Search Results (1,051)

Avian influenza infections cause substantial economic losses in the poultry industry and raise public health concerns due to viral adaptation and cross-species transmission. The frequent antigenic drift of influenza viruses further complicates the prevention and treatment of avian respiratory infections. In this study, we generated high-affinity heavy-chain variable domain (VHH) nanobodies from naïve alpaca/camelid VHH libraries using phage display combined with H9N2 influenza A virus (IAV)-infected Madin-Darby Canine Kidney (MDCK) cells. Based on binding affinity and neutralization potential, we identified seven hemagglutinin (HA)-specific and two neuraminidase (NA)-specific VHHs. Molecular docking predicted the interaction sites of HA-specific VHHs (L1-2, L1-4, A5) and NA-specific VHHs (L1-3, L2-2), providing mechanistic insights. Notably, the three HA-specific VHHs (L1-2, L1-4, A5) showed cross-reactivity to representative HA subtypes (H1, H3, and influenza B), indicating recognition of conserved epitopes across divergent influenza strains. For the first time, these camelid nanobodies were fused to the chicken IgY Fc domain, and the expression cassette was integrated into the Saccharomyces cerevisiae genome, achieving a secretion yield of 15–20 mg/L of VHH-Fc antibodies. Experimental validation confirmed that the three HA-specific VHHs-Fc constructs effectively blocked viral infection, while the two NA-specific VHH-Fc constructs (L1-3, L2-2) inhibited NA activity, demonstrating the functional efficacy of the yeast-secreted VHH–IgY Fc platform. This novel IgY Fc fusion approach offers a scalable platform with enhanced stability, extended circulation potential, and applicability in poultry. Full article

Tick-borne encephalitis virus (TBEV) is primarily transmitted by Ixodes spp. and poses significant health risks, leading to morbidity and mortality in humans. Two of the five subtypes, Siberian and Far Eastern are known to circulate in Mongolia. In 2021, Ixodes persulcatus ticks were collected from Bulgan aimag (province) using flagging and dragging methods and subsequently screened for TBEV using PCR. Positive samples underwent sequencing using an Oxford Nanopore Technologies-based hybrid capture approach, resulting in two coding-complete TBEV genomes from separate tick pools. Phylogenetic analysis classified both genomes within the Siberian subtype, grouping them with other Mongolian sequences from I. persulcatus collected in 2014, 2020, 2021, and 2023. The study sequences, PX654173 and PX654174, showed high genetic similarity (99.9% and 99.8%, respectively) to the sequence PQ479142, obtained from I. persulcatus ticks in Selenge, Mongolia, in 2021. The estimated time to most recent common ancestor (TMRCA) of the Siberian genotype was approximately 981 CE (95% HPD: 646–1347) with the emergence of a distinct Mongolian clade of TBEV around 1888 CE (95% HPD: 1834–1934). These findings highlight the value of expanded whole-genome sequencing to improve our understanding of TBEV’s genetic diversity and evolutionary history in Central Asia. Full article

The coronavirus disease 19 pandemic disrupted pediatric respiratory infections through non-pharmaceutical interventions and altered contact patterns. Long-term comparisons across the pandemic timeline in children remain limited. In this study, we analyzed 15,657 respiratory specimens from patients ≤ 18 years at Dankook University Hospital (2007–2023) using multiplex polymerase chain reaction assays targeting 15 viruses. Age-stratified positivity rates were compared across pandemic phases. Children ≤ 6 years comprised 88.61% of the study population. Human rhinovirus showed the highest detection rate (24.06%), followed by adenovirus (12.33%), respiratory syncytial virus-subtypes A and B (RSV-A: 11.13%; RSV-B: 8.65%), human parainfluenza virus-type 3 (HPIV-3; 6.21%), human metapneumovirus (HMPV; 5.33%), and enterovirus (2018–2023; EV; 10.96%). Monthly distributions differed (p < 0.001). RSV peaked in late autumn and winter; influenza and seasonal coronaviruses in winter and spring; HMPV, HPIV-3, EV, and human bocavirus in summer and fall. Positivity declined during the pandemic, rebounding in 2023, most prominently among children aged 1–6 years (84.91%). HPIV-3 and EV increased (p < 0.001). RSV-A predominated pre-pandemic, whereas RSV-B showed a non-significant relative increase post-pandemic; no subtype differences occurred during the pandemic. Findings demonstrate pathogen-specific shifts in predominance and seasonality and support ongoing surveillance and pediatric care planning. Full article

Diagnostic testing of foot-and-mouth disease virus (FMDV) currently utilizes reverse transcription quantitative PCR (RT-qPCR) to detect the presence of viral RNA and double antibody sandwich ELISAs (DAS-ELISAs) to determine viral serotype. Serotype identification is critical to support informed vaccine selection to combat outbreaks. While DAS-ELISAs are capable of serotype identification, the test suffers from low sensitivity and requires a viral isolate for successful detection. In this study, we developed FMDV-ONTAPS: an Oxford Nanopore Technologies Amplicon P1 Sequencing protocol involving reverse transcription-PCR to amplify P1 of the FMDV genome, and Nanopore sequencing of the amplicons to provide genetic data for serotype and subtype/topotype identification. FMDV isolates representing all seven serotypes were successfully sequenced with this method. Additionally, the protocol successfully provided serotype identification from a variety of specimen matrices obtained from experimentally infected animals that included milk, serum, oral and nasal swabs, tissue suspensions, vesicular fluid, and oral fluid. The limit of detection for FMDV cell culture isolates was comparable for both sequencing and RT-qPCR detection. RT-qPCR Cq values for clinical samples evaluated ranged from 8 to 28.21. Sequencing was successful for all samples except for a single tissue suspension sample (Cq of 28.21). Identification of FMDV serotype in clinical samples is critical for effective outbreak response, and Nanopore sequencing offers a timelier and more sensitive alternative to DAS-ELISAs. Full article

Human T-cell leukemia virus type-1 (HTLV-1) is endemic to numerous regions worldwide, including Central Australia. The Australo-Melanesian subtype-C is endemic within Australia and Oceania, whereas subtype-A is the most widely distributed subtype globally. The lack of an approved vaccine highlights HTLV-1 as a neglected public health issue. To inform the development of HTLV-1 Envelope (Env)-based vaccines, we assessed anti-Env antibodies in an HTLV-1c+ cohort of First Nations individuals in Central Australia. Of the 62 plasma samples from patients with confirmed HTLV-1 serological diagnosis, 76% were positive for Env binding in ELISA, but 90% neutralized HTLV-1c pseudovirus (PSV) infection. Neutralization breadth with the capability of blocking both subtype-A and subtype-C PSV infection was identified in 100% of samples tested. Proviral load was positively associated with anti-Env response, with binding epitopes mapping to the proline-rich region of gp46-SU. Env-directed IgG showed the capacity to engage Fcγ receptors key to inducing antibody-dependent cellular cytotoxicity/phagocytosis responses. Serological response was not associated with comorbidities linked to HTLV-1c in this population (bronchiectasis, chronic kidney disease, diabetes). These findings demonstrate that potent humoral immunity arises and is sustained during HTLV-1 infection, suggesting that an Env-based vaccine displaying authentically native epitopes will be capable of recapitulating these neutralizing responses. Full article

To clarify the genetic classification, diagnostic strategies, and precision treatment pathways of steroid-resistant nephrotic syndrome (SRNS), this review systematically reviews the genetic stratification system of SRNS by integrating recent advances in genetic testing technologies and pathogenesis research. It contains the pathogenic mechanisms, diagnostic protocols, and therapeutic correlations of different genetic subtypes, while summarizing current progress and clinical challenges in gene therapy. Results indicate SRNS can be categorized into genetic (38–58%) and non-genetic/immune-mediated (40–60%). A stepwise diagnostic system comprising core proteinuria gene panel testing, whole-genome sequencing (WGS), whole-exome sequencing (WES), and supplementary multi-omics/long-range sequencing is proposed, suited for populations with “typical phenotypes and moderate genetic risk”, “atypical phenotypes and high genetic suspicion”, and “complex structural/non-coding region variants” respectively. Pathogenic mechanisms directly determine therapeutic strategies: COQ2/PDSS2 mutations respond to coenzyme Q10 suplementation, while NPHS1 mutations necessitate early renal transplantation. Adeno-associated virus (AAV)-mediated gene therapy and CRISPR-Cas editing show preclinical promise but face challenges including incomplete detection coverage and clinical translation difficulties. Genetic technologies are driving SRNS management transformation from “empirical treatment” to “mechanism-oriented precision diagnosis and therapy”. Future efforts should focus on overcoming genetic testing limitations and gene therapy translation bottlenecks to enhance diagnostic and therapeutic efficacy. Full article

Human immunodeficiency virus (HIV) remains a major global health challenge, requiring accurate diagnostic testing for early detection. Chemiluminescent immunoassay screening, particularly the Architect HIV Ag/Ab Combo assay, followed by immunoblot confirmation using INNO-LIA™ has traditionally been used in many diagnostic workflows. To address these limitations, the U.S. Centers for Disease Control and Prevention (CDC) recommends the use of an HIV-1/2 antibody differentiation immunoassay, such as the Geenius HIV-1/2 Supplemental Assay, as part of the confirmatory testing algorithm. This study evaluates the performance of two rapid HIV-1/2 confirmatory assays—the Multisure HIV-1/2 Confirmatory Test and the Bio-Rad Geenius HIV-1/2 Supplemental Assay—within a CDC-aligned diagnostic framework, with the aim of assessing Multisure as a potential alternative differentiation assay. A total of 224 archived serum samples were analyzed, including true positives (n = 38), true negatives (n = 139), false positives (n = 20), and INNO-LIA™ indeterminate samples (n = 27), as defined by Architect HIV and INNO-LIA™ results. Samples were initially screened using the Architect HIV Ag/Ab Combo assay, confirmed by INNO-LIA™ and PCR, and subsequently re-tested using Multisure HIV-1/2 and Geenius HIV-1/2 assays. Diagnostic performance metrics were evaluated. Both rapid assays demonstrated 100% sensitivity and specificity when compared with INNO-LIA™. Among INNO-LIA™ indeterminate samples, Multisure HIV-1/2 classified 81.5% as negative compared with 55.6% using Geenius HIV-1/2. When compared with PCR, Multisure demonstrated higher specificity (89.2%) and positive predictive value (89.5%) than Geenius (82.9% and 84.6%). No confirmed HIV-2 infections were identified in the analyzed dataset, and HIV-1 subtype information was not available for the archived samples; therefore, conclusions regarding HIV-1/2 differentiation are based primarily on assay design and antigenic targets. Multisure HIV-1/2 demonstrated strong diagnostic performance comparable to established differentiation assays and may represent a practical alternative rapid confirmatory option within CDC-aligned HIV diagnostic workflows. Further studies including larger datasets and confirmed HIV-2 infections are warranted to further validate its clinical utility. Full article

Human Immunodeficiency Virus Type 1 (HIV-1), the causative agent of the acquired immune deficiency syndrome (AIDS), originated from zoonotic transmissions of simian immunodeficiency viruses (SIVs) infecting African great apes, following complex cross-species transmission events and virus–host co-evolution. These processes were accompanied by multiple viral adaptations, particularly within structural and accessory genes, enabling evasion of host restriction factors and long-term viral persistence. In 1988, an antisense open reading frame (ORF) overlapping the env gene was proposed and subsequently confirmed by the identification of antisense transcripts and the antisense protein (ASP). An “intact” ASP ORF (defined as >150 codons) is predominantly conserved in pandemic HIV-1 group M viruses and shows evidence of positive selection, suggesting a selective advantage. Increasing evidence supports the hypothesis that the asp gene emerged de novo during the evolution of group M and contributed to viral adaptation and global spread in humans. This review combines a narrative review of the literature with original in silico analyses of HIV-1 and SIV sequences retrieved from the Los Alamos National Laboratory database. We systematically reassessed the distribution, length variability and conservation of the ASP ORF across HIV-1 groups (M, N, O, P), subtypes, circulating recombinant forms (CRFs), unique recombinant forms (URFs) and related SIV lineages. Our updated analyses confirmed the strong association between the presence of an “intact” ASP ORF and pandemic HIV-1 group M lineages, while revealing rare but notable antisense ORFs in selected SIVcpz and SIVgor strains. By integrating evolutionary, epidemiological and sequence-based evidence, we aim to clarify the origin and maintenance of the ASP ORF and to contextualize its emergence within the broader framework of overlapping gene evolution, de novo gene birth and the selective pressures shaping viral fitness and pandemic potential. Full article

Background: Influenza B virus (IBV) undergoes continuous genetic mutations that can affect vaccine effectiveness and immune evasion. Although considerable research on IBV epidemiology exists globally, understanding of its genetic behavior in Saudi Arabia remains limited. This study characterized the molecular epidemiology of IBV in Riyadh, Saudi Arabia, during the 2024–2025 influenza season and evaluated compatibility with the current vaccine strain. Methods: Nasopharyngeal samples (n = 363) were collected from individuals presenting with influenza-like illness at King Khalid University Hospital in Riyadh. Detection and subtyping of IBV were performed using RT-PCR. Complete sequencing of the hemagglutinin (HA) and neuraminidase (NA) genes was conducted on confirmed IBV isolates (n = 7), followed by phylogenetic analysis, amino acid substitution mapping, and glycosylation site prediction. Results: Of the 363 samples analyzed, 68 (18.7%) tested positive for IBV, with the majority occurring in adult females aged 15–64 years. Phylogenetic analysis revealed that all seven IBV isolates belonged to the Victoria lineage under subclade V1A.3a.2, corresponding to the current vaccine strain and strains from the 2022–2023 epidemic season. However, molecular analysis identified two substitutions (D129N and D197E) located in antigenic loop-150 and 190-helix, respectively, in the HA polypeptide that distinguished our strains from vaccine strain B/Austria/1359417/2021. Importantly, the N-glycosylation site at position 169 (NKT), which was present in B/Riyadh/1/2010, has been lost in the IBV strains circulating during 2020–2025. Conclusions: While phylogenetic clade compatibility indicates potential vaccine efficacy, the identified amino acid variations and loss of the glycosylation site underscore the necessity for ongoing molecular surveillance to monitor antigenic changes and evaluate vaccine effectiveness within the Saudi Arabian population. Full article

Zoonotic diseases account for approximately one billion cases of illness and millions of deaths globally each year. Increasing contact between humans and competent wildlife hosts elevates the risk of zoonotic spillover. Synanthropic bird species are key players in the transmission of zoonotic pathogens, including flaviviruses such as West Nile virus (WNV) and influenza A viruses like Avian Influenza Virus (AIV). Active surveillance of sentinel birds inhabiting urban areas allows for early detection of emerging pathogens before they cause zoonotic outbreaks. Despite nesting in close proximity to humans, the role of the house martin (Delichon urbicum) in the circulation of flaviviruses and AIV remains poorly understood. Here, we analyzed the presence of antibodies against flaviviruses and AIV in a colony of house martins from southwestern Spain. In addition, we aimed to detect amplicons of the matrix and nucleoprotein genes of AIV using RT-qPCR. While none of the samples tested positive for AIV by RT-qPCR, we observed an AIV seroprevalence of 2.13% based on non-subtyped ELISA. Notably, this is the first report of AIV-seropositive D. urbicum individuals captured in Spain. Moreover, we detected a flavivirus-group seroprevalence of 24.34%, similar to rates reported in the same house martin population between 2018 and 2020, suggesting widespread circulation of flaviviruses within this synanthropic species. These results support the hypothesis that house martins may participate in the transmission of these viruses between wild bird populations and humans in urban environments. Full article

Highly pathogenic influenza A viruses of the H5 subtype continue to diversify worldwide through mutation and genetic reassortment, generating novel variants with unpredictable consequences under the One Health approach. Between 2024 and 2025, five outbreaks of avian influenza A viruses were detected in backyard poultry across Michoacán, Estado de México, and Ciudad de México. We conducted molecular and genetic characterization of five highly pathogenic H5N2 viruses isolated from these events. All cases tested positive for influenza A virus and the H5 hemagglutinin, exhibiting high pathogenicity with intravenous pathogenicity index values ranging from 2.88 to 3.0. Whole-genome sequencing revealed novel reassortants containing hemagglutinin from Eurasian H5N1 clade 2.3.4.4b and neuraminidase from the endemic Mexican H5N2 lineage. The viral genome of the isolate from Michoacán contained six segments derived from Eurasian H5N1 viruses introduced into North America in 2021–2022, while nucleoprotein and neuraminidase originated from Mexican H5N2 viruses. In contrast, viruses from Estado de México and Ciudad de México contained five H5N1-derived segments and incorporated polymerase basic protein 1, nucleoprotein, and neuraminidase from low-pathogenic H5N2 viruses circulating in 2024. Phylogenetic analyses confirmed the emergence of a distinct H5N2 Mexican sublineage, providing evidence of active viral reassortment and local evolutionary processes in Mexico. Full article

Epstein–Barr virus (EBV), a ubiquitous human herpesvirus infecting over 90% of the adult population, is causally associated with a distinct molecular subtype of gastric cancer (GC). A key mechanism by which EBV influences tumour biology is the expression of viral microRNAs (miR/miRNA) encoded within the BamHI-A rightward transcript (BART) region, although inter-patient variability in EBV-miRNA expression and its molecular significance remain incompletely defined. In this study, small RNA sequencing was performed on 11 primary gastric tumour samples to characterise EBV-derived miRNA expression, followed by quantitative RT-PCR analysis in an extended cohort of 21 tumours for targeted validation. EBV-miRNAs were detected in a subset of tumours and showed marked inter-tumour heterogeneity in abundance. EBV-miRNA-positive tumours were dominated by a conserved set of BART miRNAs, including miR-BART19-3p, miR-BART1-5p, miR-BART10-3p, miR-BART6-3p, miR-BART13-5p, and miR-BART22. These BART miRNAs displayed correlated expression patterns, characterised by concurrent elevation of multiple viral miRNA species within the same tumour samples. To link viral miRNA expression with host molecular responses, in silico virus–host interaction analysis was conducted using ViRBase to prioritise host genes associated with the detected BART miRNAs. PTEN, BCL2L11, FOXO3, and CDKN1A were identified as high-confidence targets and selected for experimental assessment. RT-qPCR analysis demonstrated differential expression of these host genes across tumours stratified by EBV BART miRNA abundance. Together, these findings identify a consistent BART miRNA pattern within this cohort. This study provides patient-level molecular evidence linking EBV-miRNA regulatory output to host gene expression states in GC. Full article

Highly pathogenic avian influenza virus of the H5N1 subtype has been infecting U.S. dairy cattle and spreading among dairy operations since March 2024. H5N1 surveillance systems for dairies are needed, but information on whether environmental sampling can inform these systems is lacking. To guide a surveillance framework, we determined the environmental sites with Influenza A virus (IAV) detection on H5N1-affected dairies (n = 25) in four states (California, Colorado, Michigan, and Ohio) and explored sample characteristics that may have influenced detection. A total of 581 samples from dairy environmental sites were characterized for IAV RNA via rRT-PCR, and classified into six categories. A total of 94 samples (16.2%) had IAV detected, and the Ct values measured from these samples were typically higher than those measured in bulk tank milk from a subset of sampled herds. A majority of IAV detections were made from the following site categories: milking equipment/personal protective equipment, parlor surfaces, and wastewater/lagoons/manure. These results suggest that environmental sites most likely to be contaminated with IAV on dairies are those with exposure to milk. Meanwhile, mixed effect logistic modeling showed that days into an outbreak that samples were collected was associated with IAV detection. These results provide a framework within which to continue the assessment of environmental sampling as a surveillance tool for dairy H5N1. This framework can be strengthened by studies that perform further IAV viral characterization and collect samples from sites prior to, during, and after H5N1 outbreak periods. Full article

Background: In Pakistan, the number of Human immunodeficiency virus (HIV) cases is increasing significantly, attributed to risk factors such as injection drug use, sexual transmission, etc. However, transmission through hemodialysis units is not well documented. In 2024, an outbreak of HIV cases in Multan, Pakistan, drew alarm from local health authorities due to reports linking it to a large public hospital in South Punjab. Here, we report the molecular epidemiological investigation of the outbreak. Methods: Twenty-five hemodialysis patients identified during the outbreak were enrolled. Blood samples were subjected to DNA extraction and polymerase chain reaction (PCR) amplification. Phylogenetic analysis was conducted using the maximum-likelihood approach in IQ-TREE. For dating phylogenetics, a maximum clade credibility tree (MCC) was constructed using the BEAST tool. The MCC tree was constructed using the Bayesian Skyline model with an uncorrelated lognormal relaxed clock. The VESPA program was used to identify amino acid signatures unique to outbreak sequences compared with Pakistani reference sequences. Results: A total of 25 patients (identified as part of the HIV outbreak) were enrolled. 96% (24 out of 25) also tested positive for Hepatitis C, while none tested positive for Hepatitis B. The age range of patients in the study was 23 to 72 years (median age: 44.88 years). In terms of gender distribution, 13 out of 25 were male. All the sequences were identified as HIV subtype CRF02_AG. Phylogenetic analysis revealed that Multan sequences formed a well-supported monophyletic cluster, indicating shared recent origin. Signature pattern analysis identified a unique molecular fingerprint at 26 nucleotide positions, whereas molecular dating placed the emergence of the cluster between 2023 and 2024, consistent with the outbreak timing. Conclusions: Findings provide biologically plausible evidence of a point-source HIV outbreak linked to lapses in infection prevention and control practices at the hemodialysis unit. Full article

This commentary provides an overview of the development of a zoonotic influenza vaccine, in response to the emergence of an H5N1 subtype virus from clade 2.3.4.4b in mid-2020. When development was initiated, the World Health Organization (WHO) had recommended four candidate vaccine viruses; the A/Astrakhan/3212/2020-like strain was selected as it provided good coverage of circulating viruses and, critically, was available. To facilitate regulatory approval, the licence of an existing zoonotic vaccine, Aflunov (A/turkey/Turkey/01/2005)—a pre-pandemic monovalent A/H5N1 adjuvanted with MF59 and manufactured using the egg-based platform—was duplicated, with the plan to submit a variation to the duplicate licence for the strain update. This was supported by a ferret immunogenicity study using pseudoviruses (allowing the work to be conducted at a lower biosafety level), in conjunction with clinical data from the original Aflunov licence application, and a US study (NCT05874713) on another candidate zoonotic vaccine manufactured using the cell-based platform. Qualification batches for characterisation studies were manufactured at-risk, until calibrated, homologous reagents were available, and the final product release and stability studies were conducted, with rolling provision of stability data to health authorities. The vaccine was initially approved with a shorter shelf-life, allowing early distribution in certain countries, with later extension of the shelf-life once data became available. In terms of procurement and logistics, early consultation between the European Commission and EU member states resulted in the award of a Framework Contract for the initial supply of 665,000 doses to 15 states. Learnings from the development of this vaccine may help to improve pandemic readiness in the future. Full article