Viral Spike Research Articles

#1902 Remdesivir ameliorates ferroptotic renal injury in hamster after SARS-CoV-2 infection

Abstract Background and Aims The purpose of this study was to see how remdesivir affected renal ferroptotic damage in hamsters following a human Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection. We evaluated the inhibitory effects of remdesivir on the ferroptosis of SARS-CoV-2 infection in a hamster infection model and in in vivo assays. Method To investigate the effect of SARS-CoV-2 on renal ferroptosis, we administered 2.5 × 104 p.f.u. SARS-CoV-2 via intranasal to hamster. In the initial study, animals were treated with remdesivir subcutaneously (s.c.) for 7 days beginning at various times in relation to virus challenge. After 5 days, kidney tissues were harvested. The effect of REMDESIVIR on SARS-CoV-2 infection and ferroptosis were evaluated by immunohistochemistry. Next, ferroptosis-related markers, glutathione peroxidase 4 (Gpx4), 4-hydroxynonenal (4-HNE), Acyl-CoA synthetase long chain family member 4 (Acsl4), nuclear factor erythroid-2-related factor 2 (Nrf2), heme oxygenase-1 (Ho-1) were evaluated by quantitative PCR. Results In hamsters, SARS-CoV-2 resulted in high viral protein expression in the renal tubules and decreased renal tubular luminal diameter in periodic acid-Schiff (PAS) stained kidney sections. Remdesivir treatment decreased SARS-CoV-2-induced viral spike protein, HO-1, ferritin heavy chain1 (FTH1), ferritin light chain (FTL), and 4-HNE expression in renal tubular epithelial cells of hamsters in a dose-dependent manner. GPX4 expression suppression after infection was ameliorated by remdesivir administration. Increased ferroptosis-related mRNA marker (Gpx4, 4-HNE, Acsl4, Nrf2, Ho-1) expression in hamster kidneys was suppressed by remdesivir treatment. Conclusion All of our data suggest that remdesivir treatment ameliorated SARS-CoV-2 infection-induced ferroptosis in the kidney in a hamster model.

Display of porcine epidemic diarrhea virus spike protein B-cell linear epitope on Lactobacillus mucosae G01 S-layer surface induce a robust immunogenicity in mice

The Porcine epidemic diarrhea virus (PEDV) presents a substantial risk to the domestic pig industry, resulting in extensive and fatal viral diarrhea among piglets. Recognizing the mucosal stimulation triggered by PEDV and harnessing the regulatory impact of lactobacilli on intestinal function, we have developed a lactobacillus-based vaccine that is carefully designed to elicit a strong mucosal immune response. Through bioinformatics analysis, we examined PEDV S proteins to identify B-cell linear epitopes that meet the criteria of being non-toxic, soluble, antigenic, and capable of neutralizing the virus. In this study, a genetically modified strain of Lactobacillus mucosae G01 (L.mucosae G01) was created by utilizing the S layer protein (SLP) as a scaffold for surface presentation. Chimeric immunodominant epitopes with neutralizing activity were incorporated at various sites on SLP. The successful expression of SLP chimeric immunodominant epitope 1 on the surface of L.mucosae G01 was confirmed through indirect immunofluorescence and transmission electron microscopy, revealing the formation of a transparent membrane. The findings demonstrate that the oral administration of L.mucosae G01, which expresses the SLP chimeric immunodominant gene epitope1, induces the production of secreted IgA in the intestine and feces of mice. Additionally, there is an elevation in IgG levels in the serum. Moreover, the levels of cytokines IL-2, IL-4, IFN-γ, and IL-17 are significantly increased compared to the negative control group. These results suggest that L. mucosae G01 has the ability to deliver exogenous antigens and elicit a specific mucosal immune response against PEDV. This investigation presents new possibilities for immunoprophylaxis against PEDV-induced diarrhea.

Multi-epitope vaccine against SARS-CoV-2 targeting the spike RBD: an immunoinformatics approach.

Aim: We designed a SARS-CoV-2 epitope vaccine based on the receptor-binding domain (RBD) in virus spike protein. Methods: RT-PCR performed on nasopharyngeal swab COVID-19 patients. After registering RBD region in the GenBank, physicochemical parameters, secondary structure, homology modeling, 3D structure of RBD region and antigenicity were determined using ProtParam ExPASy, PSIPRED, MolProbity, IEDB and Vaxijen online tools, respectively. Results: B and T cell epitopes were predicted in terms of non-allergenicity and antigenicity. MolProbity analysis provided a qualitative model for RBD. The homology model showed that most of the residues are in optimal district of energy. Conclusion: High immunogenicity score of epitopes indicates promising candidates for the development of multi-epitope vaccines. It may help to develop an effective vaccine.

SARS-CoV-2 ORF3a drives dynamic dense body formation for optimal viral infectivity.

SARS-CoV-2 uses the double-membrane vesicles as replication organelles. However, how virion assembly occurs has not been fully understood. Here we identified a SARS-CoV-2-driven membrane structure named the 3a dense body (3DB). 3DBs have unusual electron-dense and dynamic inner structures, and their formation is driven by the accessory protein ORF3a via hijacking a specific subset of the trans-Golgi network (TGN) and early endosomal membranes. 3DB formation is conserved in related bat and pangolin coronaviruses yet lost during the evolution to SARS-CoV. 3DBs recruit the viral structural proteins spike (S) and membrane (M) and undergo dynamic fusion/fission to facilitate efficient virion assembly. A recombinant SARS-CoV-2 virus with an ORF3a mutant specifically defective in 3DB formation showed dramatically reduced infectivity for both extracellular and cell-associated virions. Our study uncovers the crucial role of 3DB in optimal SARS-CoV-2 infectivity and highlights its potential as a target for COVID-19 prophylactics and therapeutics.

Non-cross-reactive epitopes dominate the humoral immune response to COVID-19 vaccination - kinetics of plasma antibodies, plasmablasts and memory B cells.

COVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our knowledge of the underlying plasmablast and memory B cell (Bmem) responses is still incomplete. Here, we determined the antibody and B cell response to COVID-19 vaccination in a naïve population and contrasted it with the response to a single influenza vaccination in a primed cohort. In addition, we analyzed the antibody and B cell responses against the four endemic human coronaviruses (HCoVs). Measurement of specific plasma IgG antibodies was combined with functional analyses of antibody-secreting plasmablasts and Bmems. SARS-CoV-2- and HCoV-specific IgG antibodies were quantified with an in-house bead-based multiplexed immunoassay. The antibody and B cell responses to COVID-19 vaccination reflected the kinetics of a prime-boost immunization, characterized by a slow and moderate primary response and a faster and stronger secondary response. In contrast, the influenza vaccinees possessed robust immune memory for the vaccine antigens prior to vaccination, and the recall vaccination moderately boosted antibody production and Bmem responses. Antibody levels and Bmem responses waned several months after the 2nd COVID-19 vaccination, but were restored upon the 3rd vaccination. The COVID-19 vaccine-induced antibodies mainly targeted novel, non-cross-reactive S1 epitopes of the viral spike protein, while cross-reactive S2 epitopes were less immunogenic. Booster vaccination not only strongly enhanced neutralizing antibodies against an original SARS-CoV-2 strain, but also induced neutralizing antibodies against the Omicron BA.2 variant. We observed a 100% plasma antibody prevalence against the S1 subunits of HCoVs, which was not affected by vaccination. Overall, by complementing classical serology with a functional evaluation of plasmablasts and memory B cells we provide new insights into the specificity of COVID-19 vaccine-induced antibody and B cell responses.

In-vitro studies of relative intracellular alterations caused by three variants of Severe Acute Respiratory Syndrome Corona Virus-2 (SARS-CoV-2)

BACKGROUND: The comparative understanding between cellular basis of transmission and clinical severities caused by Wuhan, Delta and Omicron strains of SARS-CoV-2 is important. OBJECTIVE: To conduct In-Vitro studies on cellular infection caused by different variants by performing cytotoxic assays and studying the live microscopic observations. METHODS: The swab samples positive for Wuhan, Delta and Omicron strains were inoculated in the Vero cell lines and their rate of infectivity and nature of cytopathy was observed. RESULTS: The study showed maximum inter cellular connectivity among cells infected by Delta variant, followed by Wuhan and least inter cellular connections in the cells infected by Omicron variant. The Delta strain causing highest mortality as per reported epidemiological trends, showed highest inter cellular connections and maximum cell damage supporting the fastest cell to cell spread of infection and causing most mortality among patients. CONCLUSIONS: The paper reports that numerous inter cellular connections is the cause of spread of infection among cells and maximum cell damage as the cause of clinical severities. The paper sensitizes the issue that 52 subunit of viral Spike Protein could act as fusion enzyme causing the numerous inter cellular connections. Study suggests that the S2 subunit of the viral Spike Protein could be the drug target for therapeutic intervention.

MPoMA protects against lung epithelial cell injury via p65 degradation

Numerous cases of lung injury caused by viral infection were reported during the coronavirus disease-19 pandemic. While there have been significant efforts to develop drugs that block viral infection and spread, the development of drugs to reduce or reverse lung injury has been a lower priority. This study aimed to identify compounds from a library of compounds that prevent viral infection that could reduce and prevent lung epithelial cell damage. We investigated the cytotoxicity of the compounds, their activity in inhibiting viral spike protein binding to cells, and their activity in reducing IL-8 production in lung epithelial cells damaged by amodiaquine (AQ). We identified N-(4-(4-methoxyphenoxy)-3-methylphenyl)-N-methylacetamide (MPoMA) as a non-cytotoxic inhibitor against viral infection and AQ-induced cell damage. MPoMA inhibited the expression of IL-8, IL-6, IL-1β, and fibronectin induced by AQ and protected against AQ-induced morphological changes. However, MPoMA did not affect basal IL-8 expression in lung epithelial cells in the absence of AQ. Further mechanistic analysis confirmed that MPoMA selectively promoted the proteasomal degradation of inflammatory mediator p65, thereby reducing intracellular p65 expression and p65-mediated inflammatory responses. MPoMA exerted potent anti-inflammatory and protective functions in epithelial cells against LPS-induced acute lung injury in vivo. These findings suggest that MPoMA may have beneficial effects in suppressing viral infection and preventing lung epithelial cell damage through the degradation of p65 and inhibition of the production of inflammatory cytokines.

Ligand- and Structure-Based Virtual Screening Identifies New Inhibitors of the Interaction of the SARS-CoV-2 Spike Protein with the ACE2 Host Receptor.

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.

Inhibition Mechanism of SARS-CoV-2 Infection by a Cholesterol Derivative, Nat-20(S)-yne.

The coronavirus disease 2019 (COVID-19) is caused by the etiological agent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). COVID-19, with the recurrent epidemics of new variants of SARS-CoV-2, remains a global public health problem, and new antivirals are still required. Some cholesterol derivatives, such as 25-hydroxycholesterol, are known to have antiviral activity against a wide range of enveloped and non-enveloped viruses, including SARS-CoV-2. At the entry step of SARS-CoV-2 infection, the viral envelope fuses with the host membrane dependent of viral spike (S) glycoproteins. From the screening of cholesterol derivatives, we found a new compound 26,27-dinorcholest-5-en-24-yne-3β,20-diol (Nat-20(S)-yne) that inhibited the SARS-CoV-2 S protein-dependent membrane fusion in a syncytium formation assay. Nat-20(S)-yne exhibited the inhibitory activities of SARS-CoV-2 pseudovirus entry and intact SARS-CoV-2 infection in a dose-dependent manner. Among the variants of SARS-CoV-2, inhibition of infection by Nat-20(S)-yne was stronger in delta and Wuhan strains, which predominantly invade into cells via fusion at the plasma membrane, than in omicron strains. The interaction between receptor-binding domain of S proteins and host receptor ACE2 was not affected by Nat-20(S)-yne. Unlike 25-hydroxycholesterol, which regulates various steps of cholesterol metabolism, Nat-20(S)-yne inhibited only de novo cholesterol biosynthesis. As a result, plasma membrane cholesterol content was substantially decreased in Nat-20(S)-yne-treated cells, leading to inhibition of SARS-CoV-2 infection. Nat-20(S)-yne having a new mechanism of action may be a potential therapeutic candidate for COVID-19.

Development of a Cell Culture Model for Inducible SARS-CoV-2 Replication.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) induces direct cytopathic effects, complicating the establishment of low-cytotoxicity cell culture models for studying its replication. We initially developed a DNA vector-based replicon system utilizing the CMV promoter to generate a recombinant viral genome bearing reporter genes. However, this system frequently resulted in drug resistance and cytotoxicity, impeding model establishment. Herein, we present a novel cell culture model with SARS-CoV-2 replication induced by Cre/LoxP-mediated DNA recombination. An engineered SARS-CoV-2 transcription unit was subcloned into a bacterial artificial chromosome (BAC) vector. To enhance biosafety, the viral spike protein gene was deleted, and the nucleocapsid gene was replaced with a reporter gene. An exogenous sequence was inserted within NSP1 as a modulatory cassette that is removable after Cre/LoxP-mediated DNA recombination and subsequent RNA splicing. Using the PiggyBac transposon strategy, the transcription unit was integrated into host cell chromatin, yielding a stable cell line capable of inducing recombinant SARS-CoV-2 RNA replication. The model exhibited sensitivity to the potential antivirals forsythoside A and verteporfin. An innovative inducible SARS-CoV-2 replicon cell model was introduced to further explore the replication and pathogenesis of the virus and facilitate screening and assessment of anti-SARS-CoV-2 therapeutics.

SARS-CoV-2 strains bearing Omicron BA.1 spike replicate in C57BL/6 mice.

SARS-CoV-2, the cause of the COVID pandemic, is an RNA virus with a high propensity to mutate. Successive virus variants, including variants of concern (VOC), have emerged with increased transmission or immune escape. The original pandemic virus and early variants replicated poorly, if at all, in mice at least partly due to a mismatch between the receptor binding domain on the viral spike protein and the murine angiotensin converting enzyme 2 (ACE2). Omicron VOC emerged in late 2021 harboring > 50 new mutations, 35 of them in the spike protein. This variant resulted in a very large wave of infections, even in the face of prior immunity, albeit being inherently less severe than earlier variants. Reflecting the lower severity reported in humans, Omicron displayed attenuated infection in hamsters and also in the K18-hACE2 mouse model. K18-hACE2 mice express both the human ACE2 as well as the endogenous mouse ACE2. Here we infected hACE2 knock-in mice that express only human ACE2 and no murine ACE2, or C57BL/6 wildtype mice with SARS-CoV-2 D614G (first-wave isolate), Delta or Omicron BA.1 variants and assessed infectivity and downstream innate immune responses. While replication of SARS-CoV-2 Omicron was lower in the lungs of hACE2 knock-in mice compared with SARS-CoV-2 D614G and VOC Delta, it replicated more efficiently than the earlier variants in C57BL/6 wildtype mice. This opens the opportunity to test the effect of host genetics on SARS-CoV-2 infections in wildtype mice. As a proof of principle, we tested Omicron infection in mice lacking expression of the interferon-alpha receptor-1 (IFNAR1). In these mice we found that loss of type I IFN receptor signaling resulted in higher viral loads in the lungs were detected. Finally, using a chimeric virus of first wave SARS-CoV-2 harboring the Omicron spike protein, we show that Omicron spike increase infection of C57BL/6 wildtype mice, but non-spike genes of Omicron confer attenuation of viral replication. Since this chimeric virus efficiently infected C57BL/6 wildtype mice, and replicated in their lungs, our findings illustrate a pathway for genetic mapping of virushost interactions during SARS-CoV-2 infection.

JN.1 Variant Alert: Pakistan's Precarious Healthcare System under Intense Scrutiny

The recent COVID-19 variant, JN 1, identified by the World Health Organization, is considered a notable strain stemming from the OMICRON subvariant BA.2.86. Despite its unique spike protein mutation (S:L455S), JN.1 is anticipated to have minimal impact on global public health. The singular modification in the virus's spike protein, distinguishing JN.1 from BA.2.86, suggests that existing vaccines should effectively counter both variants. The World Health Organization emphasizes the continued efficacy of current vaccines in protecting against diseases and deaths caused by JN.1, urging individuals, especially those at high risk, to maintain updated vaccinations

Study on the transient expression of infectious bronchitis virus spike protein in Nicotiana benthamiana leaves

Infectious bronchitis virus (IBV) is considered as one of the main causes of economic loss in chicken farms worldwide, especially in poultry producing nations. This virus, a member of the Coronaviridae family, is classified into different genotypes based on its surface spike glycoproteins which also play important roles in cell attachment and immune response. Due to continual genetic mutation of IBV, attenuated and inactivated vaccines show decreased effectiveness or lack of cross – protection; thus ongoing studies focus on the development of new generation vaccines to prevent new IBV outbreaks. This paper describes our study on expression of the antigenic region in S1 subunit of the spike in Nicotiana benthamiana. Two expression vectors carrying either S1 or receptor binding domain (RBD) coding gene (pCB301-S1 and pCB301-RBD) were constructed and transformed into tobacco leaves by agroinfiltration method. The RBD showed a clearly higher level of expression compared to the whole S1. Purification of RBD by immobilized metal ion chromatography and size exclusion chromatography represented a mixture of monomer, dimer and trimer glycoprotein with expected sizes in Western blot. In summary, this study demonstrated our primary success in establishing an expression model that could be used to investigate plant-based IBV recombinant vaccine.

Utilizing noncatalytic ACE2 protein mutant as a competitive inhibitor to treat SARS-CoV-2 infection.

Angiotensin converting-enzyme 2 (ACE2) is an enzyme catalyzing the conversion of angiotensin 2 into angiotensin 1-7. ACE2 also serves as the receptor of several coronaviruses, including SARS-CoV-1 and SARS-CoV-2. Therefore, ACE2 could be utilized as a therapeutic target for treating these coronaviruses, ideally lacking enzymatic function. Based on structural analysis, specific mutations were introduced to generate mutants of ACE2 and ACE2-Fc (fusion protein of ACE2 and Fc region of IgG1). The enzyme activity, binding affinity, and neutralization abilities were measured. As predicted, five mutants (AMI081, AMI082, AMI083, AMI084, AMI090) have completely depleted ACE2 enzymatic activities. More importantly, enzyme-linked receptor-ligand assay (ELRLA) and surface plasmon resonance (SPR) results showed that 2 mutants (AMI082, AMI090) maintained binding activity to the viral spike proteins of SARS-CoV-1 and SARS-CoV-2. In An in vitro neutralization experiment using a pseudovirus, SARS-CoV-2 S1 spike protein-packed lentivirus particles, was also performed, showing that AMI082 and AMI090 significantly reduced GFP transgene expression. Further, in vitro virulent neutralization assays using SARS-CoV-2 (strain name: USA-WA1/2020) showed that AMI082 and AMI090 had remarkable inhibitory effects, indicated by comparable IC50 to wildtype ACE2 (5.33 µg/mL). In addition to the direct administration of mutant proteins, an alternative strategy for treating COVID-19 is through AAV delivery to achieve long-lasting effects. Therefore, AAV5 encoding AMI082 and AMI090 were packaged and transgene expression was assessed. In summary, these ACE2 mutants represent a novel approach to prevent or treat COVID-19 and other viruses with the same spike protein.

Role of Furin Activation Sites as Receptors for Invasion of Severe Acute Respiratory Syndrome Coronavirus-2 Into Human Cells

Objective: The severe acute type of respiratory distress caused by Coronavirus disease 2019 (COVID-19) was responsible for the global pandemic of 2019. While most of the focus of vaccine/drug molecules is on the receptor, there are certain enzymes that also need to be checked. Cell surface proteases are one of these. Activation of the virus spike protein becomes more complicated when many host proteases are involved. As many Variants of Concerns have been reported in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), this study aimed to understand the proteolytic function of Furin in each, and its involvement in virus-host interaction. Material and Methods: Spike Protein sequence alignment, furin cleavage site prediction of variants: Wuhan, Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2) and Omicron (B.1.1.529), and protein-protein docking studies have been undertaken using appropriate bioinformatics tools. Results: It was observed that when compared to previous variations, the November 2021, outbreak of Omicron variant showed 50 amino acid substitutions in the Spike protein. Thus, in addition to the Angiotensin Converting Enzyme 2 (ACE-2) receptor, the role of virus binding sites to act as “Addition Receptors” for viral entry has been reported here. Conclusion: It was observed that substitution of basic amino acids in the Omicron variant may be responsible for the recognition of furin cleavage sites and the presence of furin cleavage site in the receptor binding domain (RBD) region will thus enhance viral transmission. If these sites are utilized in formulation of new drugs/vaccine molecules to target the furin hydrolyse sites, we may be able to add to the existing course of COVID-19 treatment.

COVID 19- Biochemical and Other Mechanisms associated with Neuropathy and Stroke.

The Coronavirus (SARS-C0V-2) caused COVID 19 disease, commencing from China in December 2019 and soon in 2020 its neurological manifestations were noted. The viral spike (S) protein binding with host cell receptor angiotensin-converting enzyme 2 (ACE2) with assistance of membrane fusion protease (TMPRSS) caused viral entry in host cells. We searched 47 articles using PubMed from January 2021 to January 2023. The initial pooling of virus in nasal mucosa was followed by spread to respiratory and other tissues. This review highlights the neuropathy and stroke mechanisms. The role of receptors, enzymes, proteins and genes in neuropathological mechanisms are highlighted. Vascular endothelial effects, choroid plexus and Blood Brain Barrier disruptions were noted. The epigenetic neuropathological mechanisms need to be explored further to help in designing new therapeutic modalities. The post vaccination neurological manifestations as well as the post COVID effects both need further attention to promptly diagnose and start timely treatment.

SARS-CoV-2 and Angiotensin-Converting Enzyme-2 Receptor Interaction Blocker– an In-Silico Approach

The global COVID-19 pandemic, instigated by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), has led to substantial morbidity and mortality on a worldwide scale. While COVID-19 vaccines offer hope, the emergence of mutated viral strains necessitates the development of FDA-approved drugs to address future outbreaks. Objective: To examine prospective antiviral medications through an analysis of the interaction between the spike protein of SARS-CoV-2 and Angiotensin-converting enzyme-2 (ACE-2) receptors, which play a pivotal role in facilitating viral entry into host cells. Methods: Molecular docking was employed to assess the binding affinities of various protease inhibitors with ACE-2 receptors. Natural proteases, including Furin and Transmembrane serine protease 2 (TMPRSS2), cleave viral spike proteins into S1 and S2 subunits, facilitating fusion with ACE-2 receptors. We assessed the binding energies of Indinavir, Nafamostat, Fosamprenavir, Lopinavir, and Boceprevir to inhibit this interaction with a sense of optimism for their potential therapeutic applications. Results: Our findings suggest potential treatments for COVID-19, with Indinavir, Nafamostat, Fosamprenavir, Lopinavir, and Boceprevir displaying promising binding energies of -9.6 kcal/mol, -8.4 kcal/mol, -7.7 kcal/mol, and -7.5 kcal/mol, respectively. Conclusions: While promising, further clinical trials are important to potentially evaluate the efficacy and safety of these proposed drugs in combating COVID-19 and its variants.

Temporal analysis and comparative genomic study of SARS-CoV-2 variants across Uttar Pradesh, India: Insights from the first and second COVID-19 waves

The rapid emergence of genetic variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) after late 2020, lead to origin of the term “variant of concern” and became the prime issue for the health of the human populations across the globe. Genome surveillance provides the information about the functional consequences of the variants and their divergence. Presently, we report and compare the nucleotide sequences of the isolated SARS-CoV-2 genomes at two different time-frame i.e. first wave (June- September, 2020) and second wave (April-May, 2021) from Lucknow, Uttar Pradesh, India. We employed the Oxford Nanopore MINION system and ARTIC protocol for sequencing, achieving >90x genome coverage in approximately 90% of samples. Increased mutation density in viral spike proteins were the cause of higher transmission and infectivity of second wave. The second wave exhibited a higher mutation density in viral spike proteins, likely contributing to increased transmission and infectivity. Notable spike mutations included receptor-binding domain (RBD) mutations (L452R, T478K, D614G), N-terminal domain (NTD) mutations (T17R, G142D, Δ156–157, A222V), and furin cleavage site (FCS) mutations (P681R, D950N). The functional consequences of common and unique variants among the first and second wave were evaluated by Sorting Intolerant from Tolerant (SIFT) and Protein Variant Effect Analyser (PROVEAN). Furthermore, we extensively investigated phylogenetic clustering of the genome sequences by Nextstrain and identified the contrasting divergence between first and second wave. Different sub division of delta clade (21I, 21 A, 21 J) and 21 B (kappa) with mutations primarily in spike proteins and nucleocapsid proteins, were exclusive to the second wave. Our study identified changes in spike proteins and nucleocapsid potentially contributing to increased transmission and infectivity during the second wave. Loci specific variant map for different countries across the world suggested the appearance of large number of variants associated with ORF1a during the second wave in India. Overall, the data underscores the dynamic nature of viral evolution during different phases of an outbreak, highlighting the importance of continuous genomic surveillance to track and understand viral mutations and their implications for public health interventions.

Investigation of some plant stilbenoids and their fragments for the identification of inhibitors of SARS-CoV-2 viral spike/ACE2 protein binding

In silico binding studies were conducted on the known plant-derived polyphenolic tetrameric stilbenoids, (–)-hopeaphenol (1), vaticanol B (2) and vatalbinoside A (3) and their monomeric derivative resveratrol (8), identified from several plant species. The natural products (NPs) 1–3 had been previously evaluated against the SARS-CoV-2 protein targets responsible for viral transmission and infection. The two isomeric compounds (–)-hopeaphenol and vaticanol B had displayed a high affinity for blocking the binding of the SARS-CoV-2 viral spike with the human angiotensin-converting enzyme 2 (ACE2). Molecular docking and molecular dynamics simulations have been used to attempt to explain the affinity of the compounds to the spike/ACE2 complex. The hydrophobic properties of the respective target sites were computed and compared with the physicochemical properties of the NPs to explain the affinity of each compound toward the target site. Molecular docking and computed ADMET/DMPK profiles were also used to demonstrate the potential of the NPs and their fragments as possible lead compounds for antiviral discovery. These results support the experimental data obtained to date on 1–3 and clearly identify the stilbenoid structure class as one worthy of future studies during chemical biology and/or drug discovery antiviral efforts.

Intranasal delivery of PEA-producing Lactobacillus paracasei F19 alleviates SARS-CoV-2 spike protein-induced lung injury in mice

BackgroundSARS-CoV-2 belongs to the coronaviridae family and infects human cells by directly interacting with the angiotensin-converting enzyme-2 (ACE-2) through the viral Spike Protein (SP). While vaccines are crucial, much attention has been directed towards managing the symptoms of acute respiratory distress syndrome. Our present study highlights the potential in counteracting lung inflammation triggered by SARS-CoV-2 SP of the intranasal administration of the engineered probiotic Lactobacillus paracasei F19 expressing the enzyme NAPE-PLD (pNAPE-LP) able to in situ release palmitoylethanolamide (PEA) under a super-low boost of palmitate.MethodsC57BL/6J mice undergo prophylactic treatment with intranasal pNAPE-LP/palmitate for 7 days before a 7 days challenge with intranasal SARS-CoV-2 SP. Then the capability of pNAPE-LP of colonizing the lungs and actively release PEA in situ have been determined by immunofluorescence, western blot and HPLC-MS. Moreover, the innate immune system downregulation and the histological damage rescue exerted by pNAPE-LP have been tested by immunofluorescence, hematoxylin and eosin staining, western blot analysis and ELISA test for the release of the pro-inflammatory mediators.ResultspNAPE-LP effectively colonizes mice lungs and releases the anti-inflammatory compound PEA. Moreover, pNAPE-LP exhibits a protective effect on alveolar morphology, innate immune cells infiltration and in the reduction of neutrophil count, effectively reducing lung injury induced by SARS-CoV-2 SP. This is achieved by mitigating TLR4-mediated NLRP3 activation and the downstream pro-inflammatory products such as ILs, TNFα, C-reactive protein and the myeloperoxidase activity. Interestingly we observed a global reduction ACE2 expression in the lungs.ConclusionpNAPE-LP actively protect from severe inflammatory-related symptoms in SP-challenged mice. Also, it can downregulate the expression of ACE-2 receptors at the lung site potentially preventing the spreading of the infection.