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BACKGROUND Giant viruses have brought new insights into different aspects of virus-cell interactions. The resulting cytopathic effects from these interactions are one of the main aspects of infection assessment in a laboratory routine, mainly reflecting on the morphological features of an infected cell. OBJECTIVES In this work, we follow the entire kinetics of the cytopathic effect in cells infected by viruses of the Mimiviridae family, spatiotemporally quantifying typical features such as cell roundness, loss of motility, decrease in cell area and cell lysis. METHODS Infections by Acanthamoeba polyphaga mimivirus (APMV), Tupanvirus (TPV) and M4 were carried out at multiplicity of infection (MOI) 1 and MOI 10 in Acanthamoeba castellanii. Monitoring of infections was carried out using time lapse microscopy for up to 72 hours. The images were analyzed using ImageJ software. FINDINGS The data obtained indicate that APMV is the slowest virus in inducing the cytopathic effects of rounding, decrease in cell area, mobility and cell lysis. However, it is the only virus whose MOI increase accelerates the lysis process of infected cells. In turn, TPV and M4 rapidly induce morphological and behavioral changes. MAIN CONCLUSIONS Our results indicate that mimiviruses induce different temporal responses within the host cell and that it is possible to use these kinetic data to facilitate the understanding of infection by these viruses.

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Among the most intriguing structural features in the known virosphere are mimivirus surface fibrils, proteinaceous filaments approximately 150 nm long, covering the mimivirus capsid surface. Fibrils are important to promote particle adhesion to host cells, triggering phagocytosis and cell infection. However, although mimiviruses are one of the most abundant viral entities in a plethora of biomes worldwide, there has been no comparative analysis on fibril organization and abundance among distinct mimivirus isolates. Here, we describe the isolation and characterization of Megavirus caiporensis, a novel lineage C mimivirus with surface fibrils organized as "clumps." This intriguing feature led us to expand our analyses to other mimivirus isolates. By employing a combined approach including electron microscopy, image processing, genomic sequencing, and viral prospection, we obtained evidence of at least three main patterns of surface fibrils that can be found in mimiviruses: (i) isolates containing particles with abundant fibrils, distributed homogeneously on the capsid surface; (ii) isolates with particles almost fibrilless; and (iii) isolates with particles containing fibrils in abundance, but organized as clumps, as observed in Megavirus caiporensis. A total of 15 mimivirus isolates were analyzed by microscopy, and their DNA polymerase subunit B genes were sequenced for phylogenetic analysis. We observed a unique match between evolutionarily-related viruses and their fibril profiles. Biological assays suggested that patterns of fibrils can influence viral entry in host cells. Our data contribute to the knowledge of mimivirus fibril organization and abundance, as well as raising questions on the evolution of those intriguing structures. IMPORTANCE Mimivirus fibrils are intriguing structures that have drawn attention since their discovery. Although still under investigation, the function of fibrils may be related to host cell adhesion. In this work, we isolated and characterized a new mimivirus, called Megavirus caiporensis, and we showed that mimivirus isolates can exhibit at least three different patterns related to fibril organization and abundance. In our study, evolutionarily-related viruses presented similar fibril profiles, and such fibrils may affect how those viruses trigger phagocytosis in amoebas. These data shed light on aspects of mimivirus particle morphology, virus-host interactions, and their evolution.

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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.

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BACKGROUND: Viruses are the most numerous entities on Earth and have also been central to many episodes in the history of humankind. As the study of viruses progresses further and further, there are several limitations in transferring this knowledge to undergraduate and high school students. This deficiency is due to the difficulty in designing hands-on lessons that allow students to better absorb content, given limited financial resources and facilities, as well as the difficulty of exploiting viral particles, due to their small dimensions. The development of tools for teaching virology is important to encourage educators to expand on the covered topics and connect them to recent findings. Discoveries, such as giant DNA viruses, have provided an opportunity to explore aspects of viral particles in ways never seen before. Coupling these novel findings with techniques already explored by classical virology, including visualization of cytopathic effects on permissive cells, may represent a new way for teaching virology. This work aimed to develop a slide microscope kit that explores giant virus particles and some aspects of animal virus interaction with cell lines, with the goal of providing an innovative approach to virology teaching. METHODS: Slides were produced by staining, with crystal violet, purified giant viruses and BSC-40 and Vero cells infected with viruses of the genera Orthopoxvirus, Flavivirus, and Alphavirus. Slides with amoebae infected with different species of giant viruses and stained with hemacolor reagents were also produced. RESULTS: Staining of the giant viruses allowed better visualization of the viral particles, and this technique highlights the diversity in morphology and sizes among them. Hemacolor staining enabled visualization of viral factories in amoebae, and the staining of infected BSC-40 and Vero cell monolayers with crystal violet highlights plaque-forming units. CONCLUSIONS: This kit was used in practical virology classes for the Biological Sciences course (UFMG, Brazil), and it will soon be made available at a low-cost for elementary school teachers in institutions that have microscopes. We hope this tool will foster an inspiring learning environment.

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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.

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Virophages are critical regulators of viral population dynamics and potential actors in the stability of the microbial networks. These small biological entities predate the replicative cycle of giant viruses, such as the members of the Mimiviridae family or their distant relatives, which produce within the cytoplasm of their host cells a viral factory harboring a complex biochemistry propitious to the growth of the smaller parasites. In this paper, we describe the isolation and the characterization of a new virophage, the eighth, that we named Guarani. We observed that Guarani exhibits a late replication cycle compared to its giant virus host. In addition, like all Sputnik strains, Guarani is able to infect the three lineages A, B and C of the Mimiviridae family, and affects the replication and the infectivity of its host virus. In terms of genetic content, Guarani has a 18,967 bp long double-stranded DNA genome encoding 22 predicted genes very similar to Sputnik genes, except for ORF19 and ORF12. The former is more related to Zamilon while the latter seems to be novel. The architecture of the Guarani genome is closely related to Sputnik and Zamilon strains, suggesting a common origin for all these virophages.

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BACKGROUND: Since the discovery of giant viruses infecting amoebae in 2003, many dogmas of virology have been revised and the search for these viruses has been intensified. Over the last few years, several new groups of these viruses have been discovered in various types of samples and environments.In this work, we describe the isolation of 68 giant viruses of amoeba obtained from environmental samples from Brazil and Antarctica. METHODS: Isolated viruses were identified by hemacolor staining, PCR assays and electron microscopy (scanning and/or transmission). RESULTS: A total of 64 viruses belonging to the Mimiviridae family were isolated (26 from lineage A, 13 from lineage B, 2 from lineage C and 23 from unidentified lineages) from different types of samples, including marine water from Antarctica, thus being the first mimiviruses isolated in this extreme environment to date. Furthermore, a marseillevirus was isolated from sewage samples along with two pandoraviruses and a cedratvirus (the third to be isolated in the world so far). CONCLUSIONS: Considering the different type of samples, we found a higher number of viral groups in sewage samples. Our results reinforce the importance of prospective studies in different environmental samples, therefore improving our comprehension about the circulation anddiversity of these viruses in nature.

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The inclusion of Mimiviridae members in the putative monophyletic nucleocytoplasmic large DNA virus (NCLDV) group is based on genomic and phylogenomic patterns. This shows that, along with other viral families, they share a set of genes known as core or "hallmark genes," including the gene for the major capsid protein (MCP). Although previous studies have suggested that the maturation of mimivirus MCP transcripts is dependent on splicing, there is little information about the processing of this transcript in other mimivirus isolates. Here we report the characterization of a new mimivirus isolate, called Kroon virus (KV) mimivirus. Analysis of the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates revealed a remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. In addition, sequencing of KV and Acanthamoeba polyphaga mimivirus (APMV) MCP transcripts has shown that inside the family, even related giant viruses may present different ways to process the MCP mRNA. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.IMPORTANCE Mimivirus isolates have been obtained by prospecting studies since 2003. Based on genomic and phylogenomic studies of conserved genes, these viruses have been clustered together with members of six other viral families. Although the major capsid protein (MCP) gene is an important member of the so-called "hallmark genes," there is little information about the processing and structure of this gene in many mimivirus isolates. In this work, we have analyzed the structure, synteny, and phylogenetic relationships of the MCP genes in many mimivirus isolates; these genes showed remarkable variation at position and types of intronic and exonic regions, even for mimiviruses belonging to the same lineage. These results contribute to the understanding of the genetic organization and evolution of the MCP gene in mimiviruses.

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Since the discovery of mimivirus, its unusual structural and genomic features have raised great interest in the study of its biology; however, many aspects concerning its replication cycle remain uncertain. In this study, extensive analyses of electron microscope images, as well as biological assay results, shed light on unclear points concerning the mimivirus replication cycle. We found that treatment with cytochalasin, a phagocytosis inhibitor, negatively impacted the incorporation of mimivirus particles by Acanthamoeba castellanii, causing a negative effect on viral growth in amoeba monolayers. Treatment of amoebas with bafilomicin significantly impacted mimivirus uncoating and replication. In conjunction with microscopic analyses, these data suggest that mimiviruses indeed depend on phagocytosis for entry into amoebas, and particle uncoating (and stargate opening) appears to be dependent on phagosome acidification. In-depth analyses of particle morphogenesis suggest that the mimivirus capsids are assembled from growing lamellar structures. Despite proposals from previous studies that genome acquisition occurs before the acquisition of fibrils, our results clearly demonstrate that the genome and fibrils can be acquired simultaneously. Our data suggest the existence of a specific area surrounding the core of the viral factory where particles acquire the surface fibrils. Furthermore, we reinforce the concept that defective particles can be formed even in the absence of virophages. Our work provides new information about unexplored steps in the life cycle of mimivirus.IMPORTANCE Investigating the viral life cycle is essential to a better understanding of virus biology. The combination of biological assays and microscopic images allows a clear view of the biological features of viruses. Since the discovery of mimivirus, many studies have been conducted to characterize its replication cycle, but many knowledge gaps remain to be filled. In this study, we conducted a new examination of the replication cycle of mimivirus and provide new evidence concerning some stages of the cycle which were previously unclear, mainly entry, uncoating, and morphogenesis. Furthermore, we demonstrate that atypical virion morphologies can occur even in the absence of virophages. Our results, along with previous data, allow us to present an ultimate model for the mimivirus replication cycle.

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Prior to the discovery of the mimivirus in 2003, viruses were thought to be physically small and genetically simple. Mimivirus, with its ~750-nm particle size and its ~1.2-Mbp genome, shattered these notions and changed what it meant to be a virus. Since this discovery, the isolation and characterization of giant viruses has exploded. One of the more recently discovered giant viruses, Samba virus, is a Mimivirus that was isolated from the Rio Negro in the Brazilian Amazon. Initial characterization of Samba has revealed some structural information, although the preparation techniques used are prone to the generation of structural artifacts. To generate more native-like structural information for Samba, we analyzed the virus through cryo-electron microscopy, cryo-electron tomography, scanning electron microscopy, and fluorescence microscopy. These microscopy techniques demonstrated that Samba particles have a capsid diameter of ~527 nm and a fiber length of ~155 nm, making Samba the largest Mimivirus yet characterized. We also compared Samba to a fiberless mimivirus variant. Samba particles, unlike those of mimivirus, do not appear to be rigid, and quasi-icosahedral, although the two viruses share many common features, including a multi-layered capsid and an asymmetric nucleocapsid, which may be common amongst the Mimiviruses.

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The family Mimiviridae, comprised by giant DNA viruses, has been increasingly studied since the isolation of the Acanthamoeba polyphaga mimivirus (APMV), in 2003. In this work, we describe the genome analysis of two new mimiviruses, each isolated from a distinct Brazilian environment. Furthermore, for the first time, we are reporting the genomic characterization of mimiviruses of group C in Brazil (Br-mimiC), where a predominance of mimiviruses from group A has been previously reported. The genomes of the Br-mimiC isolates Mimivirus gilmour (MVGM) and Mimivirus golden (MVGD) are composed of double-stranded DNA molecules of ∼1.2 Mb, each encoding more than 1,100 open reading frames. Genome functional annotations highlighted the presence of mimivirus group C hallmark genes, such as the set of seven aminoacyl-tRNA synthetases. However, the set of tRNA encoded by the Br-mimiC was distinct from those of other group C mimiviruses. Differences could also be observed in a genome synteny analysis, which demonstrated the presence of inversions and loci translocations at both extremities of Br-mimiC genomes. Both phylogenetic and phyletic analyses corroborate previous results, undoubtedly grouping the new Brazilian isolates into mimivirus group C. Finally, an updated pan-genome analysis of genus Mimivirus was performed including all new genomes available until the present moment. This last analysis showed a slight increase in the number of clusters of orthologous groups of proteins among mimiviruses of group A, with a larger increase after addition of sequences from mimiviruses of groups B and C, as well as a plateau tendency after the inclusion of the last four mimiviruses of group C, including the Br-mimiC isolates. Future prospective studies will help us to understand the genetic diversity among mimiviruses.

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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.

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Since the recent discovery of Samba virus, the first representative of the family Mimiviridae from Brazil, prospecting for mimiviruses has been conducted in different environmental conditions in Brazil. Recently, we isolated using Acanthamoeba sp. three new mimiviruses, all of lineage A of amoebal mimiviruses: Kroon virus from urban lake water; Amazonia virus from the Brazilian Amazon river; and Oyster virus from farmed oysters. The aims of this work were to sequence and analyze the genome of these new Brazilian mimiviruses (mimi-BR) and update the analysis of the Samba virus genome. The genomes of Samba virus, Amazonia virus and Oyster virus were 97%-99% similar, whereas Kroon virus had a low similarity (90%-91%) with other mimi-BR. A total of 3877 proteins encoded by mimi-BR were grouped into 974 orthologous clusters. In addition, we identified three new ORFans in the Kroon virus genome. Additional work is needed to expand our knowledge of the diversity of mimiviruses from Brazil, including if and why among amoebal mimiviruses those of lineage A predominate in the Brazilian environment.

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The complexity of giant virus genomes is intriguing, especially the presence of genes encoding components of the protein translation machinery such as transfer RNAs and aminoacyl-tRNA-synthetases; these features are uncommon among other viruses. Although orthologs of these genes are codified by their hosts, one can hypothesize that having these translation-related genes might represent a gain of fitness during infection. Therefore, the aim of this study was to evaluate the expression of translation-related genes by mimivirus during infection of Acanthamoeba castellanii under different nutritional conditions. In silico analysis of amino acid usage revealed remarkable differences between the mimivirus isolates and the A. castellanii host. Relative expression analysis by quantitative PCR revealed that mimivirus was able to modulate the expression of eight viral translation-related genes according to the amoebal growth condition, with a higher induction of gene expression under starvation. Some mimivirus isolates presented differences in translation-related gene expression; notably, polymorphisms in the promoter regions correlated with these differences. Two mimivirus isolates did not encode the tryptophanyl-tRNA in their genomes, which may be linked with low conservation pressure based on amino acid usage analysis. Taken together, our data suggest that mimivirus can modulate the expression of translation-related genes in response to nutrient availability in the host cell, allowing the mimivirus to adapt to different hosts growing under different nutritional conditions.

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BACKGROUND: Mimiviruses have been considered putative emerging pneumonia agents. Pneumonia is a leading cause of death related to infection throughout the world, with approximately 40% of cases presenting unknown etiology. Therefore, identifying new causative agents of community and nosocomial pneumonia is of major public health concern. OBJECTIVE: We evaluated the distribution of these viruses in samples collected from different environments of one of the largest hospitals in Brazilian Southeast. STUDY DESIGN: We analyzed, by molecular and virological approaches, the distribution of mimivirus in 242 samples collected from inanimate surfaces in different hospital facilities. RESULTS: A significant positivity of mimivirus in respiratory-isolation-facilities was observed (p<0.001). CONCLUSION: Although the role of mimivirus as etiological agents of pneumonia is still under investigation, our results demonstrates interesting correlations that strengthens the need for control over the occurrence of these viruses in hospital facilities.

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To investigate circulation of mimiviruses in the Amazon Region of Brazil, we surveyed 513 serum samples from domestic and wild mammals. Neutralizing antibodies were detected in 15 sample pools, and mimivirus DNA was detected in 9 pools of serum from capuchin monkeys and in 16 pools of serum from cattle.

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