Search Results (223)

Phytoene, a colorless precursor of carotenoids, has attracted increasing attention because of its favorable bioavailability, antioxidant activity, and potential applications in functional foods, nutraceuticals, and animal nutrition. However, its industrial utilization remains limited by low natural abundance and the dependence of current production strategies on genetic engineering or metabolic pathway manipulation. In this study, we identified and characterized a naturally occurring Brevundimonas vesicularis strain (Bv-xms2024) exhibiting pronounced phytoene accumulation without genetic modification. The strain was comprehensively characterized using morphological, biochemical, molecular, genomic, metabolomic, and transcriptional analyses. Quantitative LC–MS/MS analysis demonstrated that Bv-xms2024 accumulated phytoene to 420.42 ± 98.11 μg/g dry biomass after 96 h of cultivation, substantially exceeding the levels of downstream carotenoids, including β-carotene and astaxanthin. Optimization of cultivation parameters identified 25 °C, pH 7.0, and 96 h as the optimal conditions for phytoene accumulation, while serial passaging confirmed stable production over 20 generations. Genome annotation identified the carotenoid biosynthetic gene repertoire, while RT-qPCR analysis revealed a temporal shift from early upregulation of crtE and crtB to later upregulation of downstream pathway genes, consistent with the observed phytoene-dominant carotenoid profile. Short-term tolerance evaluations in mice and chickens revealed no observable adverse effects under the tested conditions. Collectively, these findings identify Bv-xms2024 as a promising natural microbial resource for phytoene production and provide a basis for further process development and strain-level safety evaluation. Full article

Objectives: The escalation of multidrug-resistant (MDR) clinically relevant bacterial isolates, including Escherichia coli and key ESKAPE pathogens, represents a critical global healthcare threat. While antimicrobial peptides (AMPs) offer promising alternatives, metabolic instability often limits their clinical use. This study investigated the therapeutic potential, antimicrobial efficacy, and resistance dynamics of peptide D-TN6 against highly resistant bacterial strains. Methods: D-TN6 efficacy was evaluated against 164 clinical isolates, including pan-drug-resistant, carbapenem-resistant, and polymyxin-resistant phenotypes. A 20-passage serial induction assay compared resistance development kinetics of D-TN6 against gentamicin over an extended period. Results: D-TN6 demonstrated potent efficacy against MRSA (minimum inhibitory concentration (MIC)₉₀: 1 µg/mL) and remained effective against polymyxin B-resistant Klebsiella pneumoniae (MIC₉₀: 8 µg/mL) and pan-drug-resistant Acinetobacter baumannii (MIC₉₀: 8 µg/mL). In resistance assays, while the gentamicin MIC increased 256-fold by the 17th passage, the D-TN6 MIC remained constant at 1 µg/mL throughout the 20-passage study. Conclusions: These findings underscore D-TN6 as a robust therapeutic candidate. Its efficacy against MDR strains and its feature of low propensity for resistance development—likely due to membrane disruption—position D-TN6 as a promising solution for life-threatening infections where conventional last-resort agents fail. Full article

Background/Objectives: The variability of the internal acoustic meatus (IAM) and its morphological peculiarities are of high clinical significance in otologic interventions and neurosurgery. Considering the complexity of the neurovascular structures housed by the IAM, including passage of the facial nerve, the aim of the study was to highlight the morphological peculiarities of the IAM and their clinical significance. Methods: The age peculiarities of the IAM course were studied on 18 serially, transversely sectioned human embryos and fetuses, as well as on 20 newborn and 20 adult formalin-fixed hemiheads. The morphometric parameters of the IAM (width, height, length, and angles between the anterior and posterior walls of the IAM and the longitudinal axis of the petrous ridge (LAPR) were evaluated on 82 dry temporal bones. Results: In embryos and early fetal stages of development, the course of the IAM formed an acute, posteromedially open angle with the longitudinal axis of the petrous part of the temporal bone (LAP); in newborns, it was a right angle; in adults, it was an acute, anteromedially open angle. Various shapes of the IAO, such as oval (51.2%), semioval (23.2%), quadrangular (13.4%), heart-like (3.7%), and triangular (8.5%), were highlighted. The mean width of the internal acoustic opening (IAO) was 7.7 ± 2.11 mm (right/left—8.2 ± 2.22 mm/7.2 ± 1.89 mm), with a statistically significant difference (p = 0.031). The mean height of the IAO was 5.3 ± 1.43 mm (right/left—5.6 ± 1.58 mm/5.0 ± 1.22 mm), p = 0.082. The mean length of the IAM was 10.1 ± 2.83 (right/left—10.5 ± 2.96 mm/9.7 ± 2.67 mm), p = 0.214. A partial septum was revealed in 3.6% of cases, while total septation of the IAM was observed in 2.4%. The mean angle between the anterior wall of the IAM and the longitudinal axis of the petrous ridge (AWIAM/LAPR) was 21.4 ± 13.67° (right/left—22.2 ± 14.62°/20.5 ± 12.78°), p = 0.564. The mean angle between the posterior wall of the IAM and the longitudinal axis of the petrous ridge (PWIAM/LAPR) was 82.4 ± 8.02° (right/left—84.3 ± 7.93°/80.5 ± 7.76°); p = 0.032. Conclusions: In the initial stages of an individual’s ontogenesis, the IAM forms an acute, posteromedially open angle with the LAP; in newborns—a right angle; in adults—an acute, anteromedially open angle. Both morphological characteristics and morphometric parameters of the IAO and IAM vary bilaterally. The oval, semioval, quadrangular, heart-like, and triangular shapes of the IAO were highlighted. Partial and total septation of the IAM were revealed. Only the TDAIO and PWIAM/LAPR angle were statistically significant, p < 0.05, while the bilateral correlation was negligible. The established morphological variants of the IAM could predispose to facial and vestibulocochlear nerve impairments. Full article

Porcine epidemic diarrhea virus (PEDV) G2c variants have recently emerged, posing significant challenges to swine health management. As a major coronavirus affecting the swine industry, PEDV exhibits extensive genetic variability, which has greatly complicated disease control. Current vaccines provide suboptimal protection under field conditions. Therefore, the isolation of recently circulating strains and the establishment of a robust reverse genetics system are critical for advancing the study of emerging variants and facilitating rational vaccine development. In this study, a PEDV field strain designated PEDV-BJ-2023 was isolated from diarrheic piglets in Guizhou, China. Phylogenetic analysis based on the complete genome and spike gene classified PEDV-BJ-2023 within the emerging G2c lineage. To facilitate functional studies, a full-length infectious cDNA clone was constructed using transformation-associated recombination cloning in yeast. Furthermore, an enhanced green fluorescent protein reporter virus was generated via CRISPR/Cas9-assisted homologous recombination by inserting an EGFP-2A cassette upstream of the nucleocapsid gene. The recombinant viruses displayed virion morphology and plaque characteristics similar to those of the parental wild-type PEDV-BJ-2023 strain, although the parental virus exhibited faster replication during the early stage of infection in vitro. In 5-day-old piglets, all three viruses caused severe diarrhea, weight loss, and intestinal lesions; however, recombinant viruses exhibited slightly reduced viral shedding and pathogenicity, with rPEDV-EGFP being the most attenuated. Notably, rPEDV-EGFP maintained stable EGFP expression over eight serial passages. This study establishes a reverse genetics platform for an emerging G2c PEDV strain and provides a stable fluorescent reporter virus, offering valuable tools for visualizing viral infection and investigating virus–host interactions. Full article

Background: Pseudomonas aeruginosa combines intrinsic resistance, adaptive plasticity, and biofilm formation, making treatment failure common in veterinary and human medicine. We examined whether geraniol and Tris-ethylenediaminetetraacetic acid (Tris-EDTA) modulate marbofloxacin activity during serial exposure and whether whole-genome sequencing detects genomic correlates of the observed phenotypic changes. Methods: A biofilm-producing reference P. aeruginosa ATCC 27853 strain was serially passaged for 10 days with marbofloxacin, geraniol, Tris-EDTA, and their double and triple combinations. Minimum inhibitory concentration (MIC) trajectories and fractional inhibitory concentration indices were determined. Efflux involvement was assessed by MIC testing with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) in 22 canine isolates and the reference strain. Biofilm disruption was evaluated by crystal violet and MTS assays. Whole-genome sequencing was performed for day 0 controls and selected derivatives collected on days 2, 6, and 10. Results: Marbofloxacin alone showed a progressive MIC increase from 2 to 16 µg/mL, whereas its MIC remained low in the triple combination and in the marbofloxacin–Tris-EDTA combination. No antagonism was detected. The triple combination showed the most consistent interaction profile, meeting the prespecified synergy criterion on 7 of 10 passage days, compared with 5 of 10 days for marbofloxacin–Tris-EDTA, 2 of 10 days for geraniol–Tris-EDTA, and none for marbofloxacin–geraniol. CCCP caused at most a single twofold-dilution-step decrease in MIC, not supporting a dominant efflux-driven phenotype. In MTS assays, MTS-derived relative EC₅₀ values approximated planktonic inhibitory concentrations, whereas crystal violet results were poorly reproducible. Draft genomes remained highly conserved, and only one candidate nonsynonymous sequence difference in oprN was observed in a day 2 marbofloxacin–Tris-EDTA derivative. Conclusions: Geraniol- and especially Tris-EDTA-containing combinations constrained in vitro marbofloxacin resistance escalation. The marked phenotypic separation despite minimal genomic divergence is consistent with a possible contribution from regulatory or transcriptional adaptation, although this mechanism requires direct experimental confirmation. Full article

Bacteriophage exposure can impose strong selective pressure on bacterial populations and may alter antimicrobial susceptibility beyond direct lytic effects. However, the persistence of such changes after the removal of phage pressure remains insufficiently characterized in clinical P. aeruginosa isolates. This study evaluated whether short-term exposure to the lytic bacteriophage KPP10 was associated with changes in antimicrobial susceptibility categories and whether these changes remained detectable after serial passage in phage-free medium. Five non-duplicate clinical P. aeruginosa isolates were exposed to KPP10 for 24 h at a multiplicity of infection of 10. Antimicrobial susceptibility testing was performed by disk diffusion at baseline, immediately after exposure (F24) and after four serial passages in phage-free medium. Categories were interpreted according to the EUCAST 2024 clinical breakpoints. Nine categorical susceptibility shifts were detected across six antibiotics: four toward increased susceptibility and five toward decreased susceptibility. Six shifts (66.7%) remained detectable after four serial passages, whereas three shifts (33.3%), involving ciprofloxacin, cefepime, and aztreonam in LBK20, reverted to their baseline categories. Eight of the nine shifts involved β-lactam antibiotics, and cefepime was the most frequently affected agent. These exploratory findings show that short-term KPP10 exposure was associated with isolate-specific and bidirectional changes in categorical antimicrobial susceptibility and support repeated susceptibility monitoring in future phage–antibiotic studies. Full article

Live-attenuated rotavirus (RV) vaccines are the most effective interventions for preventing RV gastroenteritis (RVGE) in young children. However, the molecular basis of attenuation remains not well understood. Here, we describe a compact but comprehensive strategy to identify RV virulence determinants by combining low-passage bidirectional selection, sequence analysis, and segment-level phenotype testing via a reverse genetics infectious system. Using the virulent murine RV strain EW, virulence was quantified by diarrhea severity/duration and body-weight gain. Serial passaging in cell culture selected an attenuated population, which regained virulence after passaging in suckling mice. Sequence comparison of the virulent and attenuated EW populations revealed only seven amino acid differences. We summarized literature describing attenuation/virulence-associated mutations in various RV group A (RVA) strains and found previous findings identical or similar to four of the seven mutations: NSP4-T45M, VP4-S470L, VP4-T612A, and VP7-T75P. Virulent- and attenuated-type EW variants of VP2, VP4, VP7, and NSP4 were introduced individually, or as NSP4/VP7 or VP4/VP7 pairs, into a simian SA11-L2 backbone using an 11-plasmid reverse genetics system. Phenotyping of rescued viruses consistently linked cell-culture–adapted VP4 to enhanced replication in vitro and reduced virulence in suckling mice. In vivo passaging strongly favored VP4 residue S470 over cell-culture-selected L470. More generally, our findings (i) underscore VP4 and VP7 as key determinants of EW virulence, (ii) provide a practical framework for identifying driver mutations underlying RVA attenuation, and (iii) highlight attenuation-associated substitutions shared across diverse RVAs. Full article

Porcine group A rotavirus (PoRVA) is a significant cause of viral diarrhea in piglets, necessitating urgent global implementation of effective control strategies. This review assesses advancements in PoRVA in vitro cultivation and amplification, crucial for PoRVA vaccine development. Traditional PoRVA cultivation commonly employs primary porcine kidney cells or finite cell lines like MA-104, posing well-documented challenges in scalability, production cost, and their ability to recapitulate the natural intestinal microenvironment. Consequently, research has increasingly focused on adapting PoRVA to alternative systems, particularly immortalized porcine cell lines or physiologically relevant porcine intestinal organoids. This adaptation process, involving serial passaging, can induce genomic alterations and virulence attenuation in piglets, essential for generating live attenuated vaccine (LAV) candidates. Modern biotechnological tools, such as reverse genetics and synthetic genomics, have expedited the creation of recombinant PoRVA strains with defined antigenic profiles and enhanced in vitro growth characteristics. However, a significant concern regarding LAV candidates derived from cell culture adaptation is the risk of virulence reversion upon pig back-passage, necessitating thorough safety and genetic stability evaluations. Nevertheless, utilizing stable cell lines or organoid platforms presents a feasible and cost-effective approach for large-scale PoRVA vaccine production. Future research should focus on identifying vaccine candidates that provide broad protection and exceptional safety, with an emphasis on cross-protection against divergent epidemic genotypes, while ensuring the economic feasibility of innovative manufacturing approaches. Full article

Background/Objectives: Staphylococcus haemolyticus is a common skin commensal that has emerged as a multidrug-resistant nosocomial pathogen, with sequence type 42 frequently implicated in clinical settings. The genetic basis of fusidic acid (FA) resistance mediated by mutations in elongation factor-G (fusA/EF-G) has not been systematically characterized in S. haemolyticus. Methods: Five representative FA-susceptible S. haemolyticus isolates were selected. In vitro FA resistance was induced by incubating each isolate on Mueller–Hinton agar containing 4 µg/mL FA at 37 °C for 48 h. Resistant colonies were recovered and fusA was sequenced by Sanger sequencing to identify mutations. Growth doubling times of EF-G mutant isolates were measured and compared with those of the parental susceptible strains. Potential fitness-compensatory changes in fusA were assessed by serial passage of selected mutants for ten successive passages and re-sequencing of fusA. Results: A total of 28 FA-resistant colonies were recovered. Sequencing identified mutations at seven nucleotide loci corresponding to ten distinct amino acid substitutions in EF-G: Q115L, L430S, E433G, G452C, H457Y, H457N, H457L, H457Q, R464L, and A655G. The mean doubling time of EF-G mutant isolates was significantly longer than that of the wild-type parental strains (mutants: 55.75 ± 7.78 min, n = 28; wild type: 40.78 ± 4.13 min, n = 5; Welch’s two-sample t test: t = −6.34, df ≈ 10.0, two-tailed p < 0.0001). Following ten serial passages, we did not detect compensatory mutations in fusA that restored the ancestral growth rate. Conclusions: FA resistance in S. haemolyticus can be rapidly induced in vitro through mutations in EF-G/fusA. Compared with previously reported EF-G mutations in other staphylococci, we identified two novel substitution sites (L430S, E433G) and previously unreported substitutions at established resistance positions (H457N, H457L, A655G). Mutations in EF-G (encoded by fusA) were associated with a measurable in vitro fitness cost; following ten serial passages, compensatory mutation was not detected in fusA. These findings support systematic surveillance of EF-G/fusA mutations in clinical S. haemolyticus isolates. Further studies should address their prevalence, stability, transmissibility, and clinical impact, particularly where FA use is increasing and patient populations are vulnerable. Full article

The antimicrobial resistance crisis has led to the use of metals and bacteriophages as possible alternatives to antibiotics. Experimental studies have examined interactions between ionic/nano-silver and bacteriophages against multidrug-resistant bacteria. However, these approaches have often failed to examine whether silver affects the stability and infectivity of bacteriophages. Here, we utilized experimental evolution to evolve resistance to ionic silver in bacteriophage T7. High ionic silver concentrations that do not represent physiological exposure conditions were used to impose strong selective pressure. Evolution of ionic silver resistance in phage T7 was rapid, as evidenced by recovery of bacteriophage growth in E. coli following repeated exposures to ionic silver, enhanced infectivity of silver-selected populations relative to parallel control and ancestral populations under increasing ionic silver concentrations, and greater suppression of E. coli growth in standard medium. Furthermore, silver resistance evolved without loss of thermal or pH stability under the conditions tested. The genomic foundation of silver resistance was relatively simple, with positive and negative natural selection differentiating the silver-selected populations from the controls and ancestral populations across serial passages in silver. Support for replication-associated adaptation under ionic silver selection may be reflected in recurrent mutations identified in genes involved in transcription, DNA replication, and genome maintenance, including T7p07 (RNA polymerase), T7p10 (DNA ligase), and T7p29 (DNA polymerase I). These findings highlight the importance of evaluating phage –silver combination strategies within an evolutionary framework that accounts for the adaptive capacity of bacteriophages under silver selection. Full article

Background/Objectives: Respiratory syncytial virus (RSV), human metapneumovirus (hMPV), and parainfluenza virus type 3 (PIV3) are leading causes of acute respiratory infections in children and the elderly, yet no licensed T-cell vaccines are available. This study aimed to develop multivalent T-cell vaccine candidates against these pathogens using a live attenuated influenza virus (LAIV) vector platform. Methods: Conserved F, N, and M proteins of RSV, hMPV, and PIV3 were identified through multiple sequence alignments. Fragments enriched with experimentally confirmed and predicted T-cell epitopes were selected using the IEDB and NetMHCpan servers. These fragments were assembled into polyepitope immunogenic cassettes, and their selected order was determined by thermodynamic analysis of mRNA secondary structures using the RNAfold Web Server. The selected cassettes were cloned into the neuraminidase (NA) gene of a cold-adapted LAIV vector. Recombinant viruses were rescued by reverse genetics and assessed for replicative fitness in embryonated chicken eggs and MDCK cells, NA enzymatic activity and genetic stability upon serial passaging. Results: Four cassettes were designed for RSV, three for hMPV, and one for PIV3, all containing fragments with multiple T-cell epitopes. Three recombinant viruses of LAIV/RSV type and three of LAIV/hMPV type were successfully rescued, while attempts to recover the remaining recombinant viruses, i.e., LAIV/RSV and LAIV/PIV3, were not successful. All rescued recombinant viruses replicated to titers comparable to the parental LAIV strain and retained the full-length insert for at least eight passages in eggs. Importantly, NA enzymatic activity of the LAIV vector was not compromised by the insertion of the polyepitope T-cell cassettes. Conclusions: We developed a panel of recombinant T cell-based vaccine candidates against RSV and hMPV using the LAIV vector platform. These recombinant viruses encode conserved T-cell epitopes of the target viruses while retaining the biological properties of LAIV strains. Taken together, these characteristics warrant further evaluation of these recombinant viruses in appropriate relevant in vitro models to directly assess their immunogenicity in terms of stimulating a T-cell response against target pathogens. Full article

Hepatitis C virus (HCV) infection remains a major global health burden, and no vaccine preventing chronic infection is available. The envelope glycoproteins, E1 and E2, form a complex essential for viral entry; however, the mechanisms governing E1/E2 assembly and stability remain incompletely defined. Here, we investigated the role of the E1/E2 transmembrane (TM) regions in HCV infectivity using chimeras of JFH1-based recombinants with isolate-specific Core-NS2 sequences in which the C-terminal TM domains of E1 (TME1), E2 (TME2), or both (TME1E2) from the H77 isolate (genotype 1a) replaced those of isolates representing genotypes 1–6. We further introduced the TM domains of S52 (genotype 3a) or J6 (genotype 2a) into H77 and included reciprocal swaps between J6 and S52. Most TM-swap chimeras displayed impaired infectivity; however, serial passaging led to partial recovery associated with adaptive mutations in E1/E2 mapping not only to the C-terminal TM regions but also to the E1 stem and the internal E1 TM region (iTME1). Extending the TME1 swap to include upstream α-helical segments improved infectivity in selected chimeras, whereas inclusion of iTME1 abolished infectivity. These findings support functional interactions between membrane-associated regions of E1/E2 and their ectodomains and highlight their relevance for E1/E2-based HCV vaccine design. Full article

Background/Objectives: Serial passage in embryonated eggs is widely used to attenuate the infectious bronchitis virus (IBV) for vaccine production; however, the evolutionary processes underlying attenuation and immunogenicity remain incompletely understood. Here, we analyzed genome-wide viral evolution during serial passages to investigate how mutations emerge, persist, are lost, or become fixed over time and how these dynamics relate to changes in pathogenicity and immunogenicity. Methods: Deep sequencing was performed on 11 representative serial passages (P2–P79) of the UY/11/CA/18 strain, including two derivative lineages: P7 VIR (virulent) and P53 VAC (attenuated and immunogenic). Results: This study identified an early adaptive phase characterized by a limited set of mutations potentially associated with genome replication, viral RNA processing, and virion assembly, including a key change in non-structural protein 14 and variants in M and 3c (E). This phase was followed by a broader expansion of the variant spectrum across replicase genes and delayed accumulation of Spike protein variants. Most Spike changes emerged during later passages and exhibited transient dynamics, and only a subset reached a high frequency after the establishment of early replicase- and structural-associated changes. Consistent with these dynamics, P7 VIR diverged before the late accumulation of Spike variants and retained a pathogenic phenotype, whereas P53 VAC diverged after the emergence of early high-frequency variants but before the extensive late-stage Spike variation observed in P79, which was associated with reduced immunogenicity. Conclusions: These findings support a multi-step model of IBV attenuation in which progressive filtering of genome-wide variation shapes distinct evolutionary outcomes during serial passages. This evolutionary framework provides insight into the relationship between attenuation and immunogenicity and may help guide the rational design of live attenuated vaccines. Full article

Background/Objectives: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is a major nosocomial pathogen. Although newer agents have reduced colistin use in high-income countries, this polymyxin remains important in many low- and middle-income settings. Colistin resistance in K. pneumoniae is most commonly associated with chromosomal alterations affecting the MgrB–PhoPQ pathway, or with plasmid-mediated mcr genes. This study aimed to investigate chromosomally mediated colistin resistance in CRKP clinical isolates from a Tunisian tertiary hospital. Methods: Between 2010 and 2015, 317 non-duplicate CRKP isolates were collected at Charles Nicolle Hospital, Tunis. Colistin MICs were determined by broth microdilution. Phenotypic tests and PCR characterized carbapenemases, extended-spectrum β-lactamases, AmpC, plasmid-mediated quinolone resistance, mcr and virulence genes. Porins (OmpK35/OmpK36) and the mgrB, phoP and phoQ loci were analyzed by SDS-PAGE and sequencing. Clonal relatedness was assessed by ERIC-PCR and multilocus sequence typing. We additionally compared colistin-resistant isolates with a panel of colistin-susceptible CRKP controls and assessed phenotypic stability after serial passages without colistin. Results: Five isolates (1.6%) were colistin-resistant. All were multidrug-resistant, produced OXA-48, and two also carried NDM-1. The isolates belonged to five distinct sequence types, including high-risk clones (ST11, ST101, ST147). No mcr genes were detected. Four isolates carried disruptive mutations in mgrB, and the remaining strain harbored inactivating mutations in both phoP and phoQ with an intact mgrB. Truncating alterations in PhoP/PhoQ and frequent loss or truncation of OmpK35/OmpK36 were observed. No mgrB/phoP/phoQ alterations were detected among colistin-susceptible controls, and colistin MICs remained stable after 7 days of drug-free passaging. Conclusions: In Tunisian CRKP, colistin resistance was associated with chromosomal alterations, predominantly involving disruption of the MgrB–PhoPQ pathway, in the absence of mcr genes. These mechanisms in both high-risk and emerging sequence types underscore the adaptability of CRKP and the need for surveillance where colistin remains an important therapeutic option. Full article

H5 subtype avian influenza virus (AIV) can infect both chickens and ducks, leading to substantial economic losses. Nevertheless, certain strains cause silent infections in ducks. In this study, a goose-origin clade 2.3.4.4h H5N6 AIV was isolated, which caused high mortality in mixed-gender white leghorn chickens but no deaths in mixed-gender mallard ducks. After independent serial in vitro passage in duck embryo fibroblasts (DEFs) and in vivo passage in specific-pathogen-free (SPF) ducks, the DEF-passage 10 (P10) virus induced markedly higher mortality rates and viral loads in SPF ducks compared to the DEF-P1 virus and the original parental virus prior to passage. Similarly, the in vivo-passaged P3 and P4 viruses exhibited significantly higher mortality rates than the P1 virus in SPF ducks, with 100% mortality and markedly increased viral titers in the organs. A whole-genome SNP analysis identified seven high-frequency mutations in the M1, NA and NS1 proteins. The NS1-F161L substitution virus exhibited significantly increased mortality rates, viral loads in multiple tissues, and a robustly induced innate immune response in ducks. Furthermore, dynamic evolutionary variations in the NS1 protein among global H5 avian influenza viruses revealed that the NS1-F161L substitution became dominant in clade 2.3.4.4b viruses in 2021 and subsequent years. Collectively, our findings demonstrate that host-driven adaptation can rapidly increase the pathogenicity of H5N6 AIVs in ducks and identify NS1-F161L as a critical virulence marker. These results offer novel insights relevant to the molecular surveillance, virulence prediction, and risk assessment of circulating H5 AIVs in waterfowl. Full article