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Here, we propose the creation of the family "Yaraviridae", a new taxon to classify a virus infecting Acanthamoeba castellanii cells. Recently, we described the discovery of a new virus infecting free-living amoebae, yaravirus, which has features that strongly differ from those of all other viruses of amoebae described to date. Yaravirus particles are about 80 nm in diameter and have a dsDNA genome of ~45 kbp containing 74 ORFs, most of which (90%) have no homologs in current databases. Together, these data support the creation of a new species ("Yaravirus brasiliense"), a new viral genus (here proposed as "Yaravirus"), and a new viral family (here proposed as "Yaraviridae") to classify yaravirus and other related viruses that may be described in the future. All of them are to be included into the existing realm Varidnaviria and the kingdom Bamfordvirae, due to the presence of a major capsid protein containing a double jelly-roll fold.

Here we report the discovery of Yaravirus, a lineage of amoebal virus with a puzzling origin and evolution. Yaravirus presents 80-nm-sized particles and a 44,924-bp dsDNA genome encoding for 74 predicted proteins. Yaravirus genome annotation showed that none of its genes matched with sequences of known organ-isms at the nucleotide level; at the amino acid level, six predicted proteins had distant matches in the nr database. Complimentary prediction of three-dimensional structures indicated possible func-tion of 17 proteins in total. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially represent-ing a divergent major capsid protein (MCP) with a predicted double jelly-roll domain. Structure-guided phylogeny of MCP suggests that Yaravirus groups together with the MCPs of Pleurochrysis endemic viruses. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.

Triggering the amoebal phagocytosis process is a sine qua non condition for most giant viruses to initiate their replication cycle and consequently to promote their progeny formation. It is well known that the amoebal phagocytosis process requires the recognition of particles of500 nm, and most amoebal giant viruses meet this requirement, such as mimivirus, pandoravirus, pithovirus, and mollivirus. However, in the context of the discovery of amoebal giant viruses in the last decade, Marseillevirus marseillevirus (MsV) has drawn our attention, because despite its ability to successfully replicate in Acanthamoeba, remarkably it does not fulfill the500-nm condition, since it presents an ∼250-nm icosahedrally shaped capsid. We deeply investigated the MsV cycle by using a set of methods, including virological, molecular, and microscopic (immunofluorescence, scanning electron microscopy, and transmission electron microscopy) assays. Our results revealed that MsV is able to form giant vesicles containing dozens to thousands of viral particles wrapped by membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggested that these giant vesicles are able to stimulate amoebal phagocytosis and to trigger the MsV replication cycle by an acidification-independent process. Also, we observed that MsV entry may occur by the phagocytosis of grouped particles (without surrounding membranes) and by an endosome-stimulated pathway triggered by single particles. Taken together, not only do our data deeply describe the main features of MsV replication cycle, but this is the first time, to our knowledge, that the formation of giant infective vesicles related to a DNA virus has been described.Triggering the amoebal phagocytosis process is a sine qua non condition required by most giant viruses to initiate their replication cycle. This process requires the recognition of particles of500 nm, and many giant viruses meet this requirement. However, MsV is unusual, as despite having particles of ∼250 nm it is able to replicate in Acanthamoeba Our results revealed that MsV is able to form giant vesicles, containing dozens to thousands of viral particles, wrapped in membranes derived from amoebal endoplasmic reticulum. Remarkably, our results strongly suggest that these giant vesicles are able to stimulate phagocytosis using an acidification-independent process. Our work not only describes the main features of the MsV replication cycle but also describes, for the first time to our knowledge, the formation of huge infective vesicles in a large DNA viruses.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has infected millions of people worldwide. SARS-CoV-2 belongs to the Betacoronavirus genus, containing the mouse hepatitis virus (MHV), an extensively studied animal coronavirus. Since MHV and SARS-CoV-2 share the same genus, MHV could offer insights relative to SARS-CoV-2 studies. MHV-3 strain causes hepatitis and cellular injury, making MHV-3 infection one of the best models for this debilitating disease. Surrogate coronaviruses have been used for virus resistance and inactivation studies, and although real-life conditions using SARS-CoV-2 should be encouraged, their use needs to be balanced with safety and costs. MHV can be manipulated under BSL2 laboratory conditions, unlike SARS-CoV-2, making it a model for studying the virucidal effects on coronaviruses. In this study, we used the betacoronavirus MHV-3 as a model to investigate the virucidal activity of an air disinfection equipment named STR Solution®, an air sterilizer with patented technology. MHV-3 was dried on different surfaces and exposed at varying distances from the STR Solution® equipment and at different exposure times. The residual infectivity was evaluated using the endpoint method. There was not a significant reduction (mean p-value = 0.4) of the viral titer under STR Solution® exposition. STR Solution® caused a slight decrease of the infectious particles' titer (> 1 log10) only under the following conditions: polypropylene at 3 m, for 1 and 3 h (1.2 log10 reduction TCID50) and Sus domesticus skin at 0.05 m, for 1 h (1.3 log10 reduction TCID50), and at 3 m for 1 h (1.2 log10 reduction TCID50). These and other studies confirm the usefulness of this model to evaluate virucidal activity.

Giant amoebal viruses were discovered in the beginning of the 21st century and broke many paradigms of traditional virology related to particle size and genome content. These viruses are associated with different amoebae species and have been found in various places on the planet. Due to their astonishing complexity, giant viruses lie in the center of a hot debate about the origin and evolution of viruses. Moreover, they can be parasitized by other viruses, named virophages, and have their own genetic mobilome. In this article, we describe the history and general features of the giant viruses and their parasites.

Importance The COVID-19 pandemic has resulted in more than 3.5 million cases and 245 thousand deaths worldwide as of May 6, 2020. Determining the extent of the presence of the virus on public surfaces is critical for understanding the potential risk of infection in these areas. Objective To evaluate the presence of SARS-CoV-2 RNA on public surfaces in a densely populated urban area in Brazil. Design and Setting A total of 101 samples were collected from different surfaces in public places in the region of Belo Horizonte with the highest number of COVID-19 cases. Samples were collected near the hospital and public transportation areas using sterile swabs, and then submitted to nucleic acid extraction and genomic detection and quantification by one-step qPCR. Results Seventeen of the 101 samples tested positive (16.8%) for SARS-CoV-2 RNA, including samples from bus stations/terminals, public squares, and sidewalks, including those near hospitals. Conclusions and Relevance Our data indicated the contamination of public surfaces by SARS-CoV-2, especially near hospital areas, highlighting 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. Key points Question Can SARS-CoV-2 be detected on surfaces in public areas used by a large proportion of the population? Findings SARS-CoV-2 RNA was detected in different locations, including bus stations/terminals, squares, and sidewalks, especially those near hospitals, in a densely populated area of Belo Horizonte, Brazil. Meaning This study highlights the need for constant monitoring for the presence of SARS-CoV-2 RNA in urban areas to support better strategies to fight the COVID-19 pandemic and prevent further infections.

Here we report the discovery of Yaravirus, a new lineage of amoebal virus with a puzzling origin and phylogeny. Yaravirus presents 80 nm-sized particles and a 44,924 bp dsDNA genome encoding for 74 predicted proteins. More than 90% (68) of Yaravirus predicted genes have never been described before, representing ORFans. Only six genes had distant homologs in public databases: an exonuclease/recombinase, a packaging-ATPase, a bifunctional DNA primase/polymerase and three hypothetical proteins. Furthermore, we were not able to retrieve viral genomes closely related to Yaravirus in 8,535 publicly available metagenomes spanning diverse habitats around the globe. The Yaravirus genome also contained six types of tRNAs that did not match commonly used codons. Proteomics revealed that Yaravirus particles contain 26 viral proteins, one of which potentially representing a novel capsid protein with no significant homology with NCLDV major capsid proteins but with a predicted double-jelly roll domain. Yaravirus expands our knowledge of the diversity of DNA viruses. The phylogenetic distance between Yaravirus and all other viruses highlights our still preliminary assessment of the genomic diversity of eukaryotic viruses, reinforcing the need for the isolation of new viruses of protists.Significance statementMost of the known viruses of amoeba have been seen to share many features that eventually prompted authors to classify them into common evolutionary groups. Here we describe Yaravirus, an entity that could represent either the first isolated virus of Acanthamoeba spp. out of the group of NCLDVs or, in alternative evolutive scenario, it is a distant and extremely reduced virus of this group. Contrary to what is observed in other isolated viruses of amoeba, Yaravirus is not represented by a large/giant particle and a complex genome, but at the same time carries an important number of previously undescribed genes, including one encoding a novel major capsid protein. Metagenomic approaches also testified for the rarity of Yaravirus in the environment.

International audience; 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 recent discovery of tupanvirus, one of the largest and most complex viruses isolated to date, has reinforced the structural and genomic complexity of the giant viruses 1. Tupanviruses have been isolated from soda lakes, known as an extreme aquatic environments, and from ocean sediments collected at a depth of 3000 meters (m) 1,2. Phylogenetic analyses have shown the clustering of the tupanvirus with members of the family Mimiviridae. However, there are many peculiarities that make the tupanviruses unique entities in the known virosphere. Since its first observation, tupanviruses showed remarkable morphological characteristics; it has optically visible particles that average about 1.2 µm in size and can reach lengths up to 2.3 µm 1. Tupanvirus has the largest host range described so far among amoebal-infecting giant viruses and can causes a shutdown of host rRNA that is likely related to host-nucleolus degradation 1,3-9. The tupanviruses replication cycle is similar to those for other mim-iviruses, in which viral particles attach to the host-cell surface and enter through phagocytosis. The viral inner membrane then fuses with the phagosome membrane, releasing the genome. A viral factory (VF) is formed, where particle morphogenesis occurs; the cycle ends with cell lysis and the release of progeny viruses 1,10. The study of the tupanvirus genome further aroused the interest of virologists, not only due of its large size (~1.5 Mb), but also because these viruses show the largest translational apparatus described. It is composed of up to 70 tRNA, 20 aminoacyl-tRNA synthetases (aaRS), 11 factors associated with translation, and factors related to tRNA/mRNA maturation and ribosome protein modification 1. In addition to the robust translation apparatus, tupanvirus also contains a gene encoding mannose-specific lectin, also called mannose-binding protein (MBP) 1. Interestingly, previous studies revealed that Acanthamoeba castellanii expresses an MBP and that free-mannose can inhibit the adhesion of A. castellanii to surfaces, suggesting that the MBP plays a role in the pathogenesis of Acanthamoeba infection 11-16 .

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.

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.

International audience; 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 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.

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.

Even nearly forty years after the eradication of smallpox, members of the Poxviridae family continue to be the focus of an increasing number of studies. Among these studies, prominently stands vaccinia virus, an orthopoxvirus that is associated with bovine vaccinia outbreaks. Although more frequently associated with infections in cattle and humans, the host range of vaccinia virus is not restricted only to these hosts. There are several instances of molecular and serological evidence of circulation of vaccinia virus among wildlife species. In addition, viral isolation has confirmed a broad spectrum of vaccinia virus hosts. In this report, we provide a brief update on the host range of Brazilian vaccinia virus, and present a case description of an outbreak in domestic buffalo calves from Northeastern Brazil that corroborates previous serological and molecular studies. Furthermore, in the present study, vaccinia virus has been isolated for the first time in buffaloes, and referred to as vaccinia virus Pernambuco (VACV-PE). Phylogenetic reconstruction was based on A56R clustered VACV-PE with vaccinia virus isolates belonging to group 1 Brazilian vaccinia virus. Furthermore, the vaccinia virus genome was detected in the milk of a lactating cow, which thereby revealed a pathway for future studies on the possible impact of vaccinia virus on buffalo milk and milk products. Taken together, these results provide the first description of clinical disease caused by vaccinia virus in buffaloes in South America. They also raise new questions about the chain of transmission of this virus.

Synthetic 1,3‐bis(aryloxy)propan‐2‐amines have been shown in previous studies to possess several biological activities, such as antifungal and antiprotozoal. In the present study, we describe the antibacterial activity of new synthetic 1,3‐bis(aryloxy)propan‐2‐amines against Gram‐positive pathogens (Streptococcus pyogenes, Enterococcus faecalis and Staphylococcus aureus) including Methicillin–resistant S. aureus strains. Our compounds showed minimal inhibitory concentrations (MIC) in the range of 2.5–10 μg/ml (5.99–28.58 μM), against different bacterial strains. The minimal bactericidal concentrations found were similar to MIC, suggesting a bactericidal mechanism of action of these compounds. Furthermore, possible molecular targets were suggested by chemical similarity search followed by docking approaches. Our compounds are similar to known ligands targeting the cell division protein FtsZ, Quinolone resistance protein norA and the Enoyl‐[acyl‐carrier‐protein] reductase FabI. Taken together, our data show that synthetic 1,3‐bis(aryloxy)propan‐2‐amines are active against Gram‐positive bacteria, including multidrug–resistant strains and can be a promising lead in the development of new antibacterial compounds for the treatment of these infections., The present study shows that some 1,3‐Bis(aryloxy)propan‐2‐amines inhibited the growth of Gram‐positive bacteria in the low micromolar range. Furthermore, these compounds were also effective in inhibiting the growth of clinical strains of methicillin–resistant Staphylococcus aureus (MRSA).

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior A descoberta dos vírus gigantes levou à quebra de paradigmas no campo da virologia devido ao tamanho das partículas desses vírus e pela sua complexidade genômica e estrutural. Na última década, a busca por novos vírus gigantes foi intensificada levando a descoberta dos marseillevírus, faustovírus, kaumoebavírus e tupanvírus. Visto a necessidade de elucidar características biológicas e moleculares desses vírus que ainda permanecem sem resposta, esse trabalho teve como objetivo fazer a predição e análise de motivos promotores em vírus da família Marseilleviridae, em faustovírus e kaumoebavírus e a caracterização do efeito citopático do tupanvírus em Acanthamoeba castellanii. A predição e análise de motivos promotores levou a primeira caracterização de um octâmero (AAATATTT) que pode atuar como promotor em vírus da família Marseilleviridae. A distribuição e localização do motivo em relação ao códon de iniciação seguiu um padrão semelhante ao demonstrado para outros promotores descritos entre os membros que compõem o NCLDV. Além disso, a relevância biológica do octâmero predito foi demonstrada e a presença de repetições desse motivo nas regiões intergênicas do genoma de marseillevírus foi associada à elevada capacidade dos marseillevírus de adquirir genes por transferência gênica lateral contribuindo para explicar o seu mosaicismo e plasticidade genômica. Já a busca de motivos promotores em faustovírus e kaumoebavírus revelou que esses vírus compartilham em abundância as mesmas sequências promotoras (TATTT e TATATA) previamente descritas em vírus da família Asfarviridae. Foi demonstrado que os motivos preditos estão presentes em mais de 65% das regiões a montante dos genes compartilhados entre faustovírus, kaumoebavírus e asfarvírus reforçando a relação filogenética entre estes vírus. Além da regulação dos processos transcricionais, um importante desafio para os vírus na natureza é a busca por células hospedeiras. Nesse contexto nós realizamos uma caracterização detalhada do efeito citopático desenvolvido por tupanvírus em cultura de Acanthamoeba castellanii o que levou a descrição de um novo tipo de interação vírus-hospedeiro. Foi demonstrado que a infecção por tupanvírus é capaz de induzir a expressão de um gene que codifica uma proteína ligadora de manose viral e celular sugerindo a importância dessa proteína no ciclo de multiplicação do tupanvírus. Curiosamente, a adição de manose livre durante a multiplicação de tupanvírus levou não só a supressão do aumento do nível de transcritos dos genes da proteína ligadora de manose viral e celular como a inibição da formação do cacho de uma maneira concentração dependente, sugerindo que a formação de cachos correlaciona com a expressão dos genes que codifica a proteína ligadora de manose tanto viral quanto celular. Finalmente, nós demonstramos que os cachos formados durante a multiplicação de tupanvírus são capazes de interagir com células não infectadas contribuindo para a disseminação viral. The discovery of giant viruses led to the breakdown of paradigms in virology field due virus particle size and genomic and structural complexity. In the last decade, the search for new giant viruses intensified leading to the discovery of marseillevirus, faustovirus, kaumoebavirus and tupanvirus. In order to elucidate the biological and molecular informations of these viruses that remain unclear, this work aimed to predict and analyze the promoter motifs in Marseilleviridae family viruses, in faustovirus and kaumoebavirus, further to characterize the cytopathic effect of tupanvirus in Acanthamoeba castellanii. The analysis of promoter motifs led to the first characterization of an octamer (AAATATTT) that can act as a promoter in the Marseilleviridae family. The distribution and localization of the motif relative to the start codon followed a pattern similar to that demonstrated for other promoters described among NCLDV members. In addition, the biological relevance of the predicted octamer was demonstrated and the motifs repetitions in intergenic regions of the marseillevirus genome was associated with the high ability of the marseillevirus to acquire genes by lateral gene transfer contributing to explain their mosaicism and genomic plasticity. The search for promoter motifs in faustovirus and kaumoebavirus revealed that these viruses share in abundance the same promoter sequences (TATTT and TATATA), as previously described in viruses of the Asfarviridae family. It has been demonstrated that the predicted motifs are present in more than 65% of the genes shared among faustovirus, kaumoebavirus and asfarvirus, reinforcing the phylogenetic relationship between these viruses. In addition to the regulation of transcriptional processes, an important challenge for viruses in nature is the search for host cells. In this context, we performed a detailed characterization of the cytopathic effect of tupanvirus in the culture of Acanthamoeba castellanii, which led to the description of a new type of virus-host interaction involving the tupanvirus. Tupanvirus infection has been shown to be capable of inducing the expression of genes encoding a viral and cellular mannose binding protein (MBP), suggesting the importance of the MBP in tupanvirus multiplication cycle. Interestingly the presence of mannose led not only to suppression of the increase in transcripts level of the viral and cellular MBP genes, but also led to the inhibition of amoebal-bunches formation at a concentration dependent, suggesting that the bunches formation correlates with the expression of the viral and cellular MBP genes. Finally, we demonstrate that the amoebal-bunches formed during multiplication of tupanviruses are able to interact with uninfected cells contributing to viral spread.

Putative promoter motifs have been described in viruses belonging to the nucleocytoplasmic large DNA viruses (NCLDVs) group; however, few studies have been conducted to search for promoter sequences in newly discovered amoebal giant viruses. Faustovirus and kaumoebavirus are two Asfarviridae-related giant viruses belonging to the NCLDVs group. The phylogenetic relationships among these viruses led us to investigate if the promoter regions previously identified in the asfarvirus genome could be shared by its amoebal virus relatives. Previous studies demonstrated the role of A/T-rich motifs as promoters of asfarvirus. In this study, we reinforce the importance of A/T rich motifs in asfarvirus and show that the TATTT and TATATA motifs are also shared in abundance by faustovirus and kaumoebavirus. Here, we demonstrate that TATTT and TATATA are mostly present in faustovirus and kaumoebavirus genomic intergenic regions (IRs) and that they are widely distributed at 0 to -100 bp upstream to the start codons. We observed that putative promoter motifs are present as one to dozens of repetitions in IRs of faustovirus, kaumoebavirus, and asfarvirus, which is similar to that described previously for marseilleviruses. Furthermore, the motifs were found in most of the upstream regions of the core genes of faustovirus, kaumoebavirus, and asfarvirus, which suggests that the motifs could already be present in the ancestor of these viruses before the irradiation of this group. Our work provides an in-depth analysis of the putative promoter motifs present in asfarvirus, kaumoebavirus, and faustovirus, which reinforces the relationship among these viruses.

Vaccinia virus (VACV), the etiological agent of bovine vaccinia (BV), is widespread in Brazil and present in most of the milk-producing regions. We conducted a horizontal study of BV in Bahia, a state of Brazil in which the production of milk is increasing. During 2011, human and bovine clinical samples were collected during outbreaks for BV diagnosis, virus isolation and molecular analysis. We collected data for epidemiological inferences. Vaccinia virus was detected in 87.7 % of the analyzed outbreaks, highlighting the effective circulation of VACV in Bahia. The molecular data showed the spreading of group 1 Brazilian VACV to Bahia. We observed a seasonal profile of BV, with its peak in the drier and cooler season. Manual milking was observed in 96 % of the visited properties, showing its importance to viral spread in herds. Under-notification of BV, ineffective animal trade surveillance, and bad milking practices have contributed to the spread of VACV in Brazil.

Vaccinia virus (VACV) has had an important role for humanity because of its use during the smallpox eradication campaign. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health problems, mainly in Brazil and India. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into at least two groups based on genetic and biological diversity: group 1 (G1) and group 2 (G2). In this study, we went to the field and investigated VACV clonal diversity directly from exanthemous lesions, during BV outbreaks. Our results demonstrate that the G1 VACV-BR were more frequently isolated. Furthermore, we were able to co-detect the two variants (G1 and G2) in the same sample. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. The detected G2 clones presented exclusive genetic and biological markers, distinct to reference isolates, including VACV-Western Reserve. Two clones presented a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes. Indeed, some SNPs and INDELs in A56R nucleotide sequences were observed among clones of the same virus population, maybe as a result of an increased mutation rate in a mixed population. These results provide information about the diversity profile in VACV populations, highlighting its importance to VACV evolution and maintenance in the environment.

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.

Outbreaks of Vaccinia virus (VACV) affecting cattle and humans have been reported in Brazil in the last 15 years, but the origin of outbreaks remains unknown. Although VACV DNA have been already detected in mice (Mus musculus), opossums (Didelphis albiventris) and dogs during VACV zoonotic outbreaks, no transmission to cattle or humans from any of these were reported during Brazilian outbreaks. In this work, we assessed the PCR positivity to VACV in blood samples of cows and other domestic mammals, wild rodents and other wild mammals, and humans from areas with or without VACV infection reports. Our results show the detection of VACV DNA in blood samples of cows, horse and opossums, raising important questions about VACV spread.

Vaccinia virus (VACV) is the etiological agent of bovine vaccinia (BV), an emerging zoonosis that has been associated with economic losses and social effects. Despite increasing reports of BV outbreaks in Brazil, little is known about the biological interactions of Brazilian VACV (VACV-BR) isolates during coinfections; furthermore, there are no tools for the diagnosis of these coinfections. In this study, a tool to co-detect two variants of VACV was developed to provide new information regarding the pathogenesis, virulence profile, and viral spread during coinfection with VACV-BR isolates. To test the quantitative polymerase chain reactions (qPCR) tool, groups of BALB/c mice were intranasally monoinfected with Pelotas virus 1—Group II (PV1-GII) and Pelotas virus 2—Group I (PV2-GI), or were coinfected with PV1-GII and PV2-GI. Clinical signs of the mice were evaluated and the viral load in lung and spleen were detected using simultaneous polymerase chain reactions (PCR) targeting the A56R (hemagglutinin) gene of VACV. The results showed that qPCR for the quantification of viral load in coinfection was efficient and highly sensitive. Coinfected mice presented more severe disease and a higher frequency of VACV detection in lung and spleen, when compared to monoinfected groups. This study is the first description of PV1 and PV2 pathogenicity during coinfection in mice, and provides a new method to detect VACV-BR coinfections.

The Poxviridae family is comprised of double-stranded DNA viruses belonging to nucleocytoplasmic large DNA viruses (NCLDV). Among the NCLDV, poxviruses exhibit the widest known host range, which is likely observed because this viral family has been more heavily investigated. However, relative to each member of the Poxviridae family, the spectrum of the host is variable, where certain viruses can infect a large range of hosts, while others are restricted to only one host species. It has been suggested that the variability in host spectrum among poxviruses is linked with the presence or absence of some host range genes. Would it be possible to extrapolate the restriction of viral replication in a specific cell lineage to an animal, a far more complex organism? In this study, we compare and discuss the relationship between the host range of poxvirus species and the abundance/diversity of host range genes. We analyzed the sequences of 38 previously identified and putative homologs of poxvirus host range genes, and updated these data with deposited sequences of new poxvirus genomes. Overall, the term host range genes might not be the most appropriate for these genes, since no correlation between them and the viruses’ host spectrum was observed, and a change in nomenclature should be considered. Finally, we analyzed the evolutionary history of these genes, and reaffirmed the occurrence of horizontal gene transfer (HGT) for certain elements, as previously suggested. Considering the data presented in this study, it is not possible to associate the diversity of host range factors with the amount of hosts of known poxviruses, and this traditional nomenclature creates misunderstandings.

In the last decades, studies have revealed multiple and strong correlations between the host and its commensal microbiota consisting of bacteria, protozoa, fungi and viruses. This associated microbiota can positively or negatively influence the course of a wide range of infections. Here, we review the interactions between the host and its viral microbiota and discuss new paradigms from an evolutionary perspective. The viral adaptation to a microbial environment in a co-evolutionary approach is highlighted, as well as viral cross transmission in the context of the barriers imposed by the indigenous microbiota. In addition to reviewing the host-microbiota-virus relationships, we focus the discussion on microbiota-virus interactions that could be applied to preventive and therapeutic treatments. Keywords: Indigenous microbiota, Virus-host interactions, Virus-microbiota interactions, Microbiota-host interactions, Evolution

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.

Viruses display a wide range of genomic profiles and, consequently, a variety of gene expression strategies. Specific sequences associated with transcriptional processes have been described in viruses, and putative promoter motifs have been elucidated for some nucleocytoplasmic large DNA viruses (NCLDV). Among NCLDV, the Marseilleviridae is a well-recognized family because of its genomic mosaicism. The marseilleviruses have an ability to incorporate foreign genes, especially from sympatric organisms inhabiting Acanthamoeba , its main known host. Here, we identified for the first time an eight-nucleotide A/T-rich promoter sequence (AAATATTT) associated with 55% of marseillevirus genes that is conserved in all marseilleviruses lineages, a higher level of conservation than that of any giant virus described to date. We instigated our prediction about the promoter motif by biological assays and by evaluating how single mutations in this octamer can impact gene expression. The investigation of sequences that regulate the expression of genes relative to lateral transfer revealed that the promoter motifs do not appear to be incorporated by marseilleviruses from donor organisms. Indeed, analyses of the intergenic regions that regulate lateral gene transfer-related genes have revealed an independent origin of the marseillevirus intergenic regions that does not match gene-donor organisms. About 50% of AAATATTT motifs spread throughout intergenic regions of the marseilleviruses are present as multiple copies. We believe that such multiple motifs are associated with increased expression of a given gene or are related to incorporation of foreign genes into the mosaic genome of marseilleviruses. IMPORTANCE The marseilleviruses draw attention because of the peculiar features of their genomes; however, little is known about their gene expression patterns or the factors that regulate those expression patterns. The limited published research on the expression patterns of the marseilleviruses and their unique genomes has led us to study the promoter motif sequences in the intergenic regions of the marseilleviruses. This work is the first to analyze promoter sequences in the genomes of the marseilleviruses. We also suggest a strong capacity to acquire foreign genes and to express those genes mediated by multiple copies of the promoter motifs available in intergenic regions. These findings contribute to an understanding of genomic expansion and plasticity observed in these giant viruses.

International audience; For many years, gene expression in the three cellular domains has been studied in an attempt to discover sequences associated with the regulation of the transcription process. Some specific transcriptional features were described in viruses, although few studies have been devoted to understanding the evolutionary aspects related to the spread of promoter motifs through related viral families. The discovery of giant viruses and the proposition of the new viral order Megavirales that comprise a monophyletic group, named nucleo-cytoplasmic large DNA viruses (NCLDV), raised new questions in the field. Some putative promoter sequences have already been described for some NCLDV members, bringing new insights into the evolutionary history of these complex microorganisms. In this review, we summarize the main aspects of the transcription regulation process in the three domains of life, followed by a systematic description of what is currently known about promoter regions in several NCLDVs. We also discuss how the analysis of the promoter sequences could bring new ideas about the giant viruses' evolution. Finally, considering a possible common ancestor for the NCLDV group, we discussed possible promoters' evolutionary scenarios and propose the term ``MEGA-box''to designate an ancestor promoter motif ('TATATAAAATTGA') that could be evolved gradually by nucleotides gain and loss and point mutations.

Vaccinia virus (VACV) is responsible for outbreaks in Brazil and has immense potential as an emerging virus. VACV can be found naturally circulating in India, Pakistan and South America, where it causes infections characterised by exanthematic lesions in buffaloes, cattle and humans. The transmission cycle of Brazilian VACV has still not been fully characterised; one of the most important gaps in knowledge being the role of wild animals. Capybaras, which are restricted to the Americas, are the world’s largest rodents and have peculiar characteristics that make them possible candidates for being part of a natural VACV reservoir. Here, we developed a method for detecting orthopoxvirus DNA in capybara stool samples, and have described for the first time the detection of orthopoxvirus DNA in capybaras samples from three different regions in Brazil. These findings strongly suggest that capybaras might be involved in the natural transmission cycle of VACV and furthermore represent a public health problem, when associated with Brazilian bovine vaccinia outbreaks. This makes infected animals an important factor to be considered when predicting and managing Brazilian VACV outbreaks.

Natural infections of Vaccinia virus (VACV)-the prototype species of the Orthopoxvirus genus, from the family Poxviridae and subfamily Chordopoxvirinae-cause an occupational emergent zoonotic disease that is primarily associated with the handling of infected dairy cattle. In humans, VACV infection is characterized by skin lesions, primarily on the hands, and accompanied by systemic symptoms such as fever, myalgia, headache, and lymphadenopathy. The diagnosis of VACV is usually performed according to the methods described for other orthopoxviruses. This unit describes the methods utilized to obtain clinical samples, the serological and molecular techniques used for diagnosis, and the isolation methods and techniques used for molecular and biological characterization of the viruses. © 2016 by John Wiley & Sons, Inc.

The aim of this protocol is to describe the replication, purification, and titration of mimiviruses. These viruses belong to the Mimiviridae family, the first member of which was isolated in 1992 from a cooling tower water sample collected during an outbreak of pneumonia in a hospital in Bradford, England. In recent years, several new mimiviruses have been isolated from different environmental conditions. These giant viruses are easily replicated in amoeba of the Acanthamoeba genus, its natural host. Mimiviruses present peculiar features that make them unique viruses, such as the particle and genome size and the genome's complexity. The discovery of these viruses rekindled discussions about their origin and evolution, and the genetic and structural complexity opened up a new field of study. Here, we describe some methods utilized for mimiviruses replication, purification, and titration. © 2016 by John Wiley & Sons, Inc.

We studied a clinical case of vaccinia virus that caused an ocular manifestation in a dairy worker in Brazil. Biologic and molecular analyses identified a co-infection with 2 isolates from different Brazilian vaccinia virus phylogenetic groups.

Bovine vaccinia (BV) is an emerging zoonosis caused by the Vaccinia virus (VACV), genus Orthopoxvirus (OPV), Poxviridae family. In general, human cases are related to direct contact with sick cattle but there is a lack of information about human-to-human transmission of VACV during BV outbreaks. In this study, we epidemiologically and molecularly show a case of VACV transmission between humans in São Francisco de Itabapoana County, Rio de Janeiro state. Our group collected samples from the patients, a 49-year-old patient and his son. Our results showed that patients had developed anti-OPV IgG or IgM antibodies and presented neutralizing antibodies against OPV. The VACV isolates displayed high identity (99.9%) and were grouped in the same phylogenetic tree branch. Our data indicate that human-to-human VACV transmission occurred during a BV outbreak, raising new questions about the risk factors of the VACV transmission chain.

The gastrointestinal tract of vertebrates harbors one of the most complex ecosystems known in microbial ecology and this indigenous microbiota almost always has a profound influence on host-parasite relationships, which can enhance or reduce the pathology of the infection. In this context, the impact of the microbiota during the infection of several viral groups remains poorly studied, including the family Poxviridae. Vaccinia virus (VACV) is a member of this family and is the causative agent of bovine vaccinia, responsible for outbreaks that affect bovines and humans. To determine the influence of the microbiota in the development of the disease caused by VACV, a comparative study using a murine model was performed. Germ-free and conventional, 6- to 7-week-old Swiss NIH mice were infected by tail scarification and intranasally with VACV. Moreover, immunosuppression and microbiota reposition were performed, to establish the interactions among the host's immune system, microbiota and VACV. The data demonstrate that the microbiota is essential for the effective immune response of mice against VACV in intranasal inoculation and to control the virus at the primary site of infection. Furthermore, this study is the first to show that Swiss conventional mice are refractory to the intranasal infection of VACV.

O Vaccinia virus (VACV) desempenhou um importante papel para a humanidade devido ao seu uso durante a campanha de erradicação da varíola. Além disso, atualmente o VACV tem sido amplamente utilizado no desenvolvimento de vacinas recombinantes. Com o fim da campanha de vacinação da varíola, outros Orthopoxvirus têm emergido em todo o mundo, tais como Cowpox virus, Monkeypox virus e VACV. VACV é o agente etiológico da vaccínia bovina (VB), uma zoonose emergente que tem sido associada a problemas econômicos, sociais, veterinários e de saúde pública, principalmente no Brasil. Apesar da importância atual e histórica do VACV, há poucas informações sobre sua circulação, prevalência, origem e manutenção no ambiente, assim como reservatórios naturais e diversidade. Os VACV brasileiros (VACV-BR) são separados em dois grupos com base na diversidade genética e biológica: grupo 1 (G1) e grupo 2 (G2). Os vírus do G2 apresentam placas de lise de maior tamanho e são altamente virulentos em modelo murino Balb/c, ao contrário dos vírus do G1. Além disso, polimorfismos demonstrados em genes de VACV, tais como A56R, A26L e C23L, têm sido utilizados em estudos filogenéticos confirmando a dicotomia entre os vírus do G1 e G2 de VACV-BR. Neste estudo, foi investigada a diversidade clonal de amostras de campo coletadas durante surtos de VB. Os resultados demonstram que clones do G1 de VACV-BR foram mais frequentemente isolados. A análise molecular e biológica corroborou estudos anteriores, confirmando a cocirculação de duas linhagens de VACV no Brasil. Além disso, foi possível detectar a cocirculação das duas variantes (G1 e G2) na mesma amostra. Dois clones apresentaram um perfil misto, apresentando características de ambos os grupos, com base na análise molecular dos genes A56R, A26L e C23L, talvez como resultado do aumento da taxa de recombinação em uma população mista. Algumas mutações, bem como inserções e deleções em sequências de nucleotídeos do gene A56R foram observadas entre clones da mesma amostra. Estes resultados fornecem informações sobre o perfil de diversidade entre clones de VACV circulando no Brasil. Vaccinia virus (VACV) played an important role for humanity because of its use during the smallpox eradication campaign. Furthermore, currently VACV has been widely used as a vector of the recombinant vaccines. Following smallpox eradication, other orthopoxviruses have emerged worldwide, such as Cowpox virus, Monkeypox virus and VACV. VACV is the etiologic agent of the bovine vaccinia (BV), an emerging zoonosis that has been associated with economic, social, veterinary and public health issues, mainly in Brazil. Despite the current and historical VACV importance, there is little information about its circulation, prevalence, origins and maintenance in the environment, natural reservoirs and diversity. Brazilian VACV (VACV-BR) are grouped into two groups, based on genetic and biological diversity: group 1 (G1) and group 2 (G2). G2 strains displays higher plaque sizes and are virulent to mice, unlike G1. Moreover, polymorphisms observed in VACV genes, such as A56R, A26L and C23L genes, have been used in phylogenetic studies and further confirmed the dichotomy between G1 and G2 VACV-BR. In this study, was investigated VACV clonal diversity from field samples, during BV outbreaks. The results demonstrated that the G1 VACV-BR viruses were more frequently isolated. Molecular and biological analysis corroborated previous reports and confirmed the co-circulation of two VACV-BR lineages. Furthermore, it was co-detect the two variants (G1 and G2) in a same sample. Two clones showed a mosaic profile, with both G1 and G2 features based on the molecular analysis of A56R, A26L and C23L genes, maybe as a result of an increased recombination rate in a mixed population. Indeed, some single nucleotide polymorphism (SNPs) and insertions and deletions (INDELs) in A56R nucleotide sequences were observed among clones of a given virus population. These results provide information about the diversity profile in VACV clones.