Viruses

The majority of emerging infectious diseases are zoonotic, having their origin in wildlife before spilling over into the human population. While small mammals are recognized as critical reservoirs for these viruses, their viral diversity remains largely uncharacterized across many African countries. We conducted molecular surveillance of synanthropic rodents and shrews in the Kibera informal settlement in Nairobi and the rural Taita Hills region of Kenya to detect and characterize potential zoonotic viruses. Tissue samples from 228 rodents and shrews were screened for six viral families using PCR assays. Rat hepatitis E virus (HEV) (Rocahepevirus ratti), a rodent-associated virus with potential for human spillover, was identified in Mus musculus and Rattus norvegicus from Kibera. NGS was conducted for the HEV positive samples, and we obtained two near-complete HEV genomes from Rattus norvegicus, which clustered within rodent-associated HEV genotypes in the phylogenetic analysis. The two sequences from the Rattus norvegicus cluster together, indicating a close genetic relationship. Paramyxoviruses belonging to the genera Jeilongvirus and Parahenipavirus were detected both from Taita and Kibera in nine different samples from Rattus norvegicus, Mus minutoides, Crocidura sp and Acomys ignitus. One paramyxovirus positive sample (Acomys ignitus) from Taita was selected for further sequencing with NGS, and a complete genome of a new jeilongvirus was assembled. Phylogenetic analysis of the detected viruses confirmed the close relation to previously known rodent-borne jeilongviruses but also revealed potentially novel jeilong- and parahenipavirus species. Our findings highlight the circulation of potentially zoonotic viruses in both urban and rural small mammals in Kenya. It emphasizes the necessity of continued genomic surveillance of zoonotic viruses to mitigate risks of their spillover into human populations. HighlightsO_LISurveillance reveals diverse rodent-borne viruses circulating in Kenya. C_LIO_LIRat-HEV was detected in Rattus norvegicus and Mus musculus from an urban low-income area. C_LIO_LIParamyxoviruses were detected across multiple rodent and shrew species, including novel Acomys ignitus jeilongvirus. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/719784v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@194e81eorg.highwire.dtl.DTLVardef@11342cdorg.highwire.dtl.DTLVardef@186ad97org.highwire.dtl.DTLVardef@eeb516_HPS_FORMAT_FIGEXP M_FIG C_FIG

Understanding the dynamics of HIV epidemics is important to control them effectively. Classical methods that mainly rely on occurrence data are limited by the fact that an unknown part of the epidemic eludes sampling. Since the early 2000s, phylodynamic methods have enabled the estimation of key epidemiological parameters from virus genetic sequence data. These methods have the advantage of being less sensitive to partial sampling and to provide insights about epidemic history that even predates the first samples. In this study, we analysed 2,205 HIV sequences from the French ANRS PRIMO C06 cohort. We identified and were able to reconstruct the temporal dynamics of two large clades that represent the HIV-1 epidemics in the country. Using Bayesian phylodynamic inference models, we found that the first clade, from subtype B, originated in the end of 1970s, grew rapidly during the 80s before decreasing from 2000 to 2015 and stagnating since then. The second clade, from circulating recombinant form CRF02_AG, emerged and spread in the 80s, grew again in the early 2000s, before declining slightly. We also estimated key epidemiological parameters associated with each clade. Finally, using numerical simulations, we investigated prospective scenarios and assessed the possibility to meet the 2030 UNAIDS targets. This is one of the rare studies to analyse the HIV epidemic in France using molecular epidemiology methods. It highlights the value of routine HIV sequence data for studying past epidemic trends or designing public health policies.

Grapevine (Vitis vinifera L.) is highly prone to viral infections that pose a significant threat to global viticulture sustainability. Traditional detection methods, such as PCR and ELISA, are limited to well-known pathogens, highlighting the need for more comprehensive and unbiased approaches. Here, we present the development of a cost-effective viral enrichment system adapted to next-generation sequencing (NGS) for the detection and characterization of grapevine viruses. Our strategy leverages hybridization-based capture using biotin-labeled cDNA probes hereafter named "Chloro-Zero") designed to selectively deplete highly abundant host transcripts particularly plastid and ribosomal RNAs while preserving viral RNA. Probe design was informed by transcriptomic analysis of V. vinifera. We evaluated different subtractor-to-target RNA ratios, observing a consistent reduction of host RNA and a moderate enrichment of viral sequences. NGS analysis revealed improved recovery of low-abundance viral transcripts, with coverage levels comparable, to a certain extent, to those obtained using previously available commercial kits, but at a significantly lower cost. Although variability in depletion efficiency was observed, the results demonstrate the potential of this scalable and locally adaptable protocol for virome profiling in grapevines. By addressing key limitations of current depletion methods, our approach facilitates the detection of emerging viral threats and supports the development of more effective certification programs and sustainable management practices. Ongoing improvements in probe design and bioinformatic workflows are expected to enhance performance, providing a robust platform for broader applications in plant virology.

Exacerbations of respiratory viral infections significantly contribute to morbidity and healthcare burden. Among these viruses, Human Rhinoviruses (HRVs) are the most frequent causative agents of upper respiratory tract infections. To date, over 150 HRV serotypes have been identified, classified into three species: HRV-A, HRV-B, and HRV-C. No antiviral therapies are currently available against this viral family, largely due to the high serotype diversity and limited cross-protection. The major group of HRVs relies on the Intercellular Adhesion Molecule-1 (ICAM-1) receptor to infect airway epithelial cells, making ICAM-1 an attractive target for broad-spectrum therapeutic interventions. Here, we report the development of nucleic acid-based aptamers designed to disrupt ICAM-1-HRV binding and thereby prevent viral infection. Aptamers are single-stranded DNA molecules that fold into precise three-dimensional structures, enabling highly specific protein recognition. Using a Systematic Evolution of Ligands by EXponential Enrichment (SELEX) approach guided by a minimal peptide mimicking the ICAM-1 viral binding interface, a library of >1024 random single-stranded DNA sequences was screened. Through iterative rounds of selection, we identified eight candidate 77-nt DNA aptamers, which were subsequently evaluated for their potential using in silico and in vitro assays, as well as functional assays in human epithelial cells. From this strategy, two lead aptamers were selected that effectively inhibited HRV-A16 replication in a concentration-dependent manner, as measured by viral titers (TCID assay) and viral RNA quantification by RT-PCR. These findings demonstrate the potential of ICAM-1-targeting aptamers as antiviral agents capable of preventing HRV entry. By targeting a host receptor and creating a protective barrier at the cell surface, this approach may offer a broadly applicable strategy against multiple HRV serotypes, paving the way for the development of novel antiviral interventions. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=131 SRC="FIGDIR/small/717810v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1f0c564org.highwire.dtl.DTLVardef@2f5035org.highwire.dtl.DTLVardef@3b063eorg.highwire.dtl.DTLVardef@116ed49_HPS_FORMAT_FIGEXP M_FIG C_FIG

A multiplex diagnostic test is evaluated for self-reported long COVID associated persistent symptoms and a poor recovery from a SARS-CoV-2 infection. A mass-standardised concentration of total antibodies (AC), high-quality (HQ) antibodies and percentage of HQ antibodies (HQ%) is assessed against a spectrum of spike proteins to the SARS-CoV-2 variants: Wuhan, , {delta}, and the Omicron variants BA.1, BA.2, BA.2.12.1, BA.2.75, BA.5, CH.1.1, BQ.1.1 and XBB.1.5 in three cohorts. A cohort of control patients (n = 46) recovered (CC) and a cohort of self-declared long COVID patients (n = 113) (LCC). A nested Receiver Operating Characteristic (ROC) analysis, performed for the variant with lowest HQ concentration in the spectrum, produced an area under the curve and AUC = 0.61 (0.53-0.70) for the CC vs LCC cohorts. For the LCC cohort, the cut-off thresholds for AC = 0.8 mg/L, HQ = 1.5 mg/L and HQ% of 34% were determined, leading to a 71% sensitivity and 66% specificity derived by the Youden metric. The cohorts may be fully classified based on ROC and outlier analysis to give an incidence of persistent virus 62% (95% CI 52% - 71%), hyperimmune 12% (95% CI 7% - 20%) and unclassified, 26% (95% CI 18% - 35%). The overall diagnostic accuracy for both the hyper and hypo immune is 69%. All clinical interventions can now be tailored for the heterogenous long COVID patient cohort.

Despite the evolution of epidemiological analysis and modeling tools, difficulties still remain, especially in developing countries, regarding the availability and use of these tools. Often expensive, requiring high technical expertise, demanding constant connectivity of several or sometimes even significant resources, these tools, although efficient, present a major gap with the operational realities of health districts. It is in this context that we introduce Episia, an open-source Python library designed and conceived to provide a framework to facilitate epidemiological analysis and modeling. It integrates a suite of compartmental epidemic models (SIR, SEIR, SEIRD) with a sensitivity analysis using the Monte Carlo method, a complete biostatistics suite validated against the OpenEpi reference standard, as well as a native DHIS2 client for automated data ingestion. Developed in Burkina Faso, it is optimized and aims not only to address these health challenges encountered in Africa but also remains a versatile tool for global health informatics.

Interpreting serological data remains challenging, particularly in low prevalence or cross reactive contexts, where antibody responses often show substantial overlap between exposed and unexposed individuals and may depart from normal distributional assumptions. Conventional cutoff based approaches often yield inconsistent or biased estimates of seroprevalence. Here, we present a decisional framework based on finite mixture models (FMMs) that enhances the robustness and interpretability of serological analyses. Beyond simply applying mixture models, our framework integrates multiple methodological innovations : (i) systematic comparison of Gaussian and skew normal mixture models to accommodate asymmetric antibody distributions; (ii) rigorous model selection using the Cramer von Mises test (p > 0.01) combined with a parsimonious score (APS) to prioritize models with well separated clusters; and (iii) hierarchical clustering of posterior probabilities to collapse latent components into biologically meaningful seronegative and seropositive groups. Applied to chikungunya virus (CHIKV) data from Bangladesh, the framework produced prevalence estimates consistent with ROC based methods while probabilistically identifying borderline cases. Validation on SARS CoV 2 and dengue datasets further demonstrated its generalizability: for SARS CoV 2, the approach identified up to five latent clusters with high sensitivity (up to 100%) and specificity (up to 100%), enabling discrimination by disease severity. For dengue, it revealed interpretable subgrouping consistent with background exposure and subclinical infection, despite limited confirmed cases. By integrating distributional flexibility, robust goodness of fit testing, and biologically guided cluster consolidation, this decisional FMM framework provides a reproducible and scalable method for serological interpretation across pathogens and epidemiological settings, addressing key limitations of threshold based classification.

Phage therapeutics are re-emerging as adjuncts or alternatives to antibiotics and their clinical translation will be enhanced with production methods that minimise downstream processing. We evaluated whether an endotoxin-reduced E. coli strain developed for production of recombinant proteins, ClearColi(R), can serve as a useful, safe phage production host without compromising yield and whether targeted receptor complementation can expand its utility. The parent strain BL21(DE3), and its lipid A modified derivative, ClearColi(R), were compared with respect to infection and generation of phage. Across a panel of 31 phage, a similar host range was observed between BL21(DE3) and ClearColi(R). To expand host range ompC was genetically engineered into the chromosome of ClearColi(R), thereby adding OmpC-dependent phage to its production capacity. Production metrics were broadly comparable between the hosts; efficiency of plating and final titres for representative phage were not significantly different; burst size varied by phage but without consistent host bias. Endotoxin activity in ClearColi(R)-propagated lysates was reduced by over 1000-fold relative to BL21(DE3), reaching the low hundreds of endotoxin units (EU) versus hundreds of thousands for BL21(DE3). Intravesical administration of ClearColi(R)-derived phage (LUC4) into pigs elicited no clinical abnormalities and no significant increases in circulating cytokines up to 48 hours after administration. ClearColi(R) allows efficient production of diverse phage with low endotoxin, reducing the requirement for downstream processing. Although its minimal LPS reduces its capacity for producing some LPS-dependent phage and its growth is slower than BL21(DE3), requiring optimisation for maximal phage titre, the safety and simplified manufacturing process support further development of endotoxin modified strains for phage production. Impact statementAntibiotic resistance is a current global problem and treatments based on phage and phage products already have a proven track record with particular bacterial infections, especially in the urinary tract. While progress is being made on in vitro phage synthesis, large scale bacteriophage preparations require a bacterial host for production, consequently toxic components in the initial lysate need to be removed or significantly diluted for safe clinical use. This is a study of the potential to utilise an endotoxin-reduced E. coli strain, ClearColi(R), to produce safer phage therapeutics. Such endotoxin modified strains should minimise the processing steps required and reduce overall production costs of a phage preparation. The research demonstrates that the endotoxin-reduced strain was able to produce a wide range of phage and for studied examples at phage titres equivalent to the more toxic parent strain. We also show that the strain can be modified to increase its host range and confirm the very low endotoxicity of basic phage lysates produced by the strain. Replicating this process to engineer additional low-toxicity bacterial production strains will accelerate the development of safer, more cost-effective phage therapeutics.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has continuously evolved since its emergence in the human population in 2019. As of 1st August 2025, more than 1,700 Omicron subvariants have been designated by the Pango nomenclature system. The Pango nomenclature system designates a new lineage based on genetic and epidemiological information of SARS-CoV-2 strains. However, there is a possibility that strains that have similar genetic backgrounds and the same phenotype are given different Pango lineage names. In this paper, we propose a new algorithm, called FindPart-w, which can identify groups of viral lineages that share the same relative effective reproduction numbers. We introduced a new lineage replacement model, called the constrained RelRe model, which constrains groups of lineages to have the same relative effective reproduction numbers. The FindPart-w algorithm searches the equality constraints that minimise the Akaike Information Criterion of constrained RelRe models. Using hypothetical observation count data created by simulation, we found that the FindPart-w algorithm can identify groups of lineages having the same relative effective reproduction number in a practical computational time. Applying FindPart-w to actual real-world data of time-stamped lineage counts from the United States, we found that the Pango lineage nomenclature system may have given different lineage names to SARS-CoV-2 strains even if they have the same relative effective reproduction number and similar genetic backgrounds. In conclusion, this study showed that viruses that had the same relative effective reproduction number were identifiable from temporal count data of viral sequences. These findings will contribute to the future development of lineage designation systems that consider both genetic backgrounds and transmissibilities of lineages.

Emerging infectious diseases and antimicrobial resistance (AMR) have surfaced as two major public health threats over the past two decades. Consequently, integrative surveillance systems capable of detecting both emerging pathogens and resistance-carrying bacteria are crucial. With advances in next-generation sequencing, simultaneous detection of pathogens and AMR is increasingly feasible. In this study, we used short-read metatranscriptomics complemented by total 16S rRNA metagenomic long-read sequencing to analyze paired oral and plasma samples from a cohort of febrile individuals at two locations in Senegal. Oral microbiomes differed in community composition between locations, and reduced diversity and richness were significantly associated with high fever. We identified at least one known pathogen in 15.33 % (23/150) of samples, with Borrelia crocidurae as the most frequently detected pathogen. We detected both pathogenic and non-pathogenic viruses in oral (10/72) and plasma (09/78) samples. Finally, we observed a high frequency of genes associated with resistance and virulence: 10% of samples expressed at least one AMR gene (ARG), and 24% expressed virulence factor genes. Resistance to widely used beta-lactam antibiotics was the most prevalent. Our findings provide critical data on oral and plasma microbiomes in the context of acute febrile illness in Senegal while expanding understanding of circulating ARGs.

PurposeThis study evaluates the Visual Hemofilter, a novel decision-support and information transfer tool designed to assist with regional citrate anticoagulation (RCA) in hemofiltration. By representing hemofilter parameters and patient blood constituents as animated icons, the tool aims to improve clinicians interpretation of blood gas results and RCA reference tables. We hypothesized that the Visual Hemofilter would enhance clinical decision-making by enabling faster and more accurate therapy adjustments, increasing clinicians confidence in their decisions, and reducing cognitive workload compared to conventional methods. MethodsWe conducted a prospective, randomized, computer-based simulation study across four intensive care units at the University Hospital Zurich. Twenty-six critical care professionals participated, each managing regional citrate anticoagulation (RCA) scenarios using either the Visual Hemofilter or conventional methods involving blood gas analysis and reference tables. Following each scenario, participants made therapy adjustments and rated their decision confidence and cognitive workload. ResultsUse of the Visual Hemofilter significantly improved decision accuracy (odds ratio [OR] 3.96; 95% CI 2.03-7.73; p < 0.0001) and reduced decision time by an average of 33 seconds (mean difference -33.3 seconds; 95% CI -39.4 to -27.2; p < 0.0001). Participants also reported greater confidence in their decisions (OR 5.41; 95% CI 2.49-11.77; p < 0.0001) and experienced lower cognitive workload (mean difference -15.05 points on the NASA-TLX scale (National Aeronautics and Space Administration-Task Load Index); 95% CI -18.99 to -11.13; p < 0.0001). ConclusionsThe Visual Hemofilter enhances clinical decision-making in RCA by increasing accuracy and speed, boosting decision confidence, and reducing cognitive workload. This technology has the potential to reduce errors and better support critical care professionals in managing complex treatment scenarios.

Sequencing the respiratory tract transcriptome has the potential to provide insights into infectious pathogens and the hosts immune response. While DNA-based sequencing is more standard in clinical laboratories due to its stability, RNA assays offer unique advantages. RNA reflects dynamic physiological changes, and for RNA viruses, viral RNA particles directly represent copies of the viral genome, enabling greater diagnostic sensitivity. However, RNAs susceptibility to degradation remains a significant challenge, particularly in RNase-rich specimens like saliva. To address this, we conducted a systematic, combinatorial evaluation of 24 distinct mNGS workflows, crossing eight nucleic acid extraction methods with three RNA-Seq library preparation protocols. Remnant saliva samples (n = 6) were pooled and spiked with MS2 phage as a control. The SARS-CoV-2 virus was spiked into half of the samples, which were extracted using the eight different extraction methods (n = 3) and compared using RNA Integrity Number equivalent (RINe) scores and RNA concentration. The extracted RNA was then processed across the three library construction methods and subjected to short-read sequencing to assess all 24 combinations head-to-head. We compared methods based on viral read recovery and found that RINe and concentration did not correlate with viral detection. The Zymo Quick-RNA Magbead kit and the Tecan Revelo RNA-Seq High-Sensitivity RNA library kit were the extraction and library-preparation kits that yielded the most SARS-CoV-2 reads, respectively. Importantly, our combinatorial analysis revealed that any small variability attributable to different nucleic acid extraction methods was heavily overshadowed by differences in quality attributable to the RNA-Seq library preparation methods. These findings challenge the reliance on conventional RNA quality metrics for clinical metagenomics and underscore the need to redefine extraction quality standards for mNGS applications. IMPORTANCEmNGS is a powerful and unbiased approach towards pathogen detection that has mostly been applied to blood and cerebrospinal fluid samples. However mNGS has recently been applied to more areas including the respiratory pathogen detection space, with potential applications in both in-patient diagnostics and public health surveillance. Saliva samples are an ideal sample type for these use cases since they can be collected non-invasively. However, saliva is also a challenging sample type due to its high RNase activity and often yields low-quality nucleic acid. This study explores the feasibility of using saliva specimens in mNGS with contrived SARS-CoV-2 samples to optimize the combination of two factors: nucleic acid extraction and RNA-seq library preparation. Exploration in this area could enhance the sensitivity of saliva-based mNGS assays, with the goal of future expansion of this specimen type in clinical diagnostics and public health surveillance. Key PointsO_LIThe choice of RNA-Seq library preparation kit has a greater impact on pathogen detection than the nucleic acid extraction method. C_LIO_LIThe combination of Zymo Quick-RNA Magbead extraction kit and TECAN Revelo RNA-Seq High Sensitivity RNA library kit recovered the highest percentage of total SARS-CoV-2 reads. C_LIO_LIRNA quantity and RINe score do not correlate with viral read capture, indicating a need for an alternative metric to assess RNA quality for downstream mNGS clinical diagnostics. C_LI

Kaposis Sarcoma Herpesvirus (KSHV), an enveloped double-stranded DNA virus, is the etiological agent of Kaposis sarcoma (KS), an endothelial cell-based tumor. KSHV is a leading cause of infection-related cancers in sub-Saharan Africa and immunocompromised individuals worldwide. Therefore, it is vital to identify the underlying mechanisms of viral infection and transmission to effectively identify specific therapeutic strategies and combat the disease. Here, we demonstrate that KSHV rewires the host cell lipidome during lytic infection. Bulk lipidomic analysis shows significant changes in the abundance of neutral lipids and phospholipids during lytic infection. We further investigated fatty acid-binding proteins (FABPs) to understand the underlying mechanisms that support KSHV pathogenesis. Using the doxycyclin-inducible iSLK.BAC16 cell line, we find that FABP genes are differentially regulated by lytic KSHV infection compared to latent infection. We report that FABP4 is significantly upregulated during lytic infection. Loss of FABP4 during lytic infection does not impact viral gene transcription however, lytic protein translation is reduced. Moreover, our intracellular and extracellular viral titers indicate that FABP4 affects maximal infectious virion production. This study highlights the role of FABP4 and its therapeutic potential as a target that facilitates KSHV infection and pathogenesis.

Pathogens that persist subclinically across many wildlife populations can drive mass mortality in others. Mass mortality is often abrupt, and the timing can be difficult to predict from host or habitat features alone. In a recent field study tracking ranavirus epizootics in wood frog (Rana sylvatica) breeding ponds, we found that no environmental or biotic feature reliably predicted die-off occurrence or timing. Instead, the trajectory of viral accumulation in the water column was the strongest dynamic predictor of mass mortality. Infected hosts shed virus throughout epizootics, but the influence of waterborne viral concentration on disease progression was apparent only near die-off onset. This pattern suggests a potential threshold-dependent feedback operating through the shared viral environment. Here, we develop a compartmental model linking waterborne viral concentration to the rate at which subclinical infections progress to clinical, high-shedding states within already-infected hosts. We show that a dose-dependent progression model generates the two-phase epizootic trajectory observed in natural die-offs: prolonged subclinical circulation followed by abrupt clinical transition after environmental virus crosses an escalation threshold. The model exhibits a sharp phase transition between subclinical circulation and mass mortality, governed mainly by the clinical-to-subclinical shedding ratio, host density, and pond volume. Existing explanations for die-off variation emphasize individual-level susceptibility, but our model demonstrates that dose-dependent environmental feedback, a mechanism not previously formalized at the population level, can generate the transition from subclinical infection to mass mortality without invoking individual variation in host susceptibility. This mechanism may apply in any system where hosts share a bounded environment, pathogen dose influences disease severity, and pathogen shedding increases with disease progression.

O_LICardiac rate and rhythm reveal how animals adapt physiologically to day-to-day challenges, with consequences for health and fitness. However, these data remain difficult to collect in wild animals, despite their relevance for individual health and fitness. C_LIO_LIHere, we present a system for collecting and transmitting long-term, fine-scaled physiological data in wild animals. We implanted Bluetooth-enabled cardiac and physiological monitor devices in three wild adult female baboons in the Amboseli ecosystem in Kenya and paired these devices with collars that enabled remote data downloads over long-range wide area network (LoRaWAN). C_LIO_LIThe system performed well over >10 months, providing the first long-term cardiac data in wild primates. The baboons showed strong circadian patterns in heart rate, heart rate variability, and activity. We also present data on one female who left her social group for unknown reasons; while alone she exhibited higher heart rate variability, lower activity, and evidence of disrupted sleep. C_LIO_LIIn sum, physiologgers paired with low-energy methods of remote data retrieval are powerful tools for investigating physiology in wild animals on timescales that extend over many months, with minimal disruption to their behavior. C_LI

We previously reported that the oxidative stress sensor Kelch-like ECH-associated protein 1 (Keap1) recognizes hepatitis B virus (HBV) X protein (HBx) to activate the NF-E2-related factor 2 (Nrf2) signaling pathway, thereby inhibiting HBV replication, and that HBx promotes K6-linked polyubiquitylation of Nrf2. However, the molecular mechanism remains unclear. Here, we investigated the role of HECT, UBA, and WWE domain-containing E3 ubiquitin ligase 1 (HUWE1) in HBx-mediated K6-linked polyubiquitylation of Nrf2 and its impact on HBV replication. Cell-based ubiquitylation assays demonstrated that HUWE1 knockdown reduced HBx-mediated K6-linked polyubiquitylation of Nrf2, while overexpression of wild-type HUWE1, but not the catalytically inactive HUWE1(C4341A) mutant, enhanced it, indicating that HUWE1 E3 ligase activity is required. Coimmunoprecipitation and proximity ligation assays demonstrated that HUWE1 interacts with HBx in the cytoplasm and binds Nrf2 only in the presence of HBx, suggesting that HBx bridges HUWE1 and Nrf2 into a ternary complex. Cycloheximide chase assays demonstrated that HUWE1 knockdown destabilized Nrf2 in HBx-expressing cells, supporting a role for HUWE1 in Nrf2 stabilization via K6-linked polyubiquitylation. Furthermore, HUWE1 knockdown or treatment with the HUWE1 inhibitor BI8626 significantly increased HBV RNA and pgRNA levels in HBV-infected cells. Collectively, these results demonstrate that HUWE1 promotes K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner to inhibit HBV replication. IMPORTANCEHepatitis B virus (HBV) chronically infects approximately 254 million people worldwide, yet host mechanisms that restrict viral replication remain incompletely understood. The Kelch-like ECH-associated protein 1 (Keap1)/ NF-E2-related factor 2 (Nrf2) signaling pathway is a central defense against oxidative stress. Under basal conditions, Nrf2 is degraded via Keap1/Cullin3-mediated K48-linked polyubiquitylation. We previously demonstrated HBV infection promotes Nrf2 stability through non-canonical K6-linked polyubiquitylation. Here, we identify the E3 ubiquitin ligase HUWE1 as the enzyme responsible for K6-linked polyubiquitylation of Nrf2. HBV X protein (HBx) recruits HUWE1 to Nrf2, forming a HUWE1/HBx/Nrf2 complex that switches Nrf2 ubiquitylation from K48 to K6, stabilizing Nrf2 and suppressing HBV replication. These findings reveal a novel antiviral mechanism exploiting a non-canonical ubiquitin code and highlight HUWE1 as a potential therapeutic target against chronic HBV infection.

Background: Although the hemagglutination inhibition (HAI) titer remains the gold standard correlate of protection against influenza, it does not fully capture the broader antibody responses that contribute to immunity. Methods: We analyzed immune responses in paired pre-infection and convalescent sera from 306 RT-PCR-confirmed A/H3N2 infections from two household studies (2014-18) in Managua, Nicaragua. Antibody responses were measured by HAI and enzyme-linked immunosorbent assays (ELISAs) against full-length hemagglutinin (HA), the HA stalk, and neuraminidase (NA). Participants were classified as HAI responders ([&ge;]4-fold HAI rise), alternate responders (no HAI rise but [&ge;]4-fold boost in [&ge;]1 ELISA), or no-response individuals (no [&ge;]4-fold rise in any assay). We compared demographic, clinical, and pre-infection antibody characteristics across these groups. We also analyzed predictors of an NA response. Results: Overall, 77% of participants had HAI seroconversion or a 4-fold rise. Among the 23% HAI non-responders, 62% had alternate antibody responses. No-response individuals had the highest pre-infection HAI and full-length HA titers (p < 0.0001), the lowest viral loads, and the fewest fever or influenza like illness (ILI) symptoms (p < 0.01). An NA response was more common among symptomatic individuals (p = 0.0483) and those with low or high baseline NA titers. Conclusions: High baseline HAI titers can limit detectable 4-fold rises and are associated with milder illness. Evaluating additional immune responses may capture a more complete picture of the host response to infection, thereby improving surveillance and informing vaccine development. Keywords: Influenza A/H3N2; Hemagglutination inhibition (HAI); Neuraminidase antibodies; symptomatic vs asymptomatic infection; correlates of protection.

AO_SCPLOWBSTRACTC_SCPLOWImproving feed efficiency in pigs is essential for reducing production costs and environmental impacts. This study examines the influence of circadian feeding rhythms and genetic polymorphisms on feed efficiency variability using two pig lines divergently selected for Residual Feed Intake (RFI) over ten generations. Feeding behavior was monitored using automatic concentrate dispensers, recording 6,494,097 visits from 3,824 pigs to analyze meal frequency, duration, and diurnal patterns. LRFI pigs ate less frequently, with larger meals and longer durations, they exhibited two distinct feeding peaks: one around 8:00 AM and a higher one at 5:00 PM and they consumed more feed during the diurnal period and less at night. HRFI pigs showed a smoother, less rhythmic feeding behavior with increased nocturnal intake. The differences between the two RFI lines became more pronounced as the number of generations of selection increased, suggesting a genetic basis. Feeding behaviors, including intake during the two main diurnal peaks, were found to be heritable (heritability estimates: 0.30-0.40) and genetic correlations were observed between feed intake and RFI, especially for intake between the two peaks. Then, we investigated the evolution of allele frequencies of single nucleotide polymorphisms (SNPs) in DNA sequences surrounding 10 core clock genes (ARNTL, CLOCK, CRY1, CRY2, NPAS2, NR1D1, PER1, PER2, PER3, RORA) along generations of selection. SNPs with significant frequency changes were mapped to regulatory regions and transposable elements, especially in HRFI line, suggesting potential functional impacts on circadian regulation. These results underscore the role of feeding behavior and genetic variation in feed efficiency, offering insights for breeding programs aimed at improving metabolic efficiency and sustainability in pig production.

This pilot study assessed the composition and diversity of the urinary microbiome from clinically confirmed UTI samples using 16S rRNA sequencing, whilst also exploring inter-individual variability of microbial community structure. We examined ten urine samples from patients with culture-positive UTIs. Demographic and clinical metadata, including age, sex, body mass index (BMI), diabetes status and recent antibiotic exposure was recorded per sample. Metagenomic DNA was extracted from microbial samples and sequenced to generate genus-level taxonomic profiling through 16S rRNA gene sequencing. Relative abundance tables were generated for each of the samples to identify dominant bacterial genera within each sample and summarize cohort level microbial patterns. To evaluate within-sample richness and evenness, alpha diversity indices (Shannon, Simpson, observed features and Chao1) were computed; beta diversity was measured using Bray-Curtis dissimilarity with principal coordinates analysis (PCoA) for graphical representation. The studys findings revealed the sex and moderate clinical diversity of the study sample; all samples were confirmed as having been taken from a UTI patient and exhibited a wide level of heterogeneity regarding the microbial composition of each urine sample. Overall, Pseudomonas was the dominant genus present, however, specific samples had approximately 50% of their microbiomes composed of Klebsiella, Proteus, and Escherichia species as well as approximately 25% of their total microbes were made up of Burkholderia spp., which are closely related to the genus of interest used during the course of this study. The observed alpha diversity of each sample displayed considerable variation for the included samples with a continuum of samples ranging from a single dominant microbe to a highly diverse mixed population producing a highly diverse polymicrobial population/bacterial composition, with some ratios of individual taxa to collective taxa of many samples repeatedly illustrating the exact nature of the specimen. Furthermore, a significant degree of Beta diversity was found between the patients, providing compelling evidence of identifiable differences among urinary microbiomes between patients with UTI. This pilot project provides a clear indication of the diversity and overall heterogeneity of urinary microbiota found in the UTI patients studied. In addition, the results of this study support the notion that the ecological complexities present within a urinary microbiome cannot necessarily be established through conventional culture methods, and that combined with molecular techniques such as 16S rRNA sequencing of bacterial DNA could be used to quantify and characterize the ecologic condition of urinary microbiota separate from the traditional high prevalence of identifiable uropathogens.

The A(H1N1)pdm09 virus remains a major global health threat. This study examined the burden of ICU admission, mortality, and associated predictors among patients with A(H1N1)pdm09 pneumonia in a leading center for infectious diseases in Vietnam. Information on demographic, clinical, and laboratory characteristics, and outcomes was retrieved from medical records of adults admitted with influenza A(H1N1)pdm09 during 2009-2019. Among 729 cases, 21.7% (158/729) developed pneumonia. Among 158 pneumonia cases, 36.7% (58/158) developed moderate-to-severe acute respiratory distress syndrome (ARDS), and 15.2% (24/158) received invasive ventilation. ICU admission and mortality rates were 48.7% (77/158, 95%CI 41.1-56.5%) and 8.2% (13/158, 95%CI 4.9-13.6%), respectively. Predictors of ICU admission included being >60 years old (adjusted OR [AOR] 13.864, 95%CI 2.185-87.956, P=0.005), comorbidities (AOR 6.527, 95%CI 1.710-24.915, P=0.006), AST (AOR 1.013, 95%CI 1.001-1.025, P=0.029), and moderate-to-severe ARDS (AOR 14.027, 95%CI 4.220-46.627, P<0.001). Predictors of mortality were invasive ventilation (AOR 55.355, 95%CI 1.486-2062.375, P=0.030) and double-dose oseltamivir or combination therapy (AOR 32.625, 95%CI 1.594-667.661, P=0.024). In conclusion, mortality is not rare in A(H1N1)pdm09 infection. Monitoring of older patients and those with comorbidities, liver enzyme elevation, or moderate-to-severe ARDS is essential for the timely detection of complications requiring intensive care.