Visualization of SARS-CoV-2 particles in naso/oropharyngeal swabs by thin section electron microscopy.

BACKGROUND: SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. METHODS: Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy. RESULTS: Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material (e.g. vesicles and needle-like crystals), and within ciliated cells. Morphology (i.e. shape, size, spike density) of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria. CONCLUSIONS: The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.

Concentration of SARS-CoV-2-Infected Cell Culture Supernatants for Detection of Virus-like Particles by Scanning Electron Microscopy.

There is currently a need for new rapid viral diagnostic electron microscopy methods. Although the gold standard remains the transmission electron microscopy (TEM) negative staining method for electron microscopic examination of samples containing a virus, difficulties can arise when the virus particle content of the sample that has to be examined is poor. Such samples include supernatants of virus-infected cells that can be difficult to examine, as sometimes only a few virus particles are released in the culture medium upon infection. In addition to TEM, scanning electron microscopy (SEM) can also be used for visualizing virus particles. One advantage of SEM over TEM is its ability to rapidly screen several large specimens, such as microscopy slides. In this study, we investigated this possibility and tested different coating molecules as well as the effect of centrifugation for analyzing SARS-CoV-2-virus-infected cell culture supernatants deposited on microscopy glass slides by SEM. We found that centrifugation of 25XConcanavalinA-coated microscopy glass slides in shell vials provided an improved method for concentrating SARS-CoV-2-virus-infected cell supernatants for virus-like particle detection by SEM.

SARS-CoV-2 Morphometry Analysis and Prediction of Real Virus Levels Based on Full Recurrent Neural Network Using TEM Images.

The SARS-CoV-2 virus is responsible for the rapid global spread of the COVID-19 disease. As a result, it is critical to understand and collect primary data on the virus, infection epidemiology, and treatment. Despite the speed with which the virus was detected, studies of its cell biology and architecture at the ultrastructural level are still in their infancy. Therefore, we investigated and analyzed the viral morphometry of SARS-CoV-2 to extract important key points of the virus's characteristics. Then, we proposed a prediction model to identify the real virus levels based on the optimization of a full recurrent neural network (RNN) using transmission electron microscopy (TEM) images. Consequently, identification of virus levels depends on the size of the morphometry of the area (width, height, circularity, roundness, aspect ratio, and solidity). The results of our model were an error score of training network performance 3.216 × 10-11 at 639 epoch, regression of -1.6 × 10-9, momentum gain (Mu) 1 × 10-9, and gradient value of 9.6852 × 10-8, which represent a network with a high ability to predict virus levels. The fully automated system enables virologists to take a high-accuracy approach to virus diagnosis, prevention of mutations, and life cycle and improvement of diagnostic reagents and drugs, adding a point of view to the advancement of medical virology.

On the Efficacy of ZnO Nanostructures against SARS-CoV-2.

In 2019, the new coronavirus disease (COVID-19), related to the severe acute respiratory syndrome coronavirus (SARS-CoV-2), started spreading around the word, giving rise to the world pandemic we are still facing. Since then, many strategies for the prevention and control of COVID-19 have been studied and implemented. In addition to pharmacological treatments and vaccines, it is mandatory to ensure the cleaning and disinfection of the skin and inanimate surfaces, especially in those contexts where the contagion could spread quickly, such as hospitals and clinical laboratories, schools, transport, and public places in general. Here, we report the efficacy of ZnO nanoparticles (ZnONPs) against SARS-CoV-2. NPs were produced using an ecofriendly method and fully characterized; their antiviral activity was tested in vitro against SARS-CoV-2, showing a decrease in viral load between 70% and 90%, as a function of the material's composition. Application of these nano-antimicrobials as coatings for commonly touched surfaces is envisaged.

Titanium dioxide particles frequently present in face masks intended for general use require regulatory control.

Although titanium dioxide (TiO2) is a suspected human carcinogen when inhaled, fiber-grade TiO2 (nano)particles were demonstrated in synthetic textile fibers of face masks intended for the general public. STEM-EDX analysis on sections of a variety of single use and reusable face masks visualized agglomerated near-spherical TiO2 particles in non-woven fabrics, polyester, polyamide and bi-component fibers. Median sizes of constituent particles ranged from 89 to 184 nm, implying an important fraction of nano-sized particles (< 100 nm). The total TiO2 mass determined by ICP-OES ranged from 791 to 152,345 µg per mask. The estimated TiO2 mass at the fiber surface ranged from 17 to 4394 µg, and systematically exceeded the acceptable exposure level to TiO2 by inhalation (3.6 µg), determined based on a scenario where face masks are worn intensively. No assumptions were made about the likelihood of the release of TiO2 particles itself, since direct measurement of release and inhalation uptake when face masks are worn could not be assessed. The importance of wearing face masks against COVID-19 is unquestionable. Even so, these results urge for in depth research of (nano)technology applications in textiles to avoid possible future consequences caused by a poorly regulated use and to implement regulatory standards phasing out or limiting the amount of TiO2 particles, following the safe-by-design principle.

SARS-CoV-2: Ultrastructural Characterization of Morphogenesis in an In Vitro System.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed-such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.

Nanoparticle encapsulation of non-genotoxic p53 activator Inauhzin-C for improved therapeutic efficacy.

The tumor suppressor protein p53 remains in a wild type but inactive form in ~50% of all human cancers. Thus, activating it becomes an attractive approach for targeted cancer therapies. In this regard, our lab has previously discovered a small molecule, Inauhzin (INZ), as a potent p53 activator with no genotoxicity. Method: To improve its efficacy and bioavailability, here we employed nanoparticle encapsulation, making INZ-C, an analog of INZ, to nanoparticle-encapsulated INZ-C (n-INZ-C). Results: This approach significantly improved p53 activation and inhibition of lung and colorectal cancer cell growth by n-INZ-C in vitro and in vivo while it displayed a minimal effect on normal human Wi38 and mouse MEF cells. The improved activity was further corroborated with the enhanced cellular uptake observed in cancer cells and minimal cellular uptake observed in normal cells. In vivo pharmacokinetic evaluation of these nanoparticles showed that the nanoparticle encapsulation prolongates the half-life of INZ-C from 2.5 h to 5 h in mice. Conclusions: These results demonstrate that we have established a nanoparticle system that could enhance the bioavailability and efficacy of INZ-C as a potential anti-cancer therapeutic.

Evolution of Electronic State and Properties of Silver Nanoparticles during Their Formation in Aqueous Solution.

The electron density of a nanoparticle is a very important characteristic of the properties of a material. This paper describes the formation of silver nanoparticles (NPs) and the variation in the electronic state of an NP's surface upon the reduction in Ag+ ions with oxalate ions, induced by UV irradiation. The calculations were based on optical spectrophotometry data. The NPs were characterized using Transmission electron microscopy and Dynamic light scattering. As ~10 nm nanoparticles are formed, the localized surface plasmon resonance (LSPR) band increases in intensity, decreases in width, and shifts to the UV region from 402 to 383 nm. The interband transitions (IBT) band (≤250 nm) increases in intensity, with the band shape and position remaining unchanged. The change in the shape and position of the LSPR band of silver nanoparticles in the course of their formation is attributable to an increasing concentration of free electrons in the particles as a result of a reduction in Ag+ ions on the surface and electron injection by CO2- radicals. The ζ-potential of colloids increases with an increase in electron density in silver nuclei. A quantitative relationship between this shift and electron density on the surface was derived on the basis of the Mie-Drude theory. The observed blue shift (19 nm) corresponds to an approximately 10% increase in the concentration of electrons in silver nanoparticles.

Ethanol Inactivation of Enveloped Viruses: Structural and Surface Chemistry Insights into Phi6.

Lipid-enveloped viruses, such as Ebola, influenza, or coronaviruses, are a major threat to human health. Ethanol is an efficient disinfectant that is widely used to inactivate these viruses and prevent their transmission. However, the interactions between ethanol and enveloped viruses leading to their inactivation are not yet fully understood. This study demonstrates the link between ethanol-induced viral inactivation and the nanostructural and chemical transformations of the model virus Phi6, an 85 nm diameter lipid-enveloped bacterial virus that is commonly used as surrogate for human pathogenic viruses. The virus morphology was investigated using small-angle X-ray scattering and dynamic light scattering and was related to its infectivity. The Phi6's surface chemistry was characterized by cryogenic X-ray photoelectron spectroscopy, and the modifications in protein structure were assessed by circular dichroism and fluorescence spectroscopy. Ethanol-triggered structural modifications were found in the lipid envelope, detaching from the protein capsid and forming coexisting nanostructures.

Destruction mechanisms of ozone over SARS-CoV-2.

In this pandemic SARS-CoV-2 crisis, any attempt to contain and eliminate the virus will also stop its spread and consequently decrease the risk of severe illness and death. While ozone treatment has been suggested as an effective disinfection process, no precise mechanism of action has been previously reported. This study aimed to further investigate the effect of ozone treatment on SARS-CoV-2. Therefore, virus collected from nasopharyngeal and oropharyngeal swab and sputum samples from symptomatic patients was exposed to ozone for different exposure times. The virus morphology and structure were monitored and analyzed through Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM), Atomic Absorption Spectroscopy (AAS), and ATR-FTIR. The obtained results showed that ozone treatment not only unsettles the virus morphology but also alters the virus proteins' structure and conformation through amino acid disturbance and Zn ion release from the virus non-structural proteins. These results could provide a clearer pathway for virus elimination and therapeutics preparation.

SARS-CoV-2 Initiates Programmed Cell Death in Platelets.

[Figure: see text].

Ultrastructural modifications induced by SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle morphogenesis and virion release.

Many studies on SARS-CoV-2 have been performed over short-time scale, but few have focused on the ultrastructural characteristics of infected cells. We used TEM to perform kinetic analysis of the ultrastructure of SARS-CoV-2-infected cells. Early infection events were characterized by the presence of clusters of single-membrane vesicles and stacks of membrane containing nuclear pores called annulate lamellae (AL). A large network of host cell-derived organelles transformed into virus factories was subsequently observed in the cells. As previously described for other RNA viruses, these replication factories consisted of double-membrane vesicles (DMVs) located close to the nucleus. Viruses released at the cell surface by exocytosis harbored the typical crown of spike proteins, but viral particles without spikes were also observed in intracellular compartments, possibly reflecting incorrect assembly or a cell degradation process.

SARS-CoV-2 identified by transmission electron microscopy in lymphoproliferative and ischaemic intestinal lesions of COVID-19 patients with acute abdominal pain: two case reports.

BACKGROUND: SARS-CoV-2 may produce intestinal symptoms that are generally mild, with a small percentage of patients developing more severe symptoms. The involvement of SARS-CoV-2 in the physiopathology of bowel damage is poorly known. Transmission electron microscopy (TEM) is a useful tool that provides an understanding of SARS-CoV-2 invasiveness, replication and dissemination in body cells but information outside the respiratory tract is very limited. We report two cases of severe intestinal complications (intestinal lymphoma and ischaemic colitis) in which the presence of SARS-CoV-2 in intestinal tissue was confirmed by TEM. These are the first two cases reported in the literature of persistence of SARS-CoV-2 demonstrated by TEM in intestinal tissue after COVID 19 recovery and SARS-CoV-2 nasopharyngeal clearance. CASE PRESENTATION: During the first pandemic peak (1st March-30th April 2020) 932 patients were admitted in Hospital Universitari Mútua Terrassa due to COVID-19, 41 (4.4%) required cross-sectional imaging techniques to assess severe abdominal pain and six of them (0.64%) required surgical resection. SARS-CoV-2 in bowel tissue was demonstrated by TEM in two of these patients. The first case presented as an ileocaecal inflammatory mass which turned to be a B-cell lymphoma. Viral particles were found in the cytoplasm of endothelial cells of damaged mucosa. In situ hybridization was negative in tumour cells, thus ruling out an oncogenic role for the virus. SARS-CoV-2 remained in intestinal tissue 6 months after nasopharyngeal clearance, suggesting latent infection. The second patient had a severe ischaemic colitis with perforation and SARS-CoV-2 was also identified in endothelial cells. CONCLUSIONS: Severe intestinal complications associated with COVID-19 are uncommon. SARS-CoV-2 was identified by TEM in two cases, suggesting a causal role in bowel damage.

Small Structural Proteins E and M Render the SARS-CoV-2 Pseudovirus More Infectious and Reveal the Phenotype of Natural Viral Variants.

The SARS-CoV-2 pseudovirus is a commonly used strategy that mimics certain biological functions of the authentic virus by relying on biological legitimacy at the molecular level. Despite the fact that spike (S), envelope (E), and membrane (M) proteins together wrap up the SARS-CoV-2 virion, most of the reported pseudotype viruses consist of only the S protein. Here, we report that the presence of E and M increased the virion infectivity by promoting the S protein priming. The S, E, and M (SEM)-coated pseudovirion is spherical, containing crown-like spikes on the surface. Both S and SEM pseudoviruses packaged the same amounts of viral RNA, but the SEM virus bound more efficiently to cells stably expressing the viral receptor human angiotensin-converting enzyme II (hACE2) and became more infectious. Using this SEM pseudovirus, we examined the infectivity and antigenic properties of the natural SARS-CoV-2 variants. We showed that some variants have higher infectivity than the original virus and that some render the neutralizing plasma with lower potency. These studies thus revealed possible mechanisms of the dissemination advantage of these variants. Hence, the SEM pseudovirion provides a useful tool to evaluate the viral infectivity and capability of convalescent sera in neutralizing specific SARS-CoV-2 S dominant variants.

Secretory Vesicles Are the Principal Means of SARS-CoV-2 Egress.

The mechanisms of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) egress, similar to those of other coronaviruses, remain poorly understood. The virus buds in intracellular compartments and is therefore thought to be released by the biosynthetic secretory pathway. However, several studies have recently challenged this hypothesis. It has been suggested that coronaviruses, including SARS-CoV-2, use lysosomes for egress. In addition, a focused ion-beam scanning electron microscope (FIB/SEM) study suggested the existence of exit tunnels linking cellular compartments rich in viral particles to the extracellular space resembling those observed for the human immunodeficiency (HIV) in macrophages. Here, we analysed serial sections of Vero cells infected with SARS-CoV-2 by transmission electron microscopy (TEM). We found that SARS-CoV-2 was more likely to exit the cell in small secretory vesicles. Virus trafficking within the cells involves small vesicles, with each generally containing a single virus particle. These vesicles then fuse with the plasma membrane to release the virus into the extracellular space. This work sheds new light on the late stages of the SARS-CoV-2 infectious cycle of potential value for guiding the development of new antiviral strategies.

Preparation of Samples for Large-Scale Automated Electron Microscopy of Tissue and Cell Ultrastructure.

Manual selection of targets in experimental or diagnostic samples by transmission electron microscopy (TEM), based on single overview and detail micrographs, has been time-consuming and susceptible to bias. Substantial information and throughput gain may now be achieved by the automated acquisition of virtually all structures in a given EM section. Resulting datasets allow the convenient pan-and-zoom examination of tissue ultrastructure with preserved microanatomical orientation. The technique is, however, critically sensitive to artifacts in sample preparation. We, therefore, established a methodology to prepare large-scale digitization samples (LDS) designed to acquire entire sections free of obscuring flaws. For evaluation, we highlight the supreme performance of scanning EM in transmission mode compared with other EM technology. The use of LDS will substantially facilitate access to EM data for a broad range of applications.

TEM virus images: Benchmark dataset and deep learning classification.

BACKGROUND AND OBJECTIVE: To achieve the full potential of deep learning (DL) models, such as understanding the interplay between model (size), training strategy, and amount of training data, researchers and developers need access to new dedicated image datasets; i.e., annotated collections of images representing real-world problems with all their variations, complexity, limitations, and noise. Here, we present, describe and make freely available an annotated transmission electron microscopy (TEM) image dataset. It constitutes an interesting challenge for many practical applications in virology and epidemiology; e.g., virus detection, segmentation, classification, and novelty detection. We also present benchmarking results for virus detection and recognition using some of the top-performing (large and small) networks as well as a handcrafted very small network. We compare and evaluate transfer learning and training from scratch hypothesizing that with a limited dataset, transfer learning is crucial for good performance of a large network whereas our handcrafted small network performs relatively well when training from scratch. This is one step towards understanding how much training data is needed for a given task. METHODS: The benchmark dataset contains 1245 images of 22 virus classes. We propose a representative data split into training, validation, and test sets for this dataset. Moreover, we compare different established DL networks and present a baseline DL solution for classifying a subset of the 14 most-represented virus classes in the dataset. RESULTS: Our best model, DenseNet201 pre-trained on ImageNet and fine-tuned on the training set, achieved a 0.921 F1-score and 93.1% accuracy on the proposed representative test set. CONCLUSIONS: Public and real biomedical datasets are an important contribution and a necessity to increase the understanding of shortcomings, requirements, and potential improvements for deep learning solutions on biomedical problems or deploying solutions in clinical settings. We compared transfer learning to learning from scratch on this dataset and hypothesize that for limited-sized datasets transfer learning is crucial for achieving good performance for large models. Last but not least, we demonstrate the importance of application knowledge in creating datasets for training DL models and analyzing their results.

Presumed SARS-CoV-2 Viral Particles in the Human Retina of Patients With COVID-19.

Importance: The presence of the SARS-CoV-2 virus in the retina of deceased patients with COVID-19 has been suggested through real-time reverse polymerase chain reaction and immunological methods to detect its main proteins. The eye has shown abnormalities associated with COVID-19 infection, and retinal changes were presumed to be associated with secondary microvascular and immunological changes. Objective: To demonstrate the presence of presumed SARS-CoV-2 viral particles and its relevant proteins in the eyes of patients with COVID-19. Design, Setting, and Participants: The retina from enucleated eyes of patients with confirmed COVID-19 infection were submitted to immunofluorescence and transmission electron microscopy processing at a hospital in São Paulo, Brazil, from June 23 to July 2, 2020. After obtaining written consent from the patients' families, enucleation was performed in patients deceased with confirmed SARS-CoV-2 infection. All patients were in the intensive care unit, received mechanical ventilation, and had severe pulmonary involvement by COVID-19. Main Outcomes and Measures: Presence of presumed SARS-CoV-2 viral particles by immunofluorescence and transmission electron microscopy processing. Results: Three patients who died of COVID-19 were analyzed. Two patients were men, and 1 was a woman. The age at death ranged from 69 to 78 years. Presumed S and N COVID-19 proteins were seen by immunofluorescence microscopy within endothelial cells close to the capillary flame and cells of the inner and the outer nuclear layers. At the perinuclear region of these cells, it was possible to observe by transmission electron microscopy double-membrane vacuoles that are consistent with the virus, presumably containing COVID-19 viral particles. Conclusions and Relevance: The present observations show presumed SARS-CoV-2 viral particles in various layers of the human retina, suggesting that they may be involved in some of the infection's ocular clinical manifestations.