Virus Particles Research Articles

The infectivity of virus particles from Wolbachia-infected Drosophila

Viruses transmitted by arthropods pose a huge risk to human health. Wolbachia is an endosymbiotic bacterium that infects various arthropods and can block the viral replication cycle of several medically important viruses. As such, it has been successfully implemented in vector control strategies against mosquito-borne diseases, including Dengue virus. Whilst the mechanisms behind Wolbachia-mediated viral blocking are not fully characterised, it was recently shown that viruses grown in the presence of Wolbachia in some Dipteran cell cultures are less infectious than those grown in the absence of Wolbachia. Here, we investigate the breadth of this mechanism by determining if Wolbachia reduces infectivity in a different system at a different scale. To do this, we looked at Wolbachia’s impact on insect viruses from two diverse virus families within the whole organism Drosophila melanogaster. Drosophila C virus (DCV; Family Dicistroviridae) and Flock House virus (FHV; Famliy Nodaviridae) were grown in adult D. melanogaster flies with and without Wolbachia strain wMelPop. Measures of the physical characteristics, infectivity, pathogenicity, and replicative properties of progeny virus particles did not identify any impact of Wolbachia on either DCV or FHV. Therefore, there was no evidence that changes in infectivity contribute to Wolbachia-mediated viral blocking in this system. Overall, this is consistent with growing evidence that the mechanisms behind Wolbachia viral blocking are dependent on the unique tripartite interactions occurring between the host, the Wolbachia strain, and the infecting virus.

Kaposi's sarcoma-associated herpesvirus (KSHV) gB dictates a low-pH endocytotic entry pathway as revealed by a dual-fluorescent virus system and a rhesus monkey rhadinovirus expressing KSHV gB.

Interaction with host cell receptors initiates internalization of Kaposi's sarcoma-associated herpesvirus (KSHV) particles. Fusion of viral and host cell membranes, which is followed by release of the viral capsid into the cytoplasm, is executed by the core fusion machinery composed of glycoproteins H (gH), L (gL), and B (gB), that is common to all herpesviruses. KSHV infection has been shown to be sensitive to inhibitors of vacuolar acidification, suggestive of low pH as a fusion trigger. To analyze KSHV entry at the single particle level we developed dual-fluorescent recombinant KSHV strains that incorporate fluorescent protein-tagged glycoproteins and capsid proteins. In addition, we generated a hybrid rhesus monkey rhadinovirus (RRV) that expresses KSHV gB in place of RRV gB to analyze gB-dependent differences in infection pathways. We demonstrated lytic reactivation and infectivity of dual-fluorescent KSHV. Confocal microscopy was used to quantify co-localization of fluorescently-tagged glycoproteins and capsid proteins. Using the ratio of dual-positive KSHV particles to single-positive capsids as an indicator of fusion events we established KSHV fusion kinetics upon infection of different target cells with marked differences in the "time-to-fusion" between cell types. Inhibition of vesicle acidification prevented KSHV particle-cell fusion, implicating low vesicle pH as a requirement. These findings were corroborated by comparison of RRV-YFP wildtype reporter virus and RRV-YFP encoding KSHV gB in place of RRV gB. While RRV wt infection of receptor-overexpressing cells was unaffected by inhibition of vesicle acidification, RRV-YFP expressing KSHV gB was sensitive to Bafilomycin A1, an inhibitor of vacuolar acidification. Single- and dual-fluorescent KSHV strains eliminate the need for virus-specific antibodies and enable the tracking of single viral particles during entry and fusion. Together with a hybrid RRV expressing KSHV gB and classical fusion assays, these novel tools identify low vesicle pH as an endocytotic trigger for KSHV membrane fusion.

A Lambda-evo (λevo) phage platform for Zika virus EDIII protein display

One of the most significant bacteriophage technologies is phage display, in which heterologous peptides are exhibited on the virion surface. This work describes the display of λ decorative protein Dλ linked to the E protein domain III of Zika virus (Dλ-ZEDIII), to the GFP protein (Dλ-GFP), or to different domain III epitopes of the EZIKV protein (Dλ-TD), exhibited on the surface of an in vitro evolved lambda phage (λevo). This phage harbors a gene D deletion and was subjected to directed evolution using Escherichia coli W3110/pDλ-ZEDIII as background. After 20 days (20 cycles of dilution), the λevo phage developed a ~ 22% genome deletion affecting the non-essential λ b region, rendering a more stable phage that exhibited fusion proteins Dλ-ZEDIII or Dλ-GFP but not Dλ-TD. Despite the λevo system was able to decorate itself with the Dλ-ZEDIII protein, the production of viral particles was ~ 1000-fold lower than the λ wild-type, due to the unexpected Dλ-ZEDIII protein aggregation into bacterial inclusion bodies. Decorated phages (106 PFU (plaque forming units)/100 µl) were inoculated into BALB/c mice, and subsequent dot blot and Western blot immunoassays proved the production of murine antibodies against ZIKV (Zika virus). This multipurpose λevo phage display platform may be used interchangeably with other more soluble peptides, providing better yields.Key points• λevo platform for displaying recombinant peptides.• Directed evolution to generate λevo with more efficient decoration.• Antigenic reaction in BALB/c mice by inoculating λevo with recombinant peptides.

Super-Resolution Goes Viral: T4 Virus Particles as Versatile 3D-Bio-NanoRulers.

In the burgeoning field of super-resolution fluorescence microscopy, significant efforts are being dedicated to expanding its applications into the 3D domain. Various methodologies have been developed that enable isotropic resolution at the nanometer scale, facilitating the visualization of 3D subcellular structures with unprecedented clarity. Central to this progress is the need for reliable 3D structures that are biologically compatible for validating resolution capabilities. Choosing the optimal standard poses a considerable challenge, necessitating, among other attributes, precisely defined geometry and the capability for specific labeling at sub-diffraction-limit distances. In this context, the use of the non-human-infecting virus, bacteriophage T4 is introduced as an effective and straightforward bio-ruler for 3D super-resolution imaging. Employing DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) along with the technique of astigmatic imaging, the icosahedral capsid of the bacteriophage T4, measuring 120nm in length and 86nm in width, and its hollow viral tail is uncovered. This level of detail in light microscopy represents a significant advancement in T4 imaging. A simple protocol for the production and preparation of samples is further outlined. Moreover, the extensive potential of bacteriophage T4 as a multifaceted 3D bio-ruler, proposing its application as a novel benchmark for 3D super-resolution imaging in biological studies is explored.

HSPG-binding peptide Pep19-2.5 is a potent inhibitor of HPV16 infection.

Peptide-based therapeutics are gaining attention for their potential to target various viral and host cell factors. One notable example is Pep19-2.5 (Aspidasept), a synthetic anti-lipopolysaccharide peptide that binds to heparan sulfate proteoglycans (HSPGs) and has demonstrated inhibitory effects against certain bacteria and enveloped viruses. This study explores, for the first time, the effectiveness of Pep19-2.5 against a non-enveloped virus, using pseudoviruses of the oncogenic human papillomavirus type 16 (HPV16) as a model. HPV16 infects epithelial cells of the skin and mucosa by using multiple cell surface receptors with initial attachment to HSPGs. Pharmacological inhibition with Pep19-2.5 in HeLa and HaCaT cells resulted in a concentration-dependent reduction of HPV16 PsV infection, with near-complete blockade observed at higher concentrations. The half-maximal inhibitory concentration (IC50) was determined to be 116 nM in HeLa cells and 183 nM in HaCaT cells, highlighting its potent antiviral activity. Our results demonstrate that Pep19-2.5 not only inhibits HPV16 PsV binding to the cell surface but also significantly reduces infection when administered post-binding. Imaging analyses revealed Pep19-2.5-dependent release of large cell-associated crowds of viral particles, suggesting interference with the transfer to secondary receptor molecules. This was corroborated by the effectiveness of Pep19-2.5 in an HSPG-negative cell line, indicating that the peptide disrupts virus binding to both primary and secondary interaction partners. Based on these findings, we propose that the antimicrobial effect of Pep19-2.5 is not limited to HSPG-dependent infections. Additionally, Pep19-2.5 may be a valuable tool for dissecting specific steps in the viral entry process.

Sensing of SARS-CoV-2-infected cells by plasmacytoid dendritic cells is modulated via an interplay between CD54/ICAM-1 and CD11a/LFA-1 αL integrin.

Type I interferons (IFN-I) represent an important component of the host's innate defense against initial SARS-CoV-2 infections. Plasmacytoid dendritic cells (pDCs) produce large quantities of IFN-I upon recognition of viral particles or infected cells. This study shows that pDCs sense infected cells more efficiently than viral particles, leading to a higher production of IFN-I. Physical contact between a pDC and an infected cell is critical to this process; the interaction is mediated via CD11a and ICAM-1 complex and potentially is bidirectional. SARS-CoV-2 variants of concern (VOCs) have evolved to limit the IFN response through different mechanisms. For the Alpha variant, reduced level of CD11a on infected cells is linked to less contact with pDCs and decreased IFN-I release. Overall, our study characterizes some of the early steps involved in pDC-mediated response against SARS-CoV-2 infection and shows that these processes are targeted by VOCs to likely limit IFN-I response and enhance viral spread.

Coacervate vesicles assembled by liquid-liquid phase separation improve delivery of biopharmaceuticals.

Vesicles play critical roles in cellular materials storage and signal transportation, even in the formation of organelles and cells. Natural vesicles are composed of a lipid layer that forms a membrane for the enclosure of substances inside. Here we report a coacervate vesicle formed by the liquid-liquid phase separation of cholesterol-modified DNA and histones. Unlike a phospholipid-based membrane-bounded vesicle, a coacervate vesicle lacks a membrane structure on the surface and is organized with a high-density liquid layer and a water-filled cavity. Through a straightforward coacervation process, we demonstrate that various biological agents, including virus particles, mRNA, cytokines and peptides, can be innocuously and directly enriched in the liquid phase. In contrast to the droplet-like coacervates that are prone to aggregation challenges, coacervate vesicles display superior kinetic stability, positioning them as a versatile delivery vehicle for biopharmaceuticals. We validate that incorporating oncolytic viruses into these coacervate vesicles endows them with potent oncolytic efficacy and elicits robust anti-tumour immune responses in mouse models.

Epithelial-to-mesenchymal transition and live cell extrusion contribute to measles virus release from human airway epithelia.

Measles virus (MeV) is a highly contagious respiratory virus transmitted via aerosols. To understand how MeV exits the airways of an infected host, we use unpassaged primary cultures of human airway epithelial cells (HAE). MeV typically remains cell-associated in HAE and forms foci of infection, termed infectious centers, by directly spreading cell-to-cell. We previously described the phenomenon in which infectious centers detach en masse from HAE and remain viable. Here, we investigate the mechanism of this cellular detachment. Via immunostaining, we observed loss of tight junction and cell adhesion proteins within infectious centers. These morphological changes indicate activation of epithelial-to-mesenchymal transition (EMT). EMT can contribute to wound healing in respiratory epithelia by mobilizing nearby cells. Inhibiting TGF-β, and thus EMT, reduced infectious center detachment. Compared with uninfected cells, MeV-infected cells also expressed increased levels of sphingosine kinase 1 (SK1), a regulator of live cell extrusion. Live cell extrusion encourages cells to detach from respiratory epithelia by contracting the actomyosin of neighboring cells. Inhibition or induction of live cell extrusion impacted infectious center detachment rates. Thus, these two related pathways contributed to infectious center detachment in HAE. Detached infectious centers contained high titers of virus that may be protected from the environment, allowing the virus to live on surfaces longer and infect more hosts.IMPORTANCEMeasles virus (MeV) is an extremely contagious respiratory pathogen that continues to cause large, disruptive outbreaks each year. Here, we examine mechanisms of detachment of MeV-infected cells. MeV spreads cell-to-cell in human airway epithelial cells (HAE) to form groups of infected cells, termed "infectious centers". We reported that infectious centers ultimately detach from HAE as a unit, carrying high titers of virus. Viral particles within cells may be more protected from environmental conditions, such as ultraviolet radiation and desiccation. We identified two host pathways, epithelial-to-mesenchymal transition and live cell extrusion, that contribute to infectious center detachment. Perturbing these pathways altered the kinetics of infectious center detachment. These pathways influence one another and contribute to epithelial wound healing, suggesting that infectious center detachment may be a usurped consequence of the host's response to infection that benefits MeV by increasing its transmissibility between hosts.

Design, optimization, and inference of biphasic decay of infectious virus particles.

Virus population dynamics are driven by counter-balancing forces of production and loss. Whereas viral production arises from complex interactions with susceptible hosts, the loss of infectious virus particles is often approximated as a first-order kinetic process. As such, experimental protocols to measure infectious virus loss are not typically designed to identify non-exponential decay processes. Here, we propose methods to evaluate if an experimental design is adequate to identify multiphasic virus particle decay and to optimize the sampling times of decay experiments, accounting for uncertainties in viral kinetics. First, we evaluate synthetic scenarios of biphasic decays, with varying decay rates and initial proportions of subpopulations. We show that robust inference of multiphasic decay is more likely when the faster decaying subpopulation predominates insofar as early samples are taken to resolve the faster decay rate. Moreover, design optimization involving non-equal spacing between observations increases the precision of estimation while reducing the number of samples. We then apply these methods to infer multiple decay rates associated with the decay of bacteriophage ('phage') ΦD9, an evolved isolate derived from phage Φ21. A pilot experiment confirmed that ΦD9 decay is multiphasic, but was unable to resolve the rate or proportion of the fast decaying subpopulation(s). We then applied a Fisher information matrix-based design optimization method to propose non-equally spaced sampling times. Using this strategy, we were able to robustly estimate multiple decay rates and the size of the respective subpopulations. Notably, we conclude that the vast majority (94%) of the phage ΦD9 population decays at a rate 16-fold higher than the slow decaying population. Altogether, these results provide both a rationale and a practical approach to quantitatively estimate heterogeneity in viral decay.

Pacmanvirus isolated from the Lost City hydrothermal field extends the concept of transpoviron beyond the family Mimiviridae.

The microbial sampling of submarine hydrothermal vents remains challenging, with even fewer studies focused on viruses. Here we report the first isolation of a eukaryotic virus from the Lost City hydrothermal field, by co-culture with the laboratory host Acanthamoeba castellanii. This virus, named pacmanvirus lostcity, is closely related to previously isolated pacmanviruses (strains A23 and S19), clustering in a divergent clade within the long-established family Asfarviridae. Its icosahedral particles are 200nm in diameter, with an electron-dense core surrounded by an inner membrane. Its genome of 395 708bp (33% G + C) is predicted to encode 473 proteins. However, besides these standard properties, pacmanvirus lostcity was found associated with a new type of selfish genetic element, 7kb in length, whose architecture and gene content are reminiscent of those of transpovirons, hitherto specific to the family Mimiviridae. Like previously described transpovirons, this element propagates as an episome within its host virus particles and exhibits partial recombination with its genome. In addition, an unrelated 2kb long episome was also associated with pacmanvirus lostcity. Together, the transpoviron and the 2kb episome might participate to exchanges between pacmanviruses and other large DNA virus families. It remains to be elucidated if the presence of these mobile genetic elements is restricted to pacmanviruses or was simply overlooked in other members of the Asfarviridae.

Membrane-Active Singlet Oxygen Photogenerators as a Paradigm for Broad-Spectrum Antivirals: The Case of Halogenated (BOron)-DIPYrromethenes.

Enveloped viruses, such as flaviviruses and coronaviruses, are pathogens of significant medical concern that cause severe infections in humans. Some photosensitizers are known to possess virucidal activity against enveloped viruses, targeting their lipid bilayer. Here we report a series of halogenated difluoroboron-dipyrromethene (BODIPYs) photosensitizers with strong virus-inactivating activity. Our structure-activity relationship analysis revealed that BODIPY scaffolds with a heavy halogen atom demonstrate significant efficacy against both tick-borne encephalitis virus (TBEV; Flaviviridae family) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; Coronaviridae family) along with high singlet oxygen quantum yields. Moreover, select compounds also inactivated other enveloped viruses, such as herpes simplex virus type 1 and monkeypox virus. The nature and length of the alkyl side chain notably influenced the virus-inactivating activity of BODIPY molecules. Furthermore, molecular dynamics studies highlighted the critical importance of the positioning of the chromophore moiety within the lipid bilayer. As membrane-targeting photosensitizers, BODIPYs interact directly with virus particles, causing damage to the viral envelope membranes. Thus, TBEV pretreated with BODIPY was completely noninfective for lab mice. Consequently, BODIPY-based photosensitizers hold potential either as broad-spectrum virus-inactivating antivirals against a variety of phylogenetically unrelated enveloped viruses or as potent inactivators of viruses for the development of vaccines for preventing life-threatening emerging viral diseases.

A comprehensive review of research advances in the study of lactoferrin to treat viral infections.

Lactoferrin (Lf) is a naturally occurring glycoprotein known for its antiviral and antibacterial properties and is present in various physiological fluids. Numerous studies have demonstrated its antiviral effectiveness against multiple viruses, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza virus (IFV), herpes simplex virus (HSV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV). Lf, a vital component of the mucosal defense system, plays a crucial role in inhibiting viral infection by binding to both host cells and viral particles, such as the Hepatitis C virus (HCV). This interaction enables Lf to keep viral particles away from their target cells, emphasizing its significance as a fundamental element of mucosal defense against viral infections. Additionally, Lf has the ability to modulate cytokine expression and enhance cellular immune responses. In the innate immune system, Lf serves as a unique iron transporter and helps suppress various pathogens like bacteria, fungi, and viruses. This article summarises the potential antiviral properties of Lf against various viruses, along with its other mentioned functions. The advancement of Lf-based therapies supports the homology of food and medicine, providing a promising avenue to address viral infections and other public health challenges.

Grappa - a machine learned molecular mechanics force field.

Simulating large molecular systems over long timescales requires force fields that are both accurate and efficient. In recent years, E(3) equivariant neural networks have lifted the tension between computational efficiency and accuracy of force fields, but they are still several orders of magnitude more expensive than established molecular mechanics (MM) force fields. Here, we propose Grappa, a machine learning framework to predict MM parameters from the molecular graph, employing a graph attentional neural network and a transformer with symmetry-preserving positional encoding. The resulting Grappa force field outperforms tabulated and machine-learned MM force fields in terms of accuracy at the same computational efficiency and can be used in existing Molecular Dynamics (MD) engines like GROMACS and OpenMM. It predicts energies and forces of small molecules, peptides, and RNA at state-of-the-art MM accuracy, while also reproducing experimentally measured values for J-couplings. With its simple input features and high data-efficiency, Grappa is well suited for extensions to uncharted regions of chemical space, which we show on the example of peptide radicals. We demonstrate Grappa's transferability to macromolecules in MD simulations from a small fast-folding protein up to a whole virus particle. Our force field sets the stage for biomolecular simulations closer to chemical accuracy, but with the same computational cost as established protein force fields.

A benzo[b]thiophene-derived inhibitor of virus particle assembly via targeting capsid protein residue Arg157.

As a biological macromolecule, the coat protein (CP) of potato virus Y (PVY) mediates the virus' primary pathogenic behaviors. It has been gradually realized that certain residues on the CP are crucial for functions such as virus particle movement and assembly. However, there are few reports of potential drugs successfully targeting these key residues with unique mechanisms of action. Here, we disclose the first new phytovirucide that acts on the key site Arg157 (R157) on the PVY CP. In this investigation, we developed a series of benzo[b]thiophene-based compounds, strategically introducing sulfonamide functionalities to enhance their antiviral performance. Through bio-screening, derivative C54 (EC50 = 69.2 μg/mL for inactive activity) emerged as notably more effective against PVY than the established antiviral agent ningnanmycin (EC50 = 79.6 μg/mL). Mechanistic studies revealed that C54 is an inhibitor of viral particle assembly by specifically binding to the CP residue R157, thereby disrupting its interaction with RNA. These results underscore the promise of C54 as a potent antiviral lead and provide a fresh perspective on the strategic design of inhibitors focusing on viral assembly processes.

Evidence that the cell glycocalyx envelops respiratory syncytial virus (RSV) particles that form on the surface of RSV-infected human airway cells.

Evidence that the cell glycocalyx envelops respiratory syncytial virus (RSV) particles that form on the surface of RSV-infected human airway cells.

Molecular Diagnosis of Phlebovirus riftense Infection Using an L-Segment-Based Real-Time RT-PCR Method.

Rift Valley fever (RVF) is one of the main vector-borne zoonotic diseases that affects a wide range of ruminants and humans in Africa and the Arabian Peninsula.Several techniques involving cell culture and molecular biology methods have been developed to diagnose RVF infection. Success partly relies on sending samples to a national or reference laboratory in good conditions and having the capacity to perform the appropriate diagnostic test in the matrix of interest during the period of viremia where high loads of viral particles are present. Conventional and duplex/multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays are currently the most rapid and sensitive molecular tests for the detection and quantification of Rift Valley fever virus (RVFV) during outbreaks. The one-step real-time duplex RT-PCR technique that is detailed in this chapter is based on the L segment of RVFV. Results must be carefully validated and interpreted, taking into account the results of both positive and negative controls.

Isoviolanthin promotes Schwann cells activity in peripheral nerve regeneration via Fhl3-mediated epithelial-mesenchymal transition-like process: An in vitro study.

Schwann cells, as crucial regenerative cells, possess the ability to facilitate axon growth following peripheral nerve injury. However, the regeneration efficiency dominated by Schwann cells is impaired by factors such as the severity of peripheral nervous injury, aging, and metabolic disease. Cause the limitations of clinical treatments, it is necessary to urgently search for new substances that could reinforce the functionality of Schwann cells and promote nerve regeneration. We represented the first evidence that isoviolanthin possesses the capability to enhance Schwann cell proliferation and migration. Then, transcriptome sequencing was employed to examine the Differential Expressed Genes (DEGs), resulting in the identification of 193 DEGs. Following this, the expression levels of the top 5 up-regulated genes were confirmed through RT-qPCR, with Fhl3 demonstrating the most significant up-regulation. Schwann cells were transduced with virus particles made in HEK-293T/17cells by transfection with lentivirus packaging plasmids containing Fhl3. A notable enhancement in Schwann cell proliferation and migration was observed following transduction. Furthermore, the Fhl3-up group exhibited a significant upregulation of Vimentin expression compared to the control group. These results suggested that isoviolanthin plays a positive role in enhancing Schwann cells' activity via increasing Fhl3 expression, and the mechanism may be related to the EMT(epithelial-mesenchymal transition)-like process.

Polycyclic aromatic polymer nanoparticles show potent infectious particle adsorption capability.

Nonspecific viral adsorption by polymer nanoparticles is more economical and superior in terms of operating cost and energy efficiency than viral adsorption using virus-specific antibodies and filtration techniques involving size exclusion in the order of tens of nanometres. In this study, we synthesised four types of polycyclic aromatic polymer (ArP) nanoparticles with different structures and evaluated their virus adsorption capability for infectious particles of the newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). ArP nanoparticles with a diameter of approximately 500 nm were prepared by one-pot precipitation polymerisation using structural isomers of bifunctional dihydroxynaphthalene (1,5-dihydroxynaphthalene and 2,6-dihydroxynaphthalene) as phenol monomers, as well as 3-hydroxybenzoic acid and 3-aminophenol as comonomers to introduce carboxylic acid and amino groups, respectively. This wide range of phenolic monomers offers a powerful molecular design capability, enabling the optimisation of surface properties for the adsorption of various infectious virus particles. The virus adsorption capacity of the ArP nanoparticles exceeded 20 000 plaque-forming units and was found to be correlated with the nitrogen (primary and secondary amines) and quinone contents on the ArP nanoparticle surface. Furthermore, a polyvinylidene difluoride membrane filter uniformly coated with the ArP nanoparticles could remove viruses by filtration in a flow system.

Cryogenic Electron Microscopy of Rift Valley Fever Virus.

Rift Valley fever virus (RVFV) is an important livestock and human pathogen. It is also a potential bioweapon owing to its ability to spread by aerosols. It is an enveloped virus containing surface protrusions composed of two viral glycoproteins, Gc and Gn; the viral core contains ribonucleoprotein complexes. We describe the use of cryogenic electron microscopy (Cryo-EM) of single particles to visualize the three-dimensional (3D) organization of RVFV. The spherical RVFV virions have a diameter of 100nm with icosahedral symmetry (T=12) and 720 prominent protrusions on their surface.In this chapter we describe the general protocols to prepare virus suspensions for Cryo-EM, image acquisition of virus particles embedded in vitreous water, and their image processing. Because of RVFV being classified as a BSL3 and potentially as a select agent, we also describe the protocol of performing Cryo-EM grid preparation and imaging in our Cryo-EM BSL3 containment, along with the general etiquette of using BSL3 containment for research, including entry into containment, dressing up for work there, manipulation with the agent, grid preparation, transfer of the frozen grids into a microscope, etc., waste disposal, and finally exit from the containment. The microscope decontamination with ClO2 is briefly described as well because that technique is not widely used in the Cryo-EM field.

Formation of Antiviral Calcium Alginate Layers Analyzed by Combining Theory, Computational Chemistry and Experiments

Alginate, a sugar polymer derived from algae, crosslinks with calcium ions to form a stable gel or film. Several studies already analyzed the antiviral properties of calcium alginate, whereby only some studies showed viral inhibition. This research investigates the biochemistry and conditions of calcium alginate networks to form gels and membranes by a combination of literature analysis, computational simulations, and spraying experiments. Cell culture assays were applied to test the potential of calcium alginate to inhibit viral entry into cells. These investigations demonstrate that protective effects on cultured cells depend on the specific alginate substance, the concentrations and the manner of deposition. The results confirmed conditions so that the calcium alginate forms effectively gel-like networks and thin membranes. Additionally, the experiments proved that over 50% of the infections of cells with viral particles can be inhibited easily by calcium alginate overlaying cells.