The consequences of viral infection on protists.

Protists encompass a vast widely distributed group of organisms, surpassing the diversity observed in metazoans. Their diverse ecological niches and life forms are intriguing characteristics that render them valuable subjects for in-depth cell biology studies. Throughout history, viruses have played a pivotal role in elucidating complex cellular processes, particularly in the context of cellular responses to viral infections. In this comprehensive review, we provide an overview of the cellular alterations that are triggered in specific hosts following different viral infections and explore intricate biological interactions observed in experimental conditions using different host-pathogen groups.

An overview of viral chitinases: General properties and biotechnological potential.

Chitin is a biopolymer profusely present in nature and of pivotal importance as a structural component in cells. It is degraded by chitinases, enzymes naturally produced by different organisms. Chitinases are proteins enrolled in many cellular mechanisms, including the remodeling process of the fungal cell wall, the cell growth process, the autolysis of filamentous fungi, and cell separation of yeasts, among others. These enzymes also have properties with different biotechnological applications. They are used to produce polymers, for biological control, biofilm formation, and as antitumor and anti-inflammatory target molecules. Chitinases are classified into different glycoside hydrolase (GH) families and are widespread in microorganisms, including viruses. Among them, the GH18 family is highly predominant in the viral genomes, being present and active enzymes in baculoviruses and nucleocytoplasmic large DNA viruses (NCLDV), especially chloroviruses from the Phycodnaviridae family. These viral enzymes contain one or more GH domains and seem to be involved during the viral replication cycle. Curiously, only a few DNA viruses have these enzymes, and studying their properties could be a key feature for biological and biotechnological novelties. Here, we provide an overview of viral chitinases and their probable function in viral infection, showing evidence of at least two distinct origins for these enzymes. Finally, we discuss how these enzymes can be applied as biotechnological tools and what one can expect for the coming years on these GHs.

Ultrastructural analysis of monkeypox virus replication in Vero cells.

In early May 2022, the first worldwide monkeypox virus (MPXV) outbreak was reported, with different clinical aspects from previously studied human monkeypox infections. Despite monkeypox medical importance, much of its biological aspects remain to be further investigated. In the present work, we evaluated ultrastructural aspects of MPXV asynchronous infections in Vero cells by transmission electron microscopy (TEM). The viral strain was isolated from a male patient infected during the 2022 outbreak. TEM analysis showed: (i) adhered intracellular mature virus particles before entry of the host cell; (ii) a reorganization of the rough endoplasmic reticulum cisternae into the so-called "mini-nuclei" structure associated with genome replication; and (iii) noticeably different sites within the viral factory presenting granular or fibrillar aspects. We also observed viral crescents, different MPXV particle morphotypes, and cellular alterations induced by infection, such as changes in the cytoskeleton structure and multimembrane vesicles abundance. Taken together, to the best of our knowledge, these results revealed for the first-time ultrastructural aspects of different steps of the MPXV cycle.

Giant Viruses as a Source of Novel Enzymes for Biotechnological Application.

The global demand for industrial enzymes has been increasing in recent years, and the search for new sources of these biological products is intense, especially in microorganisms. Most known viruses have limited genetic machinery and, thus, have been overlooked by the enzyme industry for years. However, a peculiar group of viruses breaks this paradigm. Giant viruses of the phylum Nucleocytoviricota infect protists (i.e., algae and amoebae) and have complex genomes, reaching up to 2.7 Mb in length and encoding hundreds of genes. Different giant viruses have robust metabolic machinery, especially those in the Phycodnaviridae and Mimiviridae families. In this review, we present some peculiarities of giant viruses that infect protists and discuss why they should be seen as an outstanding source of new enzymes. We revisited the genomes of representatives of different groups of giant viruses and put together information about their enzymatic machinery, highlighting several genes to be explored in biotechnology involved in carbohydrate metabolism, DNA replication, and RNA processing, among others. Finally, we present additional evidence based on structural biology using chitinase as a model to reinforce the role of giant viruses as a source of novel enzymes for biotechnological application.

Monitoring the Establishment of VOC Gamma in Minas Gerais, Brazil: A Retrospective Epidemiological and Genomic Surveillance Study.

Since its first identification in Brazil, the variant of concern (VOC) Gamma has been associated with increased infection and transmission rates, hospitalizations, and deaths. Minas Gerais (MG), the second-largest populated Brazilian state with more than 20 million inhabitants, observed a peak of cases and deaths in March-April 2021. We conducted a surveillance study in 1240 COVID-19-positive samples from 305 municipalities distributed across MG's 28 Regional Health Units (RHU) between 1 March to 27 April 2021. The most common variant was the VOC Gamma (71.2%), followed by the variant of interest (VOI) zeta (12.4%) and VOC alpha (9.6%). Although the predominance of Gamma was found in most of the RHUs, clusters of Zeta and Alpha variants were observed. One Alpha-clustered RHU has a history of high human mobility from countries with Alpha predominance. Other less frequent lineages, such as P.4, P.5, and P.7, were also identified. With our genomic characterization approach, we estimated the introduction of Gamma on 7 January 2021, at RHU Belo Horizonte. Differences in mortality between the Zeta, Gamma and Alpha variants were not observed. We reinforce the importance of vaccination programs to prevent severe cases and deaths during transmission peaks.

Amoebae: Hiding in Plain Sight: Unappreciated Hosts for the Very Large Viruses.

For decades, viruses have been isolated primarily from humans and other organisms. Interestingly, one of the most complex sides of the virosphere was discovered using free-living amoebae as hosts. The discovery of giant viruses in the early twenty-first century opened a new chapter in the field of virology. Giant viruses are included in the phylum Nucleocytoviricota and harbor large and complex DNA genomes (up to 2.7 Mb) encoding genes never before seen in the virosphere and presenting gigantic particles (up to 1.5 µm). Different amoebae have been used to isolate and characterize a plethora of new viruses with exciting details about novel viral biology. Through distinct isolation techniques and metagenomics, the diversity and complexity of giant viruses have astonished the scientific community. Here, we discuss the latest findings on amoeba viruses and how using these single-celled organisms as hosts has revealed entities that have remained hidden in plain sight for ages.

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.

Comparative Analysis of Transcriptional Regulation Patterns: Understanding the Gene Expression Profile in Nucleocytoviricota.

The nucleocytoplasmic large DNA viruses (NCLDV) possess unique characteristics that have drawn the attention of the scientific community, and they are now classified in the phylum Nucleocytoviricota. They are characterized by sharing many genes and have their own transcriptional apparatus, which provides certain independence from their host's machinery. Thus, the presence of a robust transcriptional apparatus has raised much discussion about the evolutionary aspects of these viruses and their genomes. Understanding the transcriptional process in NCLDV would provide information regarding their evolutionary history and a better comprehension of the biology of these viruses and their interaction with hosts. In this work, we reviewed NCLDV transcription and performed a comparative functional analysis of the groups of genes expressed at different times of infection of representatives of six different viral families of giant viruses. With this analysis, it was possible to observe a temporal profile of their gene expression and set of genes activated in specific phases throughout the multiplication cycle as a common characteristic of this group. Due to the lack of information regarding the transcriptional regulation process of this group of pathogens, we sought to provide information that contributes to and opens up the field for transcriptional studies of other viruses belonging to Nucleocytoviricota.

Zoonotic vaccinia virus strains belonging to different genetic clades exhibit immunomodulation abilities that are proportional to their virulence.

BACKGROUND: The vaccinia virus (VACV) isolates, Guarani P1 virus (GP1V) and Passatempo virus (PSTV), were isolated during zoonotic outbreaks in Brazil. Each one of them belongs to two different VACV clades, defined by biological aspects that include virulence in mice and phylogenetic analysis. Considering that information about how vaccinia viruses from different groups elicit immune responses in animals is scarce, we investigated such responses in mice infected either by GP1V (group 2) or PSTV (group 1), using VACV Western Reserve strain (VACV-WR) as control. METHODS: The severity of the infections was evaluated in BALB/c mice considering diverse clinical signs and defined scores, and the immune responses triggered by GP1V and PSTV infections were analysed by immune cell phenotyping and intra-cytoplasmic cytokines detection. RESULTS: We detected a reduction in total lymphocytes (CD3 +), macrophages (CD14 +), and NK cells (CD3-CD49 +) in animals infected with VACV-WR or GP1V. The VACV-WR and GP1V viruses, belonging to the most virulent group in a murine model, were able to down-modulate the cell immune responses upon mice infection. In contrast, PSTV, a virus considered less virulent in a murine model, showed little ability to down-modulate the mice immune responses. Mice infected with VACV-WR and GP1V viruses presented significant weight loss and developed lesions in their spleens, as well as damage to liver and lungs whereas mice infected with PSTV developed only moderate clinical signs. CONCLUSIONS: Our results suggest that VACV immunomodulation in vivo is clade-related and is proportional to the strain's virulence upon infection. Our data corroborate the classification of the different Brazilian VACV isolates into clades 1 and 2, taking into account not only phylogenetic criteria, but also clinical and immunological data.

Detection of SARS-CoV-2 RNA on public surfaces in a densely populated urban area of Brazil: A potential tool for monitoring the circulation of infected patients.

The world is experiencing the worst global health crisis in recent decades since December/2019 due to a new pandemic coronavirus. The COVID-19 disease, caused by SARS-CoV-2, has resulted in more than 30 million cases and 950 thousand deaths worldwide as of September 21, 2020. Determining the extent of the virus on public surfaces is critical for understanding the potential risk of infection in these areas. In this study, we investigated the presence of SARS-CoV-2 RNA on public surfaces in a densely populated urban area in Brazil. Forty-nine of 933 samples tested positive (5.25%) for SARS-CoV-2 RNA, including samples collected from distinct material surfaces, including metal and concrete, and distinct places, mainly around hospital care units and public squares. Our data indicated the contamination of public surfaces by SARS-CoV-2, suggesting the circulation of infected patients and the risk of infection for the population. Constant monitoring of the virus in urban areas is required as a strategy to fight the pandemic and prevent further infections.

Pristimerin isolated from Salacia crassifolia (Mart. Ex. Schult.) G. Don. (Celastraceae) roots as a potential antibacterial agent against Staphylococcus aureus.

ETHNOPHARMACOLOGICAL RELEVANCE: Pristimerin is a triterpenoid considered the main component of Salacia crassifolia extracts. This terpene has shown promising antitumor, anti-inflammatory, and antimicrobial effects. Likewise, S. crassifolia has been used in traditional medicine to treat cancer and as an antimicrobial and anti-inflammatory agent. AIM OF THE STUDY: This study aimed to evaluate the antibacterial activity of the hexane extract of Salacia crassifolia roots (HER) and its isolate, pristimerin, against pathogenic bacteria. MATERIALS AND METHODS: First, we evaluated the spectrum of action of HER and pristimerin by the determination of the minimum inhibitory concentration (MIC) and the minimal bactericidal concentration (MBC). Subsequently, we analyzed the time-kill curve of these plant-derived compounds against Staphylococcus aureus. Then, we examined their mode of action by three different assays: the crystal violet methodology, the release of intracellular material, and transmission electron microscopy methods (TEM). Finally, we evaluated the effect of HER and pristimerin on the pre-formed biofilm of S. aureus by the crystal violet assay, the synergistic effect by the checkerboard method, the cytotoxicity against Vero cells, and the in silico activity using the online software PASS. RESULTS: HER and pristimerin presented a narrow spectrum of action against Gram-positive bacteria (MIC 0.195-25 µg/mL), and their primary mode of action is the alteration of membrane permeability of S. aureus. Our results show that the compounds disrupted the pre-formed biofilm of S. aureus in a dose-dependent manner. Furthermore, HER and pristimerin presented a significant synergic effect after the combination with well-known antibiotics, which was associated with the ability of these phytomedicines to change membrane permeability. Regarding the cytotoxic effect, the selective index (SI) of HER ranged from 0.37 to 11.86, and the SI of pristimerin varied from 0.24 to 30.87, according to the bacteria tested. CONCLUSIONS: Overall, HER and pristimerin showed a promising antibacterial effect in vitro through the alteration of membrane permeability of S. aureus.

Analysis of a Marseillevirus Transcriptome Reveals Temporal Gene Expression Profile and Host Transcriptional Shift.

Marseilleviruses comprise a family of large double-stranded DNA viruses belonging to the proposed order "Megavirales." These viruses have a circular genome of ∼370 kbp, coding hundreds of genes. Over a half of their genes are associated with AT-rich putative promoter motifs, which have been demonstrated to be important for gene regulation. However, the transcriptional profile of Marseilleviruses is currently unknown. Here we used RNA sequencing technology to get a general transcriptional profile of Marseilleviruses. Eight million 75-bp-long nucleotide sequences were robustly mapped to all 457 genes initially predicted for Marseillevirus isolate T19, the prototype strain of the family, and we were able to assemble 359 viral contigs using a genome-guided approach with stringent parameters. These reads were differentially mapped to the genes according to the replicative cycle time point from which they were obtained. Cluster analysis indicated the existence of three main temporal categories of gene expression, early, intermediate and late, which were validated by quantitative reverse transcription polymerase chain reaction assays targeting several genes. Genes belonging to different functional groups exhibited distinct expression levels throughout the infection cycle. We observed that the previously predicted promoter motif, AAATATTT, as well as new predicted motifs, were not specifically related to any of the temporal or functional classes of genes, suggesting that other components are involved in temporally regulating virus transcription. Moreover, the host transcription machinery is heavily altered, and many genes are down regulated, including those related to translation process. This study provides an overview of the transcriptional landscape of Marseilleviruses.

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.

Isolation and genomic characterization of a new mimivirus of lineage B from a Brazilian river.

Since its discovery, the first identified giant virus associated with amoebae, Acanthamoeba polyphaga mimivirus (APMV), has been rigorously studied to understand the structural and genomic complexity of this virus. In this work, we report the isolation and genomic characterization of a new mimivirus of lineage B, named "Borely moumouvirus". This new virus exhibits a structure and replicative cycle similar to those of other members of the family Mimiviridae. The genome of the new isolate is a linear double-strand DNA molecule of ~1.0 Mb, containing over 900 open reading frames. Genome annotation highlighted different translation system components encoded in the DNA of Borely moumouvirus, including aminoacyl-tRNA synthetases, translation factors, and tRNA molecules, in a distribution similar to that in other lineage B mimiviruses. Pan-genome analysis indicated an increase in the genetic arsenal of this group of viruses, showing that the family Mimiviridae is still expanding. Furthermore, phylogenetic analysis has shown that Borely moumouvirus is closely related to moumouvirus australiensis. This is the first mimivirus lineage B isolated from Brazilian territory to be characterized. Further prospecting studies are necessary for us to better understand the diversity of these viruses so a better classification system can be established.

Trapping the Enemy: Vermamoeba vermiformis Circumvents Faustovirus Mariensis Dissemination by Enclosing Viral Progeny inside Cysts.

Viruses depend on cells to replicate and can cause considerable damage to their hosts. However, hosts have developed a plethora of antiviral mechanisms to counterattack or prevent viral replication and to maintain homeostasis. Advantageous features are constantly being selected, affecting host-virus interactions and constituting a harsh race for supremacy in nature. Here, we describe a new antiviral mechanism unveiled by the interaction between a giant virus and its amoebal host. Faustovirus mariensis infects Vermamoeba vermiformis, a free-living amoeba, and induces cell lysis to disseminate into the environment. Once infected, the cells release a soluble factor that triggers the encystment of neighbor cells, preventing their infection. Remarkably, infected cells stimulated by the factor encyst and trap the viruses and viral factories inside cyst walls, which are no longer viable and cannot excyst. This unprecedented mechanism illustrates that a plethora of antiviral strategies remains to be discovered in nature.IMPORTANCE Understanding how viruses of microbes interact with its hosts is not only important from a basic scientific point of view but also for a better comprehension of the evolution of life. Studies involving large and giant viruses have revealed original and outstanding mechanisms concerning virus-host relationships. Here, we report a mechanism developed by Vermamoeba vermiformis, a free-living amoeba, to reduce Faustovirus mariensis dissemination. Once infected, V. vermiformis cells release a factor that induces the encystment of neighbor cells, preventing infection of further cells and/or trapping the viruses and viral factories inside the cyst walls. This phenomenon reinforces the need for more studies regarding large/giant viruses and their hosts.

Microscopic Analysis of the Tupanvirus Cycle in Vermamoeba vermiformis.

Since Acanthamoeba polyphaga mimivirus (APMV) was identified in 2003, several other giant viruses of amoebae have been isolated, highlighting the uniqueness of this group. In this context, the tupanviruses were recently isolated from extreme environments in Brazil, presenting virions with an outstanding tailed structure and genomes containing the most complete set of translation genes of the virosphere. Unlike other giant viruses of amoebae, tupanviruses present a broad host range, being able to replicate not only in Acanthamoeba sp. but also in other amoebae, such as Vermamoeba vermiformis, a widespread, free-living organism. Although the Tupanvirus cycle in A. castellanii has been analyzed, there are no studies concerning the replication of tupanviruses in other host cells. Here, we present an in-depth microscopic study of the replication cycle of Tupanvirus in V. vermiformis. Our results reveal that Tupanvirus can enter V. vermiformis and generate new particles with similar morphology to when infecting A. castellanii cells. Tupanvirus establishes a well-delimited electron-dense viral factory in V. vermiformis, surrounded by lamellar structures, which appears different when compared with different A. castellanii cells. Moreover, viral morphogenesis occurs entirely in the host cytoplasm within the viral factory, from where complete particles, including the capsid and tail, are sprouted. Some of these particles have larger tails, which we named "supertupans." Finally, we observed the formation of defective particles, presenting abnormalities of the tail and/or capsid. Taken together, the data presented here contribute to a better understanding of the biology of tupanviruses in previously unexplored host cells.

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.

New Isolates of Pandoraviruses: Contribution to the Study of Replication Cycle Steps.

Giant viruses are complex members of the virosphere, exhibiting outstanding structural and genomic features. Among these viruses, the pandoraviruses are some of the most intriguing members, exhibiting giant particles and genomes presenting at up to 2.5 Mb, with many genes having no known function. In this work, we analyzed, by virological and microscopic methods, the replication cycle steps of three new pandoravirus isolates from samples collected in different regions of Brazil. Our data indicate that all analyzed pandoravirus isolates can deeply modify the Acanthamoeba cytoplasmic environment, recruiting mitochondria and membranes into and around the electron-lucent viral factories. We also observed that the viral factories start forming before the complete degradation of the cellular nucleus. Various patterns of pandoravirus particle morphogenesis were observed, and the assembly of the particles seemed to be started either by the apex or by the opposite side. On the basis of the counting of viral particles during the infection time course, we observed that pandoravirus particles could undergo exocytosis after their morphogenesis in a process that involved intense recruitment of membranes that wrapped the just-formed particles. The treatment of infected cells with brefeldin affected particle exocytosis in two of the three analyzed strains, indicating biological variability among isolates. Despite such particle exocytosis, the lysis of host cells also contributed to viral release. This work reinforces knowledge of and reveals important steps in the replication cycle of pandoraviruses.IMPORTANCE The emerging Pandoraviridae family is composed of some of the most complex viruses known to date. Only a few pandoravirus isolates have been described until now, and many aspects of their life cycle remain to be elucidated. A comprehensive description of the replication cycle is pivotal to a better understanding of the biology of the virus. For this report, we describe new pandoraviruses and used different methods to better characterize the steps of the replication cycle of this new group of viruses. Our results provide new information about the diversity and biology of these giant viruses.

"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.