Structural and Proteomic Characterization of the Initiation of Giant Virus Infection.

Since their discovery, giant viruses have expanded our understanding of the principles of virology. Due to their gargantuan size and complexity, little is known about the life cycles of these viruses. To answer outstanding questions regarding giant virus infection mechanisms, we set out to determine biomolecular conditions that promote giant virus genome release. We generated four infection intermediates in Samba virus (Mimivirus genus, lineage A) as visualized by cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-ET), and scanning electron microscopy (SEM). Each of these four intermediates reflects similar morphology to a stage that occurs in vivo. We show that these genome release stages are conserved in other mimiviruses. Finally, we identified proteins that are released from Samba and newly discovered Tupanvirus through differential mass spectrometry. Our work revealed the molecular forces that trigger infection are conserved among disparate giant viruses. This study is also the first to identify specific proteins released during the initial stages of giant virus infection.

Analysis of the Genomic Features and Evolutionary History of Pithovirus-Like Isolates Reveals Two Major Divergent Groups of Viruses.

New representatives of the phylum Nucleocytoviricota have been rapidly described in the last decade. Despite this, not all viruses of this phylum are allocated to recognized taxonomic families, as is the case for orpheovirus, pithovirus, and cedratvirus, which form the proposed family Pithoviridae. In this study, we performed comprehensive comparative genomic analyses of 8 pithovirus-like isolates, aiming to understand their common traits and evolutionary history. Structural and functional genome annotation was performed de novo for all the viruses, which served as a reference for pangenome construction. The synteny analysis showed substantial differences in genome organization between these viruses, with very few and short syntenic blocks shared between orpheovirus and its relatives. It was possible to observe an open pangenome with a significant increase in the slope when orpheovirus was added, alongside a decrease in the core genome. Network analysis placed orpheovirus as a distant and major hub with a large fraction of unique clusters of orthologs, indicating a distant relationship between this virus and its relatives, with only a few shared genes. Additionally, phylogenetic analyses of strict core genes shared with other viruses of the phylum reinforced the divergence of orpheovirus from pithoviruses and cedratviruses. Altogether, our results indicate that although pithovirus-like isolates share common features, this group of ovoid-shaped giant viruses presents substantial differences in gene contents, genomic architectures, and the phylogenetic history of several core genes. Our data indicate that orpheovirus is an evolutionarily divergent viral entity, suggesting its allocation to a different viral family, Orpheoviridae. IMPORTANCE Giant viruses that infect amoebae form a monophyletic group named the phylum Nucleocytoviricota. Despite being genomically and morphologically very diverse, the taxonomic categories of some clades that form this phylum are not yet well established. With advances in isolation techniques, the speed at which new giant viruses are described has increased, escalating the need to establish criteria to define the emerging viral taxa. In this work, we performed a comparative genomic analysis of representatives of the putative family Pithoviridae. Based on the dissimilarity of orpheovirus from the other viruses of this putative family, we propose that orpheovirus be considered a member of an independent family, Orpheoviridae, and suggest criteria to demarcate families consisting of ovoid-shaped giant viruses.

Gene duplication as a major force driving the genome expansion in some giant viruses.

IMPORTANCE: Giant viruses are noteworthy not only due to their enormous particles but also because of their gigantic genomes. In this context, a fundamental question has persisted: how did these genomes evolve? Here we present the discovery of cedratvirus pambiensis, featuring the largest genome ever described for a cedratvirus. Our data suggest that the larger size of the genome can be attributed to an unprecedented number of duplicated genes. Further investigation of this phenomenon in other viruses has illuminated gene duplication as a key evolutionary mechanism driving genome expansion in diverse giant viruses. Although gene duplication has been described as a recurrent event in cellular organisms, our data highlights its potential as a pivotal event in the evolution of gigantic viral genomes.

A long-term prospecting study on giant viruses in terrestrial and marine Brazilian biomes.

The discovery of mimivirus in 2003 prompted the search for novel giant viruses worldwide. Despite increasing interest, the diversity and distribution of giant viruses is barely known. Here, we present data from a 2012-2022 study aimed at prospecting for amoebal viruses in water, soil, mud, and sewage samples across Brazilian biomes, using Acanthamoeba castellanii for isolation. A total of 881 aliquots from 187 samples covering terrestrial and marine Brazilian biomes were processed. Electron microscopy and PCR were used to identify the obtained isolates. Sixty-seven amoebal viruses were isolated, including mimiviruses, marseilleviruses, pandoraviruses, cedratviruses, and yaraviruses. Viruses were isolated from all tested sample types and almost all biomes. In comparison to other similar studies, our work isolated a substantial number of Marseillevirus and cedratvirus representatives. Taken together, our results used a combination of isolation techniques with microscopy, PCR, and sequencing and put highlight on richness of giant virus present in different terrestrial and marine Brazilian biomes.

In-Depth Characterization of the Chikungunya Virus Replication Cycle.

The chikungunya virus has spread globally with a remarkably high attack rate. Infection causes arthralgic sequelae that can last for years. Nevertheless, there are no specific drugs or vaccines to contain the virus. Understanding the biology of the virus, such as its replication cycle, is a powerful tool to identify new drugs and comprehend virus-host interactions. Even though the chikungunya virus has been known for a long time (it was first described in 1952), many aspects of the replication cycle remain unclear. Furthermore, part of the cycle is based on observations of other alphaviruses. In this study, we used electron and scanning microscopy, as well as biological assays, to analyze and investigate the stages of the chikungunya virus replication cycle. Based on our data, we found infection cellular activities other than those usually described for the chikungunya virus replication cycle, i.e., we show particles enveloping intracellularly without budding in a membrane-delimited morphogenesis area, and we also observed virion release by membrane protrusions. Our work provides novel details regarding the biology of chikungunya virus and fills gaps in our knowledge of its replication cycle. These findings may contribute to a better understanding of virus-host interactions and support the development of antivirals. IMPORTANCE The understanding of virus biology is essential to containing virus dissemination, and exploring the virus replication cycle is a powerful tool to do this. There are many points in the biology of the chikungunya virus that need to be clarified, especially regarding its replication cycle. Our incomplete understanding of chikungunya virus infection stages is based on studies with other alphaviruses. We systematized the chikungunya virus replication cycle using microscopic imaging in the order of infection stages, as follows: entry, replication, protein synthesis, assembly/morphogenesis, and release. The imaging evidence shows novel points in the replication cycle of enveloping without budding, as well as particle release by cell membrane protrusion.

Taxonomic update for giant viruses in the order Imitervirales (phylum Nucleocytoviricota).

Large DNA viruses in the phylum Nucleocytoviricota, sometimes referred to as "giant viruses" owing to their large genomes and virions, have been the subject of burgeoning interest over the last decade. Here, we describe recently adopted taxonomic updates for giant viruses within the order Imitervirales. The families Allomimiviridae, Mesomimiviridae, and Schizomimiviridae have been created to accommodate the increasing diversity of mimivirus relatives that have sometimes been referred to in the literature as "extended Mimiviridae". In addition, the subfamilies Aliimimivirinae, Megamimivirinae, and Klosneuvirinae have been established to refer to subgroups of the Mimiviridae. Binomial names have also been adopted for all recognized species in the order. For example, Acanthamoeba polyphaga mimivirus is now classified in the species Mimivirus bradfordmassiliense.

Yaravirus: A novel 80-nm virus infecting Acanthamoeba castellanii.

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 organisms 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 function 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 representing 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.

Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus.

BACKGROUND: Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant's antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants. METHODS: In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed. RESULTS: Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host. CONCLUSION: The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a 'very difficult to manage' sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.

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.

Serologic and Molecular Evidence of Vaccinia Virus Circulation among Small Mammals from Different Biomes, Brazil.

Vaccinia virus (VACV) is a zoonotic agent that causes a disease called bovine vaccinia, which is detected mainly in milking cattle and humans in close contact with these animals. Even though many aspects of VACV infection have been described, much is still unknown about its circulation in the environment and its natural hosts/reservoirs. To investigate the presence of Orthopoxvirus antibodies or VACV DNA, we captured small rodents and marsupials in 3 areas of Minas Gerais state, Brazil, and tested their samples in a laboratory. A total of 336 animals were tested; positivity ranged from 18.1% to 25.5% in the 3 studied regions located in different biomes, including the Atlantic Forest and the Cerrado. Analysis of nucleotide sequences indicated co-circulation of VACV groups I and II. Our findings reinforce the possible role played by rodents and marsupials in VACV maintenance and its transmission chain.

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.

c-Jun integrates signals from both MEK/ERK and MKK/JNK pathways upon vaccinia virus infection.

Usurpation of the host's signalling pathways is a common strategy employed by viruses to promote their successful replication. Here we show that infection with the orthopoxvirus vaccinia virus (VACV) leads to sustained stimulation of c-Jun activity during the entire infective cycle. This stimulation is temporally regulated through MEK/ERK or MKK/JNK pathways, i.e. during the early/mid phase (1 to 6 hpi) and in the late phase (9 to 24 hpi) of the infective cycle, respectively. As a transcriptional regulator, upon infection with VACV, c-Jun is translocated from the cytoplasm to the nucleus, where it binds to the AP-1 DNA sequence found at the promoter region of its target genes. To investigate the role played by c-Jun during VACV replication cycle, we generated cell lines that stably express a c-Jun-dominant negative (DNc-Jun) mutation. Our data revealed that c-Jun is required during early infection to assist with viral DNA replication, as demonstrated by the decreased amount of viral DNA found in the DNc-Jun cells. We also demonstrated that c-Jun regulates the expression of the early growth response gene (egr-1), a gene previously shown to affect VACV replication mediated by MEK/ERK signalling. VACV-induced stimulation of the MKK/JNK/JUN pathway impacts viral dissemination, as we observed a significant reduction in both viral yield, during late stages of infection, and virus plaque size. Collectively, our data suggest that, by modulating the host's signalling pathways through a common target such as c-Jun, VACV temporally regulates its infective cycle in order to successfully replicate and subsequently spread.

Vaccinia virus dissemination requires p21-activated kinase 1.

The orthopoxvirus vaccinia virus (VACV) interacts with both actin and microtubule cytoskeletons in order to generate and spread progeny virions. Here, we present evidence demonstrating the involvement of PAK1 (p21-activated kinase 1) in the dissemination of VACV. Although PAK1 activation has previously been associated with optimal VACV entry via macropinocytosis, its absence does not affect the production of intracellular mature virions (IMVs) and extracellular enveloped virions (EEVs). Our data demonstrate that low-multiplicity infection of PAK1(-/-) MEFs leads to a reduction in plaque size followed by decreased production of both IMVs and EEVs, strongly suggesting that virus spread was impaired in the absence of PAK1. Confocal and scanning electron microscopy showed a substantial reduction in the amount of VACV-induced actin tails in PAK1(-/-) MEFs, but no significant alteration in the total amount of cell-associated enveloped virions (CEVs). Furthermore, the decreased VACV dissemination in PAK1(-/-) cells was correlated with the absence of phosphorylated ARPC1 (Thr21), a downstream target of PAK1 and a key regulatory subunit of the ARP2/3 complex, which is necessary for the formation of actin tails and viral spread. We conclude that PAK1, besides its role in virus entry, also plays a relevant role in VACV dissemination.

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.

Horizontal study of vaccinia virus infections in an endemic area: epidemiologic, phylogenetic and economic aspects.

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.

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.

Evaluating Known Zika Virus NS2B-NS3 Protease Inhibitor Scaffolds via In Silico Screening and Biochemical Assays.

The NS2B-NS3 protease (NS2B-NS3pro) is regarded as an interesting molecular target for drug design, discovery, and development because of its essential role in the Zika virus (ZIKV) cycle. Although no NS2B-NS3pro inhibitors have reached clinical trials, the employment of drug-like scaffolds can facilitate the screening process for new compounds. In this study, we performed a combination of ligand-based and structure-based in silico methods targeting two known non-peptide small-molecule scaffolds with micromolar inhibitory activity against ZIKV NS2B-NS3pro by a virtual screening (VS) of promising compounds. Based on these two scaffolds, we selected 13 compounds from an initial library of 509 compounds from ZINC15's similarity search. These compounds exhibited structural modifications that are distinct from previously known compounds yet keep pertinent features for binding. Despite promising outcomes from molecular docking and initial enzymatic assays against NS2B-NS3pro, confirmatory assays with a counter-screening enzyme revealed an artifactual inhibition of the assessed compounds. However, we report two compounds, 9 and 11, that exhibited antiviral properties at a concentration of 50 µM in cellular-based assays. Overall, this study provides valuable insights into the ongoing research on anti-ZIKV compounds to facilitate and improve the development of new inhibitors.