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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.

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 .

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.

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).

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.

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.

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.

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.