Fábio P. Dornas

Oysters as hot spots for mimivirus isolation

Viruses are ubiquitous organisms, but their role in the ecosystem and their prevalence are still poorly understood. Mimiviruses are extremely complex and large DNA viruses. Although metagenomic studies have suggested that members of the family Mimiviridae are abundant in oceans, there is a lack of information about the association of mimiviruses with marine organisms. In this work, we demonstrate by molecular and virological methods that oysters are excellent sources for mimiviruses isolation. Our data not only provide new information about the biology of these viruses but also raise questions regarding the role of oyster consumption as a putative source of mimivirus infection in humans.

Acanthamoeba polyphaga mimivirus and other giant viruses: an open field to outstanding discoveries

In 2003, Acanthamoeba polyphaga mimivirus (APMV) was first described and began to impact researchers around the world, due to its structural and genetic complexity. This virus founded the family Mimiviridae. In recent years, several new giant viruses have been isolated from different environments and specimens. Giant virus research is in its initial phase and information that may arise in the coming years may change current conceptions of life, diversity and evolution. Thus, this review aims to condense the studies conducted so far about the features and peculiarities of APMV, from its discovery to its clinical relevance.

A Brazilian Marseillevirus Is the Founding Member of a Lineage in Family Marseilleviridae

In 2003, Acanthamoeba polyphaga mimivirus (APMV) was discovered as parasitizing Acanthamoeba. It was revealed to exhibit remarkable features, especially odd genomic characteristics, and founded viral family Mimiviridae. Subsequently, a second family of giant amoebal viruses was described, Marseilleviridae, whose prototype member is Marseillevirus, discovered in 2009. Currently, the genomes of seven different members of this family have been fully sequenced. Previous phylogenetic analysis suggested the existence of three Marseilleviridae lineages: A, B and C. Here, we describe a new member of this family, Brazilian Marseillevirus (BrMV), which was isolated from a Brazilian sample and whose genome was fully sequenced and analyzed. Surprisingly, data from phylogenetic analyses and comparative genomics, including mean amino acid identity between BrMV and other Marseilleviridae members and the analyses of the core genome and pan-genome of marseilleviruses, indicated that this virus can be assigned to a new Marseilleviridae lineage. Even if the BrMV genome is one of the smallest among Marseilleviridae members, it harbors the second largest gene content into this family. In addition, the BrMV genome encodes 29 ORFans. Here, we describe the isolation and genome analyses of the BrMV strain, and propose its classification as the prototype virus of a new lineage D within the family Marseilleviridae.

A resourceful giant: APMV is able to interfere with the human type I interferon system

Acanthamoeba polyphaga mimivirus (APMV) is a giant, double-stranded virus of the Mimiviridae family that was discovered in 2003. Recent studies have shown that this virus is able to replicate in murine and human phagocytes and might be considered a putative human pathogen that causes pneumonia. However, there is little data regarding APMV and its host defense relationship. In the present study, we investigated how some components of the interferon (IFN) system are stimulated by APMV in human peripheral blood mononuclear cells (PBMCs) and how APMV replication is affected by IFN treatment. Our results demonstrated that APMV is able to replicate in human PBMCs, inducing type I Interferons (IFNs) but inhibiting interferon stimulated genes (ISG) induction by viroceptor and STAT-1 and STAT-2 dephosphorylation independent mechanisms. We also showed that APMV is resistant to the antiviral action of interferon-alpha2 (IFNA2) but is sensitive to the antiviral action of interferon-beta (IFNB1). Our results demonstrated the productive infection of professional phagocytes with APMV and showed that this virus is recognized by the immune system of vertebrates and inhibits it. It provides the first data regarding APMV and the IFN system interaction and raise new and relevant evolutional questions about the relationship between APMV and vertebrate hosts.

Mimivirus circulation among wild and domestic mammals, Amazon Region, Brazil.

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

Pan-Genome Analysis of Brazilian Lineage A Amoebal Mimiviruses

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

Mimivirus Fibrils Are Important for Viral Attachment to the Microbial World by a Diverse Glycoside Interaction Repertoire.

ABSTRACT Acanthamoeba polyphaga mimivirus (APMV) is a giant virus from the Mimiviridae family. It has many unusual features, such as a pseudoicosahedral capsid that presents a starfish shape in one of its vertices, through which the ∼1.2-Mb double-stranded DNA is released. It also has a dense glycoprotein fibril layer covering the capsid that has not yet been functionally characterized. Here, we verified that although these structures are not essential for viral replication, they are truly necessary for viral adhesion to amoebae, its natural host. In the absence of fibrils, APMV had a significantly lower level of attachment to the Acanthamoeba castellanii surface. This adhesion is mediated by glycans, specifically, mannose and N-acetylglucosamine (a monomer of chitin and peptidoglycan), both of which are largely distributed in nature as structural components of several organisms. Indeed, APMV was able to attach to different organisms, such as Gram-positive bacteria, fungi, and arthropods, but not to Gram-negative bacteria. This prompted us to predict that (i) arthropods, mainly insects, might act as mimivirus dispersers and (ii) by attaching to other microorganisms, APMV could be ingested by amoebae, leading to the successful production of viral progeny. To date, this mechanism has never been described in the virosphere. IMPORTANCE APMV is a giant virus that is both genetically and structurally complex. Its size is similar to that of small bacteria, and it replicates inside amoebae. The viral capsid is covered by a dense glycoprotein fibril layer, but its function has remained unknown, until now. We found that the fibrils are not essential for mimivirus replication but that they are truly necessary for viral adhesion to the cell surface. This interaction is mediated by glycans, mainly N-acetylglucosamine. We also verified that APMV is able to attach to bacteria, fungi, and arthropods. This indicates that insects might act as mimivirus dispersers and that adhesion to other microorganisms could facilitate viral ingestion by amoebae, a mechanism never before described in the virosphere.

Acanthamoeba polyphaga mimivirus Stability in Environmental and Clinical Substrates: Implications for Virus Detection and Isolation

Viruses are extremely diverse and abundant and are present in countless environments. Giant viruses of the Megavirales order have emerged as a fascinating research topic for virologists around the world. As evidence of their ubiquity and ecological impact, mimiviruses have been found in multiple environmental samples. However, isolation of these viruses from environmental samples is inefficient, mainly due to methodological limitations and lack of information regarding the interactions between viruses and substrates. In this work, we demonstrate the long-lasting stability of mimivirus in environmental (freshwater and saline water) and hospital (ventilator plastic device tube) substrates, showing the detection of infectious particles after more than 9 months. In addition, an enrichment protocol was implemented that remarkably increased mimivirus detection from all tested substrates, including field tests. Moreover, biological, morphological and genetic tests revealed that the enrichment protocol maintained mimivirus particle integrity. In conclusion, our work demonstrated the stability of APMV in samples of environmental and health interest and proposed a reliable and easy protocol to improve giant virus isolation. The data presented here can guide future giant virus detection and isolation studies.

Amoebas as mimivirus bunkers: increased resistance to UV light, heat and chemical biocides when viruses are carried by amoeba hosts

Amoebas of the genus Acanthamoeba are protists that are associated with human disease and represent a public health concern. They can harbor pathogenic microorganisms, acting as a platform for pathogen replication. Acanthamoeba polyphaga mimivirus (APMV), the type species of the genus Mimivirus, family Mimiviridae, represents the largest group of amoeba-associated viruses that has been described to date. Recent studies have demonstrated that APMV and other giant viruses may cause pneumonia. Amoebas can survive in most environments and tolerate various adverse conditions, including UV light irradiation, high concentrations of disinfectants, and a broad range of temperatures. However, it is unknown how the amoebal intracellular environment influences APMV stability and resistance to adverse conditions. Therefore, in this work, we evaluated the stability of APMV, either purified or carried by the amoeba host, under extreme conditions, including UV irradiation, heat and exposure to six different chemical biocides. After each treatment, the virus was titrated in amoebas using the TCID50 method. APMV was more stable in all resistance tests performed when located inside its host. Our results demonstrate that Acanthamoeba acts as a natural bunker for APMV, increasing viral resistance to extreme physical and chemical conditions. The data raise new questions regarding the survival of APMV in nature and in hospital environments.

Modulation of the expression of mimivirus-encoded translation-related genes in response to nutrient availability during Acanthamoeba castellanii infection

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