Search Results (215)

PPV5 NS1 is a nonstructural and multifunctional protein comprising helicase and nuclease domains. The helicase domain contains conserved motifs from superfamily 3 helicases, including Walker A, Walker B, Motif B’, Motif C, and Box VII, whereas the nuclease domain consists of glutamate, a HUH motif, lysine, and tyrosine. In Mexico, the reported prevalence of PPV5 is higher than in other countries, with notable amino acid differences compared with pathogenic PPVs. This study compares the helicase and nuclease domains from a full-length PPV5 NS1 sequence with porcine parvovirus 1 (PPV1) and canine parvovirus (CPV) to characterize the protein further and perform three-dimensional (3D) modeling using bioinformatic tools, including solvent-accessible surface area (SASA) and electrostatic potential assessments. The main findings highlight the ATP-binding pocket, showing electrostatic values in PPV5 that contrast with PPV1 and CPV. The electrostatic potential 3D models suggest those differences involve non-conserved regions. In particular, the PPV5 Box VII surface is predominantly negative due to a glutamate substitution at position 7. In the nuclease domain, the interaction with Mg²⁺ differs between PPV5 and pathogenic PPV. The electrostatic findings suggest that these differences may have functional implications for both domains, but confirmation must be completed with functional assays. 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

Dengue infection remains a major global health concern, with a subset of patients progressing from self-limited dengue fever to severe disease characterised by plasma leakage, shock, and organ dysfunction. The dengue non-structural protein 1 (NS1), a multifunctional glycoprotein expressed on infected cells and secreted into circulation, has emerged as a key mediator linking viral infection to immune-driven vascular pathology. This review synthesises experimental, animal, and human clinical evidence on NS1-driven immunopathogenesis, focusing on mechanisms leading to endothelial dysfunction and increased vascular permeability. NS1 modulates the complement system in a context-dependent manner, contributing to immune evasion by inhibiting terminal complement complex formation, while also promoting antibody-dependent complement activation associated with severe disease. Additionally, NS1 directly disrupts endothelial barrier integrity through disruption of adherens and tight junction architecture, Ang-2/Tie2 imbalance, activation of RhoA/ROCK (RhoA/Rho-associated coiled-coil-containing protein kinase) signalling, and enzymatic degradation of the endothelial glycocalyx, with further amplification through inflammatory mediators. In addition, evidence shows that NS1 activates innate immune signalling, perturbs platelet biology and haemostasis, and forms pro-inflammatory complexes with lipoproteins. Moreover, anti-NS1 antibodies may be both protective and pathogenic. Collectively, these data position NS1-linked pathways as rational targets for adjunctive therapies and next-generation vaccines aimed at preventing vascular leakage and severe dengue infection. 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

Senecavirus A (SVA) has rapidly emerged as a globally distributed swine pathogen, with clinical signs mimicking vesicular diseases such as Foot-and-Mouth Disease, posing challenges for timely detection and control. Here, we analyzed 329 complete SVA genomes spanning multiple continents to provide a comprehensive view of its evolutionary dynamics, recombination patterns, haplotype diversity, and global dissemination. Phylogenetic analyses revealed two major lineages: Lineage 1, consisting mainly of early strains from the United States before 2007, and Lineage 2, which emerged post-2007 and subsequently spread across the Americas and East Asia. Recombination was confined to Lineage 2 and concentrated in nonstructural regions, particularly 2C, highlighting intra-lineage genetic exchange as a driver of recent diversification. Haplotype analysis of the 3AB gene identified 170 distinct haplotypes, revealing a star-like network structure consistent with rapid population expansion from a central ancestral variant, while secondary branches reflect ongoing regional diversification. Despite this high genetic variation, genome-wide dN/dS ratios remained below one, and purifying selection was strongest in the N-terminal domains of structural and nonstructural proteins, indicating functional constraints that maintain viral fitness. Time-scaled phylogenetic reconstruction and Bayesian Skyline analysis revealed rapid lineage diversification and a marked increase in effective population size in the early 2010s. Phylogeographic inference further identified repeated introductions from the Americas into East Asia, likely facilitated by swine trade and other anthropogenic factors. Collectively, SVA evolution is driven by frequent mutation and intra-lineage recombination yet constrained by pervasive purifying selection, generating extensive genetic diversity while maintaining functional integrity, with implications for genomic surveillance and targeted control. Full article

Non-structural protein 1 (NS1) of influenza A virus is a multifunctional virulence factor and represents a promising anti-influenza target, considering its conserved and druggable structure. As antiviral target, NS1-RNA-binding domain (RBD) remains unexplored, despite its critical role in replication. In this study, we applied a “Map-and-Mutate” strategy to identify and functionally validate highly conserved and druggable regions within the NS1-RBD. Using large-scale sequence alignments and structural characterization, we integrated conservation and druggability analyses to predict conserved druggable pockets and top-ranked hot spots, mutate the five most promising residues (L15, W16, R19, R35, and L43) and study their impact on viral fitness. In vitro, the mutations W16 and R35 caused most significant reduction in viral fitness; however, L15 and R19 also impaired replication. Combined mutations involving W16 and either L15 or L43 exerted a cumulative effect, reducing viral replication, hemagglutination titers and neuraminidase activity. This study demonstrates that most residues identified and investigated using the “Map-and-Mutate” strategy negatively impact viral fitness, underscoring the approach’s value in pinpointing novel antiviral targets. Together with our prior research, this study reinforces the importance of NS1 as a promising antiviral target, providing a rationale for designing and developing therapies with a higher resilience to viral resistance. Full article

Retinoic acid-inducible gene I (RIG-I) is a crucial pattern recognition receptor for detecting viral RNA and initiating an immune response against influenza A viruses (IAVs). The activation of RIG-I in mammalian cells requires ubiquitination by two E3 ubiquitin ligases: TRIM25 and RIPLET. Using dual luciferase assays, we demonstrate that duck RIPLET enhances the activation of RIG-I, driving the IFN-β promoter activity in chicken DF-1 fibroblasts. qPCR analyses show that the co-expression of duck RIG-I and RIPLET significantly upregulates key immune genes and reduces viral RNA transcripts in DF-1 cells challenged with low pathogenic avian influenza (LPAI) H6N2. Co-immunoprecipitation and confocal microscopy studies suggest the interaction and confirm the colocalization of duck RIG-I and RIPLET in the cytoplasm. Further, we show that the non-structural protein 1 (NS1) of IAV, known for its role in immune evasion, suppression, and pathogenicity, from five different strains of IAV (PR8, BC500, CA431, D4AT, and VN1203) can all inhibit duck RIPLET activation of RIG-I, with NS1 from avian strains showing the greatest decrease in IFN-β promoter activity in chicken DF-1 cells. Overall, our research provides valuable insight into the E3 ubiquitin ligases required for RIG-I activation and susceptibility of this pathway to IAV interference across species. Full article

The detection rate of Porcine Reproductive and Respiratory Syndrome Virus type 1 (PRRSV-1) in China has been increasing, with its growing genetic diversity and evolving pathogenicity posing significant challenges to disease control. In this study, a novel PRRSV-1 strain, designated GD18-2, was identified from a pig farm in Guangdong Province that experienced an outbreak despite vaccination with a PRRSV-2 vaccine. Whole-genome sequencing indicated that the GD18-2 strain possesses a genome length of 14,932 bp and exhibits 81.4% to 83.9% nucleotide identity with classical PRRSV-1 strains. Phylogenetic analyses based on both the complete genome and the ORF5 gene indicated that GD18-2 belongs to a distinct, new lineage. A unique amino acid deletion (positions 306–357) was identified in the non-structural protein Nsp2, along with specific mutations within the hypervariable regions of the structural proteins GP3 and GP4. Pathogenicity assessment demonstrated that GD18-2 induced fever, respiratory symptoms, and mortality in piglets. In pregnant sows, it caused reproductive failure (abortion, stillbirth, weak piglets) and was capable of vertical transmission via the placenta. This study highlights the emergence of a PRRSV-1 strain with a unique genetic background and high pathogenicity in southern China, underscoring the necessity for enhanced molecular epidemiological surveillance and updated control strategies. Recombination analysis using RDP4 revealed no significant recombination events in GD18-2. Full article

Background: β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitors demonstrated amyloid-lowering efficacy but failed in phase II/III clinical trials due to adverse effects and limited disease-modifying outcomes. This study employed an integrated network pharmacology and molecular docking approach to quantitatively elucidate the multitarget mechanisms of 4 (phase II/III) discontinued BACE1 inhibitors (Verubecestat, Lanabecestat, Elenbecestat, and Umibecestat) and the preclinical compound AM-6494 in Alzheimer’s disease (AD). Methods: Drug-associated targets were intersected with AD-related genes to construct a protein–protein interaction (PPI) network, followed by topological analysis to identify hub proteins. Gene Ontology (GO) and KEGG pathway enrichment analyses were performed using statistically significant thresholds (p < 0.05, FDR-adjusted). Molecular docking was conducted using AutoDock Vina to quantify binding affinities and interaction modes between the selected compounds and the identified hub proteins. Results: Network analysis identified 10 hub proteins (CASP3, STAT3, BCL2, AKT1, MTOR, BCL2L1, HSP90AA1, HSP90AB1, TNF, and MDM2). GO enrichment highlighted key biological processes, including the negative regulation of autophagy, regulation of apoptotic signalling, protein folding, and inflammatory responses. KEGG pathway analysis revealed significant enrichment in the PI3K–AKT–MTOR signalling, apoptosis, and TNF signalling pathways. Molecular docking demonstrated strong multitarget binding, with binding affinities ranging from approximately −6.6 to −11.4 kcal/mol across the hub proteins. Umibecestat exhibited the strongest binding toward AKT1 (−11.4 kcal/mol), HSP90AB1 (−9.5 kcal/mol), STAT3 (−8.9 kcal/mol), HSP90AA1 (−8.5 kcal/mol), and MTOR (−8.3 kcal/mol), while Lanabecestat showed high affinity for AKT1 (−10.6 kcal/mol), HSP90AA1 (−9.9 kcal/mol), BCL2L1 (−9.2 kcal/mol), and CASP3 (−8.5 kcal/mol), respectively. These interactions were stabilized by conserved hydrogen bonding, hydrophobic contacts, and π–alkyl interactions within key regulatory domains of the target proteins, supporting their multitarget engagement beyond BACE1 inhibition. Conclusions: This study demonstrates that clinically failed BACE1 inhibitors engage multiple non-structural regulatory proteins that are central to AD pathogenesis, particularly those governing autophagy, apoptosis, proteostasis, and neuroinflammation. The identified ligand–hub protein complexes provide a mechanistic rationale for repurposing and optimization strategies targeting network-level dysregulation in Alzheimer’s disease, warranting further in silico refinement and experimental validation. Full article

Dengue fever remains a major mosquito–borne disease worldwide, with over 400 million infections annually and a high risk of severe complications such as dengue hemorrhagic fever. The disease is prevalent in tropical and subtropical regions, where population density and limited vector control accelerate transmission, making early and reliable diagnosis essential for outbreak prevention and disease management. Conventional diagnostic methods, including virus isolation, reverse transcription polymerase chain reaction (RT–PCR), enzyme–linked immunosorbent assays (ELISA), and serological testing, are accurate but often constrained by high cost, labor–intensive procedures, centralized laboratory requirements, and delayed turnaround times. This review examines current dengue diagnostic technologies by outlining their working principles, performance characteristics, and practical limitations, with emphasis on key target analytes such as viral RNA; nonstructural protein 1 (NS1), including DENV–2 NS1; and host antibodies. Diagnostic approaches across commonly used biofluids, including whole blood, serum, plasma, and urine, are discussed. Recent advances in biosensing technologies are reviewed, including optical, electrochemical, microwave, microfluidic, and CRISPR–based platforms, along with the integration of artificial intelligence for data analysis and diagnostic enhancement. Overall, this review highlights the need for accurate, scalable, and field–deployable diagnostic solutions to support early dengue detection and reduce the global disease burden. Full article

The four serotypes of dengue virus (DENV), types 1 to 4 (DENV-1 to DENV-4), exhibit approximately 60% identity in the encoded amino acid residues of viral proteins. Reverse transcription of RNA extracted from patient serum specimens followed by PCR amplification with serotype-specific probes is the current standard technique for DENV serotyping. However, this method is time- and cost-consuming, and rapid detection systems with low cost are desirable. Previously, we developed a prototype serotype-specific immunochromatography system. That system was composed of four strips with four corresponding distinct sample buffers, each specifically detecting a single DENV serotype. In the present study, we improved this system by combining pairs of strips into one lateral-flow cassette each, providing DENV-1 and DENV-2 detection in one device and DENV-3 and DENV-4 detection in a second device; this strategy successfully reduced the required sample volume. Furthermore, we were able to adjust the composition of the sample buffers such that a single sample buffer sufficed for all four DENV serotype detection reactions, allowing much easier handling of the devices. Evaluation of this new device against laboratory and clinical DENV isolates and clinical specimens from DENV-infected individuals showed sensitivity that was comparable to that of our previous version, yielding serotype specificity of 100%. These new devices are expected to be of use in the clinical setting, accelerating both prospective and retrospective epidemiological studies. Full article

Background: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, remains a leading cause of viral encephalitis. Current management is largely supportive, with no specific antivirals. This study evaluated the antiviral efficacy and mechanism of action of CC-90009 against JEV in vitro and in vivo. Methods: Five targeted protein degraders (TPDs) were screened for anti-JEV activity in the human neuroblastoma cell line SH-SY5Y. Time-of-addition, binding, and endocytosis assays were used to delineate the phase of action of CC-90009, a cereblon (CRBN) E3 ligase modulator (CELMoD) and molecular glue degrader. Small interfering RNA knockdown and co-immunoprecipitation (Co-IP) confirmed targets essential for its antiviral effects. The broad-spectrum activity of CC-90009 against other mosquito-borne viruses was also evaluated. In vivo efficacy was tested in a murine JEV model. Results: Of the five TPDs tested, only CC-90009 significantly inhibited JEV infection in SH-SY5Y cells, acting during both viral entry and post-entry phases without reducing adsorbed or internalised virions. CC-90009 reduced JEV RNA and non-structural protein accumulation. Knockdown of G1-to-S phase transition 1 (GSPT1), a key target of CC-90009, suppressed JEV infection and translation; Co-IP confirmed GSPT1 interaction with JEV non-structural protein 5 (NS5). CC-90009 disrupted JEV translation and replication by inducing proteasomal degradation of the GSPT1/NS5 complex, further demonstrating its broad-spectrum antiviral activity through the effective inhibition of West Nile virus and chikungunya virus. In vivo, it protected mice from JEV-induced mortality, reducing viral load, antigen levels, and brain pathology. Conclusions: CC-90009 exerts potent anti-JEV activity both in vitro and in vivo by inducing proteasomal degradation of the GSPT1/NS5 complex, thereby disrupting viral translation and replication. This targeted protein degradation strategy represents a novel host-directed antiviral approach with promising therapeutic potential against mosquito-borne viral encephalitis. Full article

Multiplexed detection of arboviruses using a 4 × 4 Si-based electrolyte-gated transistor (EGT) array functionalized with specific aptamers has been investigated. The Si-based EGTs were fabricated using conventional Si microfabrication processes. The EGTs showed excellent intrinsic electrical characteristics, including a low threshold voltage of 0.8 V, a sub-threshold swing of 75 mV/dec, and a gate leakage of <10 pA, ensuring uniform device performance with low device-to-device variation. Aptamers specific to the yellow fever virus nonstructural protein 1 (YF), dengue virus nonstructural protein 1 (DN), and chikungunya virus envelope protein 2 (CHK) were functionalized on EGT arrays to evaluate individual and multiplexed detection. In individual-target detections, concentration-dependent negative shifts in threshold voltage were observed, and relevant limits of detection (LOD) as low as 38.6 pg/mL, 95.2 pg/mL, and 1.6 ng/mL were extracted for YF, DN, and CHK, respectively. In multiplexed detections, sensitivities decreased and variations increased relative to the individual responses, resulting in higher LODs. The extracted LODs were 0.2 ng/mL, 0.6 ng/mL, and 2.8 ng/mL for YF, DN, and CHK, respectively, which are lower than those reported for other methods. These results suggest that Si-based EGT arrays are promising as a scalable, low-cost, and highly sensitive biosensing platform for multiplexed arbovirus detection and point-of-care diagnostics. Full article

Background: Latvia is one of the most endemic tick-borne encephalitis (TBE) countries in Europe. However, assessing TBE seroprevalence and true infection rates has been challenging. Current diagnostics cannot reliably distinguish between immune responses induced by natural infection from those induced by vaccination, especially in TBE endemic countries with recommended immunisation programmes. A recently developed ELISA targeting antibodies against TBEV non-structural protein 1 (NS1) offers improved specificity for natural infection and can differentiate among three clinically relevant TBEV subtypes. Methods: We conducted a cross-sectional TBEV seroprevalence study in the Latvian population during 2019–2022, consisting of two pools: 1020 residents living in different regions of Latvia and 200 random Latvian Biobank blood samples. We used the standard anti-TBEV IgG ELISA (VIDITEST, VIDIA, Czech Republic) for the screening and detection of TBEV (whole virus) IgG antibodies and the newly established research-use anti-TBEV NS1 IgG ELISA for the detection of subtype-specific TBEV NS1 IgG antibodies against three virus subtypes: European, Siberian and Far Eastern. Results: The total TBEV seroprevalence among 1020 residents was 39.7%, representing all age cohorts of the population from all regions of Latvia. In total, 33.4% of the enrolled population were vaccinated against TBE with at least one dose of the TBE vaccine. Among the unvaccinated population, 16.3% had positive TBEV-specific IgG antibodies by standard ELISA. On the contrary, NS1-specific antibodies, indicating past natural infection, were detected in only 4.3% of the overall study population. Subtype differentiation revealed infections from all three major TBEV subtypes present in Latvia. Conclusions: In conclusion, this population-based study highlights a high risk of TBE in Latvia, with substantial seroprevalence even among unvaccinated individuals. The NS1-based ELISA enhances the accuracy of TBE surveillance and offers important clinical utility by facilitating more reliable diagnosis and case classification, regardless of vaccination status. Full article

Porcine deltacoronavirus (PDCoV) is an emerging pathogen that causes severe, often fatal, diarrhea in suckling piglets and has zoonotic potential. Its nonstructural protein 12 (Nsp12), functioning as the RNA-dependent RNA polymerase (RdRp), is a central component of the viral replication–transcription complex and a critical target for host antiviral mechanisms. Here, we identified eukaryotic elongation factor 1 gamma (eEF1G) as a host interactor of PDCoV Nsp12 by immunoprecipitation-coupled mass spectrometry in IPEC-J2 cells. This interaction was confirmed by co-immunoprecipitation, pull-down assays, and confocal microscopy. Functional analyses involving siRNA knockdown and overexpression of eEF1G, combined with viral titration, strand-specific real-time quantitative PCR, and RNA immunoprecipitation assays, demonstrated that eEF1G directly binds to Nsp12. Knockdown of eEF1G significantly enhanced viral replication and increased negative-stranded RNA synthesis, whereas overexpression did not affect viral proliferation. Furthermore, eEF1G was found to bind PDCoV genomic RNA and competitively disrupt the interaction between Nsp12 and viral RNA, thereby impairing RdRp activity. Our results indicate that eEF1G acts as a novel host restriction factor that inhibits PDCoV replication by competing with Nsp12 for genomic RNA binding, ultimately blocking negative-stranded RNA synthesis. This study unveils a new antiviral mechanism and highlights a potential target for developing interventions against PDCoV. Full article