Getting Hold of the Tobamovirus Particle-Why and How? Purification Routes over Time and a New Customizable Approach.

This article develops a multi-perspective view on motivations and methods for tobamovirus purification through the ages and presents a novel, efficient, easy-to-use approach that can be well-adapted to different species of native and functionalized virions. We survey the various driving forces prompting researchers to enrich tobamoviruses, from the search for the causative agents of mosaic diseases in plants to their increasing recognition as versatile nanocarriers in biomedical and engineering applications. The best practices and rarely applied options for the serial processing steps required for successful isolation of tobamoviruses are then reviewed. Adaptations for distinct particle species, pitfalls, and 'forgotten' or underrepresented technologies are considered as well. The article is topped off with our own development of a method for virion preparation, rooted in historical protocols. It combines selective re-solubilization of polyethylene glycol (PEG) virion raw precipitates with density step gradient centrifugation in biocompatible iodixanol formulations, yielding ready-to-use particle suspensions. This newly established protocol and some considerations for perhaps worthwhile further developments could serve as putative stepping stones towards preparation procedures appropriate for routine practical uses of these multivalent soft-matter nanorods.

naRNA-LL37 composite DAMPs define sterile NETs as self-propagating drivers of inflammation.

Neutrophil extracellular traps (NETs) are a key antimicrobial feature of cellular innate immunity mediated by polymorphonuclear neutrophils (PMNs). NETs counteract microbes but are also linked to inflammation in atherosclerosis, arthritis, or psoriasis by unknown mechanisms. Here, we report that NET-associated RNA (naRNA) stimulates further NET formation in naive PMNs via a unique TLR8-NLRP3 inflammasome-dependent pathway. Keratinocytes respond to naRNA with expression of psoriasis-related genes (e.g., IL17, IL36) via atypical NOD2-RIPK signaling. In vivo, naRNA drives temporary skin inflammation, which is drastically ameliorated by genetic ablation of RNA sensing. Unexpectedly, the naRNA-LL37 'composite damage-associated molecular pattern (DAMP)' is pre-stored in resting neutrophil granules, defining sterile NETs as inflammatory webs that amplify neutrophil activation. However, the activity of the naRNA-LL37 DAMP is transient and hence supposedly self-limiting under physiological conditions. Collectively, upon dysregulated NET release like in psoriasis, naRNA sensing may represent both a potential cause of disease and a new intervention target.

The baseless mutant links protein phosphatase 2A with basal cell identity in the brown alga Ectocarpus.

The first mitotic division of the initial cell is a key event in all multicellular organisms and is associated with the establishment of major developmental axes and cell fates. The brown alga Ectocarpus has a haploid-diploid life cycle that involves the development of two multicellular generations: the sporophyte and the gametophyte. Each generation deploys a distinct developmental programme autonomously from an initial cell, the first cell division of which sets up the future body pattern. Here, we show that mutations in the BASELESS (BAS) gene result in multiple cellular defects during the first cell division and subsequent failure to produce basal structures during both generations. BAS encodes a type B″ regulatory subunit of protein phosphatase 2A (PP2A), and transcriptomic analysis identified potential effector genes that may be involved in determining basal cell fate. The bas mutant phenotype is very similar to that observed in distag (dis) mutants, which lack a functional Tubulin-binding co-factor Cd1 (TBCCd1) protein, indicating that TBCCd1 and PP2A are two essential components of the cellular machinery that regulates the first cell division and mediates basal cell fate determination.

Adhesion of Bartonella henselae to Fibronectin Is Mediated via Repetitive Motifs Present in the Stalk of Bartonella Adhesin A.

Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram-negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). Bartonella henselae targets the extracellular matrix glycoprotein fibronectin (Fn) via the Bartonella adhesin A (BadA) attaching the bacteria to the host cell. The TAA BadA is characterized by a highly repetitive passenger domain consisting of 30 neck/stalk domains with various degrees of similarity. To elucidate the motif sequences mediating Fn binding, we generated 10 modified BadA constructs and verified their expression via Western blotting, confocal laser scanning, and electron microscopy. We analyzed their ability to bind human plasma Fn using quantitative whole-cell enzyme-linked immunosorbent assays (ELISAs) and fluorescence microscopy. Polyclonal antibodies targeting a 15-mer amino acid motif sequence proved to reduce Fn binding. We suggest that BadA adheres to Fn in a cumulative effort with quick saturation primarily via unpaired ß-strands appearing in motifs repeatedly present throughout the neck/stalk region. In addition, we demonstrated that the length of truncated BadA constructs correlates with the immunoreactivity of human patient sera. The identification of BadA-Fn binding regions will support the development of new "antiadhesive" compounds inhibiting the initial adherence of B. henselae and other TAA-expressing pathogens to host cells. IMPORTANCE Trimeric autotransporter adhesins (TAAs) are important virulence factors and are widely present in various pathogenic Gram-negative bacteria. TAA-expressing bacteria cause a wide spectrum of human diseases, such as cat scratch disease (Bartonella henselae), enterocolitis (Yersinia enterocolitica), meningitis (Neisseria meningitis), and bloodstream infections (multidrug-resistant Acinetobacter baumannii). TAA-targeted antiadhesive strategies (against, e.g., Bartonella adhesin A [BadA], Yersinia adhesin A [YadA], Neisseria adhesin A [NadA], and Acinetobacter trimeric autotransporter [Ata]) might represent a universal strategy to counteract such bacterial infections. BadA is one of the best characterized TAAs, and because of its high number of (sub)domains, it serves as an attractive adhesin to study the domain-function relationship of TAAs in the infection process. The identification of common binding motifs between TAAs (here, BadA) and their major binding partner (here, fibronectin) provides a basis toward the design of novel "antiadhesive" compounds preventing the initial adherence of Gram-negative bacteria in infections.

Strongyloides spp. eliminate male-determining sperm post-meiotically.

If normal male meiosis occurs, it would be expected that 50 % of sperm lack an X chromosome (nullo X) and hence upon fertilisation, result in male progeny. However, for sexual reproduction within the free-living stages of Strongyloides spp. male offspring are absent. We had shown earlier by quantitative whole genome sequencing that within Strongyloides spp., nullo-X sperm are either absent (S. papillosus) or underrepresented (S. ratti) among mature sperm. To investigate how and when this elimination of male-determining sperm occurs, we characterised spermatogenesis and the dynamic localisation of important molecular players such as tubulin, actin and major sperm protein by DIC microscopy, immunohistochemistry, and fluorescent in situ hybridization (FISH) in S. ratti, S. papillosus and Parastrongyloides trichosuri. We found that meiotic divisions in these parasites proceeded as expected for organisms with XO males, resulting in four equally sized spermatocytes, two with and two without an X chromosome. However, mature sperm were found to almost always contain an X chromosome. We also observed structures that contained protein constituents of sperm, such as actin and major sperm protein (MSP) but no DNA. These structures resemble C. elegans residual bodies in appearance and may assume their function. We hypothesize that spermatocytes without an X-chromosome undergo some form of programmed cell death and transform into these residual body-like structures. As in C. elegans, MSP is found in fibrous body-membranous organelles (FB-MOs). Knocking down MSP by RNAi showed that MSP is essential for fertility in S. ratti, as it is in C. elegans.

The leaf beetle Chelymorpha alternans propagates a plant pathogen in exchange for pupal protection.

Many insects rely on microbial protection in the early stages of their development. However, in contrast to symbiont-mediated defense of eggs and young instars, the role of microbes in safeguarding pupae remains relatively unexplored, despite the susceptibility of the immobile stage to antagonistic challenges. Here, we outline the importance of symbiosis in ensuring pupal protection by describing a mutualistic partnership between the ascomycete Fusarium oxysporum and Chelymorpha alternans, a leaf beetle. The symbiont rapidly proliferates at the onset of pupation, extensively and conspicuously coating C. alternans during metamorphosis. The fungus confers defense against predation as symbiont elimination results in reduced pupal survivorship. In exchange, eclosing beetles vector F. oxysporum to their host plants, resulting in a systemic infection. By causing wilt disease, the fungus retained its phytopathogenic capacity in light of its symbiosis with C. alternans. Despite possessing a relatively reduced genome, F. oxysporum encodes metabolic pathways that reflect its dual lifestyle as a plant pathogen and a defensive insect symbiont. These include virulence factors underlying plant colonization, along with mycotoxins that may contribute to the defensive biochemistry of the insect host. Collectively, our findings shed light on a mutualism predicated on pupal protection of an herbivorous beetle in exchange for symbiont dissemination and propagation.

For the road: calibrated maternal investment in light of extracellular symbiont transmission.

Faithful transmission of beneficial symbionts is critical for the persistence of mutualisms. Many insect groups rely on extracellular routes that require microbial symbionts to survive outside the host during transfer. However, given a prolonged aposymbiotic phase in offspring, how do mothers mitigate the risk of symbiont loss due to unsuccessful transmission? Here, we investigated symbiont regulation and reacquisition during extracellular transfer in the tortoise beetle, Chelymorpha alternans (Coleoptera: Cassidinae). Like many cassidines, C. alternans relies on egg caplets to vertically propagate its obligate symbiont Candidatus Stammera capleta. On average, each caplet is supplied with 12 symbiont-bearing spheres where Stammera is embedded. We observe limited deviation (±2.3) in the number of spheres allocated to each caplet, indicating strict maternal control over symbiont supply. Larvae acquire Stammera 1 day prior to eclosion but are unable to do so after hatching, suggesting that a specific developmental window governs symbiont uptake. Experimentally manipulating the number of spheres available to each egg revealed that a single sphere is sufficient to ensure successful colonization by Stammera relative to the 12 typically packaged within a caplet. Collectively, our findings shed light on a tightly regulated symbiont transmission cycle optimized to ensure extracellular transfer.

Changes in Envelope Structure and Cell-Cell Communication during Akinete Differentiation and Germination in Filamentous Cyanobacterium Trichormus variabilis ATCC 29413.

Planktonic freshwater filamentous cyanobacterium Trichormus variabilis ATCC 29413 (previously known as Anabaena variabilis) can differentiate heterocysts and akinetes to survive under different stress conditions. Whilst heterocysts enable diazotrophic growth, akinetes are spore-like resting cells that make the survival of the species possible under adverse growth conditions. Under suitable environmental conditions, they germinate to produce new vegetative filaments. Several morphological and physiological changes occur during akinete formation and germination. Here, using scanning electron microscopy (SEM), we found that the mature akinetes had a wrinkled envelope, and the surface of the envelope smoothened as the cell size increased during germination. Thereupon, the akinete envelope ruptured to release the short emerging filament. Focused ion beam-scanning electron microscopy (FIB/SEM) tomography of immature akinetes revealed the presence of cytoplasmic granules, presumably consisting of cyanophycin or glycogen. In addition, the akinete envelope architecture of different layers, the exopolysaccharide and glycolipid layers, could be visualized. We found that this multilayered envelope helped to withstand osmotic stress and to maintain the structural integrity. Furthermore, by fluorescence recovery after photobleaching (FRAP) measurements, using the fluorescent tracer calcein, we found that intercellular communication decreased during akinete formation as compared with the vegetative cells. In contrast, freshly germinating filaments restored cell communication.

Long-Read Sequencing Reveals Genetic Adaptation of Bartonella Adhesin A Among Different Bartonella henselae Isolates.

Bartonella henselae is the causative agent of cat scratch disease and other clinical entities such as endocarditis and bacillary angiomatosis. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations. Human, feline, and laboratory adapted B. henselae isolates often display genomic and phenotypic differences that are related to the expression of outer membrane proteins, for example the Bartonella adhesin A (BadA). This modularly-structured trimeric autotransporter adhesin is a major virulence factor of B. henselae and is crucial for the initial binding to the host via the extracellular matrix proteins fibronectin and collagen. By using next-generation long-read sequencing we demonstrate a conserved genome among eight B. henselae isolates and identify a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes. Two of the eight tested B. henselae strains lack BadA expression because of frameshift mutations. We suggest that active recombination mechanisms, possibly via phase variation (i.e., slipped-strand mispairing and site-specific recombination) within the repetitive badA island facilitate reshuffling of homologous domain arrays. The resulting variations among the different BadA proteins might contribute to host immune evasion and enhance long-term and efficient colonisation in the differing host environments. Considering the role of BadA as a key virulence factor, it remains important to check consistently and regularly for BadA surface expression during experimental infection procedures.

Curbing gastrointestinal infections by defensin fragment modifications without harming commensal microbiota.

The occurrence and spread of multidrug-resistant pathogens, especially bacteria from the ESKAPE panel, increases the risk to succumb to untreatable infections. We developed a novel antimicrobial peptide, Pam-3, with antibacterial and antibiofilm properties to counter this threat. The peptide is based on an eight-amino acid carboxyl-terminal fragment of human ß-defensin 1. Pam-3 exhibited prominent antimicrobial activity against multidrug-resistant ESKAPE pathogens and additionally eradicated already established biofilms in vitro, primarily by disrupting membrane integrity of its target cell. Importantly, prolonged exposure did not result in drug-resistance to Pam-3. In mouse models, Pam-3 selectively reduced acute intestinal Salmonella and established Citrobacter infections, without compromising the core microbiota, hence displaying an added benefit to traditional broad-spectrum antibiotics. In conclusion, our data support the development of defensin-derived antimicrobial agents as a novel approach to fight multidrug-resistant bacteria, where Pam-3 appears as a particularly promising microbiota-preserving candidate.

New insights in the coordinated amidase and glucosaminidase activity of the major autolysin (Atl) in Staphylococcus aureus.

After bacterial cell division, the daughter cells are still covalently interlinked by the peptidoglycan network which is resolved by specific hydrolases (autolysins) to release the daughter cells. In staphylococci, the major autolysin (Atl) with its two domain enzymes, N-acetylmuramyl-L-alanine amidase (AmiA) and ß-N-acetylglucosaminidase (GlcA), resolves the peptidoglycan to release the daughter cells. Internal deletions in each of the enzyme domains revealed defined morphological alterations such as cell cluster formation in ΔamiA, ΔglcA and Δatl, and asymmetric cell division in the ΔglcA. A most important finding was that GlcA activity requires the prior removal of the stem peptide by AmiA for its activity thus the naked glycan strand is its substrate. Furthermore, GlcA is not an endo-ß-N-acetylglucosaminidase but an exo-enzyme that cuts the glycan backbone to disaccharides independent of its O-acetylation modification. Our results shed new light into the sequential peptidoglycan hydrolysis by AmiA and GlcA during cell division in staphylococci.

Optimized production strategy of the major capsid protein HPV 16L1 non-assembly variant in E. coli.

The capsid of human papillomavirus (HPV) consists of two capsid proteins - the major capsid protein L1 and the minor capsid protein L2. Assembled virus-like particles, which only consist of L1 proteins, are successfully applied as prophylactic vaccines against HPV infections. The capsid subunits are L1-pentamers, which are also reported to protect efficiently against HPV infections in animals. The recombinant production of L1 has been previously shown in E. coli, yeast, insect cells, plants and mammalian cell culture. Principally, in E. coli-based expression system L1 shows high expression yields but the protein is largely insoluble. In order to overcome this problem reported strategies address fusion proteins and overexpression of bacterial chaperones. However, an insufficient cleavage of the fusion proteins and removal of co-purified chaperones can hamper subsequent down streaming. We report a significant improvement in the production of soluble L1-pentamers by combining (I) a fusion of a N-terminal SUMO-tag to L1, (II) the heterologous co-expression of the chaperon system GroEL/ES and (III) low expression temperature. The fusion construct was purified in a 2-step protein purification including efficient removal of GroEL/ES and complete removal of the N-terminal SUMO-tag. The expression strategy was transferred to process-controlled high-cell-density fermentation with defined media according to the guidelines of good manufacturing practice. The produced L1 protein is highly pure (>95%), free of DNA (260:280 = 0.5) and pentameric. The production strategy yielded 5.73 mg of purified L1-pentamers per gram dry biomass. The optimized strategy is a suitable alternative for high yield L1-pentamer production and purification as a cheaper process for vaccine production.

CRISPR/Cas9-mediated ELANE knockout enables neutrophilic maturation of primary hematopoietic stem and progenitor cells and induced pluripotent stem cells of severe congenital neutropenia patients.

A Autosomal-dominant ELANE mutations are the most common cause of severe congenital neutropenia. Although the majority of congenital neutropenia patients respond to daily granulocyte colony stimulating factor, approximately 15 % do not respond to this cytokine at doses up to 50 µg/kg/day and approximately 15 % of patients will develop myelodysplasia or acute myeloid leukemia. "Maturation arrest," the failure of the marrow myeloid progenitors to form mature neutrophils, is a consistent feature of ELANE associated congenital neutropenia. As mutant neutrophil elastase is the cause of this abnormality, we hypothesized that ELANE associated neutropenia could be treated and "maturation arrest" corrected by a CRISPR/Cas9-sgRNA ribonucleoprotein mediated ELANE knockout. To examine this hypothesis, we used induced pluripotent stem cells from two congenital neutropenia patients and primary hematopoietic stem and progenitor cells from four congenital neutropenia patients harboring ELANE mutations as well as HL60 cells expressing mutant ELANE We observed that granulocytic differentiation of ELANE knockout induced pluripotent stem cells and primary hematopoietic stem and progenitor cells were comparable to healthy individuals. Phagocytic functions, ROS production, and chemotaxis of the ELANE KO (knockout) neutrophils were also normal. Knockdown of ELANE in the mutant ELANE expressing HL60 cells also allowed full maturation and formation of abundant neutrophils. These observations suggest that ex vivo CRISPR/Cas9 RNP based ELANE knockout of patients' primary hematopoietic stem and progenitor cells followed by autologous transplantation may be an alternative therapy for congenital neutropenia.

MERLIN: a novel BRET-based proximity biosensor for studying mitochondria-ER contact sites.

The contacts between the ER and mitochondria play a key role in cellular functions such as the exchange of lipids and calcium between both organelles, as well as in apoptosis and autophagy signaling. The molecular architecture and spatiotemporal regulation of these distinct contact regions remain obscure and there is a need for new tools that enable tackling these questions. Here, we present a new bioluminescence resonance energy transfer-based biosensor for the quantitative analysis of distances between the ER and mitochondria that we call MERLIN (Mitochondria-ER Length Indicator Nanosensor). The main advantages of MERLIN compared with available alternatives are that it does not rely on the formation of artificial physical links between the two organelles, which could lead to artifacts, and that it allows to study contact site reversibility and dynamics. We show the applicability of MERLIN by characterizing the role of the mitochondrial dynamics machinery on the contacts of this organelle with the ER.

The Acinetobacter trimeric autotransporter adhesin Ata controls key virulence traits of Acinetobacter baumannii.

Acinetobacter baumannii is a Gram-negative pathogen that causes a multitude of nosocomial infections. The Acinetobacter trimeric autotransporter adhesin (Ata) belongs to the superfamily of trimeric autotransporter adhesins which are important virulence factors in many Gram-negative species. Phylogenetic profiling revealed that ata is present in 78% of all sequenced A. baumannii isolates but only in 2% of the closely related species A. calcoaceticus and A. pittii. Employing a markerless ata deletion mutant of A. baumannii ATCC 19606 we show that adhesion to and invasion into human endothelial and epithelial cells depend on Ata. Infection of primary human umbilical cord vein endothelial cells (HUVECs) with A. baumannii led to the secretion of interleukin (IL)-6 and IL-8 in a time- and Ata-dependent manner. Furthermore, infection of HUVECs by WT A. baumannii was associated with higher rates of apoptosis via activation of caspases-3 and caspase-7, but not necrosis, in comparison to ∆ata. Ata deletion mutants were furthermore attenuated in their ability to kill larvae of Galleria mellonella and to survive in larvae when injected at sublethal doses. This indicates that Ata is an important multifunctional virulence factor in A. baumannii that mediates adhesion and invasion, induces apoptosis and contributes to pathogenicity in vivo.

Different forms of African cassava mosaic virus capsid protein within plants and virions.

One geminiviral gene encodes the capsid protein (CP), which can appear as several bands after electrophoresis depending on virus and plant. African cassava mosaic virus-Nigeria CP in Nicotiana benthamiana, however, yielded one band (~ 30 kDa) in total protein extracts and purified virions, although its expression in yeast yielded two bands (~ 30, 32 kDa). Mass spectrometry of the complete protein and its tryptic fragments from virions is consistent with a cleaved start M1, acetylated S2, and partial phosphorylation at T12, S25 and S62. Mutants for additional potentially modified sites (N223A; C235A) were fully infectious and formed geminiparticles. Separation in triton acetic acid urea gels confirmed charge changes of the CP between plants and yeast indicating differential phosphorylation. If the CP gene alone was expressed in plants, multiple bands were observed like in yeast. A high turnover rate indicates that post-translational modifications promote CP decay probably via the ubiquitin-triggered proteasomal pathway.

Bio-On-Magnetic-Beads (BOMB): Open platform for high-throughput nucleic acid extraction and manipulation.

Current molecular biology laboratories rely heavily on the purification and manipulation of nucleic acids. Yet, commonly used centrifuge- and column-based protocols require specialised equipment, often use toxic reagents, and are not economically scalable or practical to use in a high-throughput manner. Although it has been known for some time that magnetic beads can provide an elegant answer to these issues, the development of open-source protocols based on beads has been limited. In this article, we provide step-by-step instructions for an easy synthesis of functionalised magnetic beads, and detailed protocols for their use in the high-throughput purification of plasmids, genomic DNA, RNA and total nucleic acid (TNA) from a range of bacterial, animal, plant, environmental and synthetic sources. We also provide a bead-based protocol for bisulfite conversion and size selection of DNA and RNA fragments. Comparison to other methods highlights the capability, versatility, and extreme cost-effectiveness of using magnetic beads. These open-source protocols and the associated webpage (https://bomb.bio) can serve as a platform for further protocol customisation and community engagement.

Simple Synthesis of Functionalized Paramagnetic Beads for Nucleic Acid Purification and Manipulation.

The purification of nucleic acids is one of the most common procedures employed in modern molecular biology laboratories. Typically, commercial column-based protocols are utilized to isolate DNA or RNA from various sources. However, these methods not only require specialized equipment, but are also extremely expensive for high-throughput applications. Although an elegant answer to this issue can be provided by paramagnetic beads, bead-based open-source protocols have been limited in the past. Here, we provide an easy to follow step-by-step manual for the synthesis of paramagnetic beads, as well as their functionalization with either a silica- or a carboxyl-surface that can be used to replace the commercial columns with self-made magnetic beads. Together with a variety of detailed protocols for their use in high-throughput nucleic acids extractions, this bead synthesis method forms the recently published open platform Bio-On-Magnetic-Beads (BOMB), which is available on PLOS Biology ( Oberacker et al., 2019 ). Updated protocols can be found on the associated webpage (https://bomb.bio).

Structural characterization of the bacterial proteasome homolog BPH reveals a tetradecameric double-ring complex with unique inner cavity properties.

Eukaryotic and archaeal proteasomes are paradigms for self-compartmentalizing proteases. To a large extent, their function requires interplay with hexameric ATPases associated with diverse cellular activities (AAA+) that act as substrate unfoldases. Bacteria have various types of self-compartmentalizing proteases; in addition to the proteasome itself, these include the proteasome homolog HslV, which functions together with the AAA+ HslU; the ClpP protease with its partner AAA+ ClpX; and Anbu, a recently characterized ancestral proteasome variant. Previous bioinformatic analysis has revealed a novel bacterial member of the proteasome family Betaproteobacteria proteasome homolog (BPH). Using cluster analysis, we here affirmed that BPH evolutionarily descends from HslV. Crystal structures of the Thiobacillus denitrificans and Cupriavidus metallidurans BPHs disclosed a homo-oligomeric double-ring architecture in which the active sites face the interior of the cylinder. Using small-angle X-ray scattering (SAXS) and electron microscopy averaging, we found that BPH forms tetradecamers in solution, unlike the dodecamers seen in HslV. Although the highly acidic inner surface of BPH was in striking contrast to the cavity characteristics of the proteasome and HslV, a classical proteasomal reaction mechanism could be inferred from the covalent binding of the proteasome-specific inhibitor epoxomicin to BPH. A ligand-bound structure implied that the elongated BPH inner pore loop may be involved in substrate recognition. The apparent lack of a partner unfoldase and other unique features, such as Ser replacing Thr as the catalytic residue in certain BPH subfamilies, suggest a proteolytic function for BPH distinct from those of known bacterial self-compartmentalizing proteases.

Electron Microscopy Methods for Virus Diagnosis and High Resolution Analysis of Viruses.

The term "virosphere" describes both the space where viruses are found and the space they influence, and can extend to their impact on the environment, highlighting the complexity of the interactions involved. Studying the biology of viruses and the etiology of virus disease is crucial to the prevention of viral disease, efficient and reliable virus diagnosis, and virus control. Electron microscopy (EM) is an essential tool in the detection and analysis of virus replication. New EM methods and ongoing technical improvements offer a broad spectrum of applications, allowing in-depth investigation of viral impact on not only the host but also the environment. Indeed, using the most up-to-date electron cryomicroscopy methods, such investigations are now close to atomic resolution. In combination with bioinformatics, the transition from 2D imaging to 3D remodeling allows structural and functional analyses that extend and augment our knowledge of the astonishing diversity in virus structure and lifestyle. In combination with confocal laser scanning microscopy, EM enables live imaging of cells and tissues with high-resolution analysis. Here, we describe the pivotal role played by EM in the study of viruses, from structural analysis to the biological relevance of the viral metagenome (virome).