Search Results (107)

Human non-polio enteroviruses (NPEVs) cause a plethora of infections in humans, ranging from mild to severe neurological diseases including aseptic meningitis. NPEVs are the leading cause of aseptic meningitis in both children and adults worldwide. In Russia, reports of NPEV infections have surged, especially in the post-COVID era starting in 2022, with elevated infection rates into 2023. A comprehensive examination of the whole genome is crucial for understanding the evolution of NPEV genes and for predicting potential outbreaks. This study focused on identifying the circulating NPEV strains in the Ural Federal District and Western Siberia, using Sanger sequencing and next-generation sequencing (NGS) methodologies. Biological samples were collected from (n = 225) patients diagnosed with aseptic meningitis. Bioinformatics analysis targeted the nucleotide sequences of the major capsid protein (partial VP1) gene fragment, and the assembly of whole NPEV genomes. A total of 159 NPEVs were characterized, representing 70.7% of the collected samples. The main capsid variants forming the predominant genotypic profile included E30 (n = 39, 24.3%), E6 (n = 31, 19.3%), and CVA9 (n = 25, 15.6%). Using NGS, we successfully assembled 13 whole genomes for E6, E30, EV-B80, CVA9, CVB5, E11, and EV-A71 and 3 partial genomes for E6 and EV-B87. This molecular-genetic analysis provides contemporary insights into the genotypic composition, circulation patterns, and evolutionary dynamics of the dominant NPEV associated with aseptic meningitis in the Ural Federal District and Western Siberia. The laboratory-based monitoring and epidemiological surveillance for genetic changes and evolutionary studies are important for improving prevention and healthcare. Full article

Coxsackievirus B3 (CVB3) is a picornavirus that causes systemic inflammatory diseases including myocarditis, pericarditis, pancreatitis, and meningoencephalitis. We have previously reported that CVB3 induces mitochondrial fission and mitophagy while inhibiting lysosomal degradation by blocking autophagosome-lysosome fusion. This promotes the release of virus-laden mitophagosomes from host cells as infectious extracellular vesicles (EVs), enabling non-lytic viral egress. Transient receptor potential vanilloid 1 (TRPV1), a heat and capsaicin-sensitive cation channel, regulates mitochondrial dynamics by inducing mitochondrial membrane depolarization and fission. In this study, we found that TRPV1 activation by capsaicin dramatically enhances CVB3 egress from host cells via EVs. Released EVs revealed increased levels of viral capsid protein VP1, mitochondrial protein TOM70, and fission protein phospho-DRP1. Moreover, these EVs were enriched in heat shock protein HSP70, suggesting its role in facilitating infectious EV release from cells. Furthermore, TRPV1 inhibition with capsazepine and SB-366791 significantly reduced viral infection in vitro. Our in vivo studies also found that SB-366791 significantly mitigates pancreatic damage and reduces viral titers in a mouse model of CVB3 pancreatitis. Given the lack of understanding regarding factors that contribute to diverse clinical manifestations of CVB3, our study highlights capsaicin and TRPV1 as potential exacerbating factors that facilitate CVB3 dissemination via mitophagy-derived EVs. Full article

Noroviruses are a leading cause of acute gastroenteritis worldwide, with a particularly high burden among children under five years of age. We analyzed nationwide surveillance data from EnterNet-Korea collected between 2019 and 2024—covering both the pre- and post-COVID-19 pandemic periods—to assess norovirus detection rates and genotype distribution. Noroviruses were detected by RT-qPCR, and dual genotyping of capsid (G) and polymerase (P) types was performed by sequencing the ORF1–2 junction region. Among the 67,159 specimens tested, 8.4% (n = 5652) were norovirus-positive, with the highest prevalence observed in children aged 0–5 years (19.9%). In this age group, genotyping was successful in 72.4% (2633/3635) of positive cases, identifying 30 distinct genotypes (GI: 9; GII: 21). The most common strains were GII.4[P31] (38.1%), GII.4[P16] (27.1%), and GII.2[P16] (10.4%), with substantial year-to-year variation. Notably, the emergence of GII.17[P17] in late 2024 indicates shifting genotype dynamics, underscoring the need for strengthened surveillance and reconsideration of vaccine strain selection. To our knowledge, this is the first nationwide dual-typing study in Korea encompassing the COVID-19 pandemic era. These findings provide essential baseline data for integration into global surveillance systems and inform future vaccine development and public health strategies. Full article

JC virus (JCV) replicates within the nuclei of glial cells in the human brain and causes progressive multifocal leukoencephalopathy. JCV possesses a small, circular, double-stranded DNA genome, divided into early and late protein-coding regions. The non-coding control region (NCCR) functions bidirectionally for both early and late genes, and the agnogene is located downstream of TCR and upstream of three capsid proteins in the late region. Previously, in cell culture systems, we demonstrated that these capsid proteins accumulate in intranuclear domains known as promyelocytic leukemia nuclear bodies (PML-NBs), where they assemble into virus-like particles (VLPs). To investigate the agnogene’s function, VLPs were formed in its presence or absence, and differential gene expression was analyzed using microarray technology. The results revealed altered expression of histone-modifying enzymes, including methyltransferases (EHMT1, PRMT7) and demethylases (KDM2B, KDM5C, KDM6B), as well as various kinases and phosphatases. Notably, CTDP1, which dephosphorylates the C-terminal domain of an RNA polymerase II subunit, was also differentially expressed. The changes were predominant in the presence of the agnogene. These findings indicate that the agnogene and/or its protein product likely influence epigenetic regulation associated with PML-NBs, which may influence cell cycle control. Consistently, in human brain tissue, JCV-infected glial cells displayed maintenance of a diploid chromosomal complement, likely through G2 arrest. The precise mechanism of this, however, remains to be elucidated. Full article

Human noroviruses (HuNoVs) stand as the primary cause of acute viral gastroenteritis outbreaks worldwide, particularly impacting children under the age of five. In Russia, reports of norovirus gastroenteritis have surged, especially in the post-COVID-19 era starting in 2022, with elevated infection rates reported into 2024. These viruses exhibit significant mutational variability, leading to the emergence of recombinant strains that can evade immune responses. A comprehensive examination of the complete genome is crucial for understanding the evolution of norovirus genes and for predicting potential outbreaks. This research focuses on analyzing the genotypic composition of HuNoVs circulating in the Sverdlovsk region during 2024, using Sanger sequencing and next-generation sequencing (NGS). Biological samples were collected (n = 384) from patients diagnosed with norovirus infection within the region. Bioinformatics analysis targeted the nucleotide sequences of the ORF1/ORF2 fragment and the assembly of complete genomes for the GII.4 and GII.7 genotypes. In total, 220 HuNoVs were characterized, representing 57.3% of the collected samples. The main capsid variants forming the predominant genotypic profile included GII.4 (n = 88, 40%), GII.7 (n = 86, 39%), and GII.17 (n = 14, 6%). Using NGS, we successfully assembled 8 out of 10 complete genomes for noroviruses GII.4[P16] and GII.7[P7]. Non-synonymous substitutions appeared at amino acid sites corresponding to the subdomains of VP1 in these strains. This molecular–genetic analysis provides contemporary insights into the genotypic composition, circulation patterns, and evolutionary dynamics associated with the dominant genovariants GII.4[P16] and GII.7[P7]. Full article

Sindbis virus (SINV), a prototype of the Alphavirus genus (family Togaviridae), is a globally distributed arbovirus causing febrile rash and debilitating arthritis in humans. Viral structural proteins—capsid (C), E1, and E2—are fundamental to the virion’s architecture, mediating all stages from assembly to host cell entry and pathogenesis, thus representing critical targets for study. This review consolidates the historical and current understanding of SINV structural biology, tracing progress from early microscopy to recent high-resolution cryo-electron microscopy (cryo-EM) and X-ray crystallography. We detail the virion’s precise T = 4 icosahedral architecture, composed of a nucleocapsid core and an outer glycoprotein shell. Key functional roles tied to protein structure are examined: the capsid’s dual capacity as a serine protease and an RNA-packaging scaffold that interacts with the E2 cytoplasmic tail; the E1 glycoprotein’s function as a class II fusion protein driving membrane fusion; and the E2 glycoprotein’s primary role in receptor binding, which dictates cellular tropism and serves as the main antigenic target. Furthermore, we connect these molecular structures to viral evolution and disease, analyzing how genetic variation among SINV genotypes, particularly in the E2 gene, influences host adaptation, immune evasion, and the clinical expression of arthritogenic and neurovirulent disease. In conclusion, the wealth of structural data on SINV offers a powerful paradigm for understanding alphavirus biology. However, critical gaps persist, including the high-resolution visualization of dynamic conformational states during viral entry and the specific molecular determinants of chronic disease. Addressing these challenges through integrative structural and functional studies is paramount. Such knowledge will be indispensable for the rational design of next-generation antiviral therapies and broadly protective vaccines against the ongoing threat posed by SINV and related pathogenic alphaviruses. Full article

Super-resolution microscopy (SRM) has revolutionized our understanding of subcellular structures, including cell organelles and viruses. For human immunodeficiency virus (HIV), SRM has significantly advanced knowledge of viral structural biology and assembly dynamics. This review analyzes how SRM techniques (particularly PALM, STORM, STED, and SIM) have been applied over the past decade to study HIV structural components and assembly. By categorizing and comparing studies based on SRM methods, HIV components, and labeling strategies, we assess the strengths and limitations of each approach. Our analysis shows that PALM is most commonly used for live-cell imaging of HIV Gag, while STED is primarily used to study the viral envelope (Env). STORM and SIM have been applied to visualize various components, including Env, capsid, and matrix. Antibody labeling is prevalent in PALM and STORM studies, targeting Env and capsid, whereas fluorescent protein labeling is mainly associated with PALM and focused on Gag. A recent emphasis on Gag and Env points to deeper investigation into HIV assembly and viral membrane dynamics. Insights from SRM studies of HIV not only enhance virological understanding but also inform future research in therapeutic strategies and delivery systems, including extracellular vesicles. Full article

Background: Parvovirus B19 (B19V) can cause severe relapsing episodes of pure red cell aplasia in immunocompromised individuals, which are commonly treated with intravenous immunoglobulins (IVIGs). Few data are available on B19V intra-host evolution and the role of humoral immune selection. Here, we report the dynamics of genomic mutations and subsequent protein changes during relapsing infection. Methods: Longitudinal plasma samples from immunocompromised patients with relapsing B19V infection in the period 2011–2019 were analyzed using whole-genome sequencing to evaluate intra-host evolution. The impact of mutations on the 3D viral protein structure was predicted by deep neural network modeling. Results: Of the three immunocompromised patients with relapsing infections for 3 to 9 months, one patient developed two consecutive nonsynonymous mutations in the VP1/2 region: T372S/T145S and Q422L/Q195L. The first mutation was detected in multiple B19V IgG-seropositive follow-up samples and resolved after IgG seroreversion. Computational prediction of the VP1 3D structure of this mutant showed a conformational change in the proximity of the antibody binding domain. No conformational changes were predicted for the other mutations detected. Discussion: Analysis of relapsing B19V infections showed mutational changes occurring over time. Resulting amino acid changes were predicted to lead to a conformational capsid protein change in an IgG-seropositive patient. The impact of humoral response and IVIG treatment on B19V infections should be further investigated to understand viral evolution and potential immune escape. Full article

Avian influenza is an economically significant disease affecting poultry worldwide and is caused by influenza A viruses that can range from low to highly pathogenic strains. These viruses primarily target the respiratory, digestive, and nervous systems of birds, leading to severe outbreaks that threaten poultry production and pose zoonotic risks. The ectodomain of the avian influenza virus (AIV) matrix protein 2 (M2e), known for its high conservation across influenza strains, has emerged as a promising candidate for developing a universal influenza vaccine in a mouse model. However, the efficacy of such expression against poultry AIVs remains limited. The objective of this study was to evaluate the immunogenicity of nodavirus-like particles displaying the M2e proteins. In this study, three synthetic heterologous M2e genes originated from AIV strains H5N1, H9N2 and H5N2 were fused with the nodavirus capsid protein (NVC) of the giant freshwater prawn Macrobrachium rosenbergii (NVC-3xAvM2e) prior to immunogenicity characterisations in chickens. The expression vector pTRcHis-TARNA2 carrying the NVC-3xAvM2e gene cassette was introduced into E. coli TOP-10 cells. The recombinant proteins were purified, inoculated into one-week-old specific pathogen-free chickens subcutaneously and analysed. The recombinant protein NVC-3xAvM2e formed virus-like particles (VLPs) of approximately 25 nm in diameter when observed under a transmission electron microscope. Dynamic light scattering (DLS) analysis revealed that the VLPs have a polydispersity index (PDI) of 0.198. A direct ELISA upon animal experiments showed that M2e-specific antibodies were significantly increased in vaccinated chickens after the booster, with H5N1 M2e peptides having the highest mean absorbance value when compared with those of H9N2 and H5N2. A challenge study using low pathogenic AIV (LPAI) strain A/chicken/Malaysia/UPM994/2018 (H9N2) at 10^6.5 EID₅₀ showed significant viral load in the lung and cloaca, but not in the oropharyngeal of vaccinated animals when compared with the unvaccinated control group. Collectively, this study suggests that nodavirus-like particles displaying three heterologous M2e have the potential to provide protection against LPAI H9N2 in chickens, though the vaccine’s efficacy and cross-protection across different haemagglutinin (HA) subtypes should be further evaluated. Full article

Capsid assembly modulators (CAMs) are a novel class of antiviral agents in clinical development for the treatment of chronic hepatitis B. CAMs inhibit hepatitis B virus (HBV) replication by binding to a hydrophobic pocket, i.e., HAP pocket, between HBV capsid protein (Cp) dimer–dimer interfaces to misdirect its assembly into empty capsids or aberrant structures and designated as CAM-E and CAM-A, respectively. Because the emergence of CAM-resistant variants results in the failure of antiviral therapy, it is important to rationally design CAMs with a high barrier of resistance for development. To establish computational approaches for the prediction of Cp mutations that confer resistance to CAMs, we investigated the interaction of representative CAM-A and CAM-E compounds, BAY 41-4109 and JNJ-56136379, with wild-type and 35 naturally occurring mutations of Cp residues at the HAP pocket using molecular docking, prime molecular mechanics with generalized Born and surface area solvation (MM/GBSA) and molecular dynamics (MD) simulation methods. Out of nine publicly available HBV capsid or CpY132A hexamer structures in the protein database, molecular docking correctly predicted the resistance and sensitivity of more than 50% Cp mutations to JNJ-56136379 with structures 5D7Y and 5T2P-FA. MM/GBSA correctly predicted the resistance and sensitivity of more than 50% Cp mutations to BAY41-4109 with the structures 5E0I-BC and 5WRE-FA, and to JNJ-56136379 with the 5E0I-FA structure. Our work indicates that only the capsid or CpY132A hexamer structure bound with a CAM with similar chemical scaffold can be used for more accurately predicting the resistance and sensitivity of Cp mutations to a CAM molecule under investigation by molecular docking and/or MM/GBSA methods. Full article

Background: Pancreas disease (PD) is a serious disease in European salmonid aquaculture caused by salmonid alphavirus (SAV), of which six genotypes (SAV1–6) have been described. The use of inactivated virus and DNA PD vaccines is common in marine salmonid aquaculture and has contributed to a reduction of the occurrence of disease; however, outbreaks are still frequent. Methods: In this study, we compared the long-term protection after immunization of Atlantic salmon (Salmo salar) with three different clones of attenuated infectious SAV3. The clones were made by site-directed mutagenesis targeting the glycoprotein E2 to disrupt the viral attachment and/or nuclear localization signal (NLS) of the capsid protein to disrupt the viral suppression of cellular nuclear-cytosol trafficking. The resulting clones (Clones 1–3) were evaluated after injection of Atlantic salmon for infection dynamics, genetic stability, transmission, and protection against a subsequent SAV3 challenge. Results: Attenuated clones demonstrated reduced virulence, as indicated by lower viral RNA loads, diminished transmission to cohabitant fish, and minimal clinical symptoms compared to the virulent wild-type virus. The clones mutated in both capsid and E2 exhibited the most attenuation, observed as rapid clearing of the infection and showing little transmission, while the clone with glycoprotein E2 mutations displayed greater residual virulence but provided stronger protection, seen as reduced viral loads upon subsequent challenge with SAV3. Despite their attenuation, all viral clones caused significant reductions in weight gain. Conclusions: Despite promising attenuation and protection, this study highlights the trade-offs between virulence and immunogenicity in live vaccine design. Concerns over environmental risks, such as the shedding of genetically modified virus, necessitate further evaluation. Future efforts should optimize vaccine candidates to balance attenuation, immunogenicity, and minimal side effects. Full article

In the dynamic field of gene therapy, recombinant adeno-associated viruses (rAAVs) have become leading viral vectors due to their safety, long-term expression, and wide-ranging cell and tissue tropism. With numerous FDA approvals and commercial products underscoring their potential, there is a critical need for efficient production processes to achieve high vector titers and quality. A major challenge in rAAV production is the efficient packaging of the genome into the viral capsid, with empty or partially filled capsids often representing over 90% of the produced material. To tackle this issue, we engineered the replication and packaging proteins of an AAV (Rep) to boost their functionality and improve vector titers. We subjected a complex Rep library derived from the AAV serotypes 1–13 to directed evolution in an AAV producer cell line. After each round of selection, single clones were analyzed, showing enrichment of specific hybrid Rep domains. Comparative analysis of these selected clones revealed considerable differences in their ability to package AAV2-based viral genomes, with hybrid Rep proteins achieving up to a 2.5-fold increase in packaging efficiency compared to their parental counterparts. These results suggest that optimizing rep gene variants through directed evolution is an effective strategy to enhance rAAV production efficiency. Full article

Human sapovirus (HuSaV) is a significant cause of gastroenteritis. This study aims to analyze the evolutionary dynamics of the RNA-dependent RNA polymerase (RdRp) and capsid (VP1) genes of the HuSaV GI.1 and GI.2 genotypes between 1976 and 2020. Using bioinformatics tools such as the Bayesian phylogenetics software BEAST 2 package (v.2.7.6), we constructed time-scale evolutionary trees based on the gene sequences. Most of the recent common ancestors (MRCAs) of the RdRp region and VP1 gene in the present HuSaV GI.1 diverged around 1930 and 1933, respectively. The trees of the HuSaV GI.1 RdRp region and VP1 gene were divided into two clusters. Further, the MRCAs of the RdRp region and VP1 gene in HuSaV GI.2 diverged in 1960 and 1943, respectively. The evolutionary rates were higher for VP1 gene in HuSaV GI.1 than that in HuSaV GI.2, furthermore, were higher in GI.1 Cluster B than GI.1 Cluster A. In addition, a steep increase was observed in the time-scaled genome population size of the HuSaV GI.1 Cluster B. These results indicate that the HuSaV GI.1 Cluster B may be evolving more actively than other genotypes. The conformational B-cell epitopes were predicted with a higher probability in RdRp for GI.1 and in VP1 for GI.2, respectively. These results suggest that the RdRp region and VP1 gene in HuSaV GI.1 and GI.2 evolved uniquely. These findings suggest unique evolutionary patterns in the RdRp region and VP1 gene of HuSaV GI.1 and GI.2, emphasizing the need for a ‘One Health’ approach to better understand and combat this pathogen. Full article

During the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, the development of efficient serological tests for monitoring the dynamics of the disease as well as the immune response after illness or vaccination was critical. In this regard, low-cost and fast production of immunogenic antigens is essential for the rapid development of diagnostic serological kits. This study assessed the plant-based production of nucleoprotein (N) of SARS-CoV-2 and chimeric receptor-binding domain (RBD) of SARS-CoV-2 presented by hepatitis E virus capsid (HEV/RBD) and validation of the plant-derived proteins as diagnostic antigens for serological tests. The target proteins were expressed in and purified from Nicotiana benthamiana plants. The resulting yield of chimeric HEV/RBD protein reached 100 mg/kg fresh weight and 30 mg/kg fresh weight for N protein. The purified N protein and HEV/RBD protein were used to develop an indirect enzyme-linked immunosorbent assay (iELISA) for the detection of antibodies to SARS-CoV-2 in human sera. To validate the iELISA tests, a panel of 84 sera from patients diagnosed with COVID-19 was used, and the results were compared to those obtained by another commercially available ELISA kit (Dia.Pro D. B., Sesto San Giovanni, Italy). The performance of an HEV/RBD in-house ELISA showed a sensitivity of 89.58% (95% Cl: 75.23–95.37) and a specificity of 94.44% (95% Cl: 76.94–98.2). Double Recognition iELISA based on HEV/RBD and N protein is characterized by a lower sensitivity of 85.42% (95% Cl: 72.24–93.93) and specificity of 94.44% (95% Cl: 81.34–99.32) at cut-off = 0.154, compared with iELISA based on HEV/RBD. Our study confirms that N and fusion HEV/RBD proteins, which are transiently expressed in plants, can be used to detect responses to SARS-CoV-2 in human sera reliably. Our research validates the commercial potential of using plants as an expression system for recombinant protein production and their application as diagnostic reagents for serological detection of infectious diseases, hence lowering the cost of diagnostic kits. Full article

In recent years, plasma medicine has developed rapidly as a new interdisciplinary discipline. However, the key mechanisms of interactions between cold atmospheric plasma (CAP) and biological tissue are still in the exploration stage. In this study, by introducing the reactive molecular dynamics (MD) simulation, the capsid protein (CA) molecule of HIV was selected as the model to investigate the reaction process upon impact by reactive oxygen species (ROS) from CAP and protein molecules at the atomic level. The simulation results show that ground-state oxygen atoms can abstract hydrogen atoms from protein chains and break hydrogen bonds, leading to the destruction of the disulfide bonds, C–C bonds, and C–N bonds. Furthermore, the generation of alcohol-based groups resulting from the impact of ROS can alter the hydrophobicity of molecules and induce damage to the primary, secondary, and tertiary structures of proteins. The dosage effects on the reaction processes and products induced by CAP are also explored with varying numbers of ROS in the simulation box, and the influences on the broken C–H, N–H, and C–N bonds are discussed. In this study, the computational data suggest that severe damage can be caused to CA upon the impact of ROS by revealing the reaction processes and products. Full article