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Avian influenza is a highly contagious viral disease affecting domestic and wild birds, caused by influenza A viruses of the family Orthomyxoviridae. Continuous genetic surveillance is essential for monitoring viral evolution and identifying molecular determinants associated with pathogenicity and host adaptation. In this study, the PB1 and PB2 polymerase genes of highly pathogenic avian influenza (HPAI) H5N1 viruses detected in Romania between 2017 and 2024 were genetically characterized. A total of 18 organ samples collected from domestic and wild birds in several Romanian counties were analyzed by real-time RT-PCR, followed by conventional RT-PCR, Sanger sequencing, mutational analysis, and phylogenetic reconstruction. High-quality sequence data were obtained for six PB1 and eight PB2 gene fragments. Sequence analysis revealed a high degree of conservation within the PB1 gene, particularly across regions involved in viral replication, indicating strong evolutionary constraints. In contrast, the PB2 gene exhibited several amino acid substitutions, including changes at positions 464 and 678 in selected isolates. Notably, all analyzed strains lacked the mammalian adaptation marker E627K. Phylogenetic analysis clustered the Romanian isolates within Eurasian H5N1 lineages and demonstrated close genetic relationships with contemporary strains circulating in Eastern Europe and Russia. These findings highlight the genetic stability of PB1, the ongoing diversification of PB2, and the importance of continuous molecular surveillance for the early detection of viral variants with potential implications for host adaptation and zoonotic risk. Full article

Background: Equine herpesvirus type 1 (EHV-1) and equine influenza virus (EIV) are major respiratory pathogens in horses, causing significant economic losses in domesticated horses. Bacterial Artificial Chromosome (BAC) technology can be used to precisely manipulate the EHV-1 genome for the development of live-attenuated vector vaccines. Earlier, our group developed a live-attenuated EHV-1 vaccine by deleting virulence-associated genes using this technology and the mutant EHV-1 has been exploited for expressing foreign gene in the current study. Specifically, in this study, a mutant EHV-1 virus expressing the hemagglutinin (HA) gene of H3N8 EIV (sub-lineage: Florida clade 2) was generated and characterized in vitro. Methods: The HA gene of EIV (Florida clade 2) was used for antigen gene cloning. The expression cassette for the HA gene was commercially synthesized and inserted into the backbone of EHV1∆IR6 BAC using an En passant mutagenesis strategy. Recombinant clones were selected using antibiotic selection, PCR, and RFLP. Further, the recombinant virus was regenerated in RK-13 cells via transfection and characterized in vitro for plaque size, growth kinetics and immunofluorescence antibody test (IFAT). Results: PCR and RFLP confirmed the successful insertion of the HA gene into pEHV1∆IR6/gE BAC. The recombinant virus, vEHV1∆IR6/gE-HA(FC2), was successfully rescued in RK13 cells and demonstrated expression of the EIV haemagglutinin proteins by immunofluorescence assay. Although plaque size was reduced in the generated mutant virus in comparison to parental virus, the growth kinetics of the recombinant viruses were comparable to those of vEHV1∆IR6/gE. Conclusions: These findings demonstrate the successful expression of immunodominant hemagglutinin protein of EIV by recombinant EHV-1 and indicate the potential suitability of EHV-1 BAC as a vector platform for foreign gene expression. Full article

Influenza A virus is a segmented, negative-sense RNA virus. Since the early 2020s, H5 clade 2.3.4.4b viruses have spread widely across Europe, Africa, and Asia, affecting wild birds and poultry. Costa Rica reported its first H5 clade 2.3.4.4b avian influenza virus (AIV) case on 19 January 2023. This study describes an outbreak in backyard chickens and ducks. Initial serum samples collected on 24 January showed three chickens negative for AIV, while one duck tested positive by ELISA and agar gel immunodiffusion (AGID). During a second visit on 27 January, three of four chicken sera collected tested positive by ELISA and AGID. Tissue samples were positive for influenza A by qRT-PCR. Next-generation sequencing recovered five of the eight viral genomic segments, and the hemagglutinin cleavage site sequence (REKRRKR↓G) confirmed a highly pathogenic avian influenza virus (HPAIV) H5 strain. The samples were submitted to the National Veterinary Services Laboratories for confirmation. Serological testing showed reactivity to North American low pathogenic H5 antigens, and qRT-PCR amplified influenza A and N1 genes. Virus isolation and next-generation sequencing (NGS) of all eight viral genome segments were successfully performed at the WHO Collaborating Centre at St. Jude Children’s Research Hospital (SJCRH). Full article

Syndromic multiplex-PCR panels deliver rapid, comprehensive detection of pathogens and resistance determinants in acute infections, but their unrestricted use can generate a high proportion of negative results with substantial economic and stewardship implications. We retrospectively analysed all BioFire FilmArray meningitis/encephalitis (ME), upper-respiratory (RP2.1), pneumonia (PN) and gastrointestinal (GI) determinations performed at the UOSD Microbiology and Virology of P.O. “Vito Fazzi”, ASL Lecce (Apulia, Italy) between 1 July 2024 and 30 April 2026. Repeat determinations from the same patient with the same panel were identified in the laboratory information system and removed before any analysis, so that each determination analysed corresponds to a distinct diagnostic episode. Across 5381 determinations (CNS, n = 761; upper respiratory, n = 2601; lower respiratory, n = 399; gastrointestinal, n = 1620), 59.3% were negative for every target, with a steep appropriateness gradient: 90.0% negativity for the ME panel and 77.1% for the gastrointestinal panel versus 44.2% for the upper-respiratory and 26.6% for the pneumonia panel. CNS positives were predominantly viral (75%), led by enterovirus; Streptococcus pneumoniae was the only consistent bacterial agent. Human rhinovirus/enterovirus dominated the respiratory ecology; influenza A was almost entirely H3 and H1N1pdm09, but 8.1% of influenza-A–positive specimens were equivocal or non-subtypeable and were never referred for sequencing, an avoidable surveillance blind spot for novel/zoonotic (avian) influenza. Pneumonia-panel resistance markers (mecA/C–MREJ, CTX-M, KPC, NDM) clustered in Enterobacterales co-infections. The gastrointestinal panel was dominated by diarrhoeagenic Escherichia coli pathotypes (chiefly EPEC and EAEC) and Clostridioides difficile toxin, its 77% negativity identifying a second over-utilised stream. We argue for CSF-pleocytosis gating, tiered/reflex respiratory algorithms, gastrointestinal-panel gating to community-onset diarrhoea, and mandatory reflex sequencing of unsubtypeable influenza A. Full article

Background: Brucellosis remains a significant zoonotic infection affecting approximately 500,000 people worldwide annually, with no licensed human vaccine available. Recombinant influenza virus vectors expressing Brucella spp. antigens represent a promising vaccine platform. However, transitioning from laboratory constructs to clinical candidates requires validation of scalable purification methods compliant with Good Manufacturing Practice (GMP) standards. This study aimed to develop and optimize a pilot-scale purification protocol for these vectors. Methods: Recombinant influenza A viruses (H5N1) expressing Brucella spp. antigens (Omp16, Omp19, L7/L12, Cu-Zn SOD) were propagated in MDCK cell culture. The optimized purification process included: (1) clarification; (2) ultrafiltration/diafiltration (100 kDa MWCO); (3) two-step chromatography (anion-exchange Q-Sepharose^® Fast Flow and multimodal Capto™ Core 700); and (4) sterile filtration. Process validation was performed across three independent pilot-scale batches (20 L each). Results: The purification process demonstrated high reproducibility for all constructs. Final preparations met established quality criteria: infectious titer ≥ 5.2 log₁₀ TCID₅₀/mL, hemagglutination activity 7.33 ± 0.58–8.33 ± 0.58 log₂, total protein content 157–305 μg/mL, residual host cell DNA < 10 ng/dose, and bacterial endotoxin levels ≤ 0.15 IU/mL. The overall recovery of infectious virus was 20–24%, an optimal value for multi-stage bioprocessing. Preservation of the target genetic insert was confirmed in all final preparations by PCR and sequencing. Conclusions: The developed integrated purification protocol yields vectors with high purification efficiency, preserving biological activity and meeting regulatory quality requirements for residual host cell DNA and endotoxins. The technological platform demonstrated versatility, robustness (inter-batch coefficient of variation for yield did not exceed 10–12%), and scalability, establishing a foundation for preclinical and clinical studies of candidate brucellosis vaccines. Full article

Post-COVID-19, influenza A(H3N2) has re-emerged with accelerated genetic diversification. The rise in antigenically drifted subclades raises concerns regarding immune escape and vaccine mismatch. To synthesize the available evidence on the post-pandemic evolution of influenza A(H3N2), with particular attention to emerging subclades and their potential public health implications, a scoping review (PRISMA-ScR) was conducted across six databases in January 2026, including studies on genomic surveillance, antigenic characterization, and vaccine effectiveness (VE). Twenty studies were included. While clade 2a.3a.1 (J lineage) predominated, reports highlighted the rapid emergence of the antigenically distinct K subclade (formerly J.2.4.1). Genetic and laboratory assays (HI/neutralization) confirmed immune escape and reduced vaccine match. Observational estimates indicated diminished VE against drifted strains, though no consistent increase in clinical severity was observed. Post-pandemic A(H3N2) is defined by rapid drift and diversification. Emerging variants like the K subclade challenge vaccine selection and seasonal preparedness. Integrated surveillance remains vital for timely vaccine updates and mitigating public health impact. Full article

In this study, we used our own prediction models to assess the antiviral potential of pyrimidin-4(3H)-one derivatives against the A/H1N1 influenza virus strain. This assessment allows us to identify promising structures. The models are based on machine learning algorithms and molecular modeling results. In general, the prediction results are consistent with experimental data. The most promising compound, namely 12 (6-amino-2-(dimethylamino)pyrimidin-4(3H)-one), inhibits the reproduction of the A/Puerto Rico/8/34 (H1N1) influenza virus strain in vitro, likely by affecting the function of the endonuclease domain of the viral polymerase complex. Compound 12 can be used to create new PAN inhibitors by modifying its structure. Full article

Background/Objectives: Pandemic influenza remains a persistent global threat, and while vaccination is the primary preventive measure, conventional vaccines often induce narrow, strain-specific immunity. This study evaluated the immunogenicity, protective efficacy, and cross-protective potential of a CpG-adjuvanted trivalent inactivated influenza vaccine (CpG-TIV) administered intramuscularly at high and low doses in ferrets. Methods: Groups of influenza-seronegative ferrets received two intramuscular injections, 3 weeks apart, of high- or low-dose CpG-TIV or a commercial non-adjuvanted trivalent vaccine. Three weeks after the second immunization (Day 42), serum was obtained, and the ferrets were subsequently challenged intranasally with homologous H1N1 and influenza B viruses, as well as a heterologous drifted H3N2 strain. Clinical signs, body weight, nasal viral load, and lung histopathology were monitored following the viral challenge. Results: CpG-TIV induced significantly higher dose-dependent HI and IgG antibodies than the commercial unadjuvanted vaccine. High-dose CpG-TIV markedly reduced weight loss, clinical symptoms, nasal viral load (by up to 99%), and lung pathological damage. Notably, high-dose CpG-TIV provided significant cross-protection against heterologous H3N2, whereas the commercial vaccine showed no protective effect. At Day 42, HI GMTs in the high-dose group reached 500, 254, and 594 against H1N1, H3N2, and B strains, respectively, with a maximal 2.58 log10 reduction in H1N1 viral load. Conclusions: High-dose CpG-TIV demonstrates strong immunogenicity and robust dose-dependent homologous and heterologous cross-protection in ferrets. The combination of a CpG adjuvant and high-dose antigen broadens protection against drifted influenza viruses, overcoming the narrow coverage of conventional vaccines. These data support further clinical development of this broad-spectrum influenza vaccine candidate. Full article

H9N2 low-pathogenic avian influenza viruses (LPAIV) represent an ongoing zoonotic threat due to their enzootic circulation in poultry, reassortment capacity, and increasing human transmission events. This study characterized three H9N2 isolates recovered from apparently healthy poultry in a Changchun live poultry market (September–November 2022) that exhibited unprecedented genetic and phenotypic characteristics indicating enhanced zoonotic risk. Phylogenetic analysis showed a complex mosaic genome combining segments from four distinct lineages: HA from the BJ/94-like lineage (human-associated), PB1/NP/NS from the F98-like lineage, NA from the FJ/30-C-like branch, and PB2/M genes from the G1-like lineage. Bayesian molecular clock analysis estimated the most recent common ancestor at February 2022, with HL55 and HL56 diverging by May 2022, indicating rapid local viral evolution. All isolates retained hallmark LPAIV characteristics (monobasic HA cleavage site, zero intravenous pathogenicity index in chickens). However, receptor-binding assays demonstrated a critical divergence among the isolates: while HL45 exhibited exclusive avian α2-3 receptor preference, both HL55 and HL56 retained strong avian receptor binding while additionally showing measurable affinity for human α2-6 receptors-a dual-binding phenotype associated with enhanced zoonotic potential. Most significantly, the HL55 isolate successfully infected BALB/c mice without prior adaptation, causing transient upper respiratory tract replication, moderate weight loss (~9.2%), and mild disease without mortality or systemic dissemination. These findings demonstrate that the direct mammalian infectivity of this specific mosaic H9N2 lineage adds to the growing body of evidence regarding the zoonotic potential of contemporary H9N2 variants. The presence of known mammalian-adaptation markers (PB2 A588V, NA stalk deletion, HA position 226 leucine), combined with demonstrated dual receptor-binding capacity and inherent mammalian infectivity, underscores the accelerated evolutionary trajectory of H9N2 viruses toward increased zoonotic competence. These findings warrant intensified surveillance in live poultry markets, comprehensive antigenic characterization of emerging variants, and enhanced biosecurity measures to mitigate the risk of spillover events and potential pandemic emergence. Full article

The H5N1 highly pathogenic avian influenza (HPAI) virus has caused severe global losses, reaching South America in 2022 and Antarctica in 2024. Here, we synthesize outbreak reports submitted to the World Organization for Animal Health by South American countries and overseas territories in this continent, and document the virus’s unprecedented expansion into Antarctica, affecting wild birds, wild mammals, and domestic poultry. Phylogenetic and time-calibrated Bayesian analyses were performed on available genomic sequences. Over 6 million domestic birds were lost, mostly from commercial operations. Of the 11 South American countries and overseas territories that reported H5N1 to WOAH, 10 reported infections in wild birds, spanning 104 species, 59.62% of which are migratory and predominantly non-trans-equatorial. Marine mammal outbreaks followed wild bird detections, with the South American sea lion (Otaria flavescens) being the most reported species. Several Antarctic bird species with migratory behavior were also reported in South America. Genomic analyses revealed multiple introduction events, regional viral diversification, and patterns consistent with repeated cross-species spillover events. These findings highlight H5N1’s extensive ecological reach in the Southern Hemisphere and underscore the urgent need for a One Health approach that strengthens wildlife and backyard-poultry surveillance, alongside coordinated regional action to control and prevent further HPAI spread. Full article

Wild waterfowl constitute the primary natural reservoir of influenza A viruses, and wetlands at the convergence of major migratory flyways serve as critical hubs for viral genetic exchange. Baikal Siberia, situated at the intersection of the East African–West Asian, Central Asian, and East Asian–Australasian flyways, represents a unique yet understudied region in this context. Here we report the results of long-term virological surveillance of wild birds in the Lake Baikal basin conducted between 2018 and 2024. A total of 1036 cloacal swab samples from 28 bird species were screened, yielding 42 influenza A virus isolates belonging to 12 HA/NA subtype combinations: H1N1, H3N1, H3N2, H3N5, H3N6, H3N8, H4N6, H6N1, H6N2, H6N3, H6N8, and H12N5. Among the detected subtypes, H6 viruses—identified with four distinct neuraminidase combinations (N1, N2, N3, N8)—are of particular public health relevance owing to their documented capacity for dual-receptor binding and potential for zoonotic transmission to mammals, including humans. Full-genome sequencing followed by cluster analysis of internal gene segments identified 16 distinct segment constellations, indicating extensive reassortment. BLAST searches against the GISAID database revealed closest genetic relatives in Mongolia, South Korea, Japan, China, and Western Siberia, with more distant links to Bangladesh, Europe, and a possible intercontinental connection via the Pacific flyway. Maximum-likelihood phylogenetic analysis of the HA and NA segments confirmed that all isolates belong to the Eurasian genetic lineage, yet they are distributed across multiple clades rather than forming a single monophyletic group, reflecting the role of Buryatia as a mixing zone for genetically diverse viral populations. These findings substantially expand the understanding of influenza A virus ecology in the Lake Baikal basin and underscore the importance of continued surveillance at this key migratory crossroads in Northern Asia. Full article

We propose a Bayesian sequential procedure to test hypotheses concerning the relative risk between two specific treatments based on the binary data obtained from the two-arm clinical trial. Our development is based on an optimal sequential test cast within the Bayesian framework. This approach enables us to provide, in a straightforward manner based on the Stopping Rule Principle (SRP), an assessment of the various error probabilities via posterior probabilities and conditional error probabilities. An attractive feature of our approach is the relative simplicity of the calculations involved without having to resort to cumbersome iterative methods of ‘spending the alpha’ and the like. The proposed methods are illustrated using a sequential safety study of adverse events following H1N1 influenza vaccination and are compared with existing sequential approaches. The findings demonstrate that the proposed Bayesian framework provides an efficient and flexible approach for the early detection of adverse events under several different prior distributions of the parameters involved. Full article

A capillary-driven microfluidic electrical screening platform was developed using silver nanoparticle (AgNP)-decorated laser-patterned villous microstructures on a glass substrate for the analysis of H3N2-infected A549 cells. The device integrated nanosecond laser patterning, AgNP conductive thin-film formation, passive capillary transport, and direct electrical readout within a single microfluidic sensing structure. Villous-like arrays were fabricated using a 1064 nm IR pulsed laser at a fluence of 4.35 J/cm², with a repetition rate of 300 kHz, pulse overlap of 96.7% and scanning speed of 500 mm/s. The fabricated structures exhibited a diameter of 60 μm, height of 80 μm and interpillar pitches ranging from 30 to 90 μm. After AgNP deposition, the surface showed a dominant Ag content of 59.2%, confirming successful formation of conductive microstructured electrodes. The 30 μm pitch structure produced the highest current response of 22 μA at 1 V and the highest ΔI_norm of 0.053 after introduction of H3N2-infected A549 samples. Wettability and capillary transport were tunable by pitch, with contact angles (CAs) decreasing from 140° to 30° and flow velocities decreasing from 0.1 mm/s to 0.03 mm/s. Formalin-fixed H3N2-infected A549 cells were electrically distinguished from non-infected A549 controls over 10¹–10⁶ PFU/μL, with detectable responses down to 10¹ PFU/μL. These results demonstrate a label-free, self-driven, and fabrication-oriented microfluidic strategy for electrical screening of virus-associated cellular samples. Full article

The highly pathogenic H5 subtype avian influenza viruses (AIVs) pose persistent threats to the poultry industry and public health owing to their high lethality and pandemic potential. Migratory wild birds play a pivotal role in the global dissemination and genetic reassortment of the virus, serving as both natural reservoirs and long-distance vectors that drive its spatiotemporal spread. However, the extent and evolutionary drivers of antigenic divergence among H5 AIVs circulating in wild birds in East China remain poorly understood. Here, we aim to characterize the evolutionary dynamics and antigenic divergence of H5 AIVs isolated from wild birds in East China between 2013 and 2022. Whole-genome sequencing and phylogenetic analysis revealed that the isolates belonged to multiple clades, including 2.3.2.1 and 2.3.4.4, and encompassed the H5N1, H5N6, and H5N8 subtypes. Key amino acid site analysis showed that the glycosylation site patterns in the HA and NA proteins varied among clades, with some strains exhibiting gains or losses of glycosylation sites, while certain strains had acquired mutations associated with mammalian adaptation. Cross-hemagglutination inhibition (HI) assays combined with antigenic cartography demonstrated that the majority of the isolates were antigenically well-matched with the contemporaneous vaccine strains used in China, indicating that these vaccines effectively covered the predominant circulating antigenic variants at the time. Nevertheless, potential antigenic mismatches were still observed between some circulating strains and these vaccine strains. These findings suggest that wild birds in East China may contribute to the regional movement and diversification of H5 AIVs, highlighting the value of sustained surveillance for early warning and vaccine strain evaluation. Full article