Vero Cells Research Articles

Design, synthesis, and biological evaluation of novel 2'-deoxy-2'-spirooxetane-7-deazapurine nucleoside analogs as anti-SARS-CoV-2 agents.

The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused an unprecedented global public health crisis and continues to pose grave threats to human health. The efficacy of current vaccines and therapeutics is likely limited for future emerging strains due to the highly mutative nature of the virus, underscoring an urgent need for the development of new, potent antiviral agents. In this study, we report the design and synthesis of a series of novel 2'-deoxy-2'-spirooxetane-7-deazapurine nucleoside analogs as potential inhibitors of SARS-CoV-2 replication. Some of these compounds demonstrate potent antiviral activity, offering a potential new weapon for therapeutic intervention against the ever-evolving SARS-CoV-2 virus. Among the tested compounds, nucleoside analog 11q exhibited the most potent antiviral activity against SARS-CoV-2 in Vero E6 cells, with IC50 values of 0.14 μM for the wild-type strain and 0.36 μM for the BA.5 strain. Notably, compound 11q exhibits up to nine times greater inhibitory activity against wild-type SARS-CoV-2 compared to Remdesivir and also possesses a superior selectivity index. These findings suggest that compound 11q is a highly promising lead candidate for future drug development aimed at combating SARS-CoV-2.

Diketopiperazine/piperidine alkaloid as a potential broad-spectrum coronaviral entry inhibitor identified by supercomputer-based virtual screening from a large natural product-based library.

The COVID-19 pandemic has underscored the urgent need for antiviral agents capable of targeting a broad range of coronaviruses, including emerging variants of SARS-CoV-2. While vaccines have been pivotal, the search for drugs that can prevent viral entry into host cells remains crucial, especially against evolving viral forms and other coronaviruses. In this study, we investigated natural products as a source of antiviral agents, focusing on their potential to block the spike protein's receptor-binding domain (RBD). Utilizing a library of over 210,000 natural product-based compounds from the ZINC database, we employed a Snakemake workflow to screen for inhibitors against RBDs of SARS-CoV-2, its variants, SARS-CoV, and MERS-CoV. Among top N-heterocyclic candidates from virtual screening we found that one compound, i.e., ((2 R,8S)-6-(1-benzylpiperidin-4-yl)-2-naphthalen-1-yl-3,6,17-triazatetracyclo[8.7.0.03,8.011,16]heptadeca-1(10),11,13,15 tetraene-4,7-dione), inhibited SARS-CoV-2 pseudovirus and live virus entry in HEK-ACE2 and Vero E6 host cells at low micromolar IC50 values. Cell viability assays showed that this compound exerted low cytotoxicity towards HEK-ACE2 while it was not toxic against Vero E6 and MRC5 cell lines. Microscale thermophoresis revealed that this compound strongly bound to the RBDs of SARS-CoV-2, SARS-CoV-2 XBB, SARS-CoV, MERS-CoV, and HCoV-HKU1, with their Kd values increasing as sequence similarity decreased. Molecular docking studies indicated this active compound binds to the SARS-CoV-2 spike protein RBD and interacts with hotspot amino acid residues required for the RBD-ACE2 interaction and cellular infection. These findings show that this diketopiperazine/piperidine-type alkaloid can be considered for further development as a potential pan-coronavirus entry inhibitor.

Small Interference RNA Encapsulated in Liposomes: An Effective Strategy for In vitro Inhibition of SARS-CoV-2 Load

Background: The pressing need for effective SARS-CoV-2 antiviral medicines has driven research into innovative therapeutic techniques. RNA interference with small interfering RNAs (siRNAs) has shown promise as an antiviral treatment. Objective: We evaluated the effectiveness of lipid-based nanoparticles as a viable delivery platform for siRNA-based approach against SARS-CoV-2 in vitro infection. Methods: Liposomes were fabricated by microfluidics to incorporate SARS-CoV-2-specific siRNAs based on conserved sections of the Spike protein coding sequence. Nanoparticle tracking analysis was used to evaluate the nanoparticles' physicochemical features. VERO cell lines infected with SARS-CoV-2 were used to test the efficiency of siRNA-loaded liposomes. RT-PCR was used to determine the viral load by quantifying the SARS-CoV-2 genome. Results: The results showed that liposomes efficiently decreased viral load in infected cells with good physicochemical features, such as a mean particle size of about 180 nm, zeta potential of +2.5 mV and encapsulation efficiency (53.6%). Conclusion: These findings imply that lipid-based nanoparticles might be a targeted delivery strategy for siRNA-based approaches.

Antifibrotic potential of reserpine (alkaloid) targeting Keap1/Nrf2; oxidative stress pathway in CCl4-induced liver fibrosis.

The death rate due to liver cancer approaches 2 million annually, the majority is attributed to fibrosis. Currently, there is no efficient, safe, non-toxic, and anti-fibrotic drug available, suggesting room for better drug discovery. The current study aims to evaluate the anti-fibrotic role of reserpine, an alkaloid plant compound against CCl4-induced liver fibrosis. In-silico docking analysis showed the interaction of reserpine with keap1 protein with the binding energy -9.0kcal/mol. In-vitro, biochemical analysis, anti-oxidative indexes, and inflammatory cytokines analysis were performed in HepG2 cells. The non-toxic nature of the compound (<100μg/ml) was evaluated through MTT assay in HepG2 and Vero cell lines. The antifibrotic potential of the reserpine compound (dose of 0.5mg/kg) was assessed in CCl4-administered C57BL/6J mice models. Hematoxylin & Eosin and Masson staining were performed to study the morphological changes of liver tissues. Immune histochemistry (IHC) analysis was performed to evaluate the effect of reserpine on the liver fibrosis marker. The biochemical assay indicated a significant decrease in ALT, AST, and MDA levels and increased catalase enzyme post-6-week reserpine treatment in mice models. Gene expression analysis revealed that the reserpine targets oxidative stress Keap1/Nrf2 pathway and down-regulated Keap1 expression by 5-fold and up-regulated Nrf2 and Nqo1 expression by 6 and 4.5-fold respectively showing its antioxidant response. It suppressed the expression of Cyp2e1 by 2.2-fold, illustrating the compound's ability to block lipid peroxidation. Histological and immunostaining exhibited improved hepatocyte morphology and reduced collagen deposition in liver tissues due to reserpine. Reserpine treatment lowered the fibrotic markers α-SMA and Col-1 by 1.3 and 1.5 folds respectively as compared to the control group and increased the expression of miR-200a and miR-29b by 15.5 and 8.2 folds (p<0.05) while decreased miR-128-1-5p expression by 5-fold. A comprehensive In-silico, In-vitro, and In-vivo analysis revealed that reserpine has a strong anti-fibrotic effect against the CCl4-induced liver fibrosis in C57BL/6J mice model by targeting the Keap1/Nrf2 pathway.

Comprehensive analysis of the succinylome in Vero cells infected with peste des petits ruminants virus Nigeria 75/1 vaccine strain.

Peste des petits ruminants virus (PPRV) is currently the only member of the Morbillivirus caprinae species within the genus Morbillivirus of the family Paramyoxviridae. PPRV causes a highly contagious disease in small ruminants, especially goats and sheep. Succinylation is a newly identified and conserved modification and plays an important role in host cell response to pathogen infection. However, the extent and function of succinylation in Vero cells during PPRV infection remains unknown. In this study, a global profile of the succinylome in Vero cells infected with PPRV Nigeria 75/1 vaccine strain (PPRVvac) was performed by dimethylation labeling-based quantitative proteomics analysis. A total of 2633 succinylation sites derived from 823 proteins were quantified. The comparative analysis of differentially succinylated sites revealed that 228 down-regulated succinylation sites on 139 proteins and 44 up-regulated succinylation sites on 38 proteins were significantly modified in response to PPRVvac infection, seven succinylation motifs were identified. GO classification indicated that the differentially succinylated proteins (DSuPs) mainly participated in cellular respiration, biosynthetic process and transmembrane transporter activity. KEGG pathway analysis indicated that DSuPs were related to protein processing in the endoplasmic reticulum. Protein-protein interaction networks of the identified proteins provided further evidence that various ATP synthase subunits and carbon metabolism were modulated by succinylation, while the overlapped proteins between succinylation and acetylation are involved in glyoxylate and dicarboxylate metabolism. The findings of the present study provide the first report of the succinylome in Vero cells infected with PPRVvac and provided a foundation for investigating the role of succinylation alone and its overlap with acetylation in response to PPRVvac.

87. Machine Learning-Guided Generation of a Potent Combination of Broadly Neutralizing Sarbecovirus Antibodies with a High Barrier to Resistance

Abstract Background The elderly and immunocompromised remain at high risk of severe COVID-19 due to suboptimal immune responses. Monoclonal antibodies to SARS-CoV-2 spike were highly effective for both prophylaxis and treatment, but quickly became ineffective due to viral escape. Using a machine learning-guided optimization approach, we identified two antibodies against highly conserved epitopes, one targeting the spike S2 stem helix (GB-0669) and the other against the class 4 RBD site on S1 (PRO-37587) with the goal of developing a combination for prophylaxis that would be less susceptible to viral escape. Methods GB-0669 and PRO-37587 were characterized for breadth through binding and neutralization assays against a panel of SARS-CoV-2 variants and related sarbecoviruses. We also assessed the barrier to resistance, both individually and in combination using SARS-CoV-2 Omicron BA.1 in live virus escape studies in Vero E6 cells. Passaged viruses were sequenced and assessed for viral replication. The prevalence of any observed mutations was analyzed for conservation in the SARS-CoV-2 lineages and across the sarbecovirus family. Results Both antibodies exhibited potent neutralization across SARS-CoV-2 variants including those that had not yet emerged during development of our antibodies, while displaying activity against non-SARS-CoV-2 viruses. During passage with BA.1, escape from GB-0669 was observed in passage 3 (H1156Y), while escape from PRO-37587 emerged in passage 10 through two mutations (P381S, D402G) that impaired viral replication. By the end of passage 10, neither complete nor partial escape were observed for the combination of GB-0669 and PRO-37587. These escape mutations were observed in fewer than 1% of circulating variants from the beginning of the pandemic, probably due to low selective pressure and/or their impact on viral replication. With the exception of P381S, these mutations were not observed across a range of sarbecoviruses. Conclusion We demonstrate that the combination of GB-0669 and PRO-37587 has potent and broad neutralization activity. The robust viral escape profile and conservation of these epitopes suggest that this combination is more likely to remain effective in the face of ongoing viral evolution. Disclosures Alex Ramos, PhD, Generate Biomedicines: Stocks/Bonds (Private Company) Alan Leung, PhD, Generate Biomedicines: Stocks/Bonds (Private Company) Brett T. Hannigan, PhD, Generate Biomedicines: Employee|Generate Biomedicines: Stocks/Bonds (Private Company) Adam Root, MS, MBA, Generate Biomedicines: Grant/Research Support|Generate Biomedicines: Stocks/Bonds (Private Company) Heather A. Van Epps, PhD, Generate Biomedicines: Stocks/Bonds (Private Company) Kristen Hopson, Ph.D., Generate Biomedicines: Stocks/Bonds (Private Company) Gevorg Grigoryan, PhD, Generate Biomedicines, Inc.: Employee and shareholder|Generate Biomedicines, Inc.: Stocks/Bonds (Private Company) Gavin CKW Koh, PhD, AstraZeneca: Stocks/Bonds (Public Company)|Generate:Biomedicines: Stocks/Bonds (Private Company)|GlaxoSmithKline: Stocks/Bonds (Public Company) Daria Hazuda, PhD, Generate Biomedicines: Stocks/Bonds (Private Company)

Identification of sulfonylated indolo[1,2-a]quinolines as EGFR tyrosine kinase inhibitors.

Two series of indolo[1,2-a]quinolines (IQs), comprising six 6-trifluoromethylthio indolo[1,2-a]quinolines and nine 6-arenesulfonyl indolo[1,2-a]quinolines, were screened for their inhibitory activity against EGFR tyrosine kinase (EGFR-TK) using the ADP-Glo™ kinase assay. Among the 15 IQs screened, four compounds exhibited cytotoxic activity against a lung cancer cell line (A549) that was as potent as the known drug afatinib with lower cytotoxicity in Vero cells. In addition, while they displayed cytotoxic activity against a head and neck squamous cell carcinoma cell line (SCC cells), they were inactive against a colorectal cancer cell line (LS174T cells). Molecular dynamics (MD) simulations revealed that IQSO2R-I (IC50: 0.28 ± 0.05 μM) formed a stable complex with wild-type EGFR through hydrophohic interactions and hydrogen bonding with the K745 residue. Additionally, the compound complied with the extended rule of five. This class of compounds represents a novel class of EGFR-TK inhibitors, which may serve as a novel scaffold for the development of anticancer therapeutics targeting EGFR-TK.

92. Obeldesivir Reduced SARS-CoV-2 Infectious Titers in the BIRCH Phase 3 Clinical Trial

Abstract Background COVID-19 remains a serious condition, especially for those with certain risk factors. Obeldesivir (ODV) is an oral, broad spectrum, nucleoside analog prodrug inhibitor of SARS-CoV-2 RNA-dependent RNA polymerase. Methods BIRCH was a Phase 3, randomized, double-blind, placebo-controlled trial to evaluate the efficacy and safety of a 5-day ODV treatment in adults with risk factors for developing severe COVID-19. Nasal swab samples were collected on Days 1, 3, 5, 10, and 15. For participants with baseline viral load (VL) ≥106 copies/mL, samples were evaluated for SARS-CoV-2 infectious titers. In participants with baseline infectious titer ≥200 plaque forming units (PFU)/mL, Day 3 or 5, and, if available, Day 10 samples were assessed. Infectious titer assays were performed by inoculating Vero E6 cells with nasal swab sample serial dilutions, incubating for 96 hours with a carboxymethylcellulose overlay, and quantifying PFU following staining of the cell monolayer. The proportion of participants with undetectable infectious titers was compared between the ODV and placebo (PBO) groups using Fisher’s exact test. Additionally, change from baseline in infectious titer was compared between the ODV and PBO groups using a mixed model repeated measures approach. Results The proportion of participants with positive infectious titers at baseline (≥200 PFU/mL) was similar between the ODV (74/137 [54%]) and PBO (72/129 [56%]) groups. ODV treatment reduced VL at Day 5 versus PBO (–0.58 log10 copies/mL; P &amp;lt; 0.0001) and led to significantly greater reductions from baseline in infectious titers on Day 3 (−0.54 log10 PFU/mL; P = 0.0003) and Day 5 (−0.17 log10 PFU/mL; P = 0.0014) compared to PBO. At Day 3, 35/44 participants (80%) in the ODV group and 18/37 participants (49%) in the PBO group were negative for infectious virus (P = 0.0049). At Day 5, 68/68 participants (100%) in the ODV group and 56/69 participants (81%) in the PBO group were negative for infectious virus (P = 0.0001). At Day 10, the proportions of participants negative for infectious virus were similar between the groups (ODV: 47/49 [96%]; PBO: 40/41 [98%]: P = 1.000). Conclusion In BIRCH, ODV reduced SARS-CoV-2 infectious titers and VL, demonstrating inhibition of SARS-CoV-2 replication in adults with risk factors for developing severe COVID-19. Disclosures Lauren Rodriguez, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Anca Streinu-Cercel, MD PhD Prof. Infectious Diseases, Gilead Sciences, Inc.: Grant/Research Support|Gilead Sciences, Inc.: Honoraria Yiannis Koullias, MD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Afsaneh Mozaffarian, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Kim Etchevers, MS, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Shuguang Chen, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Robert H. Hyland, DPhil, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Joe Llewellyn, PharmD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Romas Geleziunas, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company) Antonella Castagna, MD, Bristol-Myers Squibb: Advisor/Consultant|Gilead Sciences, Inc.: Advisor/Consultant|Gilead Sciences, Inc.: Grant/Research Support|Gilead Sciences, Inc.: Honoraria|Janssen: Advisor/Consultant|Janssen: Grant/Research Support|Janssen: Honoraria|Merck Sharp &amp; Dohme: Advisor/Consultant|Merck Sharp &amp; Dohme: Grant/Research Support|Merck Sharp &amp; Dohme: Honoraria|ViiV Healthcare: Advisor/Consultant|ViiV Healthcare: Grant/Research Support|ViiV Healthcare: Honoraria Charlotte Hedskog, PhD, Gilead Sciences, Inc.: Employee|Gilead Sciences, Inc.: Stocks/Bonds (Public Company)

Evaluation of Zein Nanoparticles as Delivery Agents of SARS-CoV-2 Antigens

Background/Objectives: Nanovaccines have significant potential to enhance immunization strategies by improving efficacy, safety, and cost-effectiveness. In particular, organic nanoparticles hold promise for the generation of low-cost nanovaccines obtained by environmentally friendly methods. In this study, the feasibility of using zein nanoparticles (NPs) as carriers for an antigenic peptide (p30) and the receptor binding domain (RBD) from SARS-CoV-2 spike protein was explored. Methods: A synthesis method for zein NPs was established by combining previously reported techniques, and the resulting NPs were characterized in terms of morphology, particle size, polydispersity index (PDI), surface charge, and colloidal stability using dynamic light scattering (DLS) and transmission electron microscopy (TEM). Tween 20 was employed as a surfactant to enhance particle stability and prevent aggregation. Results: The zein NPs were deemed safe based on an in vitro cytotoxicity assay using Vero cells. Immunogenicity assessments demonstrated that zein NPs:p30 and zein NPs:RBD induced IgG responses in test mice, whose magnitude was comparable to those achieved with alum as an adjuvant. Conclusions: These findings support the use of zein NPs as promising vaccine delivery vehicles with adjuvant effects due to their ease and environmentally friendly synthesis, high stability, and low cost.

Chemical composition, in vitro and in silico activity of the methanolic extract derived from Vassobia breviflora against clinically relevant bacteria

ABSTRACT This study aimed to identify chemical compounds derived from Vassobia breviflora methanolic extract using ESI-ToF-MS and their antioxidant potential activity utilizing the following methods: total phenols, DPPH, and ABTS•+. The MTT assay measured cytotoxic activity, while DCFH-DA and nitric oxide assays were employed to determine reactive oxygen species (ROS) and reactive nitrogen species (RNS) levels using African green monkey kidney (VERO) and human keratinocyte (HaCat) cell lines. The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were assessed in seven clinical isolates and nine ATCC strains. Biofilm inhibition was tested against four biofilm-forming strains. The antioxidant properties of the methanolic extract were identified as follows: 35.74 mg GAE/g (gallic acid equivalents)/g for total phenols, 10.5 µg/ml for DPPH, and 50.68 µmol trolox/µg for ABTS•+. The mean inhibitory concentration (IC50) values were 622.86 µg/ml (VERO) and 784.33 µg/ml (HaCat). These concentrations did not markedly alter levels of ROS and RNS. Conversely, Bacillus cereus β-hemolytic displayed higher sensitivity to the extract, with MIC of 64 µg/ml and MBC of 128 µg/ml. Enterococcus faecium exhibited the lowest biofilm formation among the tested bacteria. The studied plant exhibited activity against all bacterial strains at concentrations lower than the IC50 VERO and HaCat cells, suggesting potential for future studies. Data present a comprehensive molecular docking analysis against the HlyIIR protein (PDB ID: 2FX0) and determined antimicrobial and endocrine-modulating potentials. Notably, lancifodilactone I and nicandrin B demonstrated the strongest binding affinities, with binding energies of −9.8 kcal/mol and −8.3 kcal/mol, respectively, and demonstrated significant antimicrobial effects against B. cereus. In addition, several compounds showed potential interactions with nuclear receptors, indicating potential endocrine-modulating effects. These findings provide insights into developing target-specific antimicrobial therapies and endocrine-modulating agents.

Substituent Effect on Cytotoxicity and Interaction Profile of DNA/BSA With Selected Terpyridine Derivatives of Ester/Ferrocene and Their Fe(III) and Ru(III) Complexes—A Comparative Analysis

ABSTRACTThe present work demonstrates the synthesis of ester and ferrocene groups containing new terpyridine‐based ligands (FRL and RL) and their selected meal complexes FR‐Fe, FR‐Ru, RL‐Fe, and RL‐Ru. Investigations on the influence of the ester and ferrocene groups on binding to DNA/BSA and anticancer activity were carried out. All these products were structurally characterized by using spectroscopic and analytical techniques. Notably, the molecular structures of both the ligands and the ester‐based Fe(III) complex were confirmed by single‐crystal X‐ray diffraction analysis. Additionally, theoretical calculations were employed to further establish the structures of the synthesized ligands and their metal complexes, which were subsequently utilized for molecular docking studies to evaluate their binding potential. The docking results revealed the best binding orientation with the lowest possible binding energy of −10.20 kcal/mol for the RL‐Fe complex with BSA and −8.30 kcal/mol for the FR‐Fe complex with DNA. Furthermore, the binding interactions of all the synthesized compounds were investigated using UV–visible absorption (UV) and fluorescence spectroscopy (FL). The binding sites and binding constants were determined using the Stern‐Volmer equation. Moreover, cytotoxicity assays were performed against lung cancer (A549), liver cancer (Hep G2), and normal Vero cell lines, with results benchmarked against cisplatin. Notably, the ligand (FRL) exhibited significant cytotoxic action with an IC50 of 54.51 μM compared to the related Fe(III) complex (FR‐Fe) (i.e., 69.86 μM) against A549 cells. But the complex FR‐Fe displayed IC50 of 56.19 μM against Hep G2 cells, demonstrating better potency over cisplatin.

Characterization of Novel Plantaricin-Derived Antiviral Peptides Against Flaviviruses

Flaviviruses, including West Nile virus, Zika virus, and Dengue virus, pose global health challenges due to their distribution, pathogenicity, and lack of effective treatments or vaccines. This study investigated the antiviral activity of novel truncated peptides derived from the two-peptide plantaricins PLNC8 αβ, PlnEF, PlnJK, and PlnA. The antiviral potential was predicted using machine learning tools, followed by in vitro evaluation against the Kunjin virus using plaque reduction assays in Vero cells. Molecular docking assessed peptide interactions with KUNV and ZIKV. Full-length and truncated peptides from PlnA, PlnE, PlnF, PlnJ, and PlnK demonstrated limited antiviral efficacy against KUNV in vitro, despite in silico predictions suggesting antiviral potential for PlnA, PlnE, and PlnJ. Large discrepancies were observed between the predicted and experimentally determined activities. However, complementary two-peptide plantaricins PlnEF and PlnJK exhibited significant synergistic effects. Furthermore, the truncated peptides PLNC8 α1-15 and PLNC8 β1-20 reduced KUNV viral load by over 90%, outperforming their full-length counterparts. Molecular docking revealed interactions of PLNC8 α and PLNC8 β, and their truncated variants, with KUNV and ZIKV, suggesting a mechanism involving viral envelope disruption. These findings highlight the potential of plantaricin-derived peptides as promising antiviral candidates against flaviviruses, warranting further investigation into their mechanisms and applications.

Imaging flow cytometry reveals the mechanism of equine arteritis virus entry and internalization

The process of viral entry into host cells is crucial for the establishment of infection and the determination of viral pathogenicity. A comprehensive understanding of entry pathways is fundamental for the development of novel therapeutic strategies. Standard techniques for investigating viral entry include confocal microscopy and flow cytometry, both of which provide complementary qualitative and quantitative data. Imaging flow cytometry, which integrates the advantages of both methodologies, offers significant potential in virological studies. In this investigation, we employed imaging flow cytometry coupled with immunostaining to monitor the entry of equine arteritis virus EAV into Vero cells via the endosomal trafficking route. Analysis provided an insight into the early infection dynamics across thousands of cells, revealing statistically significant alterations in internalization and uncoating process. Moreover, we evaluated the effectiveness of two inhibitors targeting cellular factors involved in facilitating viral entry: ammonium chloride, which disrupts endocytosis, and camostat mesylate, which inhibits the activity of serine proteases. The results demonstrated a clear distinction between effective and ineffective inhibitors. This study highlighted the potential of imaging flow cytometry to advance the study of viral entry and the evaluation of antiviral agents.

Development and characterization of a reverse genetics system for the lineage II Chicava strain of Machupo virus in a guinea pig model.

Machupo virus (MACV) is a New World mammarenavirus (hereafter referred to as "arenavirus") and the etiologic agent of Bolivian hemorrhagic fever (BHF). No vaccine or antiviral therapy exists for BHF, which causes up to 35% mortality in humans. New World arenaviruses evolve separately in different locations. To date, up to eight lineages of MACV have been identified in Bolivia. While the prototype MACV Carvallo strain belongs to lineage I discovered in the Memore Province in the 1960, the MACV lineage II strains have become the dominantly-circulating lineage in the same province since 1993. We report the development of a reverse genetics system for the MACV lineage II Chicava strain, using a pRF42 plasmid encoding the L and S segment genomic RNA under the transcriptional control of a murine DNA-dependent RNA polymerase I promoter sequence. Rescue of the recombinant MACV Chicava strain (rMACV-Chicava) was accomplished by expression of the L protein and nucleoprotein genes of the MACV Carvallo strain in trans in transfected baby hamster kidney (BHK-21) cells. We characterized the multiplication kinetics of rMACV-Chicava in African green monkey kidney epithelial Vero cells, followed by determining the virulence phenotype in outbred Hartley guinea pigs. We demonstrated that the multiplication kinetics in Vero cells, virulence phenotype in guinea pigs, and neutralizing antibody titers are indistinguishable between rMACV-Chicava and the wild-type parental virus. We conclude that rMACV-Chicava provides a useful model system to investigate the emergence of MACV lineage II strains and the guinea pig model has utility for the development of candidate vaccines and therapeutic antibodies for BHF.

Empowering African Expertise: Enhancing Safety Data Integration and Signal Detection for COVID-19 Vaccines Through the African Union Smart Safety Surveillance Joint Signal Management Group.

The COVID-19 pandemic accelerated new vaccine development. Limited safety data necessitated robust global safety surveillance to accurately identify and promptly communicate potential safety issues. The African Union Smart Safety Surveillance(AU-3S) program established theJoint Signal Management (JSM) group to support identification of potential vaccine safety concerns in five pilot countries (Ethiopia, Ghana, Kenya,Nigeria, South Africa), accounting for approximately35% of the African population. Our objective was to provide an overview of the JSM group's role in supporting signal management activities for the AU-3S program during the COVID-19 pandemic. Spontaneous, electronically reported COVID-19 vaccine adverse events following immunization (AEFI)from each country'ssafety data were integrated into the interim Data Integration and Signal Detection system. Statistical disproportionality methods were used to identify and review vaccine-event combinations (VECs) for potential safety concerns. The JSM group-which comprised pharmacovigilance and subject matter experts from National Medicine Regulatory Authorities, Expanded Programs on Immunization, and vaccine safety committees-conducted signal detection activities on cross-country safety data and provided recommendations. From April 2021 to December 2023, a total of 48,294 spontaneously reported AEFIwere analyzed for six COVID-19 vaccines (NRVV Ad [ChAdOx1 nCoV-19]; Ad26.COV2.S; Elasomeran; Tozinameran; Covid-19 vaccine [Vero Cell], Inactivated; NRVV Ad26 [Gam-Covid-Vac]) administered in Ethiopia (34.6%), Nigeria (30.3%), South Africa (16.9%), Ghana (13.5%), and Kenya (4.7%). Overall, 2,742 VECs were validated. A causal association between theCOVID-19 vaccines and thereported AEFIcannot be inferred, as data were reported spontaneously. JSM group recommendations included monitoring for further evidence, no immediate action required, engaging marketing authorization holder(s) for additional information, or sensitizing healthcare providers and/or the public about events. Although no new safety signals were identified, nine safety-related recommendations were issued, including patient and healthcare provider education. The JSM group established a scalable and replicable model for future signal management of other priority health products in low- and middle-income countries, fostering ongoing collaboration and capacity building. Knowledge and experience gained from this pilot initiative will guide stakeholders in future safety surveillance initiativeswithin the African continent.

PAV-05 Naphthoquinone Potently Inhibit Zika Virus Replication in Infected Cells.

Zika (ZIKV) is a virus transmitted by mosquitoes that can cause Guillain- Barré syndrome and congenital malformations like microcephaly. Given its explosive resurgence and the resulting epidemics in 2016, the search for effective antiviral drugs has become absolutely necessary. In this study, we examined the potential of naphthoquinone derivatives that have a sulfonamide or sulfonate group to inhibit ZIKV replication in primary cultured neurons and in Vero cells. In our in vitro studies, we found that PAV05 had low cytotoxicity with a CC50 of 329 μM ±3.6 for Vero cells and 290 μM ±3.5 for neurons. Additionally, we observed a strong inhibitory activity on viral replication with an EC50 value of EC50 of 0.92 μM ±0.15 in Vero cells, resulting in a Selectivity Index (SI) of 357. Even when added 16 hours post-infection, PAV05 maintained its inhibitory effect. When PAV05 was evaluated in sub-optimal concentrations together with Ribavirin, we observed a strong synergistic effect, with an inhibition greater than 90% even at doses of 0.5 μM. In silico tests suggested that PAV05 may have effects on ZIKV NS2B-NS3. The ZIKV inhibitor described in this study shows promise as a compound for the development of therapies against ZIKV. It may also be considered for inclusion in the portfolio of broad-spectrum antiflavivirus inhibitors.

Structure and properties of polysaccharides from tetrasporophytes of Mazzaella parksii.

The structure and anti-SARS-CoV-2 activity of sulfated polysaccharides (Mzpt) obtained in high yield (60 %) from tetrasporophytes of Mazzaella parksii were studied. Stepwise fractionation with KCl showed that Mzpt consisted of eight (MzptF1-MzptF8) carrageenans fractions, differing in structure and molecular weight. The yield of non-gelling MzptF8 was 58.1 % of the original Mzpt. According to IR and NMR spectroscopies, gelling MzptF1 and MzptF2 were mainly kappa/iota/nu-carrageenans. MzptF7 included mainly lambda-carrageenan and in smaller quantities kappa-, iota-, mu- and nu-carrageenans. MzptF8 had a complex composition and included gamma-carrageenan, unsulfated carrageenan, probably, delta-carrageenan and also structural elements of xi-, psi- and omicron-carrageenans. According to atomic force microscopy data, MzptF8 involved several polymer chains associated with each other in a disordered structure, in contrast to MzptF2, which formed three-dimensional networks. Unlike ribavirin and remdesivir, Mzpt was not cytotoxic to Vero E6 cells at concentrations >2000 μg mL-1. Mzpt was shown to inhibit SARS-CoV-2 replication in a dose-dependent manner in CPE inhibition and RT-PCR assays. IC50 was 92.0 μg mL-1, SI - 22. At concentration 250 μg mL-1, Mzpt caused the highest reduction in viral RNA levels with an inhibition coefficient of 31.1 % and exhibited significant inhibition of the early stages of virus-cell interaction.

Biocompatibility Analysis of a Herbal Amalgamation Containing Nochi, Vilvam, and Adhimadhuram Ethanolic Extracts on Vero Cell Line

Biocompatibility Analysis of a Herbal Amalgamation Containing Nochi, Vilvam, and Adhimadhuram Ethanolic Extracts on Vero Cell Line

Construction of a Vero cell line expression human KREMEN1 for the development of CVA6 vaccines

Coxsackievirus A6 (CVA6) has emerged as a major pathogen causing hand, foot and mouth disease (HFMD) outbreaks worldwide. The CVA6 epidemic poses a new challenge in HFMD control since there is currently no vaccine available against CVA6 infections. The Vero cell line has been widely used in vaccine production, particularly in the preparation of viral vaccines, including poliovirus vaccines and EV71 vaccines. Unfortunately, most CVA6 strains failed to propagate effectively on Vero cells. The expression level of virus-specific receptors on the cell membrane significantly influences viral infection. Here, a Vero cell line with stable over-expressing of KREMEN1 (KRM1), a crucial receptor for CVA6, was constructed using the lentivirus system. The cloned cell line, called Vero-KRM1_#11, could efficiently support most CVA6 infections. The propagation of CVA6-TW00141 strain on Vero-KRM1_#11 was equal to that on RD cells. After four passages, the virus batch was obtained with a titer of about 107 TCID50/mL. Moreover, the purified CVA6 particles produced from Vero-KRM1_#11 or RD cells both could induce high and comparable levels of IgG and neutralizing antibodies. Importantly, passive transfer of the antisera from CVA6-vaccined mice showed 100% preventive efficacy against CVA6 infection in mice. Therefore, KRM1-expressing cells have the potential to serve as a valuable tool for the development and production of CVA6 or polyvalent HFMD vaccines.

Dual VEGFR-2 and EGFRT790M inhibitors of phenyldiazenes: anticancer evaluations, ADMET, docking, design and synthesis.

New phenyldiazene scaffold-linked heterocyclic pyrazole, pyrimidinone, pyrimidinthione, and/or triazine rings have been developed and synthesized. Cytotoxicity of our derivatives was estimated on four cancer and VERO normal cell lines targeting EGFRT790M (epidermal growth factor receptor) and VEGFR-2 (vascular endothelial growth factor receptor-2) enzymes. Our new derivatives selectively inhibited both VEGFR-2 and EGFR as they have the essential structural requirements for inhibitors of both receptors. Derivative 14 was the most active on A549, HCT116, HepG2, and MCF-7 cancers with half-maximal inhibitory concentration (IC50) = 5.50, 9.77, 7.12, and 7.85 µM respectively. The assessed derivatives 5, 7, 8, 9, 10, 12 and 14 showed IC50 = 54.40-62.60 μM against normal VERO (normal kidney) cells with low toxicity. In addition, derivatives 14, 8, 10, 7 and 9 were discovered to be very good active inhibitors of VEGFR-2 at IC50 values of 1.15, 1.35, 140, 1.78 and 1.90 µM, respectively. Furthermore, derivatives 14, 10, 8, and 9 strongly repressed EGFRT790M with IC50 = 0.28, 0.33, 0.35, and 0.50 µM correspondingly. Additionally, the highly active compounds 8, 10, and 14 showed good ADMET profile. Our derivatives could be considered as anticancer agents with dual VEGFR-2 and EGFRT790M inhibition.