Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide. Eight genotypes have been identified so far, with genotype 3 (gt3) predominating in high-income countries and commonly linked to eating undercooked pork. In Belgium, an estimated 54,000 annual HEV infections contrast sharply with only 25–117 clinically reported cases, revealing a critical surveillance blind spot. To evaluate the potential to complement clinical surveillance, we retrospectively analysed municipal wastewater weekly for HEV (presumed genotype 3) using digital PCR during 2022–2023. Sampling covered four Antwerp wastewater treatment plants (WWTPs) in 2022 (population of approximately 503,243 inhabitants) and 13 WWTPs across Flanders and Brussels in 2023, serving a population of approximately 2.6 million inhabitants (≈23% of Belgium's population). Viral signals were normalised to pepper mild mottle virus (PMMoV) as a proxy for faecal load and aggregated across sites to describe temporal trends. HEV gt3 RNA was detectable year-round in 2022 and 2023, but amplitudes and timing varied by catchment. In 2022, the wastewater HEV RNA signal showed an early-summer surge (May–June). In 2023, it rose from late May to a July–August peak with a second, broader elevation in late October–November. Weekly clinical case data identified at the Belgian National Reference Centre did not align with wastewater signals, suggesting that current surveillance captures only symptomatic or diagnosed infections. Since wastewater surveillance reveals sustained, seasonally patterned HEV gt3 circulation and captures asymptomatic infections missed by clinical surveillance, we advocate for further wastewater-based surveillance to complement traditional epidemiological monitoring.

The gut microbiome modulates mucosal immunity and contributes to multiple diseases, but the extent to which it shapes circulating and mucosal T cell repertoires remains unclear. Here, we paired CD4+ and CD8+ T cell receptor (TCR) sequencing with fecal 16S rRNA microbiome profiling and developed adaptive immune receptor repertoire-wide association study (AIRRWAS), a framework integrating computational TCR-microbiome interaction analysis with targeted in vitro validation. Across three independent cohorts, AIRRWAS identified reproducible associations between convergent TCR clusters and 21 bacterial genera, including core commensals, probiotics, and taxa with immunomodulatory roles. Predicted clonotypes were enriched in genus-specific interaction networks and preferentially activated by matched bacterial stimuli. These findings reveal conserved microbiome-driven immune signatures across distinct repertoires and demonstrate that the gut microbiome shapes both local and circulating T cell populations, providing a resource for biomarker discovery, immune monitoring, and microbiome-targeted therapies.

Computer-aided lung sound analysis (CALSA) paired with digital auscultation provides a cheap, objective, and non-invasive measurement option for healthcare providers. An important task of CALSA algorithms is the detection of the inspiration and expiration breathing phases. In this work, we propose a system that uses a deep learning model to predict the inspiration and expiration phases from Mel-spectrograms of the lung sound. Multiple convolutional neural networks are investigated alongside vision transformer architectures. Due to the absence of standardized metrics for the evaluation of breathing cycle detection systems, we propose a novel intersection-over-union (IoU) score in order to provide a thorough evaluation. Our final system obtained a duration weighted mean IoU score of 0.82 and an F1 score of 80% for cycle-based detection. Moreover, we demonstrate that the performance of our system is on par with the cycle-labelling performance of respiratory experts and surpasses the performance of other comparable methods. The proposed system was trained, validated, and tested on diverse datasets with a respective 591 minutes, 66 minutes and 146 minutes of respiratory recordings.

The alarming rise of antimicrobial resistance and the declining efficacy of conventional antibiotics emphasize the need for preventive strategies. Within the setting of device-associated infections, antimicrobial peptides (AMPs) have been extensively studied as antimicrobial candidates, owing to their broad-spectrum activity and structural versatility, enabling integration into functional surface coatings. This review provides a comprehensive overview of AMP-based prophylactic approaches, with a particular focus on coatings for medical devices prone to biofilm formation, such as endotracheal tubes, catheters and implants. While surface immobilization of peptides can be accomplished through comparatively straightforward methodologies, the field has progressed toward sophisticated matrix-based systems that enhance stability, biocompatibility and controlled functionality. Yet, despite extensive in vitro and small-scale in vivo studies, clinical translation remains very limited and constrained by several hurdles including regulatory ambiguity and production costs. Overall, this work aims to provide an up-to-date overview of the AMP-based technologies in infection prevention research.

BACKGROUND AND AIMS:Myocarditis is a group of inflammatory diseases of the myocardium, with viral infections being the leading cause. Previous murine studies have demonstrated a detrimental effect of extensive exercise on the acute course of viral myocarditis. Recently, we were the first to report that continuation of moderate exercise during murine viral myocarditis modulates myocardial inflammation and fibrosis at the late stage of disease, yet we did not evaluate early time points. In this study, we aimed to evaluate the impact of moderate intensity training on the acute course of disease, and compare it to the effects of a high intensity protocol. METHODS AND RESULTS:Two separate experiments were performed. For the moderate intensity (Mod) endurance exercise experiment, 50 male C57BL/6J mice (11 weeks old) were randomised to 3 weeks of treadmill running (ModEEX, 18 cm/sec, daily) or not (ModSED). Two weeks into the experiment, animals received a single intraperitoneal injection with either coxsackievirus B3 (CVB) to induce viral myocarditis, or phosphate-buffered saline (PBS) vehicle. For the high intensity (Hi) endurance exercise experiment, another 20 male C57BL/6J mice (17 weeks old) were randomised to 3 weeks of treadmill running (HiEEX) or not (HiSED). After two weeks of training, all animals of the Hi experiment were injected with CVB, and the training protocol was intensified with increasing running speeds until exhaustion in the final week of training. All animals were sacrificed 6-7 days after virus or vehicle administration. All groups demonstrated complete survival except for 1 animal of the HiSED group, and showed comparable clinical signs and body weight evolution. Nor moderate, neither high intensity exercise had any significant impact on plasma troponin levels, semiquantitative scores of cardiomyocyte loss, and digital areas of necrosis. Morphologically however, HiEEX mice showed markedly less inflammatory cells in the necrotic areas of the myocardial lesions compared to HiSED mice, as was confirmed by digital quantification (x103 inflammatory cells per mm2 HiEEX: 6.24±0.32SEM vs HiSED: 8.02 ±0.36SEM, P=0.002). The same digital quantification did not show significant differences between ModEEX and ModSED lesions. Using an extensive panel of immunohistochemical inflammatory cell markers, a different composition of inflammatory cell subtypes was observed in the myocardial lesions of HiEEX compared to ModEEX mice, with a shift towards a pro-inflammatory milieu in HiEEX mice (ratio iNOS/Arg1 HiEEX: 0.49 vs ModEEX: 0.22, P=0.041 and ratio Tbet/GATA3 HiEEX: 4.75 vs ModEEX: 0.82, P=0.005). The cardiac viral load varied considerably, but no impact of exercise was observed, nor did cardiac expression of remodelling genes (Serpina3n, CTGF, and TGF-β) show an exercise effect. CONCLUSION:In the acute phase of murine viral myocarditis, lesions show significantly fewer inflammatory cells in the myocardial lesions when performing high intensity exercise during infection. Moreover, compared to moderate intensity exercise, the composition of the inflammatory infiltrate shifts towards a more pro-inflammatory phenotype.

SUMMARYWith varicella-zoster virus (VZV) being a strictly human-specific pathogen, in vitro cell culture models to study the VZV-host cell interactome predominantly rely on the use of primary human cells, immortalized cell lines, and-more recently-stem cell-derived models. In this work, based on literature reports published within the past 15 years, we attempted to summarize major lessons learned from in vitro VZV research, with a specific focus on whether and how a variety of host cells respond upon VZV infection at the cellular level. Following this specific approach, we describe the cellular events occurring following VZV infection in a neural cell type context, an immune cell type context, and a skin cell type context. Highly relevant, and for sure subject to the development of future VZV research, cell types within each of the three compartments reviewed display similarities but also significant differences in cellular response to VZV infection. Clearly, these need further clarification on a cell-type and/or VZV strain-specific level. Finally, to increase physiological relevance, we propose an integrated approach for future VZV-host cell interactome studies on a systems level by using advanced human-induced pluripotent stem cell-derived skin, peripheral, and central nervous system compartments that can be complemented with an isogenic immune cell component. Combined with the implementation of state-of-the-art multi-omics analyses, as well as electrophysiological recordings, this next-generation toolbox for advanced virus-host cell interactome studies may help to elucidate important aspects of VZV biology, including the suggested link between VZV pathology and neurodegenerative diseases.

Based on reports of ketamine seizures, self-reported consumption and harmful associated health effects, there are signs of increased ketamine use. However, monitoring population-level consumption patterns remains difficult. This study employed wastewater-based epidemiology (WBE) to address this challenge by analysing influent wastewater (IWW) from Belgium for biomarkers of ketamine consumption to evaluate spatio-temporal trends. Daily 24-h composite IWW samples were collected from 26 locations across the northern part of Belgium (Flanders and Brussels region), every Monday and Wednesday in 2023 (n = 716). Additionally, a temporal dataset from daily IWW samples from Leuven (n = 399) and Brussels (n = 285) in 2021 and 2022 was evaluated using time series analysis. Measured concentrations of ketamine, norketamine and dehydronorketamine in IWW were transformed to population-normalised mass loads (PNML, expressed in mg/day/1000 inhabitants) to assess ketamine consumption patterns. Ketamine, norketamine and dehydronorketamine were detected in respectively 98%, 96%, and 76% of all samples. Substantial consumption was observed in both urban and rural areas, with higher PNMLs in major cities and the east of Flanders. Temporal analysis showed no seasonal trends but an increase in weekend consumption, likely linked to recreational use. PNMLs increased 7- to 11-fold compared to available data in 2012. Actual consumption and direct disposal down the drain could be differentiated by the investigation of the ketamine to norketamine ratio. Ketamine use is widespread across the north of Belgium, encompassing a relatively stable, year-round consumption. In future WBE studies, it is recommended to measure both norketamine and ketamine to assess ketamine consumption and to rule out any dumping events.

Respiratory syncytial virus (RSV) continues to have a large medical and economic impact worldwide, mainly in infants, elderly, and immunocompromised patients. While several vaccines and prophylactic antibodies are now available, effective treatment options are still needed. A highly interesting target for treatment is the replication process of the virus, in which the viral polymerase complex is critical. A critical protein of this complex is the RSV large (L) protein, which harbors multiple enzymatic functions that are all interesting targets for antiviral drug discovery. Unfortunately, not all structural parts of this L protein are currently resolved, which makes antiviral drug design and optimization challenging. In this review, an overview is given of current knowledge on the RSV L structure. Furthermore, a comparison is made between the L proteins of RSV and human metapneumovirus (hMPV), which, based on their sequence similarity, could shed light on missing structural gaps. New insights into the RSV and hMPV L protein structures are given, by modeling unresolved domains with AlphaFold2 and Alphafold3. While more structural studies are needed to confirm the modeling data, there is clearly potential for development of treatments targeting the L protein, for RSV and closely related viruses.

The gut microbiome modulates mucosal immunity, yet how specific bacterial taxa shape the diversity and specificity of T cell receptor (TCR) repertoires remains poorly understood. Existing approaches emphasize single-species effects or broad immune features, without pinpointing which microbes drive specific T cell clonotypes. We present AIRRWAS, a computational framework that integrates TCR-microbiome interaction analysis with targeted in vitro validation to detect genus-level TCR convergence. Applied to three independent cohorts, AIRRWAS identified reproducible associations between convergent TCR clusters and 21 bacterial genera spanning core commensals, probiotics and taxa with immunomodulatory roles. Predicted clonotypes were enriched within the TCR–microbiome interaction network and preferentially activated by genus-matched stimuli, eliciting different functional T cell responses. These findings demonstrate that distinct repertoires can share genus-specific TCR motifs, enabling detection of shared immune signatures. AIRRWAS can map these TCR– microbiome interactions, laying the groundwork for biomarker discovery immune monitoring and the development of microbiome-targeted therapies. ### Competing Interest Statement E.C.B. is co-applicant on an Investigator Initiated research grant of Pfizer. S.L. is an academic board member of ISAPP (International Scientific Association on Probiotic and Prebiotics), has received research funding from probiotic companies and has served as consultant for Freya Biosciences, YUN and Yakult, but unrelated to this research. B. Ol. Has received grants from Takeda, Alpha Sigma, Pfizer and Abbvie. He is also a member of advisory boards at Abbvie, Janssen, Pfizer, Ferring, BMS, Lilly, Takeda and Galapagos. F.v.W. has previously served as a speaker and/or consultant for Janssen, Johnson & Johnson, and Takeda. She has received research funding from LEO Pharma, Takeda, Galapagos, Regeneron, Sanofi, and Bristol‐Myers Squibb, all unrelated to this research. P.D has received research funding from and/or has ongoing contracts with MSD, Pfizer, Gilead, and GSK, but unrelated to this research. P.M., K.L., and B.Og. are shareholders and board members of ImmuneWatch BV. Research Foundation - Flanders, https://ror.org/03qtxy027, 1SH6624N, 1SH3924N, 1861219N, G0H4520N, G031222N, G0C9620N European Union’s Horizon 2020 research and innovation programme grant agreement, 851752-CELLULO-EPI, 852600-Lacto-Be Flemish Interuniversity Council iBOF, MIMICRY Chan Zuckerberg Initiative (United States), https://ror.org/02qenvm24 ZonMw, The Dutch Organisation for knowledge and innovation in health, healthcare and well-being, https://ror.org/01yaj9a77, 09150182010023 Alexandre Suerman program BOF-FKO mandate

Respiratory syncytial virus (RSV) is a significant cause of morbidity and mortality in high-risk populations. Although prophylactic options are available, there are no effective oral therapeutics for RSV infection. Obeldesivir (ODV) is an orally bioavailable prodrug of the nucleoside analog GS-441524, which is converted intracellularly to its active nucleoside triphosphate and inhibits the RSV RNA polymerase. Here we report the potent antiviral activity of ODV against geographically and temporally diverse RSV A and B clinical isolates (EC50: 0.20-0.66 μM). Resistance selection studies with ODV and GS-441524 against RSV identify a single amino acid substitution, I777L, in the L polymerase with reduced susceptibility (3.3-3.8-fold) to ODV and GS-441524, indicating a high barrier for resistance development. In an African green monkey RSV infection model, once-daily oral ODV doses of 30 or 90 mg/kg initiated ~24 hours post-infection significantly reduces log10 viral RNA copies/mL × day area under the curve by 69-92% in the upper and lower respiratory tracts. Together, these preclinical data support the clinical evaluation of ODV for the treatment of RSV infection.

Soft lithography has long remained the state of the art to generate the necessary micropatterning for molded microfluidic (MF) chips. Previous attempts to use printed circuit boards (PCBs) as a cheap and accessible alternative to expensive lithographed molds for the production of PDMS MF chip prototypes have shown their limitations. A more in-depth exploration of using PCBs as a mold substrate and a novel methodology of using flexible PCBs to produce highly accurate MF chips is reported here for the first time. Cross sections highlight the improved accuracy of this method, and peel testing is performed to demonstrate suitable adhesion between the glass substrate and PDMS cast. Positive cell growth viability showcases this novel method as a high-accuracy, high-accessibility, low-cost prototyping method for microfluidic chips while still maintaining all favorable properties provided by the PDMS material.

The varicella-zoster virus (VZV) infects >95% of the population. VZV reactivation causes herpes zoster (HZ), known as shingles, primarily affecting the elderly and individuals who are immunocompromised. However, HZ can occur in otherwise healthy individuals. We analyzed the immune signature and risk profile in patients with HZ using a genome-wide association study across different UK Biobank HZ cohorts. Additionally, we conducted one of the largest HZ human leukocyte antigen association studies to date, coupled with transcriptomic analysis of pathways underlying HZ susceptibility. Our findings highlight the significance of the major histocompatibility complex locus for HZ development, identifying 5 protective and 4 risk human leukocyte antigen alleles. This demonstrates that HZ susceptibility is largely governed by variations in the major histocompatibility complex. Furthermore, functional analyses revealed the upregulation of type I interferon and adaptive immune responses. These findings provide fresh molecular insights into the pathophysiology and activation of innate and adaptive immune responses triggered by symptomatic VZV reactivation.

Respiratory Syncytial Virus (RSV) poses a significant global health concern as a major cause of lower respiratory tract infections (LRTIs). Over the last few years, substantial efforts have been directed towards developing vaccines and therapeutics to combat RSV, leading to a diverse landscape of vaccine candidates. Notably, two vaccines targeting the elderly and the first maternal vaccine have recently been approved. The majority of the vaccines and vaccine candidates rely solely on a prefusion-stabilized conformation known for its highly neutralizing epitopes. Although, so far, this antigen design appears to be successful for the elderly, our current understanding remains incomplete, requiring further improvement and refinement in this field. Pediatric vaccines still have a long journey ahead, and we must ensure that vaccines currently entering the market do not lose efficacy due to the emergence of mutations in RSV's circulating strains. This review will provide an overview of the current status of vaccine designs and what to focus on in the future. Further research into antigen design is essential, including the exploration of the potential of alternative RSV proteins to address these challenges and pave the way for the development of novel and effective vaccines, especially in the pediatric population.

The role of T cell receptor (TCR) diversity in infectious disease susceptibility is not well understood. We use a systems immunology approach on three cohorts of herpes zoster (HZ) patients and controls to investigate whether TCR diversity against varicella-zoster virus (VZV) influences the risk of HZ. We show that CD4+ T cell TCR diversity against VZV glycoprotein E (gE) and immediate early 63 protein (IE63) after 1-week culture is more restricted in HZ patients. Single-cell RNA and TCR sequencing of VZV-specific T cells shows that T cell activation pathways are significantly decreased after stimulation with VZV peptides in convalescent HZ patients. TCR clustering indicates that TCRs from HZ patients co-cluster more often together than TCRs from controls. Collectively, our results suggest that not only lower VZV-specific TCR diversity but also reduced functional TCR affinity for VZV-specific proteins in HZ patients leads to lower T cell activation and consequently affects the susceptibility for viral reactivation.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in young children, elderly and immunocompromised patients worldwide. The RSV fusion (F) protein, which has 5–6 N-glycosylation sites depending on the strain, is a major target for vaccine development. Two to three of these sites are located in the p27 peptide, which is considered absent in virions. Prior research from our group showed that removing the N-glycan at position 116 (N116) in p27 led to higher neutralizing antibody responses and better protection against RSV. In this study, the effect of single, double and triple N-glycan deletion mutations in F p27 was evaluated. Surprisingly, all mutants exhibited similar expressions and functionality to the wild-type F protein. All F p27 glycomutants induced neutralizing antibodies and lowered lung viral loads after an RSV challenge in a mouse model. Although N-glycans in p27 influence immune responses, their exact role in RSV biology remains unclear. Possibly, these glycans, which are mostly conserved, play a role in other aspects of virus replication and biology.

Wastewater-based epidemiology (WBE) and wastewater surveillance have become a valuable complementary data source to collect information on community-wide exposure through the measurement of human biomarkers in influent wastewater (IWW). In WBE, normalization of data with the de facto population that corresponds to a wastewater sample is crucial for a correct interpretation of spatio-temporal trends in exposure and consumption patterns. However, knowledge gaps remain in identifying and validating suitable de facto population biomarkers (PBs) for refinement of WBE back-estimations. WBE studies that apply de facto PBs (including hydrochemical parameters, utility consumption data sources, endo- and exogenous chemicals, biological biomarkers and signalling records) for relative trend analysis and absolute population size estimation were systematically reviewed from three databases (PubMed, Web of Science, SCOPUS) according to the PRISMA guidelines. We included in this review 81 publications that accounted for daily variations in population sizes by applying de facto population normalization. To date, a wide range of PBs have been proposed for de facto population normalization, complicating the comparability of normalized measurements across WBE studies. Additionally, the validation of potential PBs is complicated by the absence of an ideal external validator, magnifying the overall uncertainty for population normalization in WBE. Therefore, this review proposes a conceptual tier-based cross-validation approach for identifying and validating de facto PBs to guide their integration for i) relative trend analysis, and ii) absolute population size estimation. Furthermore, this review also provides a detailed evaluation of the uncertainty observed when comparing different de jure and de facto population estimation approaches. This study shows that their percentual differences can range up to ±200 %, with some exceptions showing even larger variations. This review underscores the need for collaboration among WBE researchers to further streamline the application of de facto population normalization and to evaluate the robustness of different PBs in different socio-demographic communities.

Bacterial pneumonia greatly contributes to the disease burden and mortality of lower respiratory tract infections among all age groups and risk profiles. Therefore, laboratory modelling of bacterial pneumonia remains important for elucidating the complex host-pathogen interactions and to determine drug efficacy and toxicity. In vitro cell culture enables for the creation of high-throughput, specific disease models in a tightly controlled environment. Advanced human cell culture models specifically, can bridge the research gap between the classical two-dimensional cell models and animal models. This review provides an overview of the current status of the development of complex cellular in vitro models to study bacterial pneumonia infections, with a focus on air-liquid interface models, spheroid, organoid, and lung-on-a-chip models. For the wide scale, comparative literature search, we selected six clinically highly relevant bacteria (Pseudomonas aeruginosa, Mycoplasma pneumoniae, Haemophilus influenzae, Mycobacterium tuberculosis, Streptococcus pneumoniae, and Staphylococcus aureus). We reviewed the cell lines that are commonly used, as well as trends and discrepancies in the methodology, ranging from cell infection parameters to assay read-outs. We also highlighted the importance of model validation and data transparency in guiding the research field towards more complex infection models.