Search Results (336)

Background/Objectives: Self-amplifying RNA (saRNA) vectors enable high-level transgene expression from minimal initial doses. While alphavirus-based saRNA systems are widely used, they suffer from limitations, including large genome size, complex replicase machinery, and cellular toxicity. Nodamura virus (NoV) offers a promising alternative due to its compact genome (3.2 kb) and low cytotoxicity. This study aimed to elucidate NoV RNA1 replication mechanisms and develop a novel NoV-based saRNA vector platform. Methods: We established a Schizosaccharomyces pombe system to investigate NoV RNA1 replication and protein A localization. N-terminal deletion mutants and ER-targeting chimeras were constructed to characterize membrane targeting determinants. Based on mechanistic insights, we developed NovaVec by inserting transgenes at the RNA3⁴²² site within the subgenomic RNA3 region. In vivo performance was evaluated using lipid nanoparticle-encapsulated NovaVec expressing nanoluciferase or monkeypox A33R antigen in BALB/c mice. Results: We identified redundant mitochondrial targeting domains (amino acids 2-15 and 16-33) in NoV protein A, where either domain was sufficient for proper localization and replication. The replication machinery could be functionally redirected to the endoplasmic reticulum while maintaining replication competence. Lipid nanoparticle-encapsulated NovaVec achieved sustained transgene expression for 54 days in mice, significantly outperforming conventional mRNA vectors that lost signal within 14 days. The NovaVec-based monkeypox A33R vaccine elicited robust antigen-specific humoral immunity with titers reaching approximately 1:12,800 following booster immunization. Conclusions: With its compact genome encoding only a single replicase protein, minimal cytopathic effects, and demonstrated capacity for long-term protein expression, NovaVec represents a highly promising next-generation saRNA platform for vaccines. 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

Macrosteles fascifrons, a representative aster leafhopper frequently detected in rice-growing environments, is an economically significant insect that inhabits rice fields and plays a role in the ecology of crop pests and disease transmission. To expand the understanding of viral diversity associated with the aster leafhopper, we analyzed its virome using deep transcriptome sequencing. In addition to several previously reported viruses, we identified two previously unreported RNA viruses, tentatively designated as Macrosteles fascifrons hepe-like virus 1 (MfHV1) and Macrosteles fascifrons rhabdovirus 1 (MfRV1). The complete genome sequences of both genomes were obtained using overlapping RT-PCR and rapid amplification of cDNA ends. Excluding the poly(A) tail, the genome of MfHV1 is 6688 nucleotides in length and exhibits a genomic organization characteristic of the family Hepeviridae, comprising three major open reading frames (ORFs) that encode a putative nonstructural polyprotein, a capsid protein, and a small accessory protein. The ORF encoding the capsid protein partially overlaps with the ORF encoding the small accessory protein, a genomic feature commonly observed in hepe-like viruses. The genome of MfRV1 is 14,984 nucleotides in length and displays the canonical genomic organization of the family Rhabdoviridae. An additional accessory ORF was identified between the putative M and G genes. Phylogenetic analysis based on polyprotein sequences placed MfHV1 within the Hepeviridae, most closely related to insect-associated hepe-like viruses, whereas MfRV1 clustered within the subfamily Deltarhabdovirinae. According to ICTV guidelines, virus classification is based on a combination of sequence divergence, phylogenetic relationships, and genome organization. MfHV1 and MfRV1 share low amino acid sequence identities with known viruses (maximum 36.07% for the MfHV1 polyprotein and 47.7% for the MfRV1 RNA-dependent RNA polymerase). Based on sequence divergence, genome organization, and phylogenetic placement, these viruses are classified as putative novel members of their respective families. This study expands the diversity of virus-associated sequences detected in M. fascifrons and provides additional genomic resources for understanding insect-associated RNA viruses. Full article

SARS-CoV-2 infection has emerged worldwide. To reduce the number of cases and limit the transmission of the virus, health and local authorities have implemented several strategies. Mass screening is a key strategy for mitigating the damage caused by this pandemic. This strategy is based on the use of qRT-PCR and pooling to diagnose SARS-CoV-2 infection. The present work explores the performance and limitations of this strategy for the molecular diagnosis of SARS-CoV-2 infection. Three important technical aspects were retained: the comparison of two commercial extraction kits (BIGFISH and BIOER), the simulation of a non-compliant nasopharyngeal swab, and the evaluation of the pooling strategy. A total of 97 SARS-CoV-2-positive nasopharyngeal samples were used. The comparison of the two extraction kits was based on threshold cycles (Ct) values. The results showed a significant difference (IC = 95%) in the Ct of the nucleocapsid gene (N; p = 0.0000384) and RNA-dependent RNA polymerase (RdRp; p = 0.0254). However, no significant difference was observed between the Internal Control gene (IC; p = 0.0723) and Envelope gene (E; p = 0.150). The Ct values resulting from the BIGFISH extraction kit were generally lower than those obtained from BIOER. In terms of sensitivity, the RT-qPCR technique allows for the detection of viral RNA up to 10⁻³ as a dilution factor. This study demonstrated that the pooling strategy is an effective diagnostic technique. Positive samples remained detectable even in pools of 1000 or even 10,000 samples. However, the size of the pool under diagnostic conditions should not exceed a limit that must be dynamically adapted to prevalence to ensure economic and analytical viability. Full article

Nucleocapsids protect viral genomes and play fundamental roles in viral assembly and infection. While many viruses adopt icosahedral or helical symmetries, negative-strand RNA viruses (NSVs) assemble their nucleocapsids with a distinct translation-based symmetry that is often considered helical because of their curvature. Our study analyzes the structural basis, assembly principles, and functional implications of the linear nucleocapsids. Structural coordinates of viruses were obtained from the Protein Data Bank (PDB) and examined using PyMOL version 1.3 to compare protein folds, RNA–protein interactions, inter-subunit contacts, and curvature properties across multiple nucleocapsids. We found that linear nucleocapsids share a similar 5H + 3H fold in their capsid proteins and encapsidate a fixed number of nucleotides per subunit, though the degree of nucleotide sequestration varies. Their architecture differs in inter-subunit interactions, determining whether empty capsids can assemble and influencing RNase sensitivity. Although these nucleocapsids may appear helical, they lack strict helical symmetry and instead display variable curvature that is modulated by environmental conditions. Relaxation of this curvature is likely required for viral RNA-dependent RNA polymerase to access the sequestered RNA genome during transcription/replication. In conclusion, linear nucleocapsids constitute a class of RNA–protein assemblies with variable curvature. The topologically conserved fold of the capsid protein enables genome protection while regulating exposure of RNA during viral RNA synthesis. Full article

The translation of nucleic acid amplification into practical point-of-care and biosensor-integrated diagnostics is still significantly impeded by the necessity for rapid sample preparation. For this reason, a broad comparison of seven commercially available kits for DNA/RNA extraction containing their temperature-related adjustments was performed. Extracts isolated from SARS-CoV-2-positive nasopharyngeal swabs, viral stocks, as well as laboratory-prepared suspensions of clinically relevant Gram-positive and Gram-negative bacteria were evaluated by recombinase polymerase amplification (RPA) and real-time PCR. In addition, the impact of transport media for SARS-CoV-2 samples was investigated. Extraction performance varied markedly according to the kit, pathogen, sample background. For SARS-CoV-2, rapid extraction was more effective for samples collected in viral transport medium than in inactivation buffer. Across bacterial targets, performance was species dependent, highlighting substantial differences in compatibility between simplified extraction workflows and downstream amplification. Among the rapid methods tested, a simplified QuickExtract protocol (95 °C, 5 min) provided the most consistent overall results, although it did not uniformly match the reference silica-based method for all targets. In conclusion, these results demonstrate that rapid nucleic acid extraction must be thoroughly evaluated as an essential element of the entire sample-to-answer workflow, rather than being chosen as a standalone preprocessing step for point-of-care molecular diagnostics. Full article

The SARS-CoV-2 public health challenges have highlighted the urgent need for coronavirus-targeting life-saving therapeutics. Given the emergence of drug-resistant strains, the development of antivirals against viral proteins beyond the commonly targeted main protease or RNA-dependent RNA polymerase is critical. The SARS-CoV-2 nonstructural protein 13 (nsp13) is a highly conserved RNA helicase and an essential component of the viral replication–transcription complex (RTC). It unwinds double-stranded RNA to facilitate viral transcription and replication, making it a strong target for drug development. To identify nsp13 inhibitors, we used an ultra-high-throughput nucleic acid unwinding assay to screen a library of FDA-approved drugs and bioactive compounds. We identified forty inhibitors with IC₅₀ values ranging from 1.4 to 10 μM. Ten were further selected for biochemical and biophysical characterization. Four of these are bound to nsp13 without interacting with the nucleic acid substrate and without inhibiting the ATPase activity of nsp13. Hydrogen–deuterium exchange coupled with Mass Spectrometry (HDX-MS) studies show compound binding causes differential exchange in two regions of nsp13. Furthermore, these compounds have antiviral activity against infectious SARS-CoV-2 in multiple cell lines, with cytotoxicity affecting, in some cases, the apparent antiviral effect. Future optimization efforts could help develop therapeutics against SARS-CoV-2 and other potential coronavirus threats. Full article

Hepatitis E virus, a single-stranded positive-sense RNA virus, is the causative agent of acute viral hepatitis in humans and can lead to chronic infection in immunocompromised individuals. In this setting, strains containing host genome insertions within the polyproline region (PPR) of the pORF1 polyprotein were characterized and shown to display an increased replication rate across all systems. Using in silico modeling of pORF1 across 25 strains, combined with molecular dynamics (MD) simulations, we explored the structural variations caused by these insertions to investigate potential mechanisms underlying the increased replication rate compared to wild-type (WT) strains. Our results showed that the insertions neither induced structural organization within the PPR nor altered its intrinsically disordered nature. MD simulations further demonstrated that the overall stability of pORF1 remained unchanged in strains with insertions compared to WT strains. On the other hand, MD analyses revealed that strains with insertions exhibited an increased number of hydrogen bonds between the PPR and two other domains of pORF1: the MetY domain and the RNA-dependent RNA polymerase (RdRp). The stability of the MetY domain of the strains in the presence of host insertion events was higher than in the WT strains. These additional hydrogen bonds could position the MetY domain and the RdRp closer together, potentially promoting more efficient viral RNA synthesis. Validation of this hypothesis will require experimental structural studies, as well as computational modeling of the proposed dodecameric pORF1 structure. Full article

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging tick-borne viral disease with high case–fatality rates in East Asia, yet no licensed vaccines are currently available. Here, we generated attenuated severe fever with thrombocytopenia syndrome virus (SFTSV) strains through serial passage in Huh-7 cells and evaluated their pathogenicity, immunogenicity, and protective efficacy. Attenuation candidates were selected based on reduced pathogenicity, estimated based on the median lethal dose (LD₅₀), and genetic sequencing was performed to identify mutations associated with attenuation. In C57BL/6 IFNAR⁻/⁻ mice, the attenuated strain exhibited markedly reduced virulence and viral loads while inducing robust virus-specific IgG, neutralizing antibody, and cellular immune responses. Notably, immunization with the attenuated strain conferred complete protection against lethal challenge with heterologous SFTSV genotypes. Genomic analysis revealed nonsynonymous mutations in the RNA-dependent RNA polymerase (RdRp), glycoprotein, and NSs genes, implicating alterations in viral replication, entry, and immune evasion. Collectively, these findings demonstrate that serial cell-culture passage can generate attenuated SFTSV strains that retain strong immunogenicity and cross-protective efficacy, supporting their potential as live-attenuated vaccine candidates for SFTS. Full article

Avian influenza, a disease caused by avian influenza virus (AIV), mainly infects birds but can also infect mammals, which poses a serious threat to public health. Therefore, thorough understanding of its pathogenic mechanism and the identification of antiviral targets are essential for the prevention, control, and treatment of AIV. The polymerase acidic protein (PA) is a core component of the viral RNA-dependent RNA polymerase complex and plays a central role in viral transcription through its cap-snatching activity during early infection. We employed a multi-omics approach combining transcriptome analysis with PA interaction proteomics to characterize host responses during AIV infection and explore the PA–host interaction network. Transcriptomics revealed a polarized host response marked by activated translation-related processes, mitochondrial energy metabolism, and innate immune signaling, alongside broad suppression of nuclear transcriptional regulation and cell cycle pathways. Immunoprecipitation–mass spectrometry identified host proteins associated with PA that were enriched in RNA metabolism, ribosome biogenesis, and protein homeostasis. Integrative analysis of transcriptomic and interactome data, along with protein–protein interaction network analysis, prioritized a subset of high-confidence PA-interacting host factors. Among these, ribosomal protein RPS27A was validated to interact with PA and to support viral replication during early infection in this study. Full article

Cyclin-dependent kinase 1 (CDK1) regulates multiple cellular processes that HSV-1 can exploit to promote its own replication, particularly during the early steps of lytic infection. We investigated whether CDK1 inhibition disrupts immediate-early (IE) gene expression and analyzed the host phosphoproteome early in infection to identify putative host factors and mechanisms that facilitate HSV-1 IE gene expression and are controlled by CDK1. Human foreskin fibroblasts (HFFs) were pre-treated with a CDK1 inhibitor and showed a 1000-fold reduction in HSV-1 replication and significant reductions in IE mRNAs and protein levels at 4 hpi. We characterized cells after CDK1 inhibition and HSV-1 infection at 3 hpi by tandem mass spectrometry and identified >5500 phosphopetides (~2600 proteins), analyzing differential phosphorylation and protein–protein interactions. We validated CDK1 inhibition by detecting phosphorylation-specific decreases in known CDK1 substrates, as well as Robust Kinase Activity Inference. Rank- and network-based analyses of our dataset highlighted several candidate proteins, linking their CDK-directed phosphorylation to HSV-1 IE gene expression. Notably, the C-terminal domain of the large subunit of RNA polymerase II (RNAPII), POLR2A, is extensively phosphorylated, and its phosphorylation is significantly reduced upon CDK1 inhibition during viral infection. Taken together, these data support a model in which CDK1 activity maintains a transcriptionally permissive cellular state required for efficient HSV-1 IE gene expression. Our data suggest that when CDK1 is pharmacologically inhibited, key transcriptional facilitators are dysregulated, impairing viral transcription and replication. Full article

Flaviviruses are arthropod-borne RNA viruses associated with significant human diseases globally. There are no effective direct-acting antivirals approved to treat these viral infections. Given its critical role in viral replication, the RNA-dependent RNA polymerase (RdRp) is a logical target for antiviral drug development. Remdesivir (formerly GS-5734), a 1′-cyano modified C-adenosine monophosphate prodrug, was the first US Food and Drug Administration (FDA) approved antiviral for coronavirus disease 2019 (COVID-19) and was also shown to inhibit flavivirus replication. GS-7682, a 4′-cyano modified C-adenosine prodrug, exhibits a broad-spectrum antiviral activity. Here, we determined the anti-flavivirus potency of both remdesivir and GS-7682 and characterized their active triphosphate forms, GS-443902 and GS-646939, respectively, against a panel of purified flavivirus RdRps. These include dengue, Japanese encephalitis, West Nile, yellow fever, and Zika. Enzyme kinetics demonstrate efficient RNA incorporation of GS-443902 and GS-646939. GS-646939 acts as an immediate chain terminator. Conversely, GS-443902 acts through a template-dependent inhibition mechanism by impeding the incorporation of the complementary UTP. Both mechanisms correlate with anti-flavivirus activity, although remdesivir is generally superior. The data demonstrate that immediate chain termination is not necessarily a preferred mechanism of action of nucleotide analogs. Template-dependent inhibition should also be considered, especially for viruses lacking intrinsic proofreading activities. Full article

Influenza A viruses remain a major public health threat due to their high mutation rates, antigenic variability, and the emergence of resistance to current antivirals, underscoring the need for novel therapeutic options. Natural compounds rich in polyphenols and flavonoids have attracted increasing attention as potential broad-spectrum antiviral agents. In this study, the activity of Rhus coriaria L. water extract against Influenza A virus in BEAS-2B human bronchial epithelial cells was investigated. Cell viability assay identified non-cytotoxic concentrations, up to 0.1 mg/mL, which were used in infection experiments. Viral replication was assessed at multiple levels by quantitative real-time PCR, western blotting, immunofluorescence and tissue culture infectious dose 50% (TCID₅₀). Treatment with R. coriaria extract resulted in a dose-dependent and statistically significant reduction of viral load. The extract decreased mRNA levels of Hemagglutin (HA), Neuraminidase (NA) and Matrix protein 2 (M2). Consistently, western blot analysis showed a decrease in major viral proteins HA, Nucleoprotein (NP), Matrix protein 1 (M1) and Polymerase Acidic protein (PA). Confocal images revealed a marked reduction in HA and PA signals, results that are statistically significant according to quantitative fluorescence evaluation. The convergence of results obtained through independent methodologies at both the transcriptional and protein levels highlight the robustness of the findings. These data provide the experimental evidence that Rhus coriaria interferes with influenza A virus replication in airway epithelial cells and support its further investigation as a promising phytochemical platform for the development of novel anti-influenza strategies. Full article

Illumina sequencing of segment 2, which encodes the polymerase basic subunit 2 (PB2) of the RNA-dependent RNA polymerase of a divergent amnoonvirus recently detected in tissues of Nile tilapia farmed in Egypt, revealed the presence of two genetic variants of the same virus: AmnoonvirusEGY1F and -H. The phylogenetic and genetic analyses presented in this study support the inclusion of both variants in the genus Tilapinevirus, family Amnoonviridae, order Articulavirales. The Egyptian strains formed distinct, well-supported clades in both nucleotide- and amino acid-based trees, showing a notable divergence from unclassified amnoonviruses and clustered with members of the genus Tilapinevirus. Within the genus Tilapinevirus, both Egyptian strains were divergent from all tilapia lake virus (TiLV) strains, whose full RNA segment 2 sequences are available in public databases, as well as the newly isolated Tilapinevirus poikilos from the fancy-tailed guppy. The Egyptian strains were also divergent from TiLV strains identified in Israel and Lake Victoria. Although the PB2 proteins of AmnoonvirusEGY1F and -H exhibited striking similarity, several mutations were detected that altered the sequence of their antigenic cell epitopes. Some of these mutations in the AmnoonvirusEGY1H strain were predicted to affect PB2-encoded functions. Collectively, findings of this study aid in the growing understanding of viral diversity and PB2 evolution in emerging amnoonviruses, particularly the role of amino acid substitutions in affecting the encoded protein structure, function, and immunogenicity. Full article

Pleurotus ostreatus, a globally cultivated oyster mushroom, is susceptible to viral infections that threaten yield and quality. This study reports the identification and characterization of three novel viruses from a symptomatic P. ostreatus strain K3: Pleurotus ostreatus deltaflexivirus 2, 3, and 4 (PoDFV2, PoDFV3, PoDFV4). Complete genome sequencing revealed that they are single-stranded, positive-sense RNA viruses with lengths of 7809 nt, 7771 nt, and 7786 nt, encoding 5, 2, and 4 open reading frames (ORFs), respectively. The largest open reading frame (ORF1) encodes a putative replication-associated polyprotein (RP) containing three conserved domains—viral RNA methyltransferase (Mtr), viral RNA helicase (Hel), and RNA-dependent RNA polymerase (RdRp). Based on genomic sequence analysis, multiple sequence alignments, and phylogenetic analysis, PoDFV2–4 were identified as novel viruses of the genus Deltaflexivirus within the family Deltaflexiviridae. PoDFV2–4 had no significant effects on mycelial growth rate, plate mycelial biomass, or laccase activity. However, they significantly inhibited mycelial cellulase activity and resulted in malformed fruiting bodies, as well as a substantial reduction in yield. Full article