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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.

International audience; 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.

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.

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.

In the last few decades, the isolation of amoebae-infecting giant viruses has challenged established principles related to the definition of virus, their evolution, and their particle structures represented by a variety of shapes and sizes. Tupanviruses are one of the most recently described amoebae-infecting viruses and exhibit a peculiar morphology with a cylindrical tail attached to the capsid. Proteomic analysis of purified viral particles revealed that virions are composed of over one hundred proteins with different functions. The putative origin of these proteins had not yet been investigated. Here, we provide evidences for multiple origins of the proteins present in tupanvirus particles, wherein 20% originate from members of the archaea, bacteria and eukarya.

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.

No ano de 2003 iniciou-se uma das fases mais importantes da virologia moderna, com o descobrimento do primeiro exemplar de vírus gigante pertencente à família Mimiviridae, sendo este denominado de Acanthamoeba polyphaga mimivirus (APMV). O isolamento do mimivírus, e sua posterior caracterização, impressionou a comunidade cientifica pelas dimensões da partícula e por aquilo que o genoma destes virus é capaz de codificar. Diante desse cenário, muitos genes/proteínas ainda são um campo aberto para estudos envolvendo aspectos evolutivos, funcionais e de caracterização de novos isolados de vírus gigantes. Nesse trabalho, o nosso objetivo foi isolar e caracterizar geneticamente amostras de mimivírus de duas lagoas urbanas localizadas no estado de Minas Gerais, Brasil. Para isso, 80 amostras de água coletadas da Lagoa da Pampulha (Belo Horizonte) e 85 da Lagoa Central (Lagoa Santa) foram processadas e inoculadas em amebas da espécie Acanthamoeba castellanii. Dois isolados foram obtidos dessas amostras, sendo denominados de Niemeyer vírus (NYMV) e Kroon vírus (KV), respectivamente. Após esse processo focamos inicialmente em fazer uma análise da presença, distribuição e perfil de relações filogenéticas de aminoacil-tRNA-sintetases apresentadas por diferentes amostras de mimivírus. Essas enzimas denotam importância por sua recente descrição em organismos virais, estando relacionadas com o processo de tradução de proteínas. Nossos dados demonstram que NYMV apresenta duplicações em três (metionil, tirosil e arginil tRNA-sintetases) das suas quatro cópias de aaRs, tendo metionil e tirosil aaRs uma maior expressão de RNA mensageiro (RNAm) quando comparadas com o protótipo APMV. As análises filogenéticas mostraram que em NYMV, cada uma das cópias duplicadas de aaRs apresenta uma origem distinta, sendo associada a diferentes grupos de mimivirus. Em uma outra parte do trabalho, buscamos obter uma melhor compreensão das diferenças estruturais, de organização e de relações filogenéticas relacionadas a região da proteína principal do capsídeo de nossos isolados, quando comparadas com outras diferentes amostras da família Mimiviridae. Os resultados demonstraram que o gene do capsídeo de mimivirus apresenta variações marcantes em termos de posição e tipos de regiões intrônicas e exônicas, até mesmo para vírus pertencentes a uma mesma linhagem. Além disso, o sequenciamento do RNA mensageiro do gene do capsídeo de KV e APMV demonstrou que, mesmo pertencendo à mesma família e linhagem, vírus gigantes relacionados podem apresentar diferentes maneiras de processar os seus transcritos para um mesmo gene. Os resultados apresentados nesse trabalho auxiliam no levantamento de novas questões acerca da origem e de pressões seletivas que envolvem o ganho e a perda de aaRs entre os vírus gigantes, além de contribuir para o entendimento da organização genética e de evolução do gene do capsídeo entre os mimivírus.