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

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

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

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

Wild-Type Yellow Fever Virus RNA in Cerebrospinal Fluid of Child.

We report a 3-year-old child who was hospitalized because of severe manifestations of the central nervous system. The child died after 6 days of hospitalization. Analysis of postmortem cerebrospinal fluid showed the presence of yellow fever virus RNA. Nucleotide sequencing confirmed that the virus was wild-type yellow fever virus.

Analyses of the Kroon Virus Major Capsid Gene and Its Transcript Highlight a Distinct Pattern of Gene Evolution and Splicing among Mimiviruses.

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.

Ubiquitous giants: a plethora of giant viruses found in Brazil and Antarctica.

BACKGROUND: Since the discovery of giant viruses infecting amoebae in 2003, many dogmas of virology have been revised and the search for these viruses has been intensified. Over the last few years, several new groups of these viruses have been discovered in various types of samples and environments.In this work, we describe the isolation of 68 giant viruses of amoeba obtained from environmental samples from Brazil and Antarctica. METHODS: Isolated viruses were identified by hemacolor staining, PCR assays and electron microscopy (scanning and/or transmission). RESULTS: A total of 64 viruses belonging to the Mimiviridae family were isolated (26 from lineage A, 13 from lineage B, 2 from lineage C and 23 from unidentified lineages) from different types of samples, including marine water from Antarctica, thus being the first mimiviruses isolated in this extreme environment to date. Furthermore, a marseillevirus was isolated from sewage samples along with two pandoraviruses and a cedratvirus (the third to be isolated in the world so far). CONCLUSIONS: Considering the different type of samples, we found a higher number of viral groups in sewage samples. Our results reinforce the importance of prospective studies in different environmental samples, therefore improving our comprehension about the circulation anddiversity of these viruses in nature.

Detection of mimivirus genome and neutralizing antibodies in humans from Brazil.

In recent years, giant viruses belonging to the family Mimiviridae have been proposed to be infectious agents in humans. In this work we provide evidence of mimivirus genome and neutralizing antibodies detection in humans.

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

UNLABELLED: 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.

High prevalence of clinically unsuspected dengue disease among children in Ribeirao Preto city, Brazil.

The aim of this study was to analyze the characteristics of Dengue virus (DENV)-infected children and the accuracy of dengue diagnosis based on clinical presentations. The inclusion criteria were children ≥1-year-old presenting febrile illness with 1-7 days of onset. Children (n = 110) aged 2-15 years were included in this study. DENV infection was confirmed with virological tests using serum, salvia, and/or urine samples. The attending pediatricians classified 56/110 (50.91%) of the children as suspected dengue cases. The DENV infection was confirmed by specific laboratory tests in 52/56 (92.9%) of the suspected dengue cases but also in 44/54 (81.5%) of the unsuspected dengue cases; total of 96/110 (87.27%) confirmed dengue cases. The clinical diagnosis gave an overall sensitivity of 54.2% (52/96) and a specificity of 71.4% (10/14). The positive predictive value of the clinical diagnosis was 92.8% and negative predictive value was 18.5%. After the third day of onset of symptoms, the DENV genome detection rate was similar in serum and saliva samples, suggesting that saliva samples represent an alternative to blood samples for early dengue diagnosis. Vaccination against Yellow fever virus did not influence the antibody response against DENV-1, DENV-2, and DENV-3, which circulated during the study period. Although the signs and symptoms were compatible with dengue, the attending pediatricians did not suspect the disease in several children. Therefore, the inclusion of virological tests for early diagnosis in the protocols for dengue surveillance would help in the implementation of prompt treatment of patients and epidemic containment strategies. J. Med. Virol. 88:1711-1719, 2016. © 2016 Wiley Periodicals, Inc.

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.

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.

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.

Samba virus: a novel mimivirus from a giant rain forest, the Brazilian Amazon.

BACKGROUND: The identification of novel giant viruses from the nucleocytoplasmic large DNA viruses group and their virophages has increased in the last decade and has helped to shed light on viral evolution. This study describe the discovery, isolation and characterization of Samba virus (SMBV), a novel giant virus belonging to the Mimivirus genus, which was isolated from the Negro River in the Brazilian Amazon. We also report the isolation of an SMBV-associated virophage named Rio Negro (RNV), which is the first Mimivirus virophage to be isolated in the Americas. METHODS/RESULTS: Based on a phylogenetic analysis, SMBV belongs to group A of the putative Megavirales order, possibly a new virus related to Acanthamoeba polyphaga mimivirus (APMV). SMBV is the largest virus isolated in Brazil, with an average particle diameter about 574 nm. The SMBV genome contains 938 ORFs, of which nine are ORFans. The 1,213.6 kb SMBV genome is one of the largest genome of any group A Mimivirus described to date. Electron microscopy showed RNV particle accumulation near SMBV and APMV factories resulting in the production of defective SMBV and APMV particles and decreasing the infectivity of these two viruses by several logs. CONCLUSION: This discovery expands our knowledge of Mimiviridae evolution and ecology.

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.

Microscopic Analysis of the Tupanvirus Cycle in Vermamoeba vermiformis.

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

Genetic Markers of Benzimidazole Resistance among Human Hookworms (Necator americanus) in Kintampo North Municipality, Ghana.

Hookworm infection causes anemia, malnutrition, and growth delay, especially in children living in sub-Saharan Africa. The World Health Organization recommends periodic mass drug administration (MDA) of anthelminthics to school-age children (SAC) as a means of reducing morbidity. Recently, questions have been raised about the effectiveness of MDA as a global control strategy for hookworms and other soil-transmitted helminths (STHs). Genomic DNA was extracted from Necator americanus hookworm eggs isolated from SAC enrolled in a cross-sectional study of STH epidemiology and deworming response in Kintampo North Municipality, Ghana. A polymerase chain reaction (PCR) assay was then used to identify single-nucleotide polymorphisms (SNPs) associated with benzimidazole resistance within the N. americanus ß-tubulin gene. Both F167Y and F200Y resistance-associated SNPs were detected in hookworm samples from infected study subjects. Furthermore, the ratios of resistant to wild-type SNP at these two loci were increased in posttreatment samples from subjects who were not cured by albendazole, suggesting that deworming drug exposure may enrich resistance-associated mutations. A previously unreported association between F200Y and a third resistance-associated SNP, E198A, was identified by sequencing of F200Y amplicons. These data confirm that markers of benzimidazole resistance are circulating among hookworms in central Ghana, with unknown potential to impact the effectiveness and sustainability of chemotherapeutic approaches to disease transmission and control.

Discovery and Further Studies on Giant Viruses at the IHU Mediterranee Infection That Modified the Perception of the Virosphere.

The history of giant viruses began in 2003 with the identification of Acanthamoeba polyphaga mimivirus. Since then, giant viruses of amoeba enlightened an unknown part of the viral world, and every discovery and characterization of a new giant virus modifies our perception of the virosphere. This notably includes their exceptional virion sizes from 200 nm to 2 µm and their genomic complexity with length, number of genes, and functions such as translational components never seen before. Even more surprising, Mimivirus possesses a unique mobilome composed of virophages, transpovirons, and a defense system against virophages named Mimivirus virophage resistance element (MIMIVIRE). From the discovery and isolation of new giant viruses to their possible roles in humans, this review shows the active contribution of the University Hospital Institute (IHU) Mediterranee Infection to the growing knowledge of the giant viruses' field.

Cedratvirus getuliensis replication cycle: an in-depth morphological analysis.

The giant viruses are the largest and most complex viruses in the virosphere. In the last decade, new members have constantly been added to this group. Here, we provide an in-depth descriptive analysis of the replication cycle of Cedratvirus getuliensis, one of the largest viruses known to date. We tracked the virion entry, the early steps of virus factory and particles morphogenesis, and during this phase, we observed a complex and unique sequential organization of immature particle elements, including horseshoe and rectangular compartments, revealed by transverse and longitudinal sections, respectively, until the formation of the final ovoid-shaped striped virion. The genome and virion proteins are incorporated through a longitudinal opening in the immature virion, followed by the incorporation of the second cork and thickening of the capsid well. Moreover, many cell modifications occur during viral infection, including intense membrane trafficking important to viral morphogenesis and release, as evidenced by treatment using brefeldin A. Finally, we observed that Cedratvirus getuliensis particles are released after cellular lysis, although we obtained microscopic evidence that some particles are released by exocytosis. The present study provides new information on the unexplored steps in the life cycle of cedratviruses.

A Large Open Pangenome and a Small Core Genome for Giant Pandoraviruses.

Giant viruses of amoebae are distinct from classical viruses by the giant size of their virions and genomes. Pandoraviruses are the record holders in size of genomes and number of predicted genes. Three strains, P. salinus, P. dulcis, and P. inopinatum, have been described to date. We isolated three new ones, namely P. massiliensis, P. braziliensis, and P. pampulha, from environmental samples collected in Brazil. We describe here their genomes, the transcriptome and proteome of P. massiliensis, and the pangenome of the group encompassing the six pandoravirus isolates. Genome sequencing was performed with an Illumina MiSeq instrument. Genome annotation was performed using GeneMarkS and Prodigal softwares and comparative genomic analyses. The core genome and pangenome were determined using notably ProteinOrtho and CD-HIT programs. Transcriptomics was performed for P. massiliensis with the Illumina MiSeq instrument; proteomics was also performed for this virus using 1D/2D gel electrophoresis and mass spectrometry on a Synapt G2Si Q-TOF traveling wave mobility spectrometer. The genomes of the three new pandoraviruses are comprised between 1.6 and 1.8 Mbp. The genomes of P. massiliensis, P. pampulha, and P. braziliensis were predicted to harbor 1,414, 2,368, and 2,696 genes, respectively. These genes comprise up to 67% of ORFans. Phylogenomic analyses showed that P. massiliensis and P. braziliensis were more closely related to each other than to the other pandoraviruses. The core genome of pandoraviruses comprises 352 clusters of genes, and the ratio core genome/pangenome is less than 0.05. The extinction curve shows clearly that the pangenome is still open. A quarter of the gene content of P. massiliensis was detected by transcriptomics. In addition, a product for a total of 162 open reading frames were found by proteomic analysis of P. massiliensis virions, including notably the products of 28 ORFans, 99 hypothetical proteins, and 90 core genes. Further analyses should allow to gain a better knowledge and understanding of the evolution and origin of these giant pandoraviruses, and of their relationships with viruses and cellular microorganisms.

Genome Characterization of the First Mimiviruses of Lineage C Isolated in Brazil.

The family Mimiviridae, comprised by giant DNA viruses, has been increasingly studied since the isolation of the Acanthamoeba polyphaga mimivirus (APMV), in 2003. In this work, we describe the genome analysis of two new mimiviruses, each isolated from a distinct Brazilian environment. Furthermore, for the first time, we are reporting the genomic characterization of mimiviruses of group C in Brazil (Br-mimiC), where a predominance of mimiviruses from group A has been previously reported. The genomes of the Br-mimiC isolates Mimivirus gilmour (MVGM) and Mimivirus golden (MVGD) are composed of double-stranded DNA molecules of ∼1.2 Mb, each encoding more than 1,100 open reading frames. Genome functional annotations highlighted the presence of mimivirus group C hallmark genes, such as the set of seven aminoacyl-tRNA synthetases. However, the set of tRNA encoded by the Br-mimiC was distinct from those of other group C mimiviruses. Differences could also be observed in a genome synteny analysis, which demonstrated the presence of inversions and loci translocations at both extremities of Br-mimiC genomes. Both phylogenetic and phyletic analyses corroborate previous results, undoubtedly grouping the new Brazilian isolates into mimivirus group C. Finally, an updated pan-genome analysis of genus Mimivirus was performed including all new genomes available until the present moment. This last analysis showed a slight increase in the number of clusters of orthologous groups of proteins among mimiviruses of group A, with a larger increase after addition of sequences from mimiviruses of groups B and C, as well as a plateau tendency after the inclusion of the last four mimiviruses of group C, including the Br-mimiC isolates. Future prospective studies will help us to understand the genetic diversity among mimiviruses.

Promoter Motifs in NCLDVs: An Evolutionary Perspective.

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.