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Background: Virginia is a large state in the USA, yet it remains unclear what percentage of the population has had natural COVID-19 infection and whether risk factors for infection have changed over time. Methods: Using a longitudinal cohort, from December 2021-July 2022 we performed follow up serology and a questionnaire on 784 individuals from across Virginia who had previously participated in a statewide COVID-19 seroepidemiology study in 2020. Children were also invited to participate and an additional 62 children also completed the study. Serology was performed using Roche nucleocapsid and spike serological assays. Results: The majority of participants were white (78.6%), over 50 years old (60.9%), and reported having received COVID-19 vaccine (93.4%). 28.6% had evidence of prior COVID-19 infection (nucleocapsid positive). Reweighted by region, age, and sex to match the Virginia census data, the seroprevalence of nucleocapsid antibodies was estimated to be 30.6% (95% CI: 24.7, 36.6). We estimated that 25–53% of COVID-19 infections were asymptomatic. Infection rates were lower in individuals > 60 years old and were higher in Blacks and Hispanics. Infection rates were also higher in those without health insurance, in those with greater numbers of household children, and in those that reported a close contact or having undergone quarantine for COVID-19. Participants from Southwest Virginia had lower seropositivity (16.2%, 95% CI 6.5, 26.0) than other geographic regions. Boosted vaccinees had lower infection rates than non-boosted vaccinees. Frequenting indoor bars was a risk factor for infection, while frequently wearing an N95 mask was protective, though the estimates of association were imprecise. Infection rates were higher in children than adults (56.5% vs. 28.6%). Infection in the parent was a risk factor for child infection. Spike antibody levels declined with time since last vaccination, particularly in those that were vaccinated but not previously infected. Neutralizing antibody positivity was high (97–99%) for wild type, alpha, beta, gamma, delta, and omicron variants. Neutralizing antibody levels were higher in the follow-up survey compared to the first survey in 2020 and among individuals with evidence of natural infection compared to those without. Conclusions: In this longitudinal statewide cohort we observed a lower-than-expected COVID-19 infection rate as of August 2022. Boosted vaccinees had lower infection rates. Children had higher infection rates and infections tracked within households. Previously identified demographic risk factors for infection tended to persist. Even after the omicron peak, a large number of Virginians remain uninfected with COVID-19, underscoring the need for ongoing vaccination strategies. [ABSTRACT FROM AUTHOR]

Introduction: In November 2021, the SARS-CoV-2 Omicron variant of concern has emerged and is currently dominating the COVID-19 pandemic over the world. Omicron displays a number of mutations, particularly in the spike protein, leading to specific characteristics including a higher potential for transmission. Although Omicron has caused a significant number of deaths worldwide, it generally induces less severe clinical signs compared to earlier variants. As its impact on blood platelets remains unknown, we investigated platelet behavior in severe patients infected with Omicron in comparison to Delta. Methods: Clinical and biological characteristics of severe COVID-19 patients infected with the Omicron (n=9) or Delta (n=11) variants were analyzed. Using complementary methods such as flow cytometry, confocal imaging and electron microscopy, we examined platelet activation, responsiveness and phenotype, presence of virus in platelets and induction of selective autophagy. We also explored the direct effect of spike proteins from the Omicron or Delta variants on healthy platelet signaling. Results: Severe Omicron variant infection resulted in platelet activation and partial desensitization, presence of the virus in platelets and selective autophagy response. The intraplatelet processing of Omicron viral cargo was different from Delta as evidenced by the distribution of spike protein-positive structures near the plasma membrane and the colocalization of spike and Rab7. Moreover, spike proteins from the Omicron or Delta variants alone activated signaling pathways in healthy platelets including phosphorylation of AKT, p38MAPK, LIMK and SPL76 with different kinetics. Discussion: Although SARS-CoV-2 Omicron has different biological characteristics compared to prior variants, it leads to platelet activation and desensitization as previously observed with the Delta variant. Omicron is also found in platelets from severe patients where it induces selective autophagy, but the mechanisms of intraplatelet processing of Omicron cargo, as part of the innate response, differs from Delta, suggesting that mutations on spike protein modify virus to platelet interactions. [ABSTRACT FROM AUTHOR]

Mutations in the SARS-CoV-2 Spike glycoprotein can affect monoclonal antibody efficacy. Recent findings report the occurrence of resistant mutations in immunocompromised patients after tixagevimab/cilgavimab treatment. More recently, the Food and Drug Agency revoked the authorization for tixagevimab/cilgavimab, while this monoclonal antibody cocktail is currently recommended by the European Medical Agency. We retrospectively reviewed 22 immunocompetent patients at high risk for disease progression who received intramuscular tixagevimab/cilgavimab as early COVID-19 treatment and presented a prolonged high viral load. Complete SARS-CoV-2 genome sequences were obtained for a deep investigation of mutation frequencies in Spike protein before and during treatment. At seven days, only one patient showed evidence of treatment-emergent cilgavimab resistance. Quasispecies analysis revealed two different deletions on the Spike protein (S:del138–144 or S:del141–145) in combination with the resistance S:K444N mutation. The structural and dynamic impact of the two quasispecies was characterized by using molecular dynamics simulations, showing the conservation of the principal functional movements in the mutated systems and their capabilities to alter the structure and dynamics of the RBD, responsible for the interaction with the ACE2 human receptor. Our study underlines the importance of prompting an early virological investigation to prevent drug resistance or clinical failures in immunocompetent patients. [ABSTRACT FROM AUTHOR]

Analytical ultracentrifugation (AUC) analysis shows that the SARS-CoV-2 trimeric Spike (S) protein adopts different quaternary conformations in solution. The relative abundance of the "open" and "close" conformations is temperature-dependent, and samples with different storage temperature history have different open/close distributions. Neutralizing antibodies (NAbs) targeting the S receptor binding domain (RBD) do not alter the conformer populations; by contrast, a NAb targeting a cryptic conformational epitope skews the Spike trimer toward an open conformation. The results highlight AUC, which is typically applied for molecular mass determination of biomolecules as a powerful tool for detecting functionally relevant quaternary protein conformations. [ABSTRACT FROM AUTHOR]

The receptor‐binding domain (RBD) of spike recognizing the receptor angiotensin‐converting enzyme 2 (ACE2) initiates membrane fusion between severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and cell membrane. Although the structure of the RBD_ACE2 complex has been well studied, its functional mechanism in membrane fusion is still not fully understood. Here, using an in vitro cell–vesicle content‐mixing assay, it is found that the cleavage at the S2' site by thrombin (Thr) protease strongly accelerates membrane fusion, compared to that of cleavage at the S1/S2 site by PreScission (3C) protease. Moreover, mutations at the RBD_ACE2 interface resulted in a positive correlation between binding affinity and fusion probability. In both the cell–vesicle and cell–cell fusion assays, by crosslinking two membranes via the neutravidin (NTV)_biotin interaction or complementary DNA strands, it is found that spike drives membrane fusion in the absence of ACE2, and a suitable distance between two membranes is critical for spike‐mediated membrane fusion. Finally, unsuitable membrane crosslinkers significantly inhibited the fusion probability in the presence of ACE2. Taken together, the results suggest that the RBD_ACE2 complex may act as a crosslinker to bridge the viral and cell membranes at a suitable distance, which is critical, but also substitutable for spike‐mediated SARS‐CoV‐2 entry. [ABSTRACT FROM AUTHOR]

Differences in host angiotensin converting enzyme 2 (ACE2) genes may affect the host range of SARS-CoV-2-related coronaviruses (SC2r-CoVs) and further determine the tropism of host ACE2 for the infection receptor. However, the factor(s) responsible for determining the host tropism of SC2r-CoVs, which may in part be determined by the tropism of host ACE2 usage, remains unclear. Here, we use the pseudoviruses with the spike proteins of two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and the cells expressing ACE2 proteins of eight different Rhinolophus bat species to show that these two spikes have different tropisms for Rhinolophus bat ACE2. Through structural analysis and cell culture experiments, we demonstrate that this tropism is determined by residue 493 of the spike and residues 31 and 35 of ACE2. Our results suggest that SC2r-CoVs exhibit differential ACE2 tropism, which may be driven by adaptation to different Rhinolophus bat ACE2 proteins. IMPORTANCE The efficiency of infection receptor use is the first step in determining the species tropism of viruses. After the coronavirus disease 2019 pandemic, a number of SARS-CoV-2-related coronaviruses (SC2r-CoVs) were identified in Rhinolophus bats, and some of them can use human angiotensin converting enzyme 2 (ACE2) for the infection receptor without acquiring additional mutations. This means that the potential of certain SC2r-CoVs to cause spillover from bats to humans is "off-the-shelf." However, both SC2r-CoVs and Rhinolophus bat species are highly diversified, and the host tropism of SC2r-CoVs remains unclear. Here, we focus on two Laotian SC2r-CoVs, BANAL-20-236 and BANAL-20-52, and determine how the tropism of SC2r-CoVs to Rhinolophus bat ACE2 is determined at the amino acid resolution level. [ABSTRACT FROM AUTHOR]

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Copyright of Journal of Physical Education (20736452) is the property of Republic of Iraq Ministry of Higher Education & Scientific Research (MOHESR) and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

A critical step in the drug design for SARS-CoV-2 is to discover its molecular targets. This study comprehensively reviewed the molecular mechanisms of SARS-CoV-2, exploring host cell tropism and interaction targets crucial for cell entry. The findings revealed that beyond ACE2 as the primary entry receptor, alternative receptors, co-receptors, and several proteases such as TMPRSS2, Furin, Cathepsin L, and ADAM play critical roles in virus entry and subsequent pathogenesis. Additionally, SARS-CoV-2 displays tropism in various human organs due to its diverse receptors. This review delves into the intricate details of receptors, host proteases, and the involvement of each organ. Polymorphisms in the ACE2 receptor and mutations in the spike or its RBD region contribute to the emergence of variants like Alpha, Beta, Gamma, Delta, and Omicron, impacting the pathogenicity of SARS-CoV-2. The challenge posed by mutations raises questions about the effectiveness of existing vaccines and drugs, necessitating consideration for updates in their formulations. In the urgency of these critical situations, repurposed drugs such as Camostat Mesylate and Nafamostat Mesylate emerge as viable pharmaceutical options. Numerous drugs are involved in inhibiting receptors and host factors crucial for SARS-CoV-2 entry, with most discussed in this review. In conclusion, this study may provide valuable insights to inform decisions in therapeutic approaches.

The COVID‐19 pandemic threatened public health around the world at the same time as highlighting the urgency of vaccine development. Subunit vaccines are safe and effective vaccine types that utilize parts of viruses to trigger the body’s immune response. Previous research has shown that fusion of the spike protein with the foldon domain (fd) achieved the trimeric form to increase the protein stability of the recombinant subunit protein spike from SARS‐CoV and MERS‐CoV, thus exceeding the immune response in the body. The study aims to observe the expression of RBD‐fd and S1‐fd recombinant proteins from the spike protein of SARS‐CoV‐2 in CHO‐K1 mammalian cells and investigate the binding activity of those proteins with hACE2 receptor, expressed in HEK293T cells using immunofluorescence staining. The plasmids were transiently transfected into the cells, followed by antibiotic selection using G418 as an initial stage to select the positive stable transformants. Protein expression was confirmed by Western blotting and showed an estimated size for monomeric RBD‐fd of 35 kDa and S1‐fd of 55 kDa. However, the trimeric form of the proteins was not observed. In addition, immunofluorescence staining showed the binding activity between the RBD‐fd and S1‐fd proteins and hACE2 expressing cell line, revealing binding and an internalization process.

Shaia SR Almalki,1 Mohammad Asrar Izhari,1 Hanan E Alyahyawi,1 Saleha Keder Alatawi,2 Faisal Klufah,1 Waled AM Ahmed,3 Raed Alharbi1 1Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha, Saudi Arabia; 2Department of Optometry, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha, Saudi Arabia; 3Department of Nursing, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha, Saudi ArabiaCorrespondence: Shaia SR Almalki, Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha, Saudi Arabia, Tel +966-556556714, Email shalmalki@bu.edu.saPurpose: Omicron (B.1.1.529) is one of the highly mutated variants of concern of SARS-CoV-2. Lineages of Omicron bear a remarkable degree of mutations leading to enhanced pathogenicity and upward transmission trajectory. Mutating Omicron lineages may trigger a fresh COVID-19 wave at any time in any region. We aimed at the whole-genome sequencing of SARS-CoV-2 to determine variants/subvariants and significant mutations which can foster virus evolution, monitoring of disease spread, and outbreak management.Methods: We used Illumina-NovaSeq 6000 for SARS-CoV-2 genome sequencing, MEGA 10.2 and nextstrain tools for phylogeny; CD-HIT program (version 4.8.1) and MUSCLE program for clustering and alignment. At the same time, UCSF Chimera was employed for protein visualization.Results: Predominant Omicron pango lineages in Al-Baha were BA.5.2/B22 (n=4, 57%), and other lineages were BA.2.12/21L (n=1, 14.28%), BV.1/22B (n=1, 14.28%) and BA.5.2.18/22B (n=1, 14.28%). 22B nextstrain clade was predominant, while only one lineage showed 21L. BA.5.2/22B, BA.5.2/22B harbored a maximum of n=24 mutations in the spike region. Twelve crucial RBD mutations: D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, and Y505H were identified except the lineage BA.5.2/22B in which F486V mutation was not observed. Critical deletions S106 in membrane protein NSP6, E31in nucleocapsid, and L24 in spike region were observed in all the lineages. Furthermore, we identified common mutations of Omicron variants of SARS-CoV-2 in therapeutic hot spot spike region: T19I, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, A653V, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, D1146D, L452R, F486V, N679K and D796Y. The effect of RBD-targeted mutations on neutralizing (NAbs) binding was considerable.Conclusion: The outcome of this first report on SARS-CoV-2 variants identification and mutation in the Al-Baha region could be used to lay down the policies to manage and impede the regional outbreak of COVID-19 effectively.Keywords: omicron, SARS-CoV-2, nextstrain clade, phylogeny, mutation, spike, RBD

SARS-CoV-2 vaccines have contributed to attenuating the burden of the COVID-19 pandemic by promoting the development of effective immune responses, thus reducing the spread and severity of the pandemic. A clinical trial with the Sputnik-V vaccine was conducted in Venezuela from December 2020 to July 2021. The aim of this study was to explore the antibody reactivity of vaccinated individuals towards different regions of the spike protein (S). Neutralizing antibody (NAb) activity was assessed using a commercial surrogate assay, detecting NAbs against the receptor-binding domain (RBD), and a plaque reduction neutralization test. NAb levels were correlated with the reactivity of the antibodies to the spike regions over time. The presence of Abs against nucleoprotein was also determined to rule out the effect of exposure to the virus during the clinical trial in the serological response. A high serological reactivity was observed to S and specifically to S1 and the RBD. S2, although recognized with lower intensity by vaccinated individuals, was the subunit exhibiting the highest cross-reactivity in prepandemic sera. This study is in agreement with the high efficacy reported for the Sputnik V vaccine and shows that this vaccine is able to induce an immunity lasting for at least 180 days. The dissection of the Ab reactivity to different regions of S allowed us to identify the relevance of epitopes outside the RBD that are able to induce NAbs. This research may contribute to the understanding of vaccine immunity against SARS-CoV-2, which could contribute to the design of future vaccine strategies.

The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a fast-spreading viral pathogen and poses a serious threat to human health. New SARS-CoV-2 variants have been arising worldwide; therefore, is necessary to explore more therapeutic options. The interaction of the viral spike (S) protein with the angiotensin-converting enzyme 2 (ACE2) host receptor is an attractive drug target to prevent the infection via the inhibition of virus cell entry. In this study, Ligand- and Structure-Based Virtual Screening (LBVS and SBVS) was performed to propose potential inhibitors capable of blocking the S receptor-binding domain (RBD) and ACE2 interaction. The best five lead compounds were confirmed as inhibitors through ELISA-based enzyme assays. The docking studies and molecular dynamic (MD) simulations of the selected compounds maintained the molecular interaction and stability (RMSD fluctuations less than 5 Å) with key residues of the S protein. The compounds DRI-1, DRI-2, DRI-3, DRI-4, and DRI-5 efficiently block the interaction between the SARS-CoV-2 spike protein and receptor ACE2 (from 69.90 to 99.65% of inhibition) at 50 µM. The most potent inhibitors were DRI-2 (IC50 = 8.8 µM) and DRI-3 (IC50 = 2.1 µM) and have an acceptable profile of cytotoxicity (CC50 > 90 µM). Therefore, these compounds could be good candidates for further SARS-CoV-2 preclinical experiments.

Spike protein sequences in SARS-CoV-2 have been employed for vaccine epitopes, but many short constituent sequences (SCSs) in the spike protein are present in the human proteome, suggesting that some anti-spike antibodies induced by infection or vaccination may be autoantibodies against human proteins. To evaluate this possibility of “molecular mimicry” in silico and in vitro, we exhaustively identified common SCSs (cSCSs) found both in spike and human proteins bioinformatically. The commonality of SCSs between the two systems seemed to be coincidental, and only some cSCSs were likely to be relevant to potential self-epitopes based on three-dimensional information. Among three antibodies raised against cSCS-containing spike peptides, only the antibody against EPLDVL showed high affinity for the spike protein and reacted with an EPLDVL-containing peptide from the human unc-80 homolog protein. Western blot analysis revealed that this antibody also reacted with several human proteins expressed mainly in the small intestine, ovary, and stomach. Taken together, these results showed that most cSCSs are likely incapable of inducing autoantibodies but that at least EPLDVL functions as a self-epitope, suggesting a serious possibility of infection-induced or vaccine-induced autoantibodies in humans. High-risk cSCSs, including EPLDVL, should be excluded from vaccine epitopes to prevent potential autoimmune disorders.

Ad26.COV2.S vaccination can lead to vaccine-induced immune thrombotic thrombocytopenia (VITT), a rare but severe adverse effect, characterized by thrombocytopenia and thrombosis. The mechanism of VITT induction is unclear and likely multifactorial, potentially including the activation of platelets and endothelial cells mediated by the vaccine-encoded spike protein (S protein). Here, we investigated the biodistribution of the S protein after Ad26.COV2.S dosing in three animal models and in human serum samples. The S protein was transiently present in draining lymph nodes of rabbits after Ad26.COV2.S dosing. The S protein was detected in the serum in all species from 1 day to 21 days after vaccination with Ad26.COV2.S, but it was not detected in platelets, the endothelium lining the blood vessels, or other organs. The S protein S1 and S2 subunits were detected at different ratios and magnitudes after Ad26.COV2.S or COVID-19 mRNA vaccine immunization. However, the S1/S2 ratio did not depend on the Ad26 platform, but on mutation of the furin cleavage site, suggesting that the S1/S2 ratio is not VITT related. Overall, our data suggest that the S-protein biodistribution and kinetics after Ad26.COV2.S dosing are likely not main contributors to the development of VITT, but other S-protein-specific parameters require further investigation.

SARS-CoV-2 virus variants of concern (VOCs) have rapidly changed their transmissibility and pathogenicity primarily through mutations in the structural proteins. Herein, we present molecular details with dynamics of the ferritin nanocages stitched with synthetic chimeras displaying the Spike receptor binding domains (RBDs). Our findings demonstrated the potential usage of ferritin-based vaccines that may effectively inhibit viral entry by blocking the Spike–ACE2 network and may induce cross-protective antibody responses. Taking the nanocage constructs into consideration, we evaluated the effects of variants on the docked interface of the SARS-CoV-2 Spike RBD with the ACE2 (angiotensin-converting enzyme 2) host cell receptor and neutralizing antibodies (Abs). Investigating the VOCs revealed that most of the mutations reported a possibly reduced structural stability within the Spike RBD domain. Point mutations have moderate or no effect for VVH-72, CR3022, and S309 Abs when bound with the Spike RBD, whereas a significant effect was observed for B38, CB6, and m396 over the surface of the H-ferritin nanocage. In addition to providing useful therapeutic approaches against COVID-19 (coronavirus disease 2019), these structural details can also be used to fight future coronavirus outbreaks.

An intensive care unit (ICU) is a special ward in the hospital for patients who require intensive care. It is equipped with many instruments monitoring patients’ vital signs and supported by the medical staff. However, continuous monitoring demands a massive workload of medical care. To ease the burden, we aim to develop an automatic detection model to monitor when brain anomalies occur. In this study, we focus on electroencephalography (EEG), which monitors the brain electroactivity of patients continuously. It is mainly for the diagnosis of brain malfunction. We propose the gated-recurrent-unit-based (GRU-based) model for detecting brain anomalies; it predicts whether the spike or sharp wave happens within a short time window. Based on the banana montage setting, the proposed model exploits characteristics of multiple channels simultaneously to detect anomalies. It is trained, validated, and tested on separated EEG data and achieves more than 90% testing performance on sensitivity, specificity, and balanced accuracy. The proposed anomaly detection model detects the existence of a spike or sharp wave precisely; it will notify the ICU medical staff, who can provide immediate follow-up treatment. Consequently, it can reduce the medical workload in the ICU significantly.

Booster doses of the SARS-CoV-2 vaccine have been recommended to improve and prolong immunity, address waning immunity over time, and contribute to the control of the COVID-19 pandemic. A heterologous booster vaccine strategy may offer advantages over a homologous approach. To compare the immunogenicity of two doses of BNT162b2 mRNA COVID-19 vaccine with a ChAdOx1-S booster dose, immunoglobulin G (IgG) anti-spike (anti-S) and anti-nucleocapsid (anti-N) antibody titers (Ab) were compared over 1 year and post-booster vaccination. Results showed that, at 3- to 9-month assessments in vaccinated subjects, an-ti-N Ab were undetectable in participants with no history of COVID-19. In contrast, anti-S Ab measurements were lower than those with COVID-19, and a decrease was observed during the 9 months of observation. After booster vaccination, no differences were found in anti-S between participants who reported a history of COVID-19 and those who did not. Anti-S levels were higher after booster vaccination measurement vs. at 9 months in participants with COVID-19 and without COVID-19, i.e., independent of an infection history. Vaccine administration elicited a response of higher anti-S IgG levels in those infected before vaccination, although levels decreased during the first nine months. IgG anti-N titers were higher in participants with a history of declared infection and who were asymptomatic. The ChAdOx1-S booster increased anti-S Ab levels in participants regardless of whether they had been infected or not to a significantly higher value than with the first two vaccines. These findings underscore the importance of booster vaccination in eliciting a robust and sustained immune response against COVID-19, regardless of the prior infection status.

Based on the gas kinetic scheme (GKS), numerical simulation research on the thermal protection system with the spike was carried out from the independent flow field and the fluid-structure interaction. The reliability of the algorithm in independent flow field was first verified by using the standard cylindrical model, and then the effectiveness and mechanism of the heat reduction of spike were analyzed by comparing the aerothermal characteristics and the corresponding flow field characteristics of the cylindrical model without/with the spike. After verification by applying GKS to the coupled simulation of flow field/structure temperature field, the heat reduction performance of spike under the fluid-structure interaction was further analyzed. The results show that the heat reduction performance of spike increases with the advancement of the fluid-structure interaction time, which is beneficial to the long-term cruise flight of the hypersonic vehicle.

Bioactive compounds against SARS-CoV-2 targets could be potential treatments for COVID-19. Inhibitors of the receptor-binding domain (RBD) on the viral spike protein can block its binding to the human angiotensin-converting enzyme type II (ACE2) receptor. This study presents ligands based on natural products and synthetic compounds, targeting multiple N501/Y501 RBDs, besides RBD-ACE2, over different regions. The selected compounds were evaluated by docking using consensus scoring, pharmacokinetics/toxicological analyses, and molecular dynamics. Additionally, N501/Y501 RBD-ACE2 interaction properties and RBD–ligand complexes were compared. We identified that coenzyme Q10, 1-stearoyl-2-arachidonoylglycerol, and palmitone showed the greatest RBD interactions. Targeting specific residues (in particular, tyrosine) in the C-, N-terminal, and central RBD sites promoted more stable protein–ligand interactions than in the N-terminal region only. Our results indicate that the molecules had more energetically favorable interactions with residues from distinct RBD regions rather than only interacting with residues in the N-terminal site. Moreover, the compounds might better interact with mutated N501Y than N501 RBDs. These hits can be optimized to leads and investigated through QSAR models and biological assays to comprehend mechanisms better. Altogether, such strategies may anticipate antiviral strategies if or when future variants and other CoVs arise.

The study aimed at the possibility of breaking the adjustment in the body muscles that are relevant to the volleyball player in terms of connectivity in the development of motor velocity through extreme and ultra-maximum force exercises. The researchers used the experimental method to solve the search problem. (24) A player in deliberate style, and the two researchers used several tools and methods, and this vocabulary was sunk for a period of.(4) weeks, 3 modules per week and 12 modules, 1 module time.(90 minutes) Motor speed measurement tests were used because of their importance in achieving the player's goal of exercise and the overwhelming strike test, which measures the performance level of offensive skills in volleyball. Tests were conducted before and after the use of extreme and super-maximum force exercises

ABSTRACTCoronavirus disease 2019 (COVID-19) vaccines have saved millions of lives. However, variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have emerged causing large numbers of breakthrough infections. These developments necessitated the rollout of COVID-19 vaccine booster doses. It has been reported that mucosal antibody levels in the upper respiratory tract, especially for secretory IgA (sIgA), correlate with protection from infection with SARS-CoV-2. However, it is still unclear how high levels of mucosal antibodies can be induced. In this study, we measured serum IgG, saliva IgG, and saliva sIgA responses in individuals who received COVID-19 mRNA booster vaccinations or who experienced breakthrough infections. We found that mRNA booster doses could induce robust serum and saliva IgG responses, especially in individuals who had not experienced infections before, but saliva sIgA responses were weak. In contrast, breakthrough infections in individuals who had received the primary mRNA vaccination series induced robust serum and saliva IgG as well as saliva sIgA responses. Individuals who had received a booster dose and then had a breakthrough infection showed low IgG induction in serum and saliva but still responded with robust saliva sIgA induction. These data suggest that upper respiratory tract exposure to antigen is an efficient way of inducing mucosal sIgA while exposure via intramuscular injection is not.IMPORTANCEAntibodies on mucosal surfaces of the upper respiratory tract have been shown to be important for protection from infection with SARS-CoV-2. Here we investigate the induction of serum IgG, saliva IgG, and saliva sIgA after COVID-19 mRNA booster vaccination or breakthrough infections.

Immune evasion by SARS-CoV-2 paired with immune imprinting from monovalent mRNA vaccines has resulted in attenuated neutralizing antibody responses against Omicron subvariants. In this study, we characterized two new XBB variants rising in circulation – EG.5.1 and XBB.2.3, for their neutralization and syncytia formation. We determined the neutralizing antibody titers in sera of individuals that received a bivalent mRNA vaccine booster, BA.4/5-wave infection, or XBB.1.5-wave infection. Bivalent vaccination-induced antibodies neutralized ancestral D614G efficiently, but to a much less extent, two new EG.5.1 and XBB.2.3 variants. In fact, the enhanced neutralization escape of EG.5.1 appeared to be driven by its key defining mutation XBB.1.5-F456L. Notably, infection by BA.4/5 or XBB.1.5 afforded little, if any, neutralization against EG.5.1, XBB.2.3 and previous XBB variants – especially in unvaccinated individuals, with average neutralizing antibody titers near the limit of detection. Additionally, we investigated the infectivity, fusion activity, and processing of variant spikes for EG.5.1 and XBB.2.3 in HEK293T-ACE2 and CaLu-3 cells but found no significant differences compared to earlier XBB variants. Overall, our findings highlight the continued immune evasion of new Omicron subvariants and, more importantly, the need to reformulate mRNA vaccines to include XBB spikes for better protection.

SARS-CoV-2, the etiological agent behind the coronavirus disease 2019 (COVID-19) pandemic, has continued to mutate and create new variants with increased resistance against the WHO-approved spike-based vaccines. With a significant portion of the worldwide population still unvaccinated and with waning immunity against newly emerging variants, there is a pressing need to develop novel vaccines that provide broader and longer-lasting protection. To generate broader protective immunity against COVID-19, we developed our second-generation vaccinia virus-based COVID-19 vaccine, TOH-VAC-2, encoded with modified versions of the spike (S) and nucleocapsid (N) proteins as well as a unique poly-epitope antigen that contains immunodominant T cell epitopes from seven different SARS-CoV-2 proteins. We show that the poly-epitope antigen restimulates T cells from the PBMCs of individuals formerly infected with SARS-CoV-2. In mice, TOH-VAC-2 vaccination produces high titers of S- and N-specific antibodies and generates robust T cell immunity against S, N, and poly-epitope antigens. The immunity generated from TOH-VAC-2 is also capable of protecting mice from heterologous challenge with recombinant VSV viruses that express the same SARS-CoV-2 antigens. Altogether, these findings demonstrate the effectiveness of our versatile vaccine platform as an alternative or complementary approach to current vaccines.

Obesity is a known risk factor for severe respiratory tract infections. In this prospective study, we assessed the impact of being obese or overweight on longitudinal SARS-CoV-2 humoral and cellular responses up to 18 months after infection. 274 patients provided blood samples at regular time intervals up to 18 months including obese (BMI ≥30, n=32), overweight (BMI 25-29.9, n=103) and normal body weight (BMI 18.5-24.9, n=134) SARS-CoV-2 patients. We determined SARS-CoV-2 spike-specific IgG, IgA, IgM levels by ELISA and neutralising antibody titres by neutralisation assay. RBD- and spike-specific memory B cells were investigated by ELISpot, spike- and non-spike-specific IFN-γ, IL-2 and IFN-γ/IL-2 secreting T cells by FluoroSpot and T cell receptor (TCR) sequencing was performed. Higher BMI correlated with increased COVID-19 severity. Humoral and cellular responses were stronger in overweight and obese patients than normal weight patients and associated with higher spike-specific IgG binding titres relative to neutralising antibody titres. Linear regression models demonstrated that BMI, age and COVID-19 severity correlated independently with higher SARS-CoV-2 immune responses. We found an increased proportion of unique SARS-CoV-2 specific T cell clonotypes after infection in overweight and obese patients. COVID-19 vaccination boosted humoral and cellular responses irrespective of BMI, although stronger immune boosting was observed in normal weight patients. Overall, our results highlight more severe disease and an over-reactivity of the immune system in overweight and obese patients after SARS-CoV-2 infection, underscoring the importance of recognizing overweight/obese individuals as a risk group for prioritisation for COVID-19 vaccination.

IntroductionAction potentials usually travel orthodromically along a neuron’s axon, from the axon initial segment (AIS) toward the presynaptic terminals. Under some circumstances action potentials also travel in the opposite direction, antidromically, after being initiated at a distal location. Given their initiation at an atypical site, we refer to these events as “ectopic action potentials.” Ectopic action potentials (EAPs) were initially observed in pathological conditions including seizures and nerve injury. Several studies have described regular-spiking (RS) pyramidal neurons firing EAPs in seizure models. Under nonpathological conditions, EAPs were reported in a few populations of neurons, and our group has found that EAPs can be induced in a large proportion of parvalbumin-expressing interneurons in the neocortex. Nevertheless, to our knowledge there have been no prior reports of ectopic firing in the largest population of neurons in the neocortex, pyramidal neurons, under nonpathological conditions.MethodsWe performed in vitro recordings utilizing the whole-cell patch clamp technique. To elicit EAPs, we triggered orthodromic action potentialswith either long, progressively increasing current steps, or with trains of brief pulses at 30, 60, or 100 Hz delivered in 3 different ways, varying in stimulus and resting period duration.ResultsWe found that a large proportion (72.7%) of neocortical RS cells from mice can fire EAPs after a specific stimulus in vitro, and that most RS cells (56.1%) are capable of firing EAPs across a broad range of stimulus conditions. Of the 37 RS neurons in which we were able to elicit EAPs, it took an average of 863.8 orthodromic action potentials delivered over the course of an average of ~81.4 s before the first EAP was seen. We observed that some cells responded to specific stimulus frequencies while less selective, suggesting frequency tuning in a subset of the cells.DiscussionOur findings suggest that pyramidal cells can integrate information over long time-scales before briefly entering a mode of self-generated firing that originates in distal axons. The surprising ubiquity of EAP generation in RS cells raises interesting questions about the potential roles of ectopic spiking in information processing, cortical oscillations, and seizure susceptibility.

SARS-CoV-2 infection induces non-physiological syncytia when its spike fusogenic protein on the surface of the host cells interacts with the ACE2 receptor on adjacent cells. Spike-induced syncytia are beneficial for virus replication, transmission, and immune evasion, and contribute to the progression of COVID-19. In this review, we highlight the properties of viral fusion proteins, mainly the SARS-CoV-2 spike, and the involvement of the host factors in the fusion process. We also highlight the possible use of anti-fusogenic factors as an antiviral for the development of therapeutics against newly emerging SARS-CoV-2 variants and how the fusogenic property of the spike could be exploited for biomedical applications. [ABSTRACT FROM AUTHOR]

Background: Measuring specific anti-SARS-CoV-2 antibodies has become one of the main epidemiological tools to survey the ongoing SARS-CoV-2 pandemic, but also vaccination response. The WHO made available a set of well-characterized samples derived from recovered individuals to allow normalization between different quantitative anti-Spike assays to defined Binding Antibody Units (BAU). Methods: To assess sero-responses longitudinally, a cohort of ninety-nine SARS-CoV-2 RT-PCR positive subjects was followed up together with forty-five vaccinees without previous infection but with two vaccinations. Sero-responses were evaluated using a total of six different assays: four measuring anti-Spike proteins (converted to BAU), one measuring anti-Nucleocapsid proteins and one SARS-CoV-2 surrogate virus neutralization. Both cohorts were evaluated using the Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and the Roche Elecsys Anti-SARS-CoV-2 anti-S1 assay. Results: In SARS-CoV-2-convalesce subjects, the BAU-sero-responses of Euroimmun Anti-SARS-CoV-2-ELISA anti-S1 IgG and Roche Elecsys Anti-SARS-CoV-2 anti-S1 peaked both at 47 (43–51) days, the first assay followed by a slow decay thereafter (> 208 days), while the second assay not presenting any decay within one year. Both assay values in BAUs are only equivalent a few months after infection, elsewhere correction factors up to 10 are necessary. In contrast, in infection-naive vaccinees the assays perform similarly. Conclusion: The results of our study suggest that the establishment of a protective correlate or vaccination booster recommendation based on different assays, although BAU-standardised, is still challenging. At the moment the characteristics of the available assays used are not related, and the BAU-standardisation is unable to correct for that. [ABSTRACT FROM AUTHOR]

The severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) virus emerged in late 2019 leading to the COVID‐19 disease pandemic that triggered socioeconomic turmoil worldwide. A precise, prompt, and affordable diagnostic assay is essential for the detection of SARS‐CoV‐2 as well as its variants. Antibody against SARS‐CoV‐2 spike (S) protein was reported as a suitable strategy for therapy and diagnosis of COVID‐19. We, therefore, developed a quick and precise phase‐sensitive surface plasmon resonance (PS‐SPR) biosensor integrated with a novel generated anti‐S monoclonal antibody (S‐mAb). Our results indicated that the newly generated S‐mAb could detect the original SARS‐CoV‐2 strain along with its variants. In addition, a SARS‐CoV‐2 pseudovirus, which could be processed in BSL‐2 facility was generated for evaluation of sensitivity and specificity of the assays including PS‐SPR, homemade target‐captured ELISA, spike rapid antigen test (SRAT), and quantitative reverse transcription polymerase chain reaction (qRT‐PCR). Experimentally, PS‐SPR exerted high sensitivity to detect SARS‐CoV‐2 pseudovirus at 589 copies/ml, with 7‐fold and 70‐fold increase in sensitivity when compared with the two conventional immunoassays, including homemade target‐captured ELISA (4 × 103 copies/ml) and SRAT (4 × 104 copies/ml), using the identical antibody. Moreover, the PS‐SPR was applied in the measurement of mimic clinical samples containing the SARS‐CoV‐2 pseudovirus mixed with nasal mucosa. The detection limit of PS‐SPR is calculated to be 1725 copies/ml, which has higher accuracy than homemade target‐captured ELISA (4 × 104 copies/ml) and SRAT (4 × 105 copies/ml) and is comparable with qRT‐PCR (1250 copies/ml). Finally, the ability of PS‐SPR to detect SARS‐CoV‐2 in real clinical specimens was further demonstrated, and the assay time was less than 10 min. Taken together, our results indicate that this novel S‐mAb integrated into PS‐SPR biosensor demonstrates high sensitivity and is time‐saving in SARS‐CoV‐2 virus detection. This study suggests that incorporation of a high specific recognizer in SPR biosensor is an alternative strategy that could be applied in developing other emerging or re‐emerging pathogenic detection platforms. [ABSTRACT FROM AUTHOR]

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Systems for distributing electricity should be able to handle requirements such as high efficiency, low voltage fluctuation, and the integration of Renewable Energy Generators (REGs) into an intelligent grid. This paper depicts the operation of a power distribution grid that represents solutions for three optimization problems by finding the acceptable power loss configuration of the grid, the second is the so-called volt/Var optimization problem, finally compensating the voltage drop through the feeders far from the main source by sharing power from nearby REGs. A suggested developed configuration for a real radial system to make it more dependable and efficient is studied. Al-Block district as part of the Mosul province Distribution System (DS) is to be integrated with REGs besides switching spare feeds (spikes) to the main radial feeds considering fault occurs. The suggested method effects are assessed by comparing the grid performance before and after adding REGs and the spike feeders, where power flow calculations are carried out to find the best configurations for the developed grid after adding the feeder. The findings prove the enhancement of voltage profiles across feeders, voltage conservation, and power efficiency. [ABSTRACT FROM AUTHOR]

Hematopoietic cell transplantation (HCT) and chimeric antigen receptor (CAR)-T cell patients are immunocompromised, remain at high risk following SARS-CoV-2 infection, and are less likely than immunocompetent individuals to respond to vaccination. As part of the safety lead-in portion of a phase 2 clinical trial in patients post HCT/CAR-T for hematological malignancies (HM), we tested the immunogenicity of the synthetic modified vaccinia Ankara-based COVID-19 vaccine COH04S1 co-expressing spike (S) and nucleocapsid (N) antigens. Thirteen patients were vaccinated 3–12 months post HCT/CAR-T with two to four doses of COH04S1. SARS-CoV-2 antigen-specific humoral and cellular immune responses, including neutralizing antibodies to ancestral virus and variants of concern (VOC), were measured up to six months post vaccination and compared to immune responses in historical cohorts of naïve healthy volunteers (HV) vaccinated with COH04S1 and naïve healthcare workers (HCW) vaccinated with the FDA-approved mRNA vaccine Comirnaty® (Pfizer, New York, NY, USA). After one or two COH04S1 vaccine doses, HCT/CAR-T recipients showed a significant increase in S- and N-specific binding antibody titers and neutralizing antibodies with potent activity against SARS-CoV-2 ancestral virus and VOC, including the highly immune evasive Omicron XBB.1.5 variant. Furthermore, vaccination with COH04S1 resulted in a significant increase in S- and N-specific T cells, predominantly CD4+ T lymphocytes. Elevated S- and N-specific immune responses continued to persist at six months post vaccination. Furthermore, both humoral and cellular immune responses in COH04S1-vaccinated HCT/CAR-T patients were superior or comparable to those measured in COH04S1-vaccinated HV or Comirnaty®-vaccinated HCW. These results demonstrate robust stimulation of SARS-CoV-2 S- and N-specific immune responses including cross-reactive neutralizing antibodies by COH04S1 in HM patients post HCT/CAR-T, supporting further testing of COH04S1 in immunocompromised populations. [ABSTRACT FROM AUTHOR]

Mucosal IgA is widely accepted as providing protection against respiratory infections, but stimulation of mucosal immunity, collection of mucosal samples and measurement of mucosal IgA can be problematic. The relationship between mucosal and circulating IgA responses is unclear, however, whole blood is readily collected and circulating antigen-specific IgA easily measured. We measured circulating IgA against SARS-CoV-2 spike (S) to investigate vaccine- and infection-induced production and correlation with protection. Circulating IgA against ancestral (Wuhan-Hu-1) and Omicron (BA.1) S proteins was measured at different time points in a total of 143 subjects with varied backgrounds of vaccination and infection. Intramuscular vaccination induced circulating anti-SARS-CoV-2 S IgA. Subjects with higher levels of vaccine-induced IgA against SARS-CoV-2 S (p = 0.0333) or receptor binding domain (RBD) (p = 0.0266) were less likely to experience an Omicron breakthrough infection. The same associations did not hold for circulating IgG anti-SARS-CoV-2 S levels. Breakthrough infection following two vaccinations generated stronger IgA anti-SARS-CoV-2 S responses (p = 0.0002) than third vaccinations but did not selectively increase circulating IgA against Omicron over ancestral S, indicating immune imprinting of circulating IgA responses. Circulating IgA against SARS-CoV-2 S following breakthrough infection remained higher than vaccine-induced levels for over 150 days. In conclusion, intramuscular mRNA vaccination induces circulating IgA against SARS-CoV-2 S, and higher levels are associated with protection from breakthrough infection. Vaccination with ancestral S enacts imprinting within circulating IgA responses that become apparent after breakthrough infection with Omicron. Breakthrough infection generates stronger and more durable circulating IgA responses against SARS-CoV-2 S than vaccination alone. [ABSTRACT FROM AUTHOR]

The COVID-19 pandemic has witnessed the emergence of diverse variants of SARS-CoV-2, with spike proteins playing a pivotal role in mutation due to their extracellular projection and exposure to immune system pressures. Clinical manifestations of COVID-19 have shown significant variation, ranging from severe symptoms requiring ICU admission or resulting in fatality to asymptomatic cases. This study aims to investigate genetic variations in the spike protein among two distinct groups of SARS-CoV-2 sequences: asymptomatic and ICU/deceased patients. The objective is to explore the viral genetic factors associated with these two clinical outcomes. Our analysis reveals that four spike protein mutations (P26S, D253G, K417N, and D614G) may be partially linked to the ICU/deceased outcome. Additionally, the Omicron and Delta variants exhibit the highest proportions of overall asymptomatic and ICU/deceased patients, respectively. Further evaluation of the ratio of asymptomatic cases to ICU/deceased within a singular variant demonstrates that the Beta and Gamma variants elicit the greatest proportion of asymptomatic and ICU/deceased cases, respectively. In conclusion, our findings suggest a possible association between four spike protein mutations and the outcome of ICU admission or death. The Gamma variants demonstrate greater lethality, while the Delta variants are associated with higher mortality rates. [ABSTRACT FROM AUTHOR]

The spread of SARS-CoV-2 variants of concern (VOCs) is of great importance since genetic changes may increase transmissibility, disease severity and reduce vaccine effectiveness. Moreover, these changes may lead to failure of diagnostic measures. Therefore, variant-specific diagnostic methods are essential. To date, genetic sequencing is the gold-standard method to discriminate between variants. However, it is time-consuming (taking several days) and expensive. Therefore, the development of rapid diagnostic methods for SARS-CoV-2 in accordance with its genetic modification is of great importance. In this study we introduce a Mass Spectrometry (MS)-based methodology for the diagnosis of SARS-CoV-2 in propagated in cell-culture. This methodology enables the universal identification of SARS-CoV-2, as well as variant-specific discrimination. The universal identification of SARS-CoV-2 is based on conserved markers shared by all variants, while the identification of specific variants relies on variant-specific markers. Determining a specific set of peptides for a given variant consists of a multistep procedure, starting with an in-silico search for variant-specific tryptic peptides, followed by a tryptic digest of a cell-cultured SARS-CoV-2 variant, and identification of these markers by HR-LC-MS/MS analysis. As a proof of concept, this approach was demonstrated for four representative VOCs compared to the wild-type Wuhan reference strain. For each variant, at least two unique markers, derived mainly from the spike (S) and nucleocapsid (N) viral proteins, were identified. This methodology is specific, rapid, easy to perform and inexpensive. Therefore, it can be applied as a diagnostic tool for pathogenic variants. [ABSTRACT FROM AUTHOR]

To investigate in silico the occurrence of epigenetic crosstalk by nucleotide sequence complementarity between the BNT162b2 mRNA vaccine and whole human genome, including coding and noncoding (nc)RNA genes. To correlate these results with those obtained with the original spike (S) gene of Severe Acute Respiratory Syndrome CoronaVirus-2 (SARS-CoV-2). The publicly available FASTA sequence of the BNT162b2 mRNA vaccine and the SARS-CoV-2 isolate Wuhan-Hu-1 S gene (NC_045512.2) were used separately as key input to the Ensembl.org library to evaluate base pair match to human GRCh38 genome. Human coding and noncoding genes harboring hits were assessed for functional activity and health effects using bioinformatics tools and GWAS databases. The BLAT analysis against the human GRCh38 genome revealed a total of 37 hits for BNT162b2 mRNA and no hits for the SARS-CoV-2 S gene. More specifically, BNT162b2 mRNA matched 19 human genes whose protein products are variously involved in enzyme reactions, nucleotide or cation binding, signaling, and carrier functions. In BLASTN analysis of ncRNA genes, BNT162b2 mRNA and SARS-CoV-2 S gene matched 17 and 24 different human genomic regions, respectively. Overall, characterization of the matched noncoding sequences revealed stronger interference with epigenetic pathways for BNT162b2 mRNA compared with the original S gene. This pivotal in silico analysis shows that SARS-CoV-2 S gene and the BNT162b2 mRNA vaccine exhibit Watson-Crick nucleotide complementarity with human coding or noncoding genes. Although they do not share the same complementarity pattern, both may disrupt epigenetic mechanisms in target cells, potentially leading to long-term complications. [ABSTRACT FROM AUTHOR]

In this study, we provided a retrospective overview in order to better define SARS-CoV-2 variants circulating in Italy during the first two years of the pandemic, by characterizing the spike mutational profiles and their association with viral load (expressed as ct values), N-glycosylation pattern, hospitalization and vaccination. Next-generation sequencing (NGS) data were obtained from 607 individuals (among them, 298 vaccinated and/or 199 hospitalized). Different rates of hospitalization were observed over time and among variants of concern (VOCs), both in the overall population and in vaccinated individuals (Alpha: 40.7% and 31.3%, Beta: 0%, Gamma: 36.5% and 44.4%, Delta: 37.8% and 40.2% and Omicron: 11.2% and 7.1%, respectively, both p-values < 0.001). Approximately 32% of VOC-infected individuals showed at least one atypical major spike mutation (intra-prevalence > 90%), with a distribution differing among the strains (22.9% in Alpha, 14.3% in Beta, 41.8% in Gamma, 46.5% in Delta and 15.4% in Omicron, p-value < 0.001). Overall, significantly less atypical variability was observed in vaccinated individuals than unvaccinated individuals; nevertheless, vaccinated people who needed hospitalization showed an increase in atypical variability compared to vaccinated people that did not need hospitalization. Only 5/607 samples showed a different putative N-glycosylation pattern, four within the Delta VOC and one within the Omicron BA.2.52 sublineage. Interestingly, atypical minor mutations (intra-prevalence < 20%) were associated with higher Ct values and a longer duration of infection. Our study reports updated information on the temporal circulation of SARS-CoV-2 variants circulating in Central Italy and their association with hospitalization and vaccination. The results underline how SARS-CoV-2 has changed over time and how the vaccination strategy has contributed to reducing severity and hospitalization for this infection in Italy. [ABSTRACT FROM AUTHOR]

Novel coronavirus disease 2019 (COVID-19), a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has brought an unprecedented public health crisis and continues to threaten humanity due to the persistent emergence of new variants. Therefore, developing more effective and broad-spectrum therapeutic and prophylactic drugs against infection by SARS-CoV-2 and its variants, as well as future emerging CoVs, is urgently needed. In this study, we screened several US FDA-approved drugs and identified phenothiazine derivatives with the ability to potently inhibit the infection of pseudotyped SARS-CoV-2 and distinct variants of concern (VOCs), including B.1.617.2 (Delta) and currently circulating Omicron sublineages XBB and BQ.1.1, as well as pseudotyped SARS-CoV and MERS-CoV. Mechanistic studies suggested that phenothiazines predominantly inhibited SARS-CoV-2 pseudovirus (PsV) infection at the early stage and potentially bound to the spike (S) protein of SARS-CoV-2, which may prevent the proteolytic cleavage of the S protein, thereby exhibiting inhibitory activity against SARS-CoV-2 infection. In summary, our findings suggest that phenothiazines can serve as a potential broad-spectrum therapeutic drug for the treatment of SARS-CoV-2 infection as well as the infection of future emerging human coronaviruses (HCoVs). [ABSTRACT FROM AUTHOR]

Nanotechnology offers new possibilities in molecular diagnostics, with nanoparticles gaining attention as biosensor upgrades. This study evaluates gold-coated silver nanoplates coated with PEG for enhanced protection, aiming to detect Spike protein with higher sensitivity, and emphasizes the importance of considering complex environments and appropriate controls for specific binding and accurate analysis. The sensitivity of antibody-coated PEGAuTSNPs as tools for immunoassays is demonstrated through fibronectin (Fn)– anti-fibronectin binding within an isolated extracellular matrix as a complex and native environment of Fn. Moreover, the optimal functionalization volume of Spike protein was determined (4 µg/mL of PEGAuTSNP). Anti-Spike was added to confirm binding, while the TJP1 protein was used as a negative control. The same experiment was used in the presence of horse serum to simulate a complex environment. According to Localized Surface Plasmon Resonance analysis and Dynamic Light Scattering size measurements, anti-Spike exhibited a stronger affinity for the nanoplates, causing TJP1 to be replaced by the antibody on the nanoplates' surface. Future research will involve exploring alternative complex environments, filtering larger molecules, and the optimization of immunoassay performance. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) regulate gene expression posttranscriptionally and are implicated in viral replication and host tropism. miRNAs can impact the viruses either by directly interacting with the viral genome or modulating host factors. Although many miRNAs have predicted binding sites in the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) viral RNA genome, little experimental validation has been done. We first identified 492 miRNAs that have binding site(s) on the spike (S) viral RNA by a bioinformatics prediction. We then validated the selected 39 miRNAs by examining S-protein levels after coexpressing the S-protein and a miRNA into the cells. Seven miRNAs were found to reduce the S-protein levels by more than 50%. Among them, miR-15a, miR-153, miR-298, miR-508, miR-1909, and miR-3130 also significantly reduced SARS-CoV-2 viral replication. SARS-CoV-2 infection decreased the expression levels of miR-298, miR-497, miR-508, miR-1909, and miR-3130, but had no significant effects on miR-15a and miR-153 levels. Intriguingly, the targeting sequences of these miRNAs on the S viral RNA showed sequence conservation among the variants of concern. Our results suggest that these miRNAs elicit effective antiviral defense against SARS-CoV-2 by modulating S-protein expression and are likely targeting all the variants. Thus, the data signify the therapeutic potential of miRNA-based therapy for SARS-CoV-2 infections. NEW & NOTEWORTHY MicroRNAs can impact viruses either by directly interacting with the virus genome or by modulating host factors. We identified that cellular miRNAs regulate effective antiviral defense against SARS-CoV-2 via modulating spike protein expression, which may offer a potential candidate for antiviral therapy. [ABSTRACT FROM AUTHOR]

With increasing resistance of SARS-CoV-2 variants to antibodies, there is interest in developing entry inhibitors that target essential receptor-binding regions of the viral Spike protein and thereby present a high bar for viral resistance. Such inhibitors could be derivatives of the viral receptor, ACE2, or peptides engineered to interact specifically with the Spike receptor-binding pocket. We compared the efficacy of a series of both types of entry inhibitors, constructed as fusions to an antibody Fc domain. Such a design can increase protein stability and act to both neutralize free virus and recruit effector functions to clear infected cells. We tested the reagents against prototype variants of SARS-CoV-2, using both Spike pseudotyped vesicular stomatitis virus vectors and replication-competent viruses. These analyses revealed that an optimized ACE2 derivative could neutralize all variants we tested with high efficacy. In contrast, the Spike-binding peptides had varying activities against different variants, with resistance observed in the Spike proteins from Beta, Gamma, and Omicron (BA.1 and BA.5). The resistance mapped to mutations at Spike residues K417 and N501 and could be overcome for one of the peptides by linking two copies in tandem, effectively creating a tetrameric reagent in the Fc fusion. Finally, both the optimized ACE2 and tetrameric peptide inhibitors provided some protection to human ACE2 transgenic mice challenged with the SARS-CoV-2 Delta variant, which typically causes death in this model within 7-9 days. [ABSTRACT FROM AUTHOR]

We investigated the impact of the fourth dose with ChAdOx1 nCoV‐19 (AstraZeneca) in the humoral immune response to SARS‐CoV‐2 during a 9‐month follow‐up period in which Omicron was the predominant variant in Brazil. IgG for the SARS‐CoV‐2 spike protein (S) and nucleocapsid (N) proteins were analyzed in samples collected before and after the fourth dose. All participants were tested monthly for SARS‐CoV‐2 infection by RT‐qPCR. The antibody response induced by the fourth dose of the coronavirus disease 2019 vaccine was evaluated and compared with the response induced by the second and third doses. The additional antibody response to the viral S protein after the fourth dose was smaller than those after the third vaccine dose. In contrast, an increase in the N IgG levels could be observed after the fourth dose compared to other vaccine doses. In the comparison of the antibody response before and after the fourth dose, an increase in both S‐and‐N IgG was noted, mainly in the positive qPCR group. We did not observe a significant decline in IgG levels after the fourth dose, as observed after the second and third doses, therefore, a sustained humoral response to both S and N proteins seems to be achieved. [ABSTRACT FROM AUTHOR]

The female spikes (fruits) of Piper longum are widely used in Ayurvedic, Siddha and Unani medicine systems to treat respiratory and digestive disorders. The spikes are rich in piperine, a pharmacologically active amide alkaloid and a potent bioavailability enhancer, which accumulates to the highest level during the dark-green stage of spike development. Plant-associated microbiota influence the plant's fitness, response, and production of economically important metabolites. Considering the economic importance of piperine and other spike-derived alkaloids, understanding microbial community dynamics during spike development would be key to bioprospecting for economically important metabolites. In the present study, the structural diversity of microbial communities associated with early (SI), mid (SII), and late (SIII) stages of spike development in P. longum has been analysed by Illumina-based amplicon sequencing of 16S rRNA gene and ITS region. Results revealed that spike development significantly drives the diversity and abundance of spike-associated microbiota, especially bacterial communities. Cyanobacteria and Ascomycota constituted the most abundant bacterial and fungal phyla, respectively, across all stages of spike development. Interestingly, Halomonas, Kushneria and Haererehalobacter were found to be exclusively associated with SIII (corresponding to economically important) stage of spike development. Sphingomonas, Mortierella, Cladosporium and Vishniacozyma constituted the core microbiome of the spike. Besides, PICRUSt analysis revealed that amino acid metabolism was the most dominant metabolic function attributed to spike-associated bacterial communities. To the best of our knowledge, this is the first study to investigate the endomicrobiome dynamics during spike development in a medicinal plant species. [ABSTRACT FROM AUTHOR]

ABSTRACT The recent emergence of the omicron variant of the SARS-CoV-2 virus with large numbers of mutations has raised concern about a potential new surge in infections. Here we use molecular dynamics to study the biophysics of the interface of the BA1 and BA2 omicron spike protein binding to (i) the ACE2 receptor protein, (ii) antibodies from all known binding regions, and (iii) the furin binding domain. Our simulations suggest that while there is a significant reduction of antibody (Ab) binding strength corresponding to escape, the omicron spikes pay a cost in terms of weaker receptor binding as measured by interfacial hydrogen bonds (H-bond). The furin cleavage domain (FCD) is the same or weaker binding than the delta variant, suggesting lower fusogenicity resulting in less viral load and disease intensity than the delta variant. IMPORTANCE The BA1 and BA2 and closely related BA2.12.2 and BA.5 omicron variants of SARS-CoV-2 dominate the current global infection landscape. Given the high number of mutations, particularly those which will lead to antibody escape, it is important to establish accurate methods that can guide developing health policy responses that identify at a fundamental level whether omicron and its variants are more threatening than its predecessors, especially delta. The importance of our work is to demonstrate that simple in silico simulations can predict biochemical binding details of the omicron spike protein that have epidemiological consequences, especially for binding to the cells and for fusing the viral membrane with the cells. In each case, we predicted weaker binding of the omicron spike, which agreed with subsequent experimental results. Future virology experiments will be needed to test these predictions further.