Search Results (6,376)

The COVID-19 pandemic highlighted the urgent need to develop effective and broad-spectrum antiviral therapies against coronaviruses. One strategy to address this concern is a combination therapy using repurposed drugs against zoonotic viruses with pandemic potential. We previously demonstrated that the combination of Remdesivir and Ivermectin is highly potent and synergistic in inhibiting the replication of murine hepatitis virus (MHV) in RAW264.7 macrophages. This study investigated the interactions between the drug combination, coronavirus and host by proteomics and RNA sequencing of MHV-infected H2.35 murine liver epithelial cells. Time-of-addition and time-of-removal assays suggested that the drug combination likely affected the synthesis of viral RNA and viral protein. This combination drastically diminished the live virus titer greater than the respective monotherapies in MHV-infected H2.35 cells (by ~4 log₁₀), as well as in SARS-CoV-2-infected VeroE6 cells and human nasal epithelial cells. Proteomic and transcriptomic analyses revealed that viral protein and RNA levels were significantly depressed upon combination treatment. The drug combination exhibited considerable negative effects upon host RNA processes and resulted in the upregulation of host protein processes (e.g., response to unfolded protein; protein insertion into ER membrane). Molecular pathways affected by the combination treatment were markedly distinct from the monotherapies and indicated that Ivermectin enhances Remdesivir by modulating critical host processes to synergistically exert its inhibitory effect on the coronavirus replication cycle. Full article

Background: The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) created ongoing challenges for molecular diagnostics and variant surveillance. Assays capable of maintaining diagnostic sensitivity across emerging variants while providing variant-related information remain essential for clinical and public health applications. This study evaluated the performance of the GXT96 X3 extraction kit in combination with the FluoroType^® SARS-CoV-2 varID Q version 1.0 assay (Hain LifeScience SA (Pty) Ltd., South Africa) for the detection, semi-quantitative assessment, and variant characterization of SARS-CoV-2 under laboratory conditions. Methods: A total of 220 samples were evaluated, including residual nasopharyngeal clinical specimens (n = 183), reference materials, and cultured SARS-CoV-2 virus dilutions. Residual specimens collected during multiple COVID-19 waves in South Africa (wild-type, Beta, Delta, and Omicron) were compared against standard-of-care (SOC) molecular assays used for routine diagnosis. RNA extraction was performed using the automated GXT96 X3 platform, followed by amplification on the FluoroCycler^® XT using the FluoroType^® SARS-CoV-2 varID Q assay targeting RdRp and N genes, with additional spike gene mutation detection for variant detection. Diagnostic accuracy, agreement (Cohen’s kappa), precision, linearity, and limit of detection (LoD) were assessed. Results: The assay demonstrated a sensitivity of 98.4% (95% CI: 94.2–99.8) and specificity of 100% (95% CI: 95.9–100.0) compared with SOC assays, with an overall agreement of κ = 0.981. Precision analysis showed acceptable reproducibility with a standard deviation of ≤1.49 and a coefficient of variation of ≤3.83%. Regression analysis demonstrated linearity across the dilution series (R² = 0.9882 for RdRp and 0.994 for N genes). The LoD was ≤100 copies/mL for the RdRp gene and 250 copies/mL for the N gene. Variant-associated spike mutations corresponded broadly with epidemiological wave patterns observed in South Africa. Conclusions: Under the evaluated laboratory conditions, the GXT96 X3 extraction platform combined with the FluoroType^® SARS-CoV-2 varID Q assay demonstrated high diagnostic accuracy and reproducibility for SARS-CoV-2 detection across a range of viral loads with additional spike gene mutation detection as an adjunct feature. Full article

Although antiviral development against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been dominated by replication-directed strategies, structural and accessory proteins offer a complementary and increasingly important opportunity for small-molecule intervention. These proteins control key processes outside the core replication machinery, including viral entry, membrane remodelling, virion assembly, egress, and host immune modulation, thereby expanding the mechanistic scope of antiviral design. However, many of these targets are membrane-associated, oligomeric, conformationally dynamic, or function through protein–protein interactions, creating distinct challenges in target validation, assay design, and chemical optimisation. In this review, we comprehensively and critically evaluate the structural and accessory proteomes of SARS-CoV-2, with a strict focus on small-molecule tractability and translational relevance. We highlight the most credible direct-acting opportunities, focusing on the membrane (M), envelope (E), and nucleocapsid (N) structural proteins, together with the accessory protein open reading frame 3a (ORF3a), for which emerging chemical matter strengthens confidence in druggability. In contrast, Spike (S) and several host-interface accessory proteins, including ORF6, ORF8, ORF9b, and ORF10, are best viewed as more selective or earlier-stage opportunities that require stronger on-target chemical validation. Emphasis is placed on structural accessibility, mechanism-based assay systems, evidence quality, cellular and in vivo activity, and developability constraints relevant to exposure at the infection site. Rather than replacing replication-directed antivirals, these non-canonical targets are best considered adjunctive or complementary components of future combination strategies designed to broaden antiviral coverage, enhance robustness, and improve pandemic preparedness. Full article

Research on oxazolones, particularly 4-alkynyloxazolones, has garnered increasing interest due to the presence of an alkynyl group, which facilitates molecular conjugation and enables diverse chemical modifications. In this study, three representative 4-alkynyloxazolone derivatives (L1–L3) were synthesized and structurally characterized through single-crystal X-ray diffraction and computational analysis to obtain a reliable structure of L1–L3 to subsequently predict in silico interactions with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein. The crystallographic results revealed high molecular planarity and multifurcated hydrogen bonding. Considering the obtained crystallographic structure, theoretical descriptors such as HOMO–LUMO energy gaps and electrostatic potential maps indicated that these compounds exhibit favorable electronic reactivity, particularly for L3, with favorable drug-like predictions. The lack of methoxy groups in L2 and L3 makes these compounds have lower predicted toxicity parameters than L1. Molecular docking calculations targeting SARS-CoV-2 spike glycoprotein in three different feasible conformations in a biological matrix, i.e., three receptor-binding domains (RBD) in down conformation, two RBD in down and one in up conformation, as well as RBD bound to the human receptor angiotensin-converting enzyme 2 (ACE2), suggested strong binding affinities and specific interactions with the RBD moiety, mainly in the up conformation. Overall, this work integrates crystallographic and computational approaches to establish the structural and in silico evaluation of spike-binding properties of early substituted 4-alkynyloxazolones, suggesting L3 as a candidate for future in vitro antiviral assays. Full article

The SARS-CoV-2 non-structural proteome remains the most clinically validated and strategically important landscape for direct-acting small-molecule antiviral drug discovery. The success of inhibitors targeting the main protease (M^pro, Nsp5) and RNA-dependent RNA polymerase (RdRp, Nsp12) has firmly established viral replication enzymes as tractable, druggable, and therapeutically relevant targets, while setting clear benchmarks for translational antiviral development. Building on this foundation, a second wave of non-structural protein (Nsp) targets has emerged with increasing translational promise, including the papain-like protease (PL^pro), the bifunctional Nsp14 proofreading and capping machinery, Nsp16 2′-O-methyltransferase, Nsp13 helicase, and Nsp15 endoribonuclease. In parallel, additional components such as Nsp1 and the Mac1 domain of Nsp3 continue to expand the antiviral design space, although they remain at earlier stages of chemical validation. In this review, we comprehensively assess SARS-CoV-2 non-structural proteins through a medicinal chemistry and translational lens, with an emphasis on structural tractability, mechanism of action, quality of chemical matter, cellular and in vivo antiviral evidence, evolutionary conservation, resistance liabilities, and developability. Particular attention is given to the features that distinguish tool compounds from genuinely actionable leads and to the opportunities for rational combination regimens that extend beyond first-generation protease- and polymerase-centred therapy. Collectively, the non-structural proteome offers the strongest foundation for next-generation and potentially broader-spectrum coronavirus antivirals with improved resilience to viral evolution. Full article

The renin–angiotensin system (RAS) regulates inflammation, tissue homeostasis, and barrier integrity in lung and colon epithelial cells. Beyond classical pathways, non-canonical components including angiotensin-converting enzyme 2 (ACE2), epidermal growth factor receptor (EGFR), insulin-like growth factor 2 receptor (IGF2R) and aminopeptidase N (ANPEP) are implicated in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and bacterial sepsis due to their roles in tissue repair and signaling. Despite their similar inflammatory and coagulopathic features, their impact on RAS-associated non-immune gene expression in epithelial tissues remains unclear. This study investigates the regulation of these targets in lung (BEAS-2B) and colon (CRL-1831) cells following exposure to recombinant SARS-CoV-2 spike protein N-terminal domain fragment (S1-NTD) and Pseudomonas aeruginosa-derived lipopolysaccharide (LPS). Cells were treated with 100 ng/mL of S1-NTD or LPS for 12–72 h. Viability was assessed via XTT assays, and molecular changes were analyzed through qRT-PCR and Western blotting. Both stimuli induced a time and dose-dependent decrease in metabolic activity. ACE2 was significantly downregulated in lung cells, while transient upregulation occurred in colon cells at 24 h. EGFR expression increased in colon cells following LPS exposure but decreased in lung cells after S1-NTD treatment. Both IGF2R and ANPEP were upregulated by S1-NTD in lung cells at 72 h, whereas colon cells showed earlier upregulation at 24–48 h. Our findings reveal that viral and bacterial stimuli elicit distinct, tissue-specific regulatory patterns in RAS-associated pathways. These alterations may contribute to epithelial barrier dysfunction and inflammation, highlighting these proteins as potential targets for managing secondary bacterial infections and inflammatory lung–gut complications in COVID-19. Full article

Coronaviruses use discontinuous transcription to generate subgenomic RNAs (sgRNAs) that encode structural and accessory proteins. However, the factors regulating sgRNA abundance in SARS-CoV-2 remain unclear. Here, we combined strand-specific RNA sequencing, RNA–RNA interaction mapping, prediction of RNA folding energies, and targeted mutagenesis to define the regulation of (–) sgRNA synthesis in SARS-CoV-2 infection. We demonstrated that the relative (–) sgRNA abundance across viral genes is stable throughout infection and largely correlates with corresponding (+) sgmRNA levels. Through meta-analysis of published SPLASH data, we found that the frequency of long-range interactions between the 5′ genomic transcription regulatory sequence TRS-Leader and downstream TRS-Body sequences correlates with sgRNA abundance. Notably, the folding energy (ΔG) of these duplexes quantitatively predicts (–) sgRNA transcript levels. Mutations in non-coding regulatory regions that altered the ΔG resulted in corresponding changes in (–) sgRNA expression, suggesting a causal role for TRS duplex stability in transcriptional regulation. Analysis of naturally occurring mutations near regulatory sites further suggests that modulation of duplex stability may also serve as an evolutionary mechanism to fine-tune viral gene expression. Together, our findings identify the pairing stability of TRS-Leader:TRS-Body as a determinant of discontinuous transcription and reveal how RNA pairing potential contributes to the regulation of (–) sgRNA synthesis in SARS-CoV-2. Full article

During the coronavirus disease-2019 (COVID-19) pandemic, wastewater-based surveillance (WBS) gained renewed importance by enabling nationwide assessment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) circulation independent of individual testing. In Germany, WBS was established by a series of initiatives, including the AMELAG project, established in 2022. As the pandemic phase waned, the extensive surveillance infrastructure—comprising around 170 wastewater treatment plants (WWTPs)—was scaled down. A subset of 53 WWTPs was selected by a hierarchical set of criteria to ensure continuation of WBS in 2025, resulting in a reduced population coverage (26% instead of 37%), preserving the monitoring of infection dynamics while improving operational efficiency. The multi-stage selection approach integrated operational experience and performance data of WWTPs collected between November 2022 and July 2024, including wastewater characteristics and laboratory quantification success, minimum population coverage across all administrative regional states of the country, and statistical quality metrics such as the frequency of outliers and implausible inflexion points or the deviation from LOESS regression trends. Additional consideration was given to sites of extended research relevance. Reducing the number of WWTPs by more than two-thirds did not result in notable deviations in the aggregated national SARS-CoV-2 viral load profile. However, the evaluation was limited to SARS-CoV-2 data, despite ongoing expansion of the WBS network to include additional pathogens. Overall, the data-driven site-selection framework, developed from scientific and operational criteria, ensures the sustainable continuation of the nationwide WBS system. Full article

Background: The World Health Organization (WHO) declared a pandemic due to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the etiologic agent responsible for Coronavirus Disease 2019 (COVID-19). Although case numbers declined after the initial outbreak, Brazil experienced slight increases in COVID-19 cases in 2024 and 2025, underscoring the persistent need for effective therapeutic interventions. Recently, proxalutamide—an androgen receptor antagonist—has been proposed as a potential therapeutic agent against COVID-19, as supported by several clinical studies. Methods: In the present work, we aimed to elucidate the molecular interactions between proxalutamide and key proteins involved in the viral fusion and replication processes of SARS-CoV-2. Computational techniques, including molecular docking and molecular dynamics simulations, were employed. Results: Our analyses indicated a high success rate, with stable conformations and favorable binding affinity values for ACE2 (−8.90 kcal/mol) and TMPRSS2 (−9.28 kcal/mol), resulting in strong docking scores. Moreover, molecular dynamics simulations confirmed the stability of these complexes, as evidenced by consistent mean square deviation values, low structural flexibility, a stable radius of gyration, and maintained surface rigidity over a 100 ns simulation period. Conclusions: These combined docking and dynamics results suggest that proxalutamide interacts firmly with the active sites, indicating high binding affinity that may interfere with SARS-CoV-2 entry. Nevertheless, experimental validation and rigorous safety assessments are warranted to confirm this potential. Full article

Coronavirus disease 2019 (COVID-19) has been associated with an increased long-term cardiovascular risk, potentially mediated by magnitude of the acute inflammatory response inflammation. Interleukin-6 (IL-6) and serum amyloid A (SAA) are key components of the inflammatory cascade and may serve as biomarkers of post-COVID cardiovascular vulnerability. This longitudinal observational study investigated the association between post- COVID-19 infection IL-6 and SAA levels and major cardiovascular events over a six-year follow-up period. A total of 97 individuals with documented prior SARS-CoV-2 infection were included. Circulating IL-6 and SAA concentrations were measured in the acute phase. The composite endpoint included incident arrhythmia, myocardial infarction, and all-cause mortality. Biomarker distributions were right-skewed and were therefore analyzed using non-parametric methods and penalized logistic regression models. During follow-up, 14.4% of participants experienced the composite endpoint. Individuals with adverse outcomes had significantly higher IL-6 and SAA levels compared with event-free participants. IL-6 demonstrated the strongest association with mortality, whereas SAA showed particularly robust associations with the composite endpoint, and with myocardial infarction. Both biomarkers independently predicted long-term adverse events. Circulating IL-6 and SAA concentrations measured during the acute phase of SARS-CoV-2 infection were analyzed in relation to long-term cardiovascular outcomes. These findings support the hypothesis that the magnitude of the acute inflammatory response during SARS-CoV-2 infection may be associated with long-term cardiovascular outcomes and suggest that combined assessment of IL-6 and SAA may have potential utility for hypothesis-generating prognostic signal requiring validation, pending validation in larger studies. Full article

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has greatly impacted public health due to high rates of transmissibility and mutation during the COVID-19 pandemic. Macrodomain 1 (Mac1) of non-structural protein 3 remained well conserved across variants and is critical to suppression of host immune response to infection, making Mac1 a promising target for therapeutic development. Mac1 binds and cleaves the post-translational modification ADP-ribose and is hypothesized to have a downstream effect on the host interferon response, but the exact cellular targets of Mac1 are still unknown. Characterizing the substrates of Mac1 ADP-ribosyl hydrolase activity using a catalytically inactive mutant N40D can reveal critical virus–host interactions to identify protein targets of Mac1 and reveal mechanisms of host interferon suppression. Here, we performed affinity enrichment with WT Mac1 and Mac1 N40D in HEK293T and A549 cells and quantified changes in protein interactions by TMT-multiplexed tandem mass spectrometry. We identified interactions between Mac1 and ADP-ribosylated substrates involved in DNA damage response, cytoskeletal components, and cell cycle regulation. Additionally, several members of the TRiC complex involved in protein folding were selectively enriched with mutant Mac1 from A549 cells. These findings suggest a novel role of Mac1 in regulating host protein folding. Full article

Background: Wastewater-based surveillance (WBS) has emerged as a valuable tool for population-level monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission, yet the interplay between sampling strategies and disease prevalence in shaping detection performance remains ambiguous. We investigated how grab and composite sampling influence SARS-CoV-2 ribonucleic acid (RNA) detection dynamics and predictive lag times across high- and low-prevalence communities in Selangor, Malaysia. Methods: A 28-week longitudinal study was conducted in Selangor, Malaysia, comparing grab and composite wastewater sampling in communities with high and low Coronavirus disease 2019 (COVID-19) prevalence. SARS-CoV-2 RNA in 348 samples was quantified using digital Reverse Transcription Polymerase Chain Reaction (RT-dPCR), and viral lineages were characterized by Nanopore sequencing. Detection sensitivity and lead times relative to reported cases were evaluated. Results: In low-prevalence settings, grab sampling showed higher detection sensitivity than composite sampling (92.0% vs. 70.0%), whereas both methods achieved similarly high detection in high-prevalence areas (>97.0%). Lag-time analysis indicated that grab sampling in high-prevalence settings was significantly associated with case trends at potential two-week lead (p = 0.024), while composite sampling in low-prevalence settings showed the strongest association at a potential one-week lead (p = 0.0022). Overall, lag structures varied by both sampling strategy and prevalence context. Both sampling approaches captured the replacement of Omicron sublineages (XBB.1.5, XBB.1.9.1, XBB.1.16) and identified additional circulating variants, including EG.5, that were not captured in the available clinical sequencing dataset during the same period. Conclusions: These findings reveal that local transmission intensity is associated with the utility of different sampling designs. Context-specific optimization of WBS sampling strategies enhances sensitivity, reduces detection lag, and strengthens early warning and genomic-tracking capacity in public health surveillance frameworks. Full article

This study examined alterations in serum redox biomarkers before and one month after administration of the coronavirus disease 2019 (COVID-19) booster (third) doses across four vaccine regimens. A longitudinal cohort of 410 adults was analyzed following homologous Pfizer-BioNTech, Sinopharm [Vero Cell]-Inactivated, Sputnik V, or heterologous Sinopharm/Pfizer vaccination. Serum total proteins, albumin, total thiols, nitrites, ferric-reducing antioxidant power (FRAP), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity were measured, with DPPH interpreted as an ex vivo surrogate of serum radical-scavenging capacity. Additional analyses included stratification by prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, multivariable regression, correlation analysis, effect-size estimation, and sensitivity testing. Booster vaccination was associated with modest but consistent decreases in DPPH activity, albumin, and total proteins, whereas FRAP, nitrite, and total thiol levels remained stable. This pattern supports a transient shift in antioxidant buffering capacity but, by itself, does not exclude oxidative stress, as direct oxidative damage markers were not assessed. The most pronounced changes were observed in Sinopharm-based regimens, particularly in the heterologous Sinopharm/Pfizer group. Prior SARS-CoV-2 infection did not materially alter the qualitative response pattern, whereas older age and comorbidities were associated with greater declines in DPPH activity and albumin. Overall, the findings indicate a modest, transient redox-associated response following booster-induced immune activation and suggest that host-related factors, such as age and comorbidity burden, may accentuate short-term changes in antioxidant buffering capacity. Full article

Background/Objectives: Maintenance hemodialysis patients are particularly vulnerable to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This study aimed to evaluate clinical outcomes and identify admission laboratory biomarkers associated with in-hospital mortality in hospitalized hemodialysis patients with coronavirus disease 2019 (COVID-19). Methods: We conducted a retrospective observational study including 130 adult hemodialysis patients with confirmed SARS-CoV-2 infection. Clinical characteristics and admission laboratory parameters were analyzed in relation to in-hospital outcomes using comparative, multivariable logistic regression, and receiver operating characteristic (ROC) curve analyses. Results: The overall in-hospital mortality rate was 34.6%. The median age of the cohort was 66 years, with 64.6% male patients. Non-survivors showed significantly higher levels of inflammatory and tissue-injury markers, including C-reactive protein (CRP) (p < 0.001) and lactate dehydrogenase (LDH) (p < 0.001), together with lower serum albumin (p < 0.001), platelet count (p < 0.001), and lymphocyte levels (p = 0.03). In multivariable analysis, cardiovascular disease, respiratory disease, dyspnea, and ambulatory origin were independently associated with mortality. ROC analysis identified platelet count as the best individual predictor (area under the curve [AUC] = 0.767). An exploratory composite risk score demonstrated excellent discriminative performance (AUC = 0.902). Conclusions: Admission inflammatory and hematological biomarkers are strongly associated with adverse outcomes in hospitalized hemodialysis patients with COVID-19. The integration of clinical and laboratory parameters into a composite risk score may improve early risk stratification and support clinical decision-making in this high-risk population. Full article