AbstractAfrican swine fever (ASF) is a highly pathogenic swine infectious disease caused by African swine fever virus (ASFV), with a mortality rate approaching 100% in domestic pigs and causing severe economic losses to the global pig industry. Despite the recent approval of live-attenuated ASF vaccines in limited regions such as Vietnam, universally safe, globally authorized commercial vaccines and effective antiviral therapeutics remain unavailable, creating an urgent demand for innovative anti-ASFV intervention strategies. In this study, epigallocatechin gallate (EGCG) was identified to exert prominent inhibitory effects on ASFV proliferation in vitro. Viral life cycle assays indicated that EGCG mainly targets the internalization and post-entry genome replication stages. Molecular docking combined with bio-layer interferometry (BLI) confirmed high-affinity direct binding between EGCG and two indispensable ASFV proteins, p72 and p1192R. Furthermore, EGCG dose-dependently activates the AMP-activated protein kinase (AMPK) signaling pathway, downregulates lipid synthesis-related genes, and reverses ASFV-induced lipid droplet accumulation and abnormal increases in total cholesterol (TC), triglycerides (TG), and free fatty acids (FFAs), thereby disrupting the lipid metabolic microenvironment required for viral replication. Collectively, EGCG suppresses ASFV replication through dual mechanisms: targeting key viral proteins and regulating host lipid metabolism. This study highlights EGCG as a promising anti-ASFV candidate and offers a novel theoretical basis for the development of anti-ASFV agents.
Fundamental aspects of the biology of the third most prevalent human malaria species Plasmodium malariae, including the basis for its capacity to establish chronic, low-density infections associated with debilitating quartan fevers, remain poorly characterized, hampering control efforts. Here, we combined ex- vivo flowcytometric profiling of P. malariae isolates with infection studies in a recently developed humanised mouse model. We demonstrate a five-fold higher tropism of P. malariae compared to P. falciparum for immature red blood cells, challenging a nearly century-old paradigm, and—for the first time—provide evidence for the parasite’s capacity to home to and infect host bone marrow. Collectively, these findings reveal key life cycle features of this enigmatic and neglected human malaria parasite.
East and Central Africa experiences heightened threats for Ebola Virus Disease (EVD), with the latest outbreak caused by the Bundibugyo ebolavirus (BDBV) reported in the Democratic Republic of the Congo (DRC) and Uganda on 15th May 2026. The BDBV outbreak comes just over a year following declaration of Sudan Virus Disease (SVD) outbreak in Uganda on 30th January 2025. Following the 2025 outbreak, a race against the clock mobilisation occurred over the subsequent four days to launch a ring vaccination trial for a candidate vaccine against SVD. During this four-day period, the investigation team leveraged ongoing engagements with trial sponsors to activate prefinancing mechanisms and unlock in-kind financing from the government of Uganda. Further, the team activated a prepositioned research protocol to secure rapid ethics and regulatory clearance, and quickly accessed investigation product within the country, supportive ethics/regulatory systems, presence of logistical and data management systems established during the 2022 outbreak, collaborative relationship between the trial and the Ministry of Health outbreak response teams. Finally, previously trained field and community engagement personnel were mobilised overnight, offered refresher training and rapidly deployed to the field. In conclusion, the unprecedented four-day turnaround was made possible by the research, logistical and staffing capacities established during the 2022 SVD outbreak, an achievement that offers a blueprint for rapid evaluation of medical countermeasures during outbreaks.
Seasonal influenza A/H3N2 poses a significant public health burden due to its rapid antigenic evolution, necessitating frequent vaccine updates. This study systematically analysed the antigenic evolution of 14 H3N2 vaccine strains (2000-2022) using pseudovirus-based neutralisation assays and antigenic cartography. Vaccine strains exhibited cross-neutralisation patterns, with higher reactivity among strains from adjacent years, forming six antigenic clusters. Key amino acid mutations (e.g., 144 K, 189N) were identified as critical drivers of antigenic variation, with clustering-specific effects on neutralisation sensitivity. Based on these findings, a consensus HA immunogen (consensus up-HA) was designed by combining mutations at key sites. This immunogen demonstrated broad-spectrum neutralisation against pseudoviruses representing circulating strains from 2011 to 2024, including recent variants. These results highlight the potential of targeted HA modifications to develop broadly cross-reactive H3N2 vaccine candidate, offering broader and more durable neutralising activity against evolving H3N2 strains.
AbstractWastewater-based surveillance (WBS) has become an important public-health tool for tracking community-level circulation of emerging and re-emerging pathogens, but wastewater measurements are not directly interpretable public-health indicators. Signals recovered from sewer systems are shaped by sampling variability, environmental and laboratory noise, population-dependent bias, and high-dimensional molecular complexity. Machine learning (ML) is increasingly used to extract predictive and decision-relevant information from these data, but the strength of the supporting evidence varies substantially. This narrative review examines how strongly current applications are supported by published evidence. Sources were identified through iterative searches of three literature-search sources updated to June 2026 and classified by role, analytical task, and strength of evidence. To keep the analytical scope consistent, we use a pragmatic four-layer classification: classical statistical models, conventional machine learning, deep learning, and hybrid or mechanistically informed approaches. Applications are reviewed across five analytical tasks: predictive modeling and forecasting, anomaly and fluctuation detection, data harmonization and normalization, high-dimensional interpretation, and integrated decision support. Among 35 sources reporting analytical or modeling work, 25 analyzed SARS-CoV-2 alone; demonstrated early-warning evidence came predominantly from L1 methods, and most target-task combinations remain prospective. Even within SARS-CoV-2 surveillance, reported performance was context-dependent. We examine the data-quality, methodological, and implementation barriers that limit broader application and validation. Future priorities include standardized and collaborative frameworks, multimodal data fusion, mechanistic and digital-twin modeling with explicit uncertainty assessment, and transparent and actionable analytics, with broader surveillance applications constrained by governance and public acceptability.
Resistance to Leishmania (L.) major depends on the development of a L. major-specific Th1 response, while Th2 differentiation results in susceptibility. We previously showed that the early microenvironment of infected skin delivers important signals for T cell differentiation. We found increased expression levels of coagulation factor X (F10 and FX for protein, respectively) 16 h after infection in the skin of resistant as compared to susceptible mice. Activated FX is a ligand of proteinase-activated receptor-2 (PAR2), a modulator of inflammatory responses.To assess the role of PAR2, we analyzed the course of L. major infection in PAR2-deficient (PAR2-/-) mice on a resistant C57BL/6 background. PAR2-/- mice developed significantly larger lesions and harboured more parasites in footpads and draining lymph nodes compared to wild-type mice. In addition, their antigen-specific T cell response in was shifted towards Th2.Conversely, early treatment of susceptible BALB/c mice with a PAR2-agonist reduced parasite loads in footpads and spleens and shifted the T cell response towards Th1. This was accompanied by significantly higher expression of the Th1-promoting cytokines IL-6, IL-12, and TNF-α in the infected skin. We conclude that PAR2 activation favours Th1-differentiation and resistance in experimental leishmaniasis due to an altered initial microenvironment with increased expression of Th1-promoting cytokines in the infected skin.
Klebsiella pneumoniae carbapenemase-3 (KPC-3) remains rare in South Korea, where KPC-2 is the dominant carbapenemase, making the repeated detection of a concentrated blaKPC-3 signal over five years notable. We performed genomic analyses of blaKPC-3-harbouring K. pneumoniae from a regional healthcare network. Two chromosomally distinct lineages with concordant capsule loci (ST628/KL15 and ST48/KL62) presented multidrug-resistant phenotypes, and the virulence-associated loci were confined to ST48. Single-nucleotide polymorphism (SNP) analyses revealed near-clonal relatedness within lineages, with 0-38 pairwise SNPs among ST628 isolates and 8 SNPs between the two ST48 isolates. Core-genome multilocus sequence typing (cgMLST) supported this structure, as ST628 isolates were assigned to complex type 19149 with 0-7 allelic differences, and ST48 isolates were assigned to complex type 19150 with 5 allelic differences. These patterns support vertical spread via clonal expansion across multiple facilities. Despite substantial chromosomal separation, most isolates carried the same IncFII(K) plasmid backbone and blaKPC-3, and they were nearly indistinguishable from a plasmid previously reported in South Korea. One isolate carried blaKPC-3 on a distinct multireplicon IncFIB(K)/IncFII(K) plasmid, indicating that the signal was not confined to a single plasmid backbone. In both plasmids, blaKPC-3 was embedded within Tn4401b. These findings indicate that a rare blaKPC-3 genotype can persist regionally through sustained clonal dissemination and that cross-lineage linkage is compatible with past horizontal transfer involving a conserved plasmid. These findings underscore the need for subtype-resolved, regionally coordinated genomic surveillance in connected healthcare networks to detect uncommon carbapenemase variants early.
West Nile virus (WNV) is a zoonotic Orthoflavivirus transmitted by mosquitoes that is responsible for outbreaks of meningitis and encephalitis worldwide. Driven by climate change, WNV has expanded as a global public health concern, particularly in temperate regions. However, there are still no specific approved therapies, reinforcing the need for antiviral development. Previous works have documented that WNV multiplication strictly depends on certain cellular lipids. To identify novel lipid-related therapeutic targets, we analyzed the infection driven alterations in the CNS lipidome, the primary tissue supporting WNV replication. Our results indicated that the major alterations in the brain lipid content of WNV-infected mice corresponded to triacylglycerols (TAGs). Moreover, transcriptomic analysis showed that infected brains underwent changes in the expression of TAG metabolism. Supplementation with exogenous fatty acids increased lipid droplets (LD) content and promoted viral replication in cell culture models. On the contrary, pharmacological intervention in TAG metabolism using diacylglycerol acyltransferase inhibitors (DGATi) suppressed WNV multiplication in cell culture models. As a proof-of-concept of the therapeutic potential of DGATi, treatment of mice with A922500 reduced viral burden in the brain and proinflammatory cytokine production. Overall, our results unveil the importance of LDs and glycerolipid metabolism for WNV and highlight the potential of therapeutic interventions targeting this pathway to control viral replication and neuroinflammation.
Arthritogenic alphaviruses, like o’nyong-nyong virus (ONNV), cause debilitating musculoskeletal diseases and are geographically expanding. To predict their emergence, we seek to better understand evolutionary mechanisms that enable changes in virus tropism. Here, we identify adaptive mutations in the ONNV non-structural proteins (nsPs) that arose during cellular serial passaging and enabled ONNV to infect non-permissive Lunet cells. Using shotgun proteomics, we show that this human hepatoma cell line lacks the four-and-a-half-LIM domain protein 1 (FHL1), an essential host factor in ONNV RNA replication. Individual single nucleotide mutations in the nsP1 ring-aperture membrane-binding and oligomerization domain, the nsP3 macrodomain, and the nsP3 opal stop codon overcome FHL1 deficiency in Lunet cells by enhanced RNA replication. These findings demonstrate how subtle genomic changes in nsPs can profoundly influence alphavirus replication and tropism.
For Brucella spp., the ability to invade and survive within host macrophages is essential for causing chronic infections in their mammalian hosts. In this study, a genome-wide CRISPR knockout screen was performed for the first time in human THP-1 macrophages to identify host genes mediating resistance to Brucella invasion and intracellular survival. Results showed that the screening identified 35 candidate genes, 11 of which were selected to generate monoclonal knockout cell lines for functional validation. This study demonstrated that knockout of WDR4, ZNF532, or MTHFD1 significantly restricted Brucella invasion and early intracellular survival. In addition, TRAPPC2 knockout restricted Brucella invasion and, crucially, its intracellular survival throughout infection, exerting the most potent antibacterial effect. Mechanistically, TRAPPC2 deficiency suppresses Brucella infection by inhibiting autophagosome formation in macrophages. Furthermore, TRAPPC2 knockout decreases macrophage apoptosis and improves host cell viability following Brucella infection. These results provide therapeutic targets for combating Brucella infection and offer novel insights into the molecular mechanisms associated with Brucella-induced chronic infections.
The continuous antigenic drift of zoonotic H7 avian influenza viruses remains a significant global public health threat. While monoclonal antibodies (mAbs) targeting the hemagglutinin (HA) head offer potent neutralization, their efficacy is often undermined by rapid viral escape. In this study, we systematically mapped the antigenic architecture of the H7 HA head domain using a panel of 30 murine mAbs. By integrating epitope mapping with longitudinal evolutionary analysis of over 2,500 isolates, we identified highly conserved, functionally constrained residues, specifically G70, G132, N167, and M173, that remained nearly invariant over decades. Leveraging these insights, we engineered a chimeric bispecific antibody (BsAb-H7) to simultaneously engage two distinct, non-overlapping conserved epitopes. BsAb-H7 demonstrated broad neutralization breadth across divergent H7N9, H7N7, and H7N3 subtypes in vitro and exhibited a superior in vivo pharmacokinetic profile. In lethal murine challenge models, BsAb-H7 provided robust prophylactic and therapeutic protection against heterologous H7N7 infection. Notably, localized intranasal administration significantly outperformed systemic delivery by accelerating viral clearance and resolving pulmonary immunopathology during delayed intervention windows. These results demonstrate that bispecific targeting of conserved HA epitopes effectively enhances antiviral breadth and limits immune escape, providing a promising strategy for the development of broadly protective therapeutics against rapidly evolving H7 influenza viruses.
Pertussis causes recurrent outbreaks worldwide. Clinical surveillance can be affected by healthcare-seeking behaviour, diagnostic practices, testing sensitivity, and reporting delays. This study evaluated the utility of wastewater surveillance (WWS) by quantifying Bordetella-associated IS481 and IS1001 DNA concentrations. Weekly wastewater samples were collected from four treatment plants in Osaka Prefecture, Japan, between April 2023 and July 2025, and IS481- and IS1001-associated DNA concentrations were measured using quantitative PCR. The putative B. pertussis-associated IS481 DNA concentration increased markedly from 2024 onwards. Both threshold-based onset detection and cross-correlation analysis indicated that the wastewater IS481 DNA concentration preceded reported cases by one to several months. In contrast, the B. parapertussis-associated IS1001 DNA concentration exhibited a transient peak in 2023, which aligned with contemporaneous clinical reports but remained at low levels thereafter. These findings suggest that WWS may detect increases in pertussis-related activity earlier than clinical reporting and highlight the potential of WWS as a complementary tool to clinical surveillance for monitoring respiratory infectious diseases.
The global emergence of hypervirulent Klebsiella pneumoniae (hvKP) and extensively drug-resistant (XDR) phenotypes has precipitated a severe public health crisis with limited therapeutic options. In particular, the hypervirulent ST65 and ST23 lineages, alongside the carbapenem-resistant ST11 lineage, have been reported to be emerging globally with the potential for continued large-scale dissemination. Despite the urgent need for preventive strategies, the regulatory networks governing hvKP virulence and their potential as targets for live attenuated vaccines remain inadequately explored. In this study, we elucidate a pathogenic mechanism driven by the transcription factor RpoE. Deletion of the rpoE gene in hvKP significantly attenuated virulence by directly downregulating the expression of type 3 fimbriae (T3F) and subsequently impairing biofilm formation. Based on this marked virulence attenuation, we evaluated the ΔrpoE strain as a potential live attenuated vaccine candidate. Remarkably, immunization elicited protective immune responses involving functional antibody responses and CD4+ T cell-associated immunity, conferring host protection against the tested ST11, ST23, and ST65 clinical isolates. Our findings define an RpoE-T3F regulatory axis involved in hvKP virulence and support ΔrpoE as a potential live attenuated vaccine candidate with protective efficacy against these tested clinically relevant KP strains.
Nipah virus (NiV) is a highly lethal zoonotic henipavirus that causes severe respiratory disease and encephalitis and remains a priority pathogen for outbreak preparedness. Timely diagnosis is essential for containment, yet nucleic acid-based testing is often impractical in outbreak-prone, resource-limited settings. Here, we report the development of a rapid antigen test for NiV based on a cellulose nanobead (CNB)-enhanced lateral flow immunoassay (LFIA) targeting the viral nucleocapsid protein (NP). Anti-NiV NP antibodies were isolated from human synthetic phage-display libraries (scFv and Fab) by iterative bio-panning, reformatted as full-length IgGs, and screened using ELISA and immunofluorescence assays. Four lead antibodies were advanced to a sandwich-pairing matrix, which identified an optimized capture-detection configuration (mAb #5 as capture; mAb #2 as detection). Bio-layer interferometry confirmed nanomolar binding affinities for the selected antibodies (KD = 39.50 nM for mAb #5; KD = 26.36 nM for mAb #2). The finalized CNB-LFIA produced results within 15 min and achieved an analytical limit of detection of 1 ng/mL for recombinant NiV NP. The assay observed no cross-reactivity with SARS-CoV-2, SARS-CoV, MERS-CoV, or influenza A, whereas weak cross-reactivity for measles virus NP was observed only at a high antigen concentration (2 µg/mL). Using γ-irradiated cultured NiV quantified by RT-ddPCR, the LFIA detected viral load down to 4.1 × 107 copies/mL (4.1 × 10⁶ copies/reaction) by a portable reader and 8.3 × 107 copies/mL (8.3 × 10⁶ copies/reaction) by the naked eye. Collectively, this CNB-LFIA provides rapid, equipment-free NiV detection, supporting its potential utility for point-of-care diagnosis in resource-limited settings.
Human noroviruses (HuNoV) are the most common cause of viral gastroenteritis worldwide, causing sporadic cases and outbreaks. Noroviruses that infect wild and domestic animals, including pigs and dogs, are genetically related to human noroviruses, increasing the potential for cross-species transmission. To investigate the potential of noroviruses to cross the species barrier, we used porcine, canine, and avian precision-cut intestinal slices. We show that human norovirus virus-like particles (VLPs) bind to pig and dog intestinal tissue and are taken up with a similar efficiency as the porcine and canine noroviruses, respectively. In contrast, no binding or internalization of human, porcine, or canine noroviruses was detected in chicken tissue. We further showed that human norovirus replicates in pig intestinal tissue. In contrast, while animal noroviruses attached to human intestinal cells, intracellular uptake was limited. This suggests that human-to-animal transmission is more likely than animal-to-human transmission and that viral uptake likely presents a species barrier.
The H9N2 avian influenza virus (AIV) has caused substantial economic losses to the global poultry industry and poses a zoonotic threat to humans. Vaccination constitutes a pivotal strategy for the prevention and control of H9N2 AIVs. However, the ongoing antigenic evolution of the viruses pose a persistent challenge to the protective efficacy of existing vaccines. Therefore, the development of a broadly protective H9N2 influenza vaccine capable of eliciting cross-reactive immune responses is crucial for mitigating both the disease burden and the risk of pandemics. Here, we developed a bivalent chimeric inactivated vaccine, designated cHANA, by combining two individually rescued chimeric inactivated viruses, cHANA1 and cHANA2. Each recombinant virus carries one set of Epigraph-designed HA and NA immunogens, and the two sets were computationally optimized from global H9N2 HA and NA sequence datasets to complement each other in epitope coverage across the H9N2 viral population. Compared to the WHO-recommended candidate vaccine virus (CVV), AL/39, cHANA elicited more potent cross-reactive antibody responses and T cell immunity in mice. Furthermore, it elicited effective cross-protection against lethal challenge with heterologous H9N2 virus and significantly reduced pulmonary viral loads of mice. By conferring broad protective immunity, this vaccine represents a promising universal vaccine candidate for controlling H9N2 outbreaks.
OBJECTIVES:This study analysed a large French collection of carbapenemase-producing Klebsiella oxytoca complex (KoC) isolates (2017-2023) to map species and carbapenemase distributions and identify emerging lineages. We further investigated virulence traits to understand the epidemiological success of the dominant ST-2 lineage. METHODS:We conducted a retrospective study of all KoC isolates received at the French National Reference Center for Antimicrobial Resistance from 2017 to 2023. Whole-genome sequencing was performed to assess species assignment, phylogeny, and sequence types. Virulence analyses included biofilm formation assays (on abiotic surfaces and eukaryotic cells) and Galleria mellonella infection models. RESULTS:K. oxytoca sensu stricto remained the most prevalent species among the KoC. OXA-48-like enzymes were the most common carbapenemases, and two major lineages predominated: ST-141 and, far ahead, ST-2. The success of ST-2 appears linked to its low virulence combined with a strong capacity for long-term persistence. CONCLUSION:Our findings confirm that K. oxytoca comprises a complex of distinct species whose taxonomy warrants continued revision. ST-2 circulates widely in France and, despite its limited virulence, shows remarkable persistence, making it an epidemiological important lineage.
Pemivibart, a class 1/4 monoclonal antibody (mAb), is currently the only FDA-authorized SARS-CoV-2 mAb under an Emergency Use Authorization (EUA) in clinical use. The emergence of subvariants, including KP.3.1.1 and XFG, raises concerns about antibody efficacy. SA55, a fully human class 1/4 mAb, is currently under clinical trial, including a nasal spray formulation. Using the well-validated organoid-based neutralization assays, we compared the potency and breadth of Pemivibart and SA55. Our results demonstrated a significant decrease in Pemivibart's activity against KP.3.1.1 and XFG, with an approximately 80-fold increase in IC50 relative to the ancestral strain. Given KP.3.1.1's strong reliance on the TMPRSS2 pathway for cell entry, we further demonstrated that combinational treatment with Pemivibart and the broad-spectrum S2 antibody results in potent neutralization. Notably, SA55 maintained potent neutralization (IC50 ≤ 40 ng/mL) across all tested variants. Topical administration, which models nasal spray, dramatically suppressed viral replication of BA.5.2 and XFG in organoid models. Our findings evidence the high potency of SA55, supporting its potential for clinical application against emerging SARS-CoV-2 variants.
Subtype H9N2 of the avian influenza virus (AIV) poses a growing threat to the poultry industry and public health. However, since 2014, there has been no systematic study of its genetic evolution, genotype, spatial dynamics, and pathogenicity in China. Therefore, we performed a large-scale sequence analysis of the genome of Chinese H9N2 viruses from 2014 to 2025 using public databases and laboratory isolates. We identified 52 different genotypes in 1,591 H9N2 viruses, including 4 previously recognized genotypes (G6, G57, G58, and G68) and 48 newly defined genotypes (G118-G163) in this study. G57 and G118 were the main epidemic genotypes in China from 2014 to 2025. Bayesian phylogeographic analysis showed that there were 12 obvious migration paths for the spread of H9N2 AIVs in China from 2017 to 2022. In particular, the South China region was the main transmission centre. H9N2 AIV continues to circulate in chickens and ducks in China and spreads to other hosts. The H9N2 AIVs with the G57 and G118 genotypes could effectively replicate in MDCK, CEF, A549, and HBE cells with titres of 0.97-8.5 lgTCID50/mL. The G57 and G118 genotypes H9N2 viruses preferentially bound to α-2,6-linked sialic acid glycopolymers (human receptors), and effectively replicate in multiple organs of mice and chickens and cause pathological changes in the lungs. Thus, it is necessary to strengthen the monitoring and prevention of H9N2 AIVs in China.
Pigs serve as mixing vessels for influenza A viruses, facilitating reassortment and the emergence of variants with zoonotic potential. Since the 2009 pandemic, human-origin influenza gene segments have been repeatedly detected in swine populations, contributing to the generation of genetically diverse reassortant viruses. However, assessing the infection phenotypes and epithelial host responses of newly emerging reassortant swine influenza viruses remains challenging using conventional in vitro and in vivo approaches. Here, we established long-term expanding three-dimensional porcine airway organoids (pAOs) as a platform for the phenotypic and transcriptomic characterization of reassortant influenza viruses. We compared the infection phenotypes of a recently identified reassortant H1N1 virus, SNU01/H1N1/2023, with those of a classical swine-lineage H1N1 strain, GC0503/H1N1/2005, and the 2009 pandemic strain, CA04/H1N1/2009. All viruses productively infected pAOs, while SNU01 yielded higher levels of infectious progeny than the comparator strains. Bulk transcriptomic profiling at 24 h post-infection revealed that SNU01 infection was associated with an epithelial host-response profile more similar to CA04 than to GC0503, characterized by stronger epithelial antiviral and inflammatory transcriptional activation. Notably, SNU01 infection was associated with distinct cytoskeleton-associated transcriptional programs that were not prominent in the comparator infections. These findings demonstrate that porcine airway organoids can distinguish strain-associated differences in viral replication and epithelial host responses, providing a tractable platform for the comparative characterization of reassortant influenza viruses.
