Optimization and Characterization of Quercetin Vitamin C Nano-Phytosome Formulation

Objective: To design an optimal formulation for quercetin and vitamin C nano-phytosome. Method(s): Nano-phytosomes are prepared by the thin layer hydration technique using a 2-level-5-factor design experimental. A total of 32 experimental formulas were used for data analysis. The ratio of quercetin: soy lecithin (X1), the ratio of quercetin: cholesterol (X2), stirring speed (X3), stirring temperature (X4), and stirring time (X5) were used as independent factors, while globule size as a dependent factor. Data analysis was carried out by Design Expert12 application. Characterization of the optimal formula included physicochemical evaluation, globule size analysis, zeta potential, polydispersity index, entrapment efficiency, Transition Electron Microscopy (TEM) analysis, and FTIR analysis. Result(s): The optimal formula consisted of quercetin: vitamin C: lecithin: cholesterol ratio of 1: 1: 1.046: 0.105 mol;stirring speed 763.986 rpm;stirring time of 59 min, at temperature 51.73 degreeC which produced 59.26 nm average globule size, PDI value 0.66;zeta potential value-35.93+/-0.95 mV and average SPAN value 0.61. This formulation showed entrapment efficiency of quercetin 91.69+/-0.18 % and vitamin C 90.82+/-0.13 %. The TEM and FITR analysis showed the morphological of the globules and interactions between the drugs, soy lecithin, and cholesterol to form nano-phytosomes. Conclusion(s): The conditions to obtain the optimal formula for quercetin vitamin C nano-phytosome consisted of quercetin: vitamin C: lecithin: cholesterol ratio of 1: 1: 1.046: 0.105 mol;stirring speed 763.986 rpm;stirring time of 59 min, and at temperature 51.73 degreeC.Copyright © 2023 The Authors.

Investigation on anti-Corona viral potential of Yarrow tea.

Achillea millefolium (Yarrow) is a herbaceous plant of Greek origin noted to treat pneumonia, common cold, cough, and other respiratory disorders. The flowers and leaves are the core part used to prepare herbal tea that gains the world's recognition as medicinal tea. Coronavirus disease is spreading across the globe, and numerous approaches are lodged to treat virus-induced lung inflammation. Here, we used the network pharmacology, metabolite analysis, docking and molecular simulation and MM-PBSA analysis to comprehend the biochemical basis of the health-boosting impact of Yarrow tea. Next, we performed the microscopic and dynamic light scattering (DLS) analysis of yarrow-treated ChAdOx1 nCoV-19 to evaluate the virucidal activity of the Yarrow. The present study investigates the druggability, metabolites and potential interaction of the title tea with genes associated with Covid-19-induced pathogenesis. Towards this, 1022 gene hits were obtained, 30 are mutually shared. Network Pharmacology and microarray gene expression analysis find the connection of PTGS2 in relieving the virus-induced inflammation. Yarrow constituents Luteolin may inhibit or down-regulate the Cyclooxygenase II (PTGS2), a plausible mechanism underlying the Yarrow's anti-inflammatory actions. Further, the Yarrow's virucidal activity was assessed towards Transmission Electron Microscopic (TEM). The Yarrow treated SARS-nCoV-2 cell exhibits the disintegration of the virus membrane. This work provides a scientific basis for further elucidating the mechanism underlying Achillea millefolium's antiviral and anti-inflammatory properties.

Cilia impairment in bronchial epithelial cells detected in autopsy material of SARS-CoV-2-infected patient.

Recent studies indicate that cilia impairment, accompanied by the axonema loss and the basal body misorientation, is a common pathological feature of SARS-CoV-2-infected bronchial epithelial cells. However, these data were obtained using either cultured cells, or animal models, while in human postmortem material, cilia impairment has not been described yet. Here, we present direct observation of cilia impairment in SARS-CoV-2-infected bronchial epithelial cells using transmission electron microscopy of the autopsy material. We were able to observe only single infected cells with cilia impairment in one of twelve examined specimens, while the large number of desquamated bronchial epithelial cells with undisturbed ciliary layer was visible in the bronchial lumens. Thus, it seems that in the lungs of infected patients, the majority of bronchial cells do not die as a direct result of infection, which may explain the rarity of this finding in the autopsy material.

Pulmonary surfactant and COVID-19: A new synthesis

Chapter 1: COVID-19 pathogenesis poses paradoxes difficult to explain with traditional physiology. For instance, since type II pneumocytes are considered the primary cellular target of SARS-CoV-2;as these produce pulmonary surfactant (PS), the possibility that insufficient PS plays a role in COVID-19 pathogenesis has been raised. However, the opposite of predicted high alveolar surface tension is found in many early COVID-19 patients: paradoxically normal lung volumes and high compliance occur, with profound hypoxemia. That 'COVID anomaly' was quickly rationalised by invoking traditional vascular mechanisms-mainly because of surprisingly preserved alveolar surface in early hypoxemic cases. However, that quick rejection of alveolar damage only occurred because the actual mechanism of gas exchange has long been presumed to be non-problematic, due to diffusion through the alveolar surface. On the contrary, we provide physical chemical evidence that gas exchange occurs by an process of expansion and contraction of the three-dimensional structures of PS and its associated proteins. This view explains anomalous observations from the level of cryo-TEM to whole individuals. It encompasses results from premature infants to the deepest diving seals. Once understood, the COVID anomaly dissolves and is straightforwardly explained as covert viral damage to the 3D structure of PS, with direct treatment implications. As a natural experiment, the SARS-CoV-2 virus itself has helped us to simplify and clarify not only the nature of dyspnea and its relationship to pulmonary compliance, but also the fine detail of the PS including such features as water channels which had heretofore been entirely unexpected.Copyright ©

Plitidepsin Is a Broad-Spectrum Antiviral Shaping the Cell Proteostatic Balance

Background: Different viruses employ similar pathways for replication, revealing key intracellular hotspots to target with host-directed therapies and achieve a broad-spectrum antiviral activity. Plitidepsin is a clinically approved antitumoral agent that blocks the elongation factor eEF1A required for protein translation. This drug counteracts SARS-CoV-2 replication and shows a favorable safety profile in COVID-19 patients. Yet, the precise antiviral mechanism of action of plitidepsin remains unknown. Method(s): Here we used a deep quantitative proteomic analysis to measure the impact of plitidepsin on the proteome of SARS-CoV-2-infected Vero E6 cells. This was complemented with transmission electron microscopy assays, which unraveled the subcellular and morphological changes associated to plitidepsin treatment. In addition, we performed functional in vitro assays to dissect the antiviral activity of plitidepsin against SARS-CoV-2 and other viruses. Result(s): We found that this drug inhibited the synthesis of all SARS-CoV-2 proteins in a dose-dependent manner. These included the R1AB polyproteins, which facilitate the synthesis of non-structural proteins involved in the formation of double membrane vesicles (DMV) required for viral replication. Plitidepsin reduced DMV formation and the morphogenesis of new viruses, having a greater impact on viral than on host proteins. Less than 14% of the cellular proteome was significantly affected by plitidepsin, inducing the up-regulation of key molecules associated with protein biosynthesis, such as the translation initiation factors eIF4A2 and eIF2S3. Therefore, plitidepsin induced a compensatory state that rescued protein translation. This proteostatic response explains how cells preserve the cellular proteome after treatment with a translation inhibitor such as plitidepsin. In addition, it suggests that plitidepsin could inhibit other RNA-dependent and non-integrated DNA viruses, as we confirmed in vitro using Zika virus, Hepatitis C virus replicon and Herpes simplex virus. However, the compensatory proteostasis induced by plitidespin also explains why this drug failed to inhibit the replication of integrated DNA proviruses such as HIV-1. Conclusion(s): Unraveling the mechanism of action of host-directed therapies like plitidepsin is imperative to define the indications and antiviral profile of these compounds. This knowledge will be key to develop broad-spectrum treatments and have them ready to deploy when future pandemic viruses break through.

Influence of Nitrogen-Doped Carbon Dot and Silver Nanoparticle Modified Carbon Paste Electrodes on the Potentiometric Determination of Tobramycin Sulfate: A Comparative Study

Two inexpensive and simple methods for synthesis of carbon nanodots were applied and compared to each other, namely a hydrothermal and microwave-assisted method. The synthesized carbon nanodots were characterized using transmission electron microscopy (TEM), ultraviolet-visible (UV-Vis), photoluminescence (PL), Fourier transform-infrared spectroscopy (FTIR), and X-ray diffraction (XRD). The synthesized microwave carbon nanodots had smaller particle size and were thus chosen for better electrochemical performance. Therefore, they were used for our modification process. The proposed electrodes performance characteristics were evaluated according to the IUPAC guidelines, showing linear response in the concentration range 10−6–10−2, 10−7–10−2, and 10−8–10−2 M of tobramycin with a Nernstian slope of 52.60, 58.34, and 57.32 mV/decade for the bare, silver nanoparticle and carbon nanodots modified carbon paste electrodes, respectively. This developed potentiometric method was used for quantification of tobramycin in its co-formulated dosage form and spiked human plasma with good recovery percentages and without interference of the co-formulated drug loteprednol etabonate and excipients.

Diagnostic testing in people with primary ciliary dyskinesia around the world: where do we stand?

Background: In the past, few patients with primary ciliary dyskinesia (PCD) were diagnosed with the test combination recommended by guidelines (nasal nitric oxide (nNO), genetic testing, and biopsy for electron or video microscopy) [Halbeisen, ERJ, 2019]. In a large international participatory study of people with PCD, we assessed the current situation. Method(s): We used data from COVID-PCD, an international study of people with PCD, who participated between 2020 and 2022. Participants described their diagnostic tests in an online questionnaire, and we used logistic regression to identify explanatory factors. Result(s): 728 participated (median age 27 years, IQR: 12-43;60% female). Among them, 92% reported that any diagnostic testing had been done: 49% nNO, 59% genetics, and 75% biopsy for electron or video microscopy. Most did not know whether the sample had been analysed with TEM or video microscopy. Biopsy was most frequent in all countries except in North America where genetic testing predominated. Only 36% of participants reported all three tests. This proportion was highest in Germany (61%) and lowest in Australia (19%). Recently diagnosed patients reported more tests (nNO OR 1.7, 95%CI 1.1-2.6, genetics OR 4.5, 95%CI 2.9-6.9), and those with situs inversus less (nNO OR 0.5, 95%CI 0.3-0.7, biopsy OR 0.4, 95%CI 0.3-0.7, genetics OR 0.7, 95%CI 0.5-0.97). Conclusion(s): Diagnostic testing in people with PCD differed by country and few reported having undergone all recommended tests. Patients diagnosed long ago should be recalled for supplementary testing to improve diagnostic characterisation as a prerequisite for personalised medicine.

Identification of selected earthworm species of Northeast India using DNA barcoding

Aim: This study aimed at the species identification of selected indigenous earthworms of Manipur and Assam, Northeast India along with an exotic species using morpho-anatomical study and DNA barcoding. Methodology: Indigenous species of earthworms were collected from Imphal and Jorhat, North-eastern part of India. The exotic species of earthworm were collected from Indian Council of Agricultural Research Complex, Manipur. The samples were collected by digging and hand sorting method. Identification of samples was done by both conventional and molecular methods. Molecular characterization was accomplished through PCR amplification of the mitochondrial cytochrome oxidase I (COI) genes. Automatic sequencing reactions were performed for the amplified PCR products on ABI3100 Genetic Analyser (Applied Biosystems). Result(s): Out of five specimens (EM1, EM2, EM4, EG5 and EM6) examined through morpho-anatomical studies, three were identified to species level while the other two were identified to their genus level only. Out of EM1 and EM2 specimens in the genus Perionyx as per the morpho-anatomical studies, DNA barcoding could deduce the EM2 specimen up to the species level as P. excavatus. The exotic EM6 specimen morphologically identified as Eisenia fetida showed 99% COI gene sequence similarity with both E. fetida and E. andrei but its sequence divergence with E. andrei was less than 1%, so, it belonged to E. andrei. Interpretation(s): This study shows the reliability of clubbing DNA barcoding experiments with classical taxonomy in supplementing and strengthening the traditional taxonomy for accurate identification of earthworms.Copyright © Triveni Enterprises, Lucknow (India)

Development of thermosensitive hydrogel of Amphotericin-B and Lactoferrin combination-loaded PLGA-PEG-PEI nanoparticles for potential eradication of ocular fungal infections: In-vitro, ex-vivo and in-vivo studies.

The most prevalent conditions among ocular surgery and COVID-19 patients are fungal eye infections, which may cause inflammation and dry eye, and may cause ocular morbidity. Amphotericin-B eye drops are commonly used in the treatment of ocular fungal infections. Lactoferrin is an iron-binding glycoprotein with broad-spectrum antimicrobial activity and is used for the treatment of dry eye, conjunctivitis, and ocular inflammation. However, poor aqueous stability and excessive nasolacrimal duct draining impede these agens' efficiency. The aim of this study was to examine the effect of Amphotericin-B, as an antifungal against Candida albicans, Fusarium, and Aspergillus flavus, and Lactoferrin, as an anti-inflammatory and anti-dry eye, when co-loaded in triblock polymers PLGA-PEG-PEI nanoparticles embedded in P188-P407 ophthalmic thermosensitive gel. The nanoparticles were prepared by a double emulsion solvent evaporation method. The optimized formula showed particle size (177.0 ± 0.3 nm), poly-dispersity index (0.011 ± 0.01), zeta-potential (31.9 ± 0.3 mV), and entrapment% (90.9 ± 0.5) with improved ex-vivo pharmacokinetic parameters and ex-vivo trans-corneal penetrability, compared with drug solution. Confocal laser scanning revealed valuable penetration of fluoro-labeled nanoparticles. Irritation tests (Draize Test), Atomic force microscopy, cell culture and animal tests including histopathological analysis revealed superiority of the nanoparticles in reducing signs of inflammation and eradication of fungal infection in rabbits, without causing any damage to rabbit eyeballs. The nanoparticles exhibited favorable pharmacodynamic features with sustained release profile, and is neither cytotoxic nor irritating in-vitro or in-vivo. The developed formulation might provide a new and safe nanotechnology for treating eye problems, like inflammation and fungal infections.

Macrophage membrane-biomimetic adhesive polycaprolactone nanocamptothecin for improving cancer-targeting efficiency and impairing metastasis

The recent remarkable success and safety of mRNA lipid nanoparticle technology for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines has stimulated intensive efforts to expand nanoparticle strategies to treat various diseases. Numerous synthetic nanoparticles have been developed for pharmaceutical delivery and cancer treatment. However, only a limited number of nanotherapies have enter clinical trials or are clinically approved. Systemically administered nanotherapies are likely to be sequestered by host mononuclear phagocyte system (MPS), resulting in suboptimal pharmacokinetics and insufficient drug concentrations in tumors. Bioinspired drug-delivery formulations have emerged as an alternative approach to evade the MPS and show potential to improve drug therapeutic efficacy. Here we developed a biodegradable polymer-conjugated camptothecin prodrug encapsulated in the plasma membrane of lipopolysaccharide-stimulated macrophages. Polymer conjugation revived the parent camptothecin agent (e.g., 7-ethyl-10-hydroxy-camptothecin), enabling lipid nanoparticle encapsulation. Furthermore, macrophage membrane cloaking transformed the nonadhesive lipid nanoparticles into bioadhesive nanocamptothecin, increasing the cellular uptake and tumor-tropic effects of this biomimetic therapy. When tested in a preclinical murine model of breast cancer, macrophage-camouflaged nanocamptothecin exhibited a higher level of tumor accumulation than uncoated nanoparticles. Furthermore, intravenous administration of the therapy effectively suppressed tumor growth and the metastatic burden without causing systematic toxicity. Our study describes a combinatorial strategy that uses polymeric prodrug design and cell membrane cloaking to achieve therapeutics with high efficacy and low toxicity. This approach might also be generally applicable to formulate other therapeutic candidates that are not compatible or miscible with biomimetic delivery carriers. © 2022 The Authors

Controlled release nanomedicine (CRNM) of aspirin using "biomimetic niosomal nanoparticles (BNNs)”for Covid-19 and cardiovascular treatment: DOE based optimization

Biomimetic strategies can be adopted to improve biopharmaceutical aspects. Subsequently, Biomimetic reconstitutable pegylated amphiphilic lipid nanocarriers have high translational potential for systemic controlled drug delivery;however, such an improvised system for systemic aspirin delivery exploring nanotechnology is not available. Systemic administration of aspirin and its controlled delivery can significantly control blood clotting events, leading to stroke, which has immediate applications in cardiovascular diseases and Covid-19. In this work, we are developing aspirin sustained release pegylated amphiphilic self-assembling nanoparticles to develop reconstitutable aspirin injections by solvent-based co-precipitation method with phase inversion technique that leads to novel "biomimetic niosomal nanoparticles (BNNs).” DOE led optimization is done to develop Design of space for optimized particles. Upon reconstitution of solid powder, the particle size was 144.8 ± 12.90 nm with a surface charge of -29.2 ± 2.24 mV. The entrapment efficiency was found to be 49 ± 0.15%, wherein 96.99 ± 1.57% of the drug was released in 24hr showing super case II transport-based drug release mechanism. The formulation has the least hemolysis while showing significant suppression of platelet aggregation. MTT assay does not show any significant cytotoxicity. This is a potential nanoparticle that can be explored for developing aspirin injection, which is not available.

Surface control approach for growth of cerium oxide on flower-like molybdenum disulfide nanosheets enables superior removal of uremic toxins

Due to the high incidence of kidney disease, there is an urgent need to develop wearable artificial kidneys. This need is further exacerbated by the coronavirus disease 2019 pandemic. However, the dialysate regeneration system of the wearable artificial kidney has a low adsorption capacity for urea, which severely limits its application. Therefore, nanomaterials that can effectively remove uremic toxins, especially urea, to regenerate dialysate are required and should be further investigated and developed. Herein, flower-like molybdenum disulphide (MoS2) nanosheets decorated with highly dispersed cerium oxide (CeO2) were prepared (MoS2/CeO2), and their adsorption performances for urea, creatinine, and uric acid were studied in detail. Due to the open interlayer structures and the combination of MoS2 and CeO2, which can provide abundant adsorption active sites, the MoS2/CeO2 nanomaterials present excellent uremic toxin adsorption activities. Further, uremic toxin adsorption capacities were also assessed using a self-made fixed bed device under dynamic conditions, with the aim of developing MoS2/CeO2 for the practical adsorption of uremic toxins. In addition, the biocompatibility of MoS2/CeO2 was systematically analyzed using hemocompatibility and cytotoxicity assays. Our data suggest that MoS2/CeO2 can be safely used for applications requiring close contact with blood. Our findings confirm that novel 2-dimensional nanomaterial adsorbents have significant potential for dialysis fluid regeneration. © 2022

Measurement of Adenovirus-Based Vector Heterogeneity.

Adenovirus vectors have become an important class of vaccines with the recent approval of Ebola and COVID-19 products. In-process quality attribute data collected during Adenovirus vector manufacturing has focused on particle concentration and infectivity ratios (based on viral genome: cell-based infectivity), and data suggest only a fraction of viral particles present in the final vaccine product are efficacious. To better understand this product heterogeneity, lab-scale preparations of two Adenovirus viral vectors, (Chimpanzee adenovirus (ChAdOx1) and Human adenovirus Type 5 (Ad5), were studied using transmission electron microscopy (TEM). Different adenovirus morphologies were characterized, and the proportion of empty and full viral particles were quantified. These proportions showed a qualitative correlation with the sample's infectivity values. Liquid chromatography-mass spectrometry (LC-MS) peptide mapping was used to identify key adenovirus proteins involved in viral maturation. Using peptide abundance analysis, a ∼5-fold change in L1 52/55k abundance was observed between low-(empty) and high-density (full) fractions taken from CsCl ultracentrifugation preparations of ChAdOx1 virus. The L1 52/55k viral protein is associated with DNA packaging and is cleaved during viral maturation, so it may be a marker for infective particles. TEM and LC-MS peptide mapping are promising higher-resolution analytical characterization tools to help differentiate between relative proportions of empty, non-infectious, and infectious viral particles as part of Adenovirus vector in-process monitoring, and these results are an encouraging initial step to better differentiate between the different product-related impurities.

Structural and Immunoreactivity Properties of the SARS-CoV-2 Spike Protein upon the Development of an Inactivated Vaccine.

Inactivated vaccines are promising tools for tackling the COVID-19 pandemic. We applied several protocols for SARS-CoV-2 inactivation (by ß-propiolactone, formaldehyde, and UV radiation) and examined the morphology of viral spikes, protein composition of the preparations, and their immunoreactivity in ELISA using two panels of sera collected from convalescents and people vaccinated by Sputnik V. Transmission electron microscopy (TEM) allowed us to distinguish wider flail-like spikes (supposedly the S-protein's pre-fusion conformation) from narrower needle-like ones (the post-fusion state). While the flails were present in all preparations studied, the needles were highly abundant in the ß-propiolactone-inactivated samples only. Structural proteins S, N, and M of SARS-CoV-2 were detected via mass spectrometry. Formaldehyde and UV-inactivated samples demonstrated the highest affinity/immunoreactivity against the convalescent sera, while ß-propiolactone (1:2000, 36 h) and UV-inactivated ones were more active against the sera of people vaccinated with Sputnik V. A higher concentration of ß-propiolactone (1:1000, 2 h) led to a loss of antigenic affinity for both serum panels. Thus, although we did not analyze native SARS-CoV-2 for biosafety reasons, our comparative approach helped to exclude some destructive inactivation conditions and select suitable variants for future animal research. We believe that TEM is a valuable tool for inactivated COVID-19 vaccine quality control during the downstream manufacturing process.

Ultrastructural Changes in Autopsy Tissues of COVID-19 Patients.

INTRODUCTION: The COVID-19 pandemic resulted in substantial morbidity and mortality across the world. The prognosis was found to be poor in patients with co-morbidities such as diabetes, hypertension, interstitial lung disease, etc. Although biochemical studies were done in patient samples, no study has been reported from the Indian subcontinent about ultrastructural changes in the vital organs of COVID-19 patients. The present study was, therefore, conducted to understand the ultrastructural changes in the lung, liver, and brain of the deceased patients. METHODS: The present study was conducted on samples obtained from reverse transcription-polymerase chain reaction (RT-PCR)-positive patients who were admitted to a tertiary care hospital in Western India. Core needle biopsies were done in eight fatal cases of COVID-19. The samples were taken from the lungs, liver, and brain and subjected to light microscopy, immunohistochemistry (IHC), and transmission electron microscopy (TEM). Clinical details and biochemical findings were also collected.  Results: The study participants included seven males and one female. The presenting complaints included fever, breathlessness, and cough. Light microscopy revealed diffuse alveolar damage in the lungs. Further, a positive expression of SARS-CoV-2 nucleocapsid protein was observed in the pulmonary parenchyma of five patients. Also, the TEM microphotograph showed viral particles of size up to 80nm localized in alveolar epithelial cells. However, no viral particles were found in liver or brain samples. In the liver, macrovesicular steatosis and centrizonal congestion with loss of hepatocytes were observed in light microscopy. CONCLUSION:  This is the first study in the Indian population showing the in-situ presence of viral particles in core biopsies from fatal cases of COVID-19. As evident from the results, histology and ultrastructural changes in the lung correlated with the presence of viral particles. The study revealed a positive correlation between the damage in the lungs and the presence of viral particles.

Insights from the Infection Cycle of VSV-ΔG-Spike Virus.

Fundamental key processes in viral infection cycles generally occur in distinct cellular sites where both viral and host factors accumulate and interact. These sites are usually termed viral replication organelles, or viral factories (VF). The generation of VF is accompanied by the synthesis of viral proteins and genomes and involves the reorganization of cellular structure. Recently, rVSV-ΔG-spike (VSV-S), a recombinant VSV expressing the SARS-CoV-2 spike protein, was developed as a vaccine candidate against SARS-CoV-2. By combining transmission electron microscopy (TEM) tomography studies and immuno-labeling techniques, we investigated the infection cycle of VSV-S in Vero E6 cells. RT-real-time-PCR results show that viral RNA synthesis occurs 3-4 h post infection (PI), and accumulates as the infection proceeds. By 10-24 h PI, TEM electron tomography results show that VSV-S generates VF in multi-lamellar bodies located in the cytoplasm. The VF consists of virus particles with various morphologies. We demonstrate that VSV-S infection is associated with accumulation of cytoplasmatic viral proteins co-localized with dsRNA (marker for RNA replication) but not with ER membranes. Newly formed virus particles released from the multi-lamellar bodies containing VF, concentrate in a vacuole membrane, and the infection ends with the budding of particles after the fusion of the vacuole membrane with the plasma membrane. In summary, the current study describes detailed 3D imaging of key processes during the VSV-S infection cycle.

Near-Native Visualization of SARS-CoV-2 Induced Membrane Remodeling and Virion Morphogenesis.

Infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, leads to profound remodeling of cellular membranes, promoting viral replication and virion assembly. A full understanding of this drastic remodeling and the process of virion morphogenesis remains lacking. In this study, we applied room temperature transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to visualize the SARS-CoV-2 replication factory in Vero cells, and present our results in comparison with published cryo-EM studies. We obtained cryo-EM-like clarity of the ultrastructure by employing high-pressure freezing, freeze substitution (HPF-FS) and embedding, allowing room temperature visualization of double-membrane vesicles (DMVs) in a near-native state. In addition, our data illustrate the consecutive stages of virion morphogenesis and reveal that SARS-CoV-2 ribonucleoprotein assembly and membrane curvature occur simultaneously. Finally, we show the tethering of virions to the plasma membrane in 3D, and that accumulations of virus particles lacking spike protein in large vesicles are most likely not a result of defective virion assembly at their membrane. In conclusion, this study puts forward a room-temperature EM technique providing near-native ultrastructural information about SARS-CoV-2 replication, adding to our understanding of the interaction of this pandemic virus with its host cell.

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.

Lung Transplant Recipients with SARS-CoV2 Infection and Bnt162b2 Vaccination Induce Circulating Exosomes with Spike Protein S2 Capable of Inducing Immune Responses to SARS0-CoV2 in Mice

Purpose: Exosomes are small vesicles which are released by cells into body fluids. We have demonstrated the presence of circulating exosomes with viral antigens in lung transplant recipients (LTxRs) diagnosed with respiratory viral infections. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection results Covid-19 disease and SARS-CoV2 infection of LTxRs can be severe with poor clinical outcomes. The goal of this single center study is to determine the development of antibody responses specific to SARS-CoV2 in LTxRs, characterize the immune and molecular markers in the circulating exosomes induced and its role in eliciting immunity. Method(s): To determine that antibody responses and induction of circulating exosomes we enrolled LTxRs with SARS-CoV2 infection (n=50), following 2 doses of vaccination (n=100). Exosomes were isolated from plasma by exosome precipitation kit followed by 0.2 micron filtration and size determination by NanoSight300. Exosomes were subjected to transmission electron microscopy for spike (CSP) and nucleocapsid (CNP) antigens. Exosomes were also characterized by western blot for immune and molecular markers (NFkB, CIITA, 20S proteasome, beta catenin and VWF). C57BL/6 mice were immunized with circulating exosomes isolated from LTxRs with infection. Result(s): 78% of SARS-CoV2 infected LTxRs developed antibodies to CSP and CNP as opposed to normal infected individuals. In contrast, only 55% vaccinated LTxRs developed antibodies to SARS-CoV2 spike. Exosomes from SARS-CoV2 infected and vaccinated individuals contained CSP S2, CNP and immune and molecular markers. Transmission electron microscopy also revealed the presence of CSP and CNP on exosomes. C57BL/6 mice immunized with exosomes carrying CSP developed antibodies to SARS-CoV2 spike antigens. Severe inflammation and lung lesions were also demonstrated in the lungs of mice immunized with exosomes carrying CSP. Conclusion(s): In conclusion, we demonstrated that SARS-CoV2 infected and vaccinated LTxRs induced circulating exosomes with SARS-CoV2 CSP. In addition, exosomes contained important immune activating molecules suggesting that the exosomes induced by SARS-CoV2 may have a physiological role in inducing immune responses. Immunization of mice with exosomes from SARS-CoV2 infected and vaccinated LTxRs not only induced SARS-CoV2 spike specific antibody but also resulted in inflammation and lung lesions in the immunized animals.