An unconventional strategy for purifying recombinant SARS-CoV-2 spike protein.

The soluble domain of the trimeric SARS-CoV-2 spike protein is a promising candidate for a COVID-19 vaccine. Purification of this protein from mammalian cell culture supernatant using conventional resin-based chromatography is challenging as its large size (∼550 kDa) restricts its access and mobility within the pores of the resin particles. This reduces binding capacity and process robustness very significantly as extremely low flow rates need to be used during purification. Convection-based ion-exchange membrane chromatography has been found to be suitable in this respect. However, the high ionic strength of mammalian cell culture supernatant makes it difficult to bind this protein on charged membranes without dilution with a suitable buffer. An unconventional strategy involving size-exclusion chromatography as the first step, followed by cation exchange membrane chromatography as the second step is proposed in this paper. In the size exclusion chromatography step, the spike protein is excluded from the pores and can therefore be isolated in the void volume fraction. This step removes small molecule impurities and also serves as a desalting and buffer exchange step, making the partially purified material suitable for the cation exchange membrane chromatography step. The proposed process is variant-independent, fast and scalable and addresses some of the challenges associated with the currently used purification methods.

SARS-CoV-2 spike protein induces the cytokine release syndrome by stimulating T cells to produce more IL-2.

Introduction: Cytokine release syndrome (CRS) is one of the leading causes of mortality in patients with COVID-19 caused by the SARS-CoV-2 coronavirus. However, the mechanism of CRS induced by SARS-CoV-2 is vague. Methods: Using spike protein combined with IL-2, IFN-γ, and TNF-α to stimulate human peripheral blood mononuclear cells (PBMCs) to secrete CRS-related cytokines, the content of cytokines in the supernatant was detected, and the effects of NK, T, and monocytes were analyzed. Results: This study shows that dendritic cells loaded with spike protein of SARS-CoV-2 stimulate T cells to release much more interleukin-2 (IL-2,) which subsequently cooperates with spike protein to facilitate PBMCs to release IL-1ß, IL-6, and IL-8. These effects are achieved via IL-2 stimulation of NK cells to release tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), as well as T cells to release IFN-γ Mechanistically, IFN-γ and TNF-α enhance the transcription of CD40, and the interaction of CD40 and its ligand stabilizes the membrane expression of toll-like receptor 4 (TLR4) that serves as a receptor of spike protein on the surface of monocytes. As a result, there is a constant interaction between spike protein and TLR4, leading to continuous activation of nuclear factor-κ-gene binding (NF-κB). Furthermore, TNF-α also activates NF-κB signaling in monocytes, which further cooperates with IFN-γ and spike protein to modulate NF-κB-dependent transcription of CRS-related inflammatory cytokines. Discussion: Targeting TNF-α/IFN-γ in combination with TLR4 may represent a promising therapeutic approach for alleviating CRS in individuals with COVID-19.

Neutralisation Resistance of SARS-CoV-2 Spike-Variants is Primarily Mediated by Synergistic Receptor Binding Domain Substitutions.

AbstractThe evolution of SARS-CoV-2 has led to the emergence of numerous variants of concern (VOCs), marked by changes in the viral spike glycoprotein, the primary target for neutralising antibody (nAb) responses. Emerging VOCs, particularly omicron sub-lineages, show resistance to nAbs induced by prior infection or vaccination. The precise spike protein changes contributing to this resistance remain unclear in infectious cell culture systems. In the present study, a large panel of infectious SARS-CoV-2 mutant viruses, each with spike protein changes found in VOCs, including omicron JN.1 and its derivatives KP.2 and KP.3, was generated using a reverse genetic system. The susceptibility of these viruses to antibody neutralisation was measured using plasma from convalescent and vaccinated individuals. Synergistic roles of combined substitutions in the spike receptor binding domain (RBD) were observed in neutralisation resistance. However, recombinant viruses with the entire spike protein from a specific VOC showed enhanced resistance, indicating that changes outside the RBD are also significant. In silico analyses of spike antibody epitopes suggested changes in neutralisation could be due to altered antibody binding affinities. Assessing ACE2 usage for entry through anti-ACE2 antibody blocking and ACE2 siRNA revealed that omicron BA.2.86 and JN.1 mutant viruses were less dependent on ACE2 for entry. However, surface plasmon resonance analysis showed increased affinity for ACE2 for both BA.2.86 and JN.1 compared to the ancestral spike. This detailed analysis of specific changes in the SARS-CoV-2 spike enhances understanding of coronavirus evolution, particularly regarding neutralising antibody evasion and ACE2 entry receptor dependence.

Development of a safe and broad-spectrum attenuated PEDV vaccine candidate by S2 subunit replacement.

Porcine epidemic diarrhea (PED) has caused serious economic losses to the swine livestock industry. Due to the rapid variation in the PEDV) genome, especially the spike (S) protein, the cross-protection ability of antibodies between different vaccine strains is weakened. Hence, the rapid development of safe, broad-spectrum and highly effective attenuated PEDV vaccine still needs further research. Here, we found that the replacement of the S2 subunit had little effect on S protein immunogenicity. Moreover, the chimeric virus (YN-S2DR13), the S protein of the YN strain was replaced by the DR13 S2 subunit, which lost its trypsin tropism and increased its propagation ability (approximately 1 titer) in Vero cells. Then, the pathogenesis of YN-S2DR13 was evaluated in neonatal piglets. Importantly, quantitative real-time PCR, histopathology, and immunohistochemistry confirmed that the virulence of YN-S2DR13 was significantly reduced compared with that of YN. Immunization with YN-S2DR13 induced neutralizing antibodies against both YN and DR13 in weaned piglets. In vitro passaging data also showed that YN-S2DR13 had good genetic stability. Collectively, these results suggest that YN-S2DR13 has significant advantages as a novel vaccine candidate, including a capacity for viral propagation to high titers with no trypsin requirement and the potential to provide protection against both PEDV G1 and G2 strains infections. Our results also suggests that S2 subunit replacement using reverse genetics can be a rapid strategy for the rational design of live attenuated vaccines for PEDV. IMPORTANCE: Emerging highly virulent porcine epidemic diarrhea virus (PEDV) G2 strains has caused substantial economic losses worldwide. Vaccination with a live attenuated vaccine is a promising method to prevent and control PED because it can induce a strong immune response (including T- and B-cell immunity). Previous studies have demonstrated that the S2 subunit of the PEDV spike (S) protein is the determinant of PEDV trypsin independence. Here, we evaluated the pathogenicity, tissue tropism, and immunogenicity of the chimeric virus (YN-S2DR13) via animal experiments. We demonstrated that YN-S2DR13 strain, as a trypsin independent strain, increased intracellular proliferation capacity, significantly reduced virulence, and induced broad-spectrum neutralization protection against PEDV G1 and G2 strains. In vitro passaging data also validated the stability of YN-S2DR13. Our results showed that generating a chimeric PEDV strain that is trypsin-independent by replacing the S2 subunit is a promising approach for designing a live attenuated vaccine for PEDV in the future.

Design of the conserved epitope peptide of SARS-CoV-2 spike protein as the broad-spectrum COVID-19 vaccine.

Our previous study has found that monoclonal antibodies targeting a conserved epitope peptide spanning from residues 1144 to 1156 of SARS-CoV-2 spike (S) protein, namely S(1144-1156), can broadly neutralize all of the prevalent SARS-CoV-2 strains, including the wild type, Alpha, Epsilon, Delta, and Gamma variants. In the study, S(1144-1156) was conjugated with bovine serum albumin (BSA) and formulated with Montanide ISA 51 adjuvant for inoculation in BALB/c mice to study its potential as a vaccine candidate. Results showed that the titers of S protein-specific IgGs and the neutralizing antibodies in mouse sera against various SARS-CoV-2 variants, including the Omicron sublineages, were largely induced along with three doses of immunization. The significant release of IFN-γ and IL-2 was also observed by ELISpot assays through stimulating vaccinated mouse splenocytes with the S(1144-1156) peptide. Furthermore, the vaccination of the S(1143-1157)- and S(1142-1158)-EGFP fusion proteins can elicit more SARS-CoV-2 neutralizing antibodies in mouse sera than the S(1144-1156)-EGFP fusion protein. Interestingly, the antisera collected from mice inoculated with the S(1144-1156) peptide vaccine exhibited better efficacy for neutralizing Omicron BA.2.86 and JN.1 subvariants than Omicron BA.1, BA.2, and XBB subvariants. Since the amino acid sequences of the S(1144-1156) are highly conserved among various SARS-CoV-2 variants, the immunogen containing the S(1144-1156) core epitope can be designed as a broadly effective COVID-19 vaccine. KEY POINTS: • Inoculation of mice with the S(1144-1156) peptide vaccine can induce bnAbs against various SARS-CoV-2 variants. • The S(1144-1156) peptide stimulated significant release of IFN-γ and IL-2 in vaccinated mouse splenocytes. • The S(1143-1157) and S(1142-1158) peptide vaccines can elicit more SARS-CoV-2 nAbs in mice.

Identification of a potent SARS-CoV-2 neutralizing nanobody targeting the receptor-binding domain of the spike protein.

SARS-CoV-2 and its variants continue to pose a significant threat to public health. Nanobodies (Nbs) that inhibit the interaction between the receptor-binding domain (RBD) of the spike protein and the host cell receptor angiotensin-converting enzyme 2 (ACE2) are promising drug candidates. In this study, we report the discovery and structural characterization of a potent Nb that targets the RBD. By screening a phage display alpaca naive Nbs library using the RBD as bait, we identified sixteen candidate Nbs. Of these, nine exhibited nanomolar to micromolar binding affinity and strong neutralizing activity against pseudotyped SARS-CoV-2 viruses, with NbS4 showing the highest neutralization potency. The crystal structure of the SARS-CoV-2 RBD in complex with NbS4 revealed that this Nb binds to a site partially overlapping the ACE2 binding region. Importantly, the key binding residues of NbS4 in the RBD are conserved across most known variants, making it a promising candidate for COVID-19 treatment.

SARS-CoV-2 Spike Protein Exacerbates Thromboembolic Cerebrovascular Complications in Humanized ACE2 Mouse Model.

COVID-19 increases the risk for acute ischemic stroke, yet the molecular mechanisms are unclear and remain unresolved medical challenges. We hypothesize that the SARS-CoV-2 spike protein exacerbates stroke and cerebrovascular complications by increasing coagulation and decreasing fibrinolysis by disrupting the renin-angiotensin-aldosterone system (RAAS). A thromboembolic model was induced in humanized ACE2 knock-in mice after one week of SARS-CoV-2 spike protein injection. hACE2 mice were treated with Losartan, an angiotensin receptor (AT1R) blocker, immediately after spike protein injection. Cerebral blood flow and infarct size were compared between groups. Vascular-contributes to cognitive impairments and dementia was assessed using a Novel object recognition test. Tissue factor-III and plasminogen activator inhibitor-1 were measured using immunoblotting to assess coagulation and fibrinolysis. Human brain microvascular endothelial cells (HBMEC) were exposed to hypoxia with/without SARS-CoV-2 spike protein to mimic ischemic conditions and assessed for inflammation, RAAS balance, coagulation, and fibrinolysis. Our results showed that the SARS-CoV-2 spike protein caused an imbalance in the RAAS that increased the inflammatory signal and decreased the RAAS protective arm. SARS-CoV-2 spike protein increased coagulation and decreased fibrinolysis when coincident with ischemic insult, which was accompanied by a decrease in cerebral blood flow, an increase in neuronal death, and a decline in cognitive function. Losartan treatment restored RAAS balance and reduced spike protein-induced effects. SARS-CoV-2 spike protein exacerbates inflammation and hypercoagulation, leading to increased neurovascular damage and cognitive dysfunction. However, the AT1R blocker, Losartan, restored the RAAS balance and reduced COVID-19-induced thromboembolic cerebrovascular complications.

Synthesis of Low-Molecular-Weight Fucoidan Analogue and Its Inhibitory Activities against Heparanase and SARS-CoV-2 Infection.

Heparan sulfate (HS) is ubiquitous on cell surfaces and is used as a receptor by many viruses including SARS-CoV-2. However, increased activity of the inflammatory enzyme heparanase (HPSE), which hydrolyses HS, in patients with COVID-19 not only increases the severity of symptoms but also may facilitate the spread of the virus by degrading HS on the cell surface. Therefore, synthetic HPSE blockades, which can bind to SARS-CoV-2 spike protein (SARS-CoV-2-S) and inhibit viral entry, have attracted much attention. This study investigated the development of a new dual-targeting antiviral agent against HPSE and SARS-CoV-2-S using fucoidan as a structural motif. It was found that all-sulfated fucoidan derivative 10, which exhibited the highest binding affinity to SARS-CoV-2-S among 13 derivatives, also showed the highest inhibitory activity against HPSE. Based on this, a newly designed and synthesized fucoidan analogue 16, in which the octyl group of 10 was changed to a cholestanyl group, was found to show higher activity than 10 but did not inhibit factor Xa associated with undesired anticoagulant effects. The binding affinity of 16 to SARS-CoV-2-S was significantly increased approximately 400-fold over that of 10. Furthermore, 16 effectively inhibited infection by the SARS-CoV-2 Wuhan strain and two Omicron subvariants.

The Y498T499-SARS-CoV-2 spike (S) protein interacts poorly with rat ACE2 and does not affect the rat lung.

The rat is a useful laboratory model for respiratory diseases. SARS-CoV-2 proteins, such as the spike (S) protein, can induce inflammation. This study has investigated the ability of the Q498Y, P499T (QP-YT) amino acid change, described in the S-protein of the mouse-adapted laboratory SARS-CoV-2 MA strain, to interact with rat angiotensin converting enzyme-2 (ACE2) and stimulate responses in rat lungs. A real-time S-ACE2 quantitative fusion assay shows that ancestral and L452R S-proteins fuse with human but not rat ACE2 expressed on HEK293 (human embryonic kidney-293) cells. The QP-YT S-protein retains the ability to fuse with human ACE2 and increases the binding to rat ACE2. Although lower lung of the rat contains both ACE2 and TMPRSS2 (transmembrane serine protease 2) target cells, intratracheal delivery of ancestral or QP-YT S-protein pseudotyped lentivirus did not induce measurable respiratory changes, inflammatory infiltration or innate mRNA responses. Isolation of primary cells from rat alveoli demonstrated the presence of cells expressing ACE2 and TMPRSS2. Infection of these cells, however, with ancestral or QP-YT S-protein pseudotyped lentivirus was not observed, and the QP-YT S-protein pseudotyped lentivirus poorly infected HEK293 cells expressing rat ACE2. Analysis of the amino acid changes across the S-ACE2 interface highlights not only the Y498 interaction with H353 as a likely facilitator of binding to rat ACE2 but also other amino acids that could improve this interaction. Thus, rat lungs contain cells expressing receptors for SARS-CoV-2, and the QP-YT S-protein variant can bind to rat ACE2, but this does not result in infection or stimulate responses in the lung. Further, amino acid changes in S-protein may enhance this interaction to improve the utility of the rat model for defining the role of the S-protein in driving lung inflammation.

Real-space heterogeneous reconstruction, refinement, and disentanglement of CryoEM conformational states with HetSIREN.

Single-particle analysis by Cryo-electron microscopy (CryoEM) provides direct access to the conformation of each macromolecule. However, the image's signal-to-noise ratio is low, and some form of classification is usually performed at the image processing level to allow structural modeling. Classical classification methods imply the existence of a discrete number of structural conformations. However, new heterogeneity algorithms introduce a novel reconstruction paradigm, where every state is represented by a lower number of particles, potentially just one, allowing the estimation of conformational landscapes representing the different structural states a biomolecule explores. In this work, we present a novel deep learning-based method called HetSIREN. HetSIREN can fully reconstruct or refine a CryoEM volume in real space based on the structural information summarized in a conformational latent space. The unique characteristics that set HetSIREN apart start with the definition of the approach as a real space-based only method, a fact that allows spatially focused analysis, but also the introduction of a novel network architecture specifically designed to make use of meta-sinusoidal activations, with proven high analytics capacities. Continuing with innovations, HetSIREN can also refine the pose parameters of the images at the same time that it conditions the network with prior information/constraints on the maps, such as Total Variation and L 1 denoising, ultimately yielding cleaner volumes with high-quality structural features. Finally, but very importantly, HetSIREN addresses one of the most confusing issues in heterogeneity analysis, as it is the fact that real structural heterogeneity estimation is entangled with pose estimation (and to a lesser extent with CTF estimation), in this way, HetSIREN introduces a novel encoding architecture able to decouple pose and CTF information from the conformational landscape, resulting in more accurate and interpretable conformational latent spaces. We present results on computer-simulated data, public data from EMPIAR, and data from experimental systems currently being studied in our laboratories. An important finding is the sensitivity of the structure and dynamics of the SARS-CoV-2 Spike protein on the storage temperature.

Discovery and characterization of a pan-betacoronavirus S2-binding antibody.

The continued emergence of deadly human coronaviruses from animal reservoirs highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq), we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryoelectron microscopy (cryo-EM) structure of 54043-5 bound to the prefusion S2 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses in vitro, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

AlphaFold2 Modeling and Molecular Dynamics Simulations of the Conformational Ensembles for the SARS-CoV-2 Spike Omicron JN.1, KP.2 and KP.3 Variants: Mutational Profiling of Binding Energetics Reveals Epistatic Drivers of the ACE2 Affinity and Escape Hotspots of Antibody Resistance.

The most recent wave of SARS-CoV-2 Omicron variants descending from BA.2 and BA.2.86 exhibited improved viral growth and fitness due to convergent evolution of functional hotspots. These hotspots operate in tandem to optimize both receptor binding for effective infection and immune evasion efficiency, thereby maintaining overall viral fitness. The lack of molecular details on structure, dynamics and binding energetics of the latest FLiRT and FLuQE variants with the ACE2 receptor and antibodies provides a considerable challenge that is explored in this study. We combined AlphaFold2-based atomistic predictions of structures and conformational ensembles of the SARS-CoV-2 spike complexes with the host receptor ACE2 for the most dominant Omicron variants JN.1, KP.1, KP.2 and KP.3 to examine the mechanisms underlying the role of convergent evolution hotspots in balancing ACE2 binding and antibody evasion. Using the ensemble-based mutational scanning of the spike protein residues and computations of binding affinities, we identified binding energy hotspots and characterized the molecular basis underlying epistatic couplings between convergent mutational hotspots. The results suggested the existence of epistatic interactions between convergent mutational sites at L455, F456, Q493 positions that protect and restore ACE2-binding affinity while conferring beneficial immune escape. To examine immune escape mechanisms, we performed structure-based mutational profiling of the spike protein binding with several classes of antibodies that displayed impaired neutralization against BA.2.86, JN.1, KP.2 and KP.3. The results confirmed the experimental data that JN.1, KP.2 and KP.3 harboring the L455S and F456L mutations can significantly impair the neutralizing activity of class 1 monoclonal antibodies, while the epistatic effects mediated by F456L can facilitate the subsequent convergence of Q493E changes to rescue ACE2 binding. Structural and energetic analysis provided a rationale to the experimental results showing that BD55-5840 and BD55-5514 antibodies that bind to different binding epitopes can retain neutralizing efficacy against all examined variants BA.2.86, JN.1, KP.2 and KP.3. The results support the notion that evolution of Omicron variants may favor emergence of lineages with beneficial combinations of mutations involving mediators of epistatic couplings that control balance of high ACE2 affinity and immune evasion.

Characterizations of angiotensin-converting enzyme-2 (ACE2) peptidase activity.

Angiotensin (Ang) II (1-8) is a potent vasoconstrictor known for its role in hypertension. Angiotensin-converting enzyme (ACE2) converts Ang II (1-8) to a vasodilator Ang (1-7) by removing the carboxy-terminal Phe. ACE2 more recently gained attention as the receptor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that caused the coronavirus disease 2019 (COVID-19) pandemic. Given the pathophysiological importance of ACE2, the present study examined the mechanism of ACE2 catalytic activity by comparing the ability of angiotensin molecules of various lengths to compete with the artificial fluorogenic substrate. The Fluorimetric SensoLyte 390 ACE2 Activity Assay uses an Mca/Dnp fluorescence resonance energy transfer peptide as the substrate. Results showed that the natural substrate Ang II (1-8) competed with the fluorogenic substrate, reducing the fluorescence signals. Deletion of C-terminal Phe resulted in the loss of the ability to compete with the artificial substrate, as shown by the actions of Ang (1-7), Ang (2-7), and Ang (5-7). By contrast, the loss of N-terminal Asp potentiated the ability to compete with the substrate as seen by the action of Ang III (2-8). However, the loss of two amino acids (Asp-Arg) from the N-terminus reduced the ability to compete with the substrate as observed by the actions of Ang IV (3-8) and Ang (5-8). Ang I (1-10) and Ang (1-9) did not strongly compete with the substrate. Interestingly, shorter peptides Ang (1-5) and Ang (1-4) potentiated the ACE2 activity. These results suggest that Ang II and Ang III are the best natural substrates for ACE2.

Emerging severe acute respiratory syndrome coronavirus 2 variants and their impact on immune evasion and vaccine-induced immunity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants harboring mutations in the structural protein, especially in the receptor binding domain (RBD) of spike protein, have raised concern about potential immune escape. The spike protein of SARS-CoV-2 plays a vital role in infection and is an important target for neutralizing antibodies. The mutations that occur in the structural proteins, especially in the spike protein, lead to changes in the virus attributes of transmissibility, an increase in disease severity, a notable reduction in neutralizing antibodies generated and thus a decreased response to vaccines and therapy. The observed multiple mutations in the RBD of the spike protein showed immune escape because it increases the affinity of spike protein binding with the ACE-2 receptor of host cells and increases resistance to neutralizing antibodies. Cytotoxic T-cell responses are crucial in controlling SARS-CoV-2 infections from the infected tissues and clearing them from circulation. Cytotoxic T cells efficiently recognized the infected cells and killed them by releasing soluble mediator's perforin and granzymes. However, the overwhelming response of T cells and, subsequently, the overproduction of inflammatory mediators during severe infections with SARS-CoV-2 may lead to poor outcomes. This review article summarizes the impact of mutations in the spike protein of SARS-CoV-2, especially mutations of RBD, on immunogenicity, immune escape and vaccine-induced immunity, which could contribute to future studies focusing on vaccine design and immunotherapy.

Development of feline infectious peritonitis diagnosis system by using CatBoost algorithm.

This study employed machine learning techniques to predict the rate of feline infectious peritonitis (FIP) diagnoses, with a specific focus on mutations in the spike protein gene of the feline coronavirus (FCoV). FIP is a fatal viral disease affecting the peritoneum of cats and is primarily caused by mutations in FCoV. Its diagnosis largely relies on evaluations of various biomarkers and clinical symptoms. The current analysis of FCoV spike protein gene mutations exhibits certain limitations. To address this problem, the present study employed a large dataset-comprising information on FCoV copy numbers, spike protein mutation outcomes, and related clinical data-and used machine learning models to analyze the association between spike protein gene mutations and FIP diagnosis. Various algorithms were used to establish highly accurate predictive models, namely logistic regression, random forest, decision tree, neural network, support vector machine, gradient boosting tree, and categorical boosting (CatBoost) algorithms. The model obtained using the CatBoost algorithm was discovered to have accuracy of 0.9541. Accordingly, a highly accurate predictive model was developed to enable early diagnosis of FIP and improve the rate of survival in cats. The application of machine learning technology in this study yielded research findings that provide veterinarians with effective tools for managing and preventing FIP, a painful and deadly disease for cats. This study is a pioneering work in the systematic application of multiple machine learning models to the prediction of FIP and comparison of performance results to improve diagnostic accuracy and efficiency. This study is the first of its kind in the field of FIP.

Plasmon-enhanced fluorescence and electrochemical aptasensor for SARS-CoV-2 Spike protein detection.

In this work, we combined plasmon-enhanced fluorescence and electrochemical (PEF-EC) transduction mechanisms to realize a highly sensitive dual-transducer aptasensor. To implement two traducers in one biosensor, a novel large-scale nanoimprint lithography process was introduced to fabricate gold nanopit arrays (AuNpA) with unique fringe structures. Light transmitting through the AuNpA samples exhibited a surface plasmon polariton peak overlapping with the excitation peak of the C7 aptamer-associated fluorophore methylene blue (MB). We observed a five and seven-times higher average fluorescence intensity over the AuNpA and fringe structure, respectively, in comparison to a plane Au film. Furthermore, the MB fluorophore was simultaneously utilized as a redox probe for electrochemical investigations and is described here as a dual transduction label for the first time. The novel dual transducer system was deployed for the detection of SARS-CoV-2 Spike protein via a C7 aptamer in combination with a strand displacement protocol. The PEF transducer exhibited a detection range from 1 fg/mL to 10 ng/mL with a detection limit of 0.07 fg/mL, while the EC traducer showed an extended dynamic range from 1 fg/mL to 100 ng/mL with a detection limit of 0.15 fg/mL. This work provides insights into an easy-to-perform, large-scale fabrication process for nanostructures enabling plasmon-enhanced fluorescence, and the development of an advanced but universal aptasensor platform.

Nanopore sequencing provides snapshots of the genetic variation within salmonid alphavirus-3 (SAV3) during an ongoing infection in Atlantic salmon (Salmo salar) and brown trout (Salmo trutta).

Frequent RNA virus mutations raise concerns about evolving virulent variants. The purpose of this study was to investigate genetic variation in salmonid alphavirus-3 (SAV3) over the course of an experimental infection in Atlantic salmon and brown trout. Atlantic salmon and brown trout parr were infected using a cohabitation challenge, and heart samples were collected for analysis of the SAV3 genome at 2-, 4- and 8-weeks post-challenge. PCR was used to amplify eight overlapping amplicons covering 98.8% of the SAV3 genome. The amplicons were subsequently sequenced using the Nanopore platform. Nanopore sequencing identified a multitude of single nucleotide variants (SNVs) and deletions. The variation was widespread across the SAV3 genome in samples from both species. Mostly, specific SNVs were observed in single fish at some sampling time points, but two relatively frequent (i.e., major) SNVs were observed in two out of four fish within the same experimental group. Two other, less frequent (i.e., minor) SNVs only showed an increase in frequency in brown trout. Nanopore reads were de novo clustered using a 99% sequence identity threshold. For each amplicon, a number of variant clusters were observed that were defined by relatively large deletions. Nonmetric multidimensional scaling analysis integrating the cluster data for eight amplicons indicated that late in infection, SAV3 genomes isolated from brown trout had greater variation than those from Atlantic salmon. The sequencing methods and bioinformatics pipeline presented in this study provide an approach to investigate the composition of genetic diversity during viral infections.

Ethanol Inhalation as a Method to Denature the Spike Protein of SARS-CoV-2.

The SARS-CoV-2 virus caused the 2019 COVID pandemic by infecting almost eight hundred million people worldwide. Because it was a new viral infection, there were no vaccines or small molecule medications that could prevent or treat the disease.  This chapter provides some details for an obscure treatment for COVID-19, that has decades of anti-viral activity data both in vitro and in vivo in the literature. The medicinal molecules are compared to other small molecules that were identified as possible medications for COVID-19.  We developed a computational method that ranks small molecules and their ability to penetrate mucus in the lungs of a COVID-19 patient. Our focus is ethanol as a COVID-19 treatment. The results discussed here are based on Lipinski Rules and QSAR computational methods as well as in vitro and in vivo data. These parameters indicate that ethanol should be a strong candidate for future evaluations.

Investigation of SARS-CoV-2 IgG Binding Capability to Variants of the SARS-CoV-2 Virus.

The SARS-CoV-2 pandemic has confirmed that the ability to rapidly mutate may be extremely beneficial for a virus. Not long after the first wave, new variants emerged with altered infectivity, disease severity, and mortality. These new strains most notably had numerous mutations of the spike (S) protein, a surface protein responsible for binding to and entering the host cell. The Delta and Omicron strains demonstrated increased immune evasion and improved binding affinity to the host cell receptor, angiotensin-converting enzyme 2 (ACE2). This study examines the ability of wild-type SARS-CoV-2 IgG to bind Delta and Omicron antigens, as well as their functional binding capabilities to two different S-ACE2 complexes. Twenty SARS-CoV-2 positive samples from patients who had recovered from infection with ancestral SARS-CoV-2 in the first wave of COVID-19 and 10 pre-pandemic control samples were studied. SARS-CoV-2 exposed patients showed significantly higher levels of IgG to SARS-CoV-2 S1/RBD (p < 0.001), N protein (p < 0.001), and Omicron spike variant (p = 0.01), but not to Delta spike variant (p = 0.966) when compared with controls. Furthermore, patient samples showed significantly greater inhibition of SARS-CoV-2 S1/RBD and E484K spike to ACE2 binding (p < 0.001 and p = 0.015, respectively). Conversely, there was no correlation between the binding inhibition of S1/RBD and E484K spike to ACE2 receptor. This study shows there is considerable cross-reactivity of IgG generated by wild-type SARS-CoV-2 infection to the Delta and Omicron variants.

The Inhibiting Effect of GB-2, (+)-Catechin, Theaflavin, and Theaflavin 3-Gallate on Interaction between ACE2 and SARS-CoV-2 EG.5.1 and HV.1 Variants.

The ongoing COVID-19 pandemic, caused by SARS-CoV-2, continues to pose significant global health challenges. The results demonstrated that GB-2 at 200 µg/mL effectively increased the population of 293T-ACE2 cells with low RBD binding for both SARS-CoV-2 Omicron EG.5.1 and HV.1 variants by dual-color flow cytometry, indicating its ability to inhibit virus attachment. Further investigation revealed that (+)-catechin at 25 and 50 µg/mL did not significantly alter the ACE2-RBD interaction for the EG.5.1 variant. In contrast, theaflavin showed inhibitory effects at both 25 and 50 µg/mL for EG.5.1, while only the higher concentration was effective for HV.1. Notably, theaflavin 3-gallate exhibited a potent inhibition of ACE2-RBD binding for both variants at both concentrations tested. Molecular docking studies provided insight into the binding mechanisms of theaflavin and theaflavin 3-gallate with the RBD of EG.5.1 and HV.1 variants. Both compounds showed favorable docking scores, with theaflavin 3-gallate demonstrating slightly lower scores (-8 kcal/mol) compared to theaflavin (-7 kcal/mol) for both variants. These results suggest stable interactions between the compounds and key residues in the RBD, potentially explaining their inhibitory effects on virus attachment. In conclusion, GB-2, theaflavin, and theaflavin 3-gallate demonstrate significant potential as inhibitors of the ACE2-RBD interaction in Omicron variants, highlighting their therapeutic promise against COVID-19. However, these findings are primarily based on computational and in vitro studies, necessitating further in vivo research and clinical trials to confirm their efficacy and safety in humans.