present on the mimivirus capsid are one of the most intriguing features of the known virosphere. They are 150-nm-long structures attached to a 450-nm capsid, resulting in a particle with a hairy appearance. Surface fibrils have also been described in the capsids of other nucleocytoviruses, although they may differ substantially among them. In this mini review for non-experts, we compile the most important available information on surface fibrils of nucleocytoviruses, discussing their putative functions, composition, length, organization, and origins.

Taxonomic update for giant viruses in the order Imitervirales (phylum Nucleocytoviricota).

Large DNA viruses in the phylum Nucleocytoviricota, sometimes referred to as "giant viruses" owing to their large genomes and virions, have been the subject of burgeoning interest over the last decade. Here, we describe recently adopted taxonomic updates for giant viruses within the order Imitervirales. The families Allomimiviridae, Mesomimiviridae, and Schizomimiviridae have been created to accommodate the increasing diversity of mimivirus relatives that have sometimes been referred to in the literature as "extended Mimiviridae". In addition, the subfamilies Aliimimivirinae, Megamimivirinae, and Klosneuvirinae have been established to refer to subgroups of the Mimiviridae. Binomial names have also been adopted for all recognized species in the order. For example, Acanthamoeba polyphaga mimivirus is now classified in the species Mimivirus bradfordmassiliense.

A rapid genome-wide analysis of isolated giant viruses using MinION sequencing.

Following the discovery of Acanthamoeba polyphaga mimivirus, diverse giant viruses have been isolated. However, only a small fraction of these isolates have been completely sequenced, limiting our understanding of the genomic diversity of giant viruses. MinION is a portable and low-cost long-read sequencer that can be readily used in a laboratory. Although MinION provides highly error-prone reads that require correction through additional short-read sequencing, recent studies assembled high-quality microbial genomes only using MinION sequencing. Here, we evaluated the accuracy of MinION-only genome assemblies for giant viruses by re-sequencing a prototype marseillevirus. Assembled genomes presented over 99.98% identity to the reference genome with a few gaps, demonstrating a high accuracy of the MinION-only assembly. As a proof of concept, we de novo assembled five newly isolated viruses. Average nucleotide identities to their closest known relatives suggest that the isolates represent new species of marseillevirus, pithovirus and mimivirus. The assembly of subsampled reads demonstrated that their taxonomy and genomic composition could be analysed at the 50× sequencing coverage. We also identified a pithovirus gene whose homologues were detected only in metagenome-derived relatives. Collectively, we propose that MinION-only assembly is an effective approach to rapidly perform a genome-wide analysis of isolated giant viruses.

From Mimivirus to Mirusvirus: The Quest for Hidden Giants.

Our perception of viruses has been drastically evolving since the inception of the field of virology over a century ago. In particular, the discovery of giant viruses from the Nucleocytoviricota phylum marked a pivotal moment. Their previously concealed diversity and abundance unearthed an unprecedented complexity in the virus world, a complexity that called for new definitions and concepts. These giant viruses underscore the intricate interactions that unfold over time between viruses and their hosts, and are themselves suspected to have played a significant role as a driving force in the evolution of eukaryotes since the dawn of this cellular domain. Whether they possess exceptional relationships with their hosts or whether they unveil the actual depths of evolutionary connections between viruses and cells otherwise hidden in smaller viruses, the attraction giant viruses exert on the scientific community and beyond continues to grow. Yet, they still hold surprises. Indeed, the recent identification of mirusviruses connects giant viruses to herpesviruses, each belonging to distinct viral realms. This discovery substantially broadens the evolutionary landscape of Nucleocytoviricota. Undoubtedly, the years to come will reveal their share of surprises.

Asfarviruses and Closely Related Giant Viruses.

Acanthamoeba polyphaga mimivirus, so called because of its "mimicking microbe", was discovered in 2003 and was the founding member of the first family of giant viruses isolated from amoeba. These giant viruses, present in various environments, have opened up a previously unexplored field of virology. Since 2003, many other giant viruses have been isolated, founding new families and taxonomical groups. These include a new giant virus which was isolated in 2015, the result of the first co-culture on Vermamoeba vermiformis. This new giant virus was named "Faustovirus". Its closest known relative at that time was African Swine Fever Virus. Pacmanvirus and Kaumoebavirus were subsequently discovered, exhibiting phylogenetic clustering with the two previous viruses and forming a new group with a putative common ancestor. In this study, we aimed to summarise the main features of the members of this group of giant viruses, including Abalone Asfarvirus, African Swine Fever Virus, Faustovirus, Pacmanvirus, and Kaumoebavirus.

Giant Virus Infection Signatures Are Modulated by Euphotic Zone Depth Strata and Iron Regimes of the Subantarctic Southern Ocean.

Viruses can alter the abundance, evolution, and metabolism of microorganisms in the ocean, playing a key role in water column biogeochemistry and global carbon cycles. Large efforts to measure the contribution of eukaryotic microorganisms (e.g., protists) to the marine food web have been made, yet the in situ activities of the ecologically relevant viruses that infect these organisms are not well characterized. Viruses within the phylum Nucleocytoviricota ("giant viruses") are known to infect a diverse range of ecologically relevant marine protists, yet how these viruses are influenced by environmental conditions remains under-characterized. By employing metatranscriptomic analyses of in situ microbial communities along a temporal and depth-resolved gradient, we describe the diversity of giant viruses at the Southern Ocean Time Series (SOTS), a site within the subpolar Southern Ocean. Using a phylogeny-guided taxonomic assessment of detected giant virus genomes and metagenome-assembled genomes, we observed depth-dependent structuring of divergent giant virus families mirroring dynamic physicochemical gradients in the stratified euphotic zone. Analyses of transcribed metabolic genes from giant viruses suggest viral metabolic reprogramming of hosts from the surface to a 200-m depth. Lastly, using on-deck incubations reflecting a gradient of iron availability, we show that modulating iron regimes influences the activity of giant viruses in the field. Specifically, we show enhanced infection signatures of giant viruses under both iron-replete and iron-limited conditions. Collectively, these results expand our understanding of how the water column's vertical biogeography and chemical surroundings affect an important group of viruses within the Southern Ocean. IMPORTANCE The biology and ecology of marine microbial eukaryotes is known to be constrained by oceanic conditions. In contrast, how viruses that infect this important group of organisms respond to environmental change is less well known, despite viruses being recognized as key microbial community members. Here, we address this gap in our understanding by characterizing the diversity and activity of "giant" viruses within an important region in the sub-Antarctic Southern Ocean. Giant viruses are double-stranded DNA (dsDNA) viruses of the phylum Nucleocytoviricota and are known to infect a wide range of eukaryotic hosts. By employing a metatranscriptomics approach using both in situ samples and microcosm manipulations, we illuminated both the vertical biogeography and how changing iron availability affects this primarily uncultivated group of protist-infecting viruses. These results serve as a foundation for our understanding of how the open ocean water column structures the viral community, which can be used to guide models of the viral impact on marine and global biogeochemical cycling.

Evidence for [2Fe-2S]2+ and Linear [3Fe-4S]1+ Clusters in a Unique Family of Glycine/Cysteine-Rich Fe-S Proteins from Megavirinae Giant Viruses.

We have discovered a protein with an amino acid composition exceptionally rich in glycine and cysteine residues in the giant virus mimivirus. This small 6 kDa protein is among the most abundant proteins in the icosahedral 0.75 µm viral particles; it has no predicted function but is probably essential for infection. The aerobically purified red-brownish protein overproduced inEscherichia coli contained both iron and inorganic sulfide. UV/vis, EPR, and Mössbauer studies revealed that the viral protein, coined GciS, accommodated two distinct Fe-S clusters: a diamagnetic S = 0 [2Fe-2S]2+ cluster and a paramagnetic S = 5/2 linear [3Fe-4S]1+ cluster, a geometry rarely stabilized in native proteins. Orthologs of mimivirus GciS were identified within all clades of Megavirinae, a Mimiviridae subfamily infecting Acanthamoeba, including the distantly related tupanviruses, and displayed the same spectroscopic features. Thus, these glycine/cysteine-rich proteins form a new family of viral Fe-S proteins sharing unique Fe-S cluster binding properties.

Amoebal Tubulin Cleavage Late during Infection Is a Characteristic Feature of Mimivirus but Not of Marseillevirus.

Mimivirus and Marseillevirus infections of Acanthamoeba castellanii, like most other viral infections, induce cytopathic effects (CPE). The details of how they bring about CPE and to what extent and how they modify the host cytoskeletal network are unclear. In this study, we compared the rearrangement of the host cytoskeletal network induced by Mimivirus and Marseillevirus upon infection. We show that while both Mimivirus and Marseillevirus infections of A. castellanii cells cause retraction of acanthopodia and depolymerization of the host actin filament network, the Mimivirus infection also results in characteristic cleavage of the host tubulin, a phenomenon not previously reported with any intracellular pathogens. Furthermore, we show that the amoebal tubulin cleavage during Mimivirus infection is a post-replicative event. Because time-lapse microscopy showed that Mimivirus infection leads to the bursting of cells, releasing the virus, we hypothesize that tubulin cleavage together with actin depolymerization during the later stages of Mimivirus assembly is essential for cell lysis due to apoptotic/necrotic cell death. We also characterize the Mimivirus-encoded gp560, a Zn metalloprotease, however, the purified gp560 protein was unable to cleave the commercially available porcine brain tubulin. While protein synthesis is essential for causing the morphological changes in the case of Mimivirus, the proteins which are packaged in the viral capsid along with the genome are sufficient to induce CPE in the case of Marseillevirus. IMPORTANCE In general, intracellular pathogens target the cytoskeletal network to enable their life cycle inside the host. Pathogen-induced changes in the host cell morphology usually accompany global changes in the cytoskeleton resulting in cytopathic effects. While viruses have been shown to use the host actin cytoskeleton for entry and transport during early infection, the role of microtubules in the viral life cycle is only beginning to emerge. Here, we show that the giant viruses Mimivirus and Marseillevirus both induce depolymerization of the actin filament, Mimivirus also causes a characteristic cleavage of tubulin not previously reported for any intracellular pathogen. Because tubulin cleavage occurs late during infection, we hypothesize that tubulin cleavage aids in cell death and lysis rather than establishing infection. The different strategies used by viruses with similar host niches may help them survive in competition.

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.

The giant mimivirus 1.2 Mb genome is elegantly organized into a 30-nm diameter helical protein shield.

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the ~0.5 µm icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography, and proteomics revealed that it is encased into a ~30-nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal.

Mimiviruses: Giant viruses with novel and intriguing features (Review).

The Mimivirus is a giant virus that infects amoebae and was long considered to be a bacterium due to its size. The viral particles are composed of a protein capsid of ~500 nm in diameter, which is enclosed in a polysaccharide layer in which ~120­140 nm long fibers are embedded, resulting in an overall diameter of 700 nm. The virus has a genome size of 1.2 Mb DNA, and surprisingly, replicates only in the cytoplasm of the infected cells without entering the nucleus, which is a unique characteristic among DNA viruses. Their existence is undeniable; however, as with any novel discovery, there is still uncertainty concerning their pathogenicity mechanisms in humans and the nature of the Mimivirus virophage resistance element system (MIMIVIRE), a term given to describe the immune network of the Mimivirus, which closely resembles the CRISPR­Cas system. The scope of the present review is to discuss the recent developments derived from structural and functional studies performed on the distinctive characteristics of the Mimivirus, and from studies concerning their putative clinical relevance in humans.

Detection of Mimivirus from respiratory samples in tuberculosis-suspected patients.

Acanthamoeba polyphaga mimivirus (APMV), a species of amoeba-infecting giant viruses, has recently emerged as human respiratory pathogens. This study aimed to evaluate the presence of Mimivirus in respiratory samples, collected from tuberculosis (TB)-suspected patients. The study was performed on 10,166 clinical respiratory samples from April 2013 to December 2017. Mimivirus was detected using a suicide nested-polymerase chain reaction (PCR) and real-time PCR methods. Of 10,166 TB-suspected patients, 4 (0.04%) were positive for Mimivirus, including Mimivirus-53, Mimivirus-186, Mimivirus-1291, and Mimivirus-1922. Three out of four patients, hospitalized in the intensive care unit (ICU), were mechanically ventilated. All patients had an underlying disease, and the virus was detected in both sputum and bronchoalveolar lavage samples. In conclusion, Mimivirus was isolated from TB-suspected patients in a comprehensive study. The present results, similar to previous reports, showed that Mimiviruses could be related to pneumonia. Further studies in different parts of the world are needed to additional investigate the clinical importance of Mimivirus infection.

Isolation and Identification of a Large Green Alga Virus (Chlorella Virus XW01) of Mimiviridae and Its Virophage (Chlorella Virus Virophage SW01) by Using Unicellular Green Algal Cultures.

Virophages are a group of small double-stranded DNA viruses that infect protist hosts and parasitize the viral factory of host giant/large viruses to propagate. Here, we discover a novel cell-virus-virophage (CVv) tripartite interaction system by using unicellular micro-green algae (Chlorella sp.) as eukaryotic hosts for the first time. Viral particles, resembling known virophages and large alga viruses, are detected in culture supernatants and inside algal cells. Complete genomic sequences of the virophage (Chlorella virus virophage SW01 [CVv-SW01]; 24,744 bp) and large virus (Chlorella virus XW01 [CV-XW01]; 407,612 bp) are obtained from the cocultures. Both genomic and phylogenetic analyses show that CVv-SW01 is closely related to virophages previously found in Dishui Lake. CV-XW01 shares the greatest number of homologous genes (n = 82) with Cafeteria roenbergensis virus (CroV) and phylogenetically represents the closest relative to CroV. This is the first report of a large green alga virus being affiliated with a heterotrophic zooplankton-infecting Cafeteriavirus of the family Mimiviridae. Moreover, the codon usage preferences of CV-XW01 and CVv-SW01 are highly similar to those of CroV and its virophage Mavirus, respectively. The discovery of such a novel CVv system with the green alga Chlorella sp. as the single cellular eukaryotic host paves a way to further investigate the potential interaction mechanism of CVv and its significance in the ecology of green algae and the evolution of large/giant viruses and their parasitic viruses. IMPORTANCE Parasitic virophages are small unicellular eukaryotic dsDNA viruses that rely on the viral factories of coinfecting giant/large dsDNA viruses for propagation. Presently, the identified eukaryotic hosts of isolated virophages were restricted to a free-living amoeba, Acanthamoeba polyphaga, and a widespread marine heterotrophic flagellate, Cafeteria roenbergensis. In this study, we successfully discovered and identified a novel tripartite interaction system comprised of a micro-green alga (Chlorella sp.), Mimiviridae large green alga virus, and virophage at the coculture level, with Chlorella sp. as the eukaryotic host, based on combination analysis of infection, morphotype, genome, and phylogeny. The large green alga virus CV-XW01 represents the closest relative to the Mimiviridae giant virus Cafeteria roenbergensis virus, host virus of the virophage Mavirus, as well as a novel large virus of Mimiviridae that infects a non-protozoan protist host. The virophage CVv-SW01 highly resembles Mavirus in its codon usage frequency and preference, although they are phylogenetically distantly related. These findings give novel insights into the diversity of large/giant viruses and their virophages.

The Discovery of a New Mimivirus Isolate in Association with Virophage-Transpoviron Elements in Brazil Highlights the Main Genomic and Evolutionary Features of This Tripartite System.

Mimiviruses are giant viruses of amoeba that can be found in association with virophages. These satellite-like viruses are dependent on the mimivirus viral factory to replicate. Mimiviruses can also be associated with linear DNA molecules called transpovirons. Transpovirons and virophages are important drivers of giant virus evolution although they are still poorly studied elements. Here, we describe the isolation and genomic characterization of a mimivirus/virophage/transpoviron tripartite system from Brazil. We analyzed transmission electron microscopy images and performed genome sequencing and assembly, gene annotation, and phylogenetic analysis. Our data confirm the isolation of a lineage A mimivirus (1.2 Mb/1012 ORFs), called mimivirus argentum, and a sputnik virophage (18,880 bp/20 ORFs). We also detected a third sequence corresponding to a transpoviron from clade A (6365 bp/6 ORFs) that presents small terminal inverted repeats (77 nt). The main genomic features of mimivirus argentum and of its virophage/transpoviron elements corroborates with what is described for other known elements. This highlights that this triple genomic and biological interaction may be ancient and well-conserved. The results expand the basic knowledge about unique and little-known elements and pave the way to future studies that might contribute to a better understanding of this tripartite relationship.

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.

Kinetic Analysis of Acanthamoeba castellanii Infected with Giant Viruses Quantitatively Revealed Process of Morphological and Behavioral Changes in Host Cells.

Most virus-infected cells show morphological and behavioral changes, which are called cytopathic effects. Acanthamoeba castellanii, an abundant, free-living protozoan, serves as a laboratory host for some viruses of the phylum Nucleocytoviricota-the giant viruses. Many of these viruses cause cell rounding in the later stages of infection in the host cells. Here, we show the changes that lead to cell rounding in the host cells through time-lapse microscopy and image analysis. Time-lapse movies of A. castellanii cells infected with Mimivirus shirakomae, kyotovirus, medusavirus, or Pandoravirus japonicus were generated using a phase-contrast microscope. We updated our phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) and used it to analyze these time-lapse movies. Image analysis revealed that the process leading to cell rounding varies among the giant viruses; for example, M. shirakomae infection did not cause changes for some time after the infection, kyotovirus infection caused an early decrease in the number of cells with typical morphologies, and medusavirus and P. japonicus infection frequently led to the formation of intercellular bridges and rotational behavior of host cells. These results suggest that in the case of giant viruses, the putative reactions of host cells against infection and the putative strategies of virus spread are diverse. IMPORTANCE Quantitative analysis of the infection process is important for a better understanding of viral infection strategies and virus-host interactions. Here, an image analysis of the phase-contrast time-lapse movies displayed quantitative differences in the process of cytopathic effects due to the four giant viruses in Acanthamoeba castellanii, which were previously unclear. It was revealed that medusavirus and Pandoravirus japonicus infection led to the formation of a significant number of elongated particles related to intercellular bridges, emphasizing the importance of research on the interaction of viruses with host cell nuclear function. Mimivirus shirakomae infection did not cause any changes in the host cells initially, so it is thought that the infected cells can actively move and spread over a wider area, emphasizing the importance of observation in a wider area and analysis of infection efficiency. These results suggest that a kinetic analysis using the phase-contrast-based kinetic analysis algorithm for amoebae (PKA3) reveals the infection strategies of each giant virus.

Structure and physiology of giant DNA viruses.

Although giant viruses have existed for millennia and possibly exerted great evolutionary influence in their environment. Their presence has only been noticed by virologists recently with the discovery of Acanthamoeba polyphaga mimivirus in 2003. Its virion with a diameter of 500 nm and its genome larger than 1 Mpb shattered preconceived standards of what a virus is and triggered world-wide prospection studies. Thanks to these investigations many giant virus families were discovered, each with its own morphological peculiarities and genomes ranging from 0.4 to 2.5 Mpb that possibly encode more than 400 viral proteins. This review aims to present the morphological diversity, the different aspects observed in host-virus interactions during replication, as well as the techniques utilized during their investigation.

The Virophage Family Lavidaviridae.

Double-stranded (ds) DNA viruses of the family Lavidaviridae, commonly known as virophages, are a fascinating group of eukaryotic viruses that depend on a coinfecting giant dsDNA virus of the Mimiviridae for their propagation. Instead of replicating in the nucleus, virophages multiply in the cytoplasmic virion factory of a coinfecting giant virus inside a phototrophic or heterotrophic protistal host cell. Virophages are parasites of giant viruses and can inhibit their replication, which may lead to increased survival rates of the infected host cell population. The genomes of virophages are 17-33 kilobase pairs (kbp) long and encode 16-34 proteins. Genetic signatures of virophages can be found in metagenomic datasets from various saltwater and freshwater environments around the planet. Most virophages share a set of conserved genes that code for a major and a minor capsid protein, a cysteine protease, a genome-packaging ATPase, and a superfamily 3 helicase, although the genomes are otherwise diverse and variable. Lavidaviruses share genes with other mobile genetic elements, suggesting that horizontal gene transfer and recombination have been major forces in shaping these viral genomes. Integrases are occasionally found in virophage genomes and enable these DNA viruses to persist as provirophages in the chromosomes of their viral and cellular hosts. As we watch the genetic diversity of this new viral family unfold through metagenomics, additional isolates are still lacking and critical questions regarding their infection cycle, host range, and ecology remain to be answered.