Pathogens

Phylogenetic group B2 Escherichia coli is associated with urinary tract infections and other pathologies, but the basis for this phylogenetic skew is not understood. One aspect of urinary tract infections is binding to and entering uroepithelial cells. To test whether a phylogenetic skew exists for cell invasion, we examined invasion of 10 E. coli strains from three phylogenetic groups into CRL2169 and HTB-9 cells, which are derived from grade 1 and grade 2 bladder carcinomas, respectively. The top four strains that invaded CRL2169 were from group B2: three of these strains had more flagella gene transcripts than the other seven strains. The seven strains that invaded HTB-9 were from different phylogenetic groups. For the model uropathogenic group B2 strain UTI89, which expresses pili over flagella, loss of flagella or pili impacted invasion into CRL2169 to similar extents, but loss of pili had a greater effect on invasion into HTB-9 and a murine infection model than loss of flagella. A hyperflagellated variant of a group A strain did not invade either cell line better than the parental strain. Reported transcript differences, which were confirmed experimentally, showed that CRL2169 was more differentiated. The endocytosis stimulator tanshinone enhanced invasion into HTB-9, but not into CRL2169, which suggests differences in endocytic pathways and is consistent with differences in differentiation states. If the initial or recurring event in urinary tract infection is invasion into differentiated urothelial cells, as opposed to tight junctions, then the role of flagella may have been underestimated.

This novel study detected persistent low level infections of Elephant Endotheliotropic Herpesviruses (EEHV), that can cause highly pathogenic Elephant Hemorrhagic Disease (EHD) in Loxodonta and Elephas, and co-infection of presumed less pathogenic Elephant Gammaherpesviruses (EGHV), in skin nodule biopsies, saliva and tissues collected from 43 wild L. africana (savannah elephant) in Botswana, Kenya, South Africa and Zimbabwe; in saliva from 25 wild L. cyclotis (forest elephant) in Gabon; and in saliva collected over seven years from 7 wild-born L.africana at Six Flags Safari Park, USA; and in saliva, blood and tissues from an additional 200 L. africana in USA zoos. DNA from these samples was extracted in our USA laboratories and amplified by conventional polymerase chain reaction using three-round nested primer sets designed specifically to screen for known EEHV and EGHV genes loci and to discover new species and subtypes. Sanger sequencing of purified DNA from nearly all samples yielded unambiguous positive genetic matches to previously known Loxodonta-associated EEHV2, EEHV3A, EEHV3B, EEHV6, EEHV7A, and EGHV1B, EGHV2, EGHV3B, EGHV4B, EGHV5B and discovered novel types EEHV3C-H and EEHV7B and the prototype EGHV1B. Many of the primer sets used could also have detected known Elephas-associated EEHV1A, EEHV1B, EEHV4, and EEHV5 if present in these samples, but they did not. Our extensive library of EEHV and EGHV sequences from wild and zoo Loxodonta, (as well as from 100 zoo Elephas maximus not discussed in this review), is a significant contribution to the elephant virology community, particularly for comparing subtypes types of EEHV found in pathogenic cases of EHD in zoos as well as determining and comparing species and subtypes of EEHV present in existing zoo herds, and in individual elephants being transported between zoos, and for importation of wild elephants into existing zoo herds.

Toxoplasma gondii is a globally prevalent zoonotic parasite with multiple life stages and transmission routes, including ingestion and transplacental transmission. It is a major cause of abortion in sheep, goats and pigs, among other production animals, worldwide. While Type II strains are common in livestock in North America and Europe, non-archetypal, non-clonal genotypes are highly prevalent in South America. This study aimed to determine the molecular epidemiology of T. gondii strains causing sheep abortion in Uruguay. Phylogenomic analyses confirmed significant divergence among typed strains and revealed similarities with genotypes previously detected in the human population. Two novel strains, were isolated and characterized, uncovering the connection between their genetic makeup and phenotypes. Differences in virulence could be correlated to differences in gene copy number of the pseudo kinase ROP5 - further highlighting this virulence factor as relevant in wild strains. Whole-genome sequencing further confirmed the divergence among Uruguayan isolates, uncovering at least three distinct evolutionary origins. Overall, our findings highlight the circulation of virulent non-clonal lineages with links to human infections and underscore the importance of furthering genomic surveillance in South America to better understand Toxoplasmas transmission dynamics, pathogenic potential, and zoonotic risk.

Helminthiases remain a major global health burden, and limitations of current anthelmintic therapies highlight the need for new pharmacological targets. In this study, we examined the effects of ion-channel and cytoskeletal modulators on bovine lung protoscoleces (PSCs) of Echinococcus granulosus sensu lato. Compounds acting on ion channels (praziquantel, amiloride, and amlodipine) and cytoskeletal components (albendazole and cytochalasin D) were evaluated using a semi-automated motility assay, methylene blue exclusion to assess viability, and scanning electron microscopy (SEM) to characterize structural damage. All compounds produced concentration-dependent reductions in PSCs motility. Amlodipine was the most potent inhibitor of motility, whereas praziquantel and cytochalasin D produced pronounced tegumental alterations and strong correlations between motility impairment and parasite death. In contrast, amiloride markedly reduced motility with comparatively minor effects on viability, indicating a primarily paralytic effect. Cytoskeletal disruption induced severe structural damage and parallel declines in motility and viability. SEM analysis revealed extensive tegumental collapse, loss of glycocalyx, and microtrichial damage in PSCs exposed to cytoskeletal and calcium-modulating agents. These findings highlight cytoskeletal organization and calcium-dependent ion fluxes as key physiological vulnerabilities in E. granulosus. Comparative analysis of these pharmacological targets provides mechanistic insight into how disruptions in cytoskeletal dynamics and cation homeostasis compromise parasite motility and survival.

IntroductionThe vaginal microbiome is an important factor affecting both HIV risk and reproductive health in women living with HIV, and women of African descent are disproportionately affected. Here we use 16S rRNA sequencing data to predict BV in HIV-positive women living in Africa and delineate microbial features that are important for accurate prediction in this cohort compared to two HIV negative cohorts. MethodsOur study population was comprised of a cohort of HIV-positive women living in Tanzania, and two cohorts of HIV-negative women living in the United States. Using OTU variables from data in the Tanzanian cohort, we used random forest, logistic regression, support vector machine, and multi-layer perceptron algorithms to predict BV outcome. To evaluate our models, we compared predicted BV outcome to BV outcome determined using Nugent scoring. ResultsIn evaluating model performance, all four models predicted BV outcome better for the HIV-negative cohorts than the HIV-positive cohorts. Overall, models were better at predicting negative BV outcomes for low Nugent scores than positive BV outcomes for high Nugent scores. Evaluation of significant predictor variables of BV in each cohort revealed that shared features existed between the Tanzanian and Symptomatic cohorts, but not among all three cohorts. Upon comparing performance of models in predicting BV outcome for Black women in HIV-positive and HIV-negative cohorts, we observed that all four models perform better at predicting BV in the HIV-negative cohorts. ConclusionsThe lower predictive performance observed for this Tanzanian HIV positive cohort, coupled with the difference in microbial communities important for accurate prediction suggest that distinct microbial communities in women who are living with HIV may create challenges that affect the accuracy of BV diagnosis and treatment. This study highlights the need for diagnostic tools that consider unique biological and epidemiological factors of populations to address health disparities.

BackgroundInfectious bovine keratoconjunctivitis is the most important cattle ocular disease worldwide. The infection is primarily caused by Moraxella bovis and is a highly contagious disease that significantly affects cattle welfare. Currently, antibiotic medication is the primary treatment for infectious bovine keratoconjunctivitis. However, with rising concerns over antibiotic resistance, we propose developing a more targeted therapeutic strategy using bacteriophages (phages). Materials and MethodsWe have isolated the first known Moraxella bovis phages, characterised them according to their genome sequence, local virulence index and with transmission electron microscopy. The host ranges were assessed using 41 clinical M. bovis strains isolated from infected cows. ResultsFour phages were isolated and characterised. Comparative analysis identified a high degree of genomic similarity between the phages MB15, MB16, MB26 and MB43. MB43 was the most distinct, with the smallest host range phenotype. ConclusionsThe isolated phages show therapeutic potential for further development against Moraxella infections.

Vaginal dysbiosis (VD), characterized by low abundance of vaginal lactobacilli and increased bacterial community diversity, is implicated in multiple adverse reproductive outcomes and is an emerging target for preventive interventions, including live biotherapeutic products (LBPs). The most common clinical presentation of VD is bacterial vaginosis (BV), but at least half of people are asymptomatic. We investigated how two commonly used diagnostic criteria for BV, namely Amsel and Nugent, align with 16s rRNA gene sequencing-defined community state types (CSTs) demonstrating VD. We analyzed screening specimens from a Phase 1b randomized trial of LBP conducted at two sites (CAPRISA, South Africa; MGH, USA), as well as a single follow-up visit from enrolled participants. Using sequencing-based CST as the reference and multinomial mixed-effects logistic models, we evaluated the association of Amsel BV and Nugent BV with CST IV (including subtypes IV-A and IV-B) and tested for site-specific effects. Amsel BV was significantly associated with CST IV-A, and IV-B; however, the strength of association was significantly diminished at CAPRISA compared to MGH, pointing to site-specific assessment differences or underlying biological variation. Nugent BV yielded stronger associations with CST IV-A, and IV-B and showed no evidence of a site-specific interaction, indicating consistent performance across sites. These findings indicate that diagnostic performance for VD varies by framework: Amsel criteria are susceptible to geographical site effects, whereas Nugent score demonstrates stronger and more site-agnostic associations. For clinical studies targeting VD, Nugent scoring and/or sequencing-based approaches should be prioritized for VD endpoint definition and stratification.

Cochliomyia hominivorax, New world Screwworm (NWS), has become a reemerging veterinary concern in the United States due to the recent northward expansion of fly detections as far as northern Mexico. Rapid, accurate and validated detection pipelines need to be developed in the case of an incursion into the United States. Confirmatory cases are evaluated by morphological identification with no paired test to verify identifications. With the frequency of submissions of non-ideal samples, particularly from fly traps, a molecular tool would be necessary for species identification. In this manuscript, we develop and assess a pipeline including three real-time PCR assays targeting the ribosomal RNA and five sets of Sanger primers targeting the mitochondrial genome that would be used as a paired tool with morphological identification. Two of the assessed real-time PCR assays are highly specific, sensitive and repeatable requiring <1 copy per reaction for detection. Four of the five Sanger primer sets were assessed, optimized and results evaluated for potential use in preliminary geographic analysis of specimens. This workflow will expedite screening of samples, provide a method to verify results using different tools and help understand genetic variations within the mitochondria for NWS outbreaks.

BackgroundLeptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., which persist in soil and water environments for extended periods of time. The mechanisms enabling this environmental survival remain elusive. Free-living amoebae (FLA) are widespread protozoa that act as reservoirs or "Trojan horses" for numerous bacterial pathogens, protecting them from stress and contributing to their persistence. Whether pathogenic Leptospira exploit similar interactions with FLA has not been resolved. Methodology/Principal FindingsUsing live confocal microscopy, flow cytometry, and gentamicin protection assays, we investigated the interactions between pathogenic (Leptospira interrogans) and saprophytic (Leptospira biflexa) leptospires with three FLA species: Acanthamoeba castellanii, Dictyostelium discoideum, and Hartmannella vermiformis. While rapid internalization was observed, entry was only partially dependent on actin-driven processes and was enhanced by the presence of live bacteria. Following internalization, bacteria persisted for at least 48h as indicated by colony-forming assays. However, no evidence of intracellular replication was detected. The number of fluorescently labeled leptospires progressively declined over time, providing further evidence of leptospires survival without multiplication. Finally, analysis of environmental soils in New Caledonia showed co-occurrence of FLA and Leptospira. Soil-derived FLA also internalized pathogenic Leptospira in vitro, showing that these interactions extend to natural isolates. Conclusions/SignificanceOur results demonstrate that free-living amoebae internalize both pathogenic and saprophytic leptospires and allow their transient persistence without replication. By providing protection and prolonging viability in soil environments, FLA may contribute to the ecological maintenance of Leptospira. These findings pinpoint FLA as potential environmental reservoirs that could play a role in shaping leptospires survival strategies relevant for transmission and host infection. Author SummaryFor bacteria living in soils and freshwater environments, survival depends on their ability to adapt to complex ecological landscapes populated by numerous predators and competitors. In such habitats, interactions with other microorganisms are unavoidable and may shape long-term survival strategies. Pathogenic Leptospira, the bacteria responsible for leptospirosis, can persist for long periods outside their hosts, yet the ecological mechanisms supporting this environmental survival remain poorly understood. In soil and freshwater ecosystems, microscopic predators known as free-living amoebae commonly feed on bacteria. However, several bacterial pathogens can survive inside these amoebae and use them as temporary shelters. Because ancestral Leptospira were soil-dwelling saprophytes, interactions with amoebae likely represent an ancient ecological relationship in which successful survival strategies may have evolved and remain conserved in present-day pathogenic species. With this perspective in mind, we used microscopy approaches and bacterial viability assays to investigate whether Leptospira interacts with amoebae. We found that several amoeba species rapidly engulf both pathogenic and non-pathogenic Leptospira. Once internalized, the bacteria remained viable for up to two days but did not multiply. We also detected both amoebae and Leptospira in the same soil samples and showed that environmental amoebae could internalize the bacteria. These findings suggest that amoebae may act as temporary shelters for Leptospira, helping them persist in soils and water and potentially contributing to the environmental stage of leptospirosis transmission.

Chronic kidney disease is a progressive condition characterized by the accumulation of nitrogenous waste products, including urea, creatinine, and uric acid, leading to significant morbidity in advanced stages. Current management strategies, such as dialysis, are effective but associated with substantial clinical and socioeconomic burdens, highlighting the need for alternative approaches to reduce circulating toxins. In this study, we evaluated a novel formulation of psyllium-based granules functionalized with specific antibody combinations targeting urea, creatinine, and uric acid. The aim was to assess the biochemical effects, as well as the binding and sequestration efficiency, of these formulations under controlled experimental conditions. A randomized, double blind controlled in vitro study was conducted using serum samples obtained from twenty patients with uremia undergoing dialysis. Three formulations, labeled S1, S2, and S3, were evaluated. All tested formulations resulted in statistically significant reductions in urea, creatinine, and uric acid concentrations compared with baseline values. Among them, the S1 formulation demonstrated the highest binding efficiency, reducing urea by 70% {+/-} 7%, creatinine by 80% about 4%, and uric acid by 52% about 11%. Linear regression analysis confirmed a statistically significant association between the S1 formulation and reductions in these biochemical parameters. These findings suggest that antibody functionalized granules can effectively bind and sequester nitrogenous waste products under in vitro conditions. This approach may represent a potential strategy for reducing uremic toxin burden, either as a complementary method or as a future alternative to existing renal replacement therapies. Further studies, including in vivo validation, dose optimization, and controlled clinical trials, are required to establish safety, efficacy, and translational applicability.

Multiplex bead-based immunoassays (MIAs) are promising tools for simultaneously detecting humoral immunity to multiple targets, potentially playing a crucial role in serosurveillance and vaccine response assessments. However, evaluation of assay performance is paramount prior to widespread use. This study presents a performance evaluation of a pan-filovirus MIA through characterization of the analytical range for the EBOV glycoprotein (GP) target and assessments of assay precision and antigen discrimination. The precision of the MIA was evaluated by comparing the detection of anti-filovirus antibodies at two independent laboratory sites: the University of Hawaii, Honolulu (UH), and the Institut National de Recherche Biomedicale (INRB) in Kinshasa, Democratic Republic of the Congo (DRC). Forty-six samples from Yambuku, DRC, including Ebola virus Disease (EVD) survivors and close contacts, were tested at both sites. Additionally, 858 samples were tested in DRC before and after vaccination with a prophylactic EVD vaccine, ERVEBO. Results demonstrated low variability between laboratories, with intra-assay and inter-laboratory coefficients of variation below predefined thresholds for all filovirus targets included in the multiplex panel. Analyte correlations between sites were high (r2=0.86-0.92). Longitudinal analysis detected increased EBOV GP reactivity following vaccination, while reactivity to non-vaccine filovirus antigens remained stable, consistent with minimal cross-reactivity in a vaccinated cohort. These findings suggest that this pan-filovirus MIA produces reproducible results across distinct laboratory settings and may serve as a useful tool for comparative serologic investigations, serosurveillance, and evaluation of EBOV vaccine-associated antibody responses. IMPORTANCE STATEMENTThis study investigates the functionality and intra-laboratory consistency of a novel multiplex bead-based immunoassay with pan-filovirus targets. As part of the evaluation process, the feasibility of using the assay in resource-limited settings was demonstrated in the Democratic Republic of the Congo. This assay holds significant promise as a tool for detecting filovirus-specific antibody responses. By leveraging its multiplex capabilities, it may be used for widespread serosurveillance of high-consequence pathogens, including the pan-filovirus antigens such as Ebolavirus and Marburg virus already incorporated in the assay, as well as other targets of interest.

This study aimed to evaluate DARO Systems detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) against serum and oral fluid surveillance methods within a controlled study consisting of one PRRSV infected seeder pig and 46 naive nursery pigs. Findings showed DARO Systems comprehensive herd-level surveillance approach detected PRRSV earlier than traditional testing methods.

Climate change and warmer oceans will amplify the impacts on public health of waterborne harmful microorganisms. Phagotherapy offers a promising alternative; but as of today, phages can only be administered to patients when delivered along with antibiotics. Understanding possible interactions between these agents - indifference, synergy or antagonism - is thus a pivotal point. While several methods exist for characterizing such interaction, consensus on a reference method is still lacking. In this work, we screen and compare several in vitro characterization methods, using as a model nt-1, a phage of Vibrio natriegens, and studying its interaction with cefotaxime, a 3G cephalosporine. The different methods highlight different aspects of the interaction, depending whether they focus on phage or bacterial biomass. Overall, we see evidence of antagonism between the studied phage and antibiotic: this antagonism is at its optimum for antibiotic concentration of minimum inhibitory concentration (MIC)/2. Given the non-linear nature of interaction, it appears essential to use multiplexed methods and to cross technics. AUTHOR SUMMARYCurrently, antimicrobial resistance results in close to one million victims per year worldwide. In response to this alarming situation, new antimicrobial drugs and alternative therapies with innovative mechanisms have to be developed, such as phage therapy. It relies on the use of specific bacterial viruses, called bacteriophages (phages), that are therefore natural antibacterial agents. This therapy is strongly investigated for its potential to stop bacteria whenever antibiotics are no longer effective. Phage therapy is a highly personalized approach especially because of the narrow specificity of phages. Understanding how the efficiency of phages could be improved by the use of other antimicrobials, such as antibiotics, is essential in the fight against pathogens. Using a combination of a phage and an antibiotic, instead of only an antibiotic, imposes to think about new in-vitro tests for susceptibility testing. In the particular case of Vibrio bacteria, a common genus of waterborne pathogens, we investigated the efficiency of a phage in presence of cefotaxime, a last resort antibiotic, through different in-vitro methods, in liquid phase as well as on agar media. We observed a decreased efficiency of the phage, in other words an antagonism, especially at the lowest concentrations.

Tick-borne diseases represent a significant threat to human and animal health worldwide. In the United States, the blacklegged tick, Ixodes scapularis (I. scapularis), serves as a competent vector for several bacterial pathogens, including Ehrlichia muris eauclairensis (EME). The I. scapularis embryonic cell line (ISE6) is a valuable tool for propagating tick-borne pathogens and studying tick-pathogen interactions. In this study, we examined the cellular complexity of ISE6 cells and their response to EME infection. Single-cell RNA sequencing revealed 15 distinct cell clusters present. Although ISE6 cells are heterogeneous, they do not display transcriptional similarity to any known tick tissues. Notably, this lack of similarity did not influence their susceptibility to EME infection. Our results demonstrated that EME infection induces time-dependent transcriptional changes in ISE6 cells: early infection is characterized by upregulation of genes associated with stress adaptation, mitochondrial function, and metabolic pathways, whereas late infection leads to broad downregulation of genes involved in the cell cycle, DNA replication, and cytoskeletal organization. These findings enhance our understanding of ehrlichial interactions with ISE6 cells and reinforce the utility of this cell line as a resource for isolating and propagating arthropod endosymbionts and tick-borne pathogens. IMPORTANCEThis study provides a single-cell resolution framework for interpreting tick cell line biology during infection with a medically relevant ehrlichial pathogen. Using scRNA-seq, we show that the I. scapularis embryonic-derived ISE6 cell line comprises multiple transcriptionally distinct cell states, yet these states do not map cleanly onto canonical tick tissue signatures, even when compared against a curated reference tissue atlas. Despite this heterogeneity, EME broadly infects ISE6 cell population, indicating that susceptibility is not restricted to a specific cell type. We further define a time-dependent arthropod vector response in which early infection is marked by activation of stress and metabolic adaptation response, followed by late-stage inhibition of key signaling, transcriptional, and proliferative pathways as bacterial burden increases. Together, these findings strengthen the biological interpretation of ISE6 as an in vitro model for tick-pathogen interactions and provide a resource for future mechanistic studies of ehrlichial persistence, replication, and vector competence.

BackgroundThe global rise of multidrug-resistant (MDR) bacteria represents a critical public health threat, and Romania ranks amongst the most affected countries in Europe. As conventional therapy increasingly fails, bacteriophage therapy has re-emerged as a promising alternative to antibiotics. Urban rivers, contaminated with resistant bacterial strains, represent an underexplored and accessible reservoir for the isolation of lytic phages with therapeutic potential. MethodsTwo bacteriophages, 17M_Ec17_D and 22C_Ec22_D, were isolated from the Dambovita River, Bucharest, Romania, using MDR E. coli as host bacteria. Phage characterization included plaque morphology, transmission electron microscopy, and host range assessment by spot assay against 30 MDR E. coli isolates. Whole genome sequencing was performed on Illumina MiSeq and Oxford Nanopore Technologies MinION platforms, followed by bioinformatic analysis including taxonomic classification, lifestyle prediction, and functional annotation. ResultsBoth phages formed clear plaques and were classified as Kayfunavirus (17M_Ec17_D, Podoviridae-like) and Kagunavirus (22C_Ec22_D, Siphoviridae-like) with nucleotide similarities of 89.2% and 71.4% to their closest relatives, respectively, suggesting both are candidates for novel species. Host range analysis revealed lytic activity against 13% and 10% of tested MDR isolates, with complementary infection profiles. Genomic analysis confirmed a strictly lytic lifestyle for both phages, supported by the presence of holin and spanin genes and the absence of lysogenic modules, antibiotic resistance genes, and virulence factors. ConclusionsTo the best of our knowledge, this is the first study conducted in Romania to isolate and genomically characterize lytic bacteriophages targeting MDR E. coli. The characterized phages represent safe therapeutic candidates whose complementary host ranges suggest potential application as part of phage cocktail to broaden antimicrobial coverage against MDR infections.

Human leptospirosis is a disease of public health importance in Thailand, but the animal species involved in the transmission cycle have not been fully uncovered. This study investigated Leptospira infection in dogs and terrestrial micromammals in rural Nan Province, Thailand, and the pathogen diversity. Sera from 95 seemingly healthy dogs and kidney samples from 399 micromammals were analyzed using real-time PCR for Leptospira detection, followed by conventional PCR and sequencing of infecting Leptospira. We investigated environmental factors associated with Leptospira infection in micromammals, using data collected during trapping. Real-time PCR revealed ongoing infection in 8.4% (8/95) of dogs and 10.0% (40/399) of terrestrial micromammals, with 12 infected species including Bandicota indica, Berylmys berdmorei, Berylmys bowersi, Mus cervicolor, Mus cookii, and Hylomys suillus. In this qPCR-positive micromammals, three pathogenic Leptospira species were identified: L. interrogans, L. weilii, and L. borgpetersenii. This represents the first confirmed detection of L. weilii in rodents in Thailand. Infected micromammals were found in agricultural and forest habitats but not in human settlements. Our study demonstrates potential complex leptospirosis epidemiology in rural Thailand, with multiple species serving as pathogenic Leptospira reservoirs across diverse habitats, and some shared pathogen diversity with human leptospirosis cases in Thailand. Free-roaming dogs may serve as bridge hosts, transmitting zoonotic Leptospira from micromammals to humans by visiting both animal habitats and human settlements. These findings emphasize the need for integrated One Health surveillance approaches to control leptospirosis in rural communities.

Background & AimsAntiviral therapies targeting hepatitis B virus (HBV) suppress viral replication, but rarely achieve functional cure. Understanding HBV-host cell interaction is crucial for developing novel therapeutic approaches. Here, we report host cell proteins associated with HBV virions and filamentous subviral particles (fSVPs) and characterize one of them, apolipoprotein C1 (ApoC1), mechanistically. MethodsHighly purified HBV virions and fSVPs were obtained by sequential use of several biophysical methods. Particles were analyzed by mass spectrometry and associated proteins were evaluated phenotypically using an HBV infection model. The top hit, ApoC1 was characterized in detail. ResultsAssociated with virions and fSVPs, we identified in addition to known chaperones such as HSP90AB1 and HSC70, several apolipoprotein-related factors. RNAi-based phenotypic validation identified strongest effects for ApoC1, likely due to two complementary effects. First, ApoC1 depletion reduced intracellular cholesterol level impairing HBV infection and SVP production, which was compensated by exogenous cholesterol substitution. Second, ApoC1 that is mainly enriched in high-density lipoprotein (HDL), associates with HBV virions and fSVPs and increases HBV infectivity. The same was found for hepatitis D virus (HDV), a satellite virus utilizing HBV envelopes. Supplementation of exogenous HDL enhanced infection most likely via scavenger receptor class B type 1 (SR-B1), the natural HDL receptor. Consistently, inhibition of SR-B1 suppressed HBV and HDV infection. ConclusionsWe established a method for obtaining highly purified HBV virions and fSVPs and identified the HDL component ApoC1 to associate with both particle types. ApoC1 promotes HBV and HDV infection most likely via SR-B1 facilitating viral entry.

The incidence of vancomycin-resistant Enterococcus (VRE) is rising in hospitals in Canada, and resistance to last-resort antimicrobials including linezolid complicates treatment options for multidrug-resistant isolates. Recent reports from around the globe indicate that both linezolid and vancomycin resistance genes can be co-carried and mobilized by linear plasmids (named pELF) in Enterococcus species, often on the same backbone. We aimed to investigate linezolid resistance and linear plasmid prevalence in VRE bloodstream infection isolates collected by the Canadian Nosocomial Infection Surveillance Program from 2009 to 2024. We found that screening for pELF linear plasmid ends in short reads was a reliable way to predict linear plasmid presence in large-scale surveillance data (100 % accuracy on 85 reference samples). Almost half of the isolates in our collection were predicted to carry pELF plasmids (45.4 %, 941/2071) and we found that this proportion has increased from 2018 (32.2 %, 59/183) to 72 % of isolates between 2021 and 2024 (2021: 68.5 % (115/168); 2022: 71.6 % (146/204); 2023: 72.8 % (166/228); 2024: 71.6 % (235/328)). This trend of increasing linear plasmid carriage is evident from 2018 to 2024 across the dominant emerging sequence types (ST80, ST17, ST117). Linezolid resistance based on phenotypic antimicrobial susceptibility testing was low (1.0 %, 21/2071). Using long read sequencing, we characterized the linezolid resistant isolates and confirmed pELF plasmid presence in 13/21 (61.9 %) isolates. Six isolates harboured pELF plasmids encoding linezolid resistance genes (optrA, cfr(D), poxtA) and five of these also encoded vancomycin resistance genes (vanA). We compared these six plasmids to 39 public plasmid sequences and clustered them using MOB-suite and pling. Overall, this study provides further examples of the co-carriage of vancomycin and linezolid resistance genes on mobile linear plasmids and shows that linear plasmid prevalence is detectable and increasing across VRE in Canada. IMPACT STATEMENTGiven the increasing prevalence of multidrug-resistant hospital-acquired pathogens, resistance to last-resort antibiotics is a global public health threat. Linezolid is a last-resort antibiotic used to treat vancomycin-resistant Enterococcus isolates, and the dissemination of linezolid resistance genes is significantly facilitated by mobile elements that can transfer between unrelated strains and species. Linezolid resistance genes have recently been described on linear plasmids and are often co-localized with other resistance genes on the same plasmid backbone. Consequently, understanding the features and distribution of linear plasmids and those harbouring linezolid resistance genes is crucial for pathogen surveillance and mitigation of resistance. In this work, we used long-read and short-read sequencing to characterize genomic epidemiology of linear plasmids across 16 years of Enterococcus surveillance data in Canada. This study furthers knowledge of linear plasmids by demonstrating that they are relatively common across vancomycin-resistant Enterococcus blood isolates and by providing more examples of co-localized vancomycin and linezolid resistance genes on the same linear plasmid backbone. DATA SUMMARYSequencing data and genome sequences were deposited in National Centre for Biotechnology BioProject PRJNA1279082, and accessions are listed in Table S1. Supplementary materials for this study are available at the Figshare portal through DOI: XXX.

SARS{square}CoV{square}2 infection is associated with marked changes of the upper respiratory tract mycobiome. URT mycobiome Changes in non-hospitalized patients however, remains poorly defined. We performed shotgun metagenomic sequencing of 95 upper respiratory tract swab samples from 48 symptomatic SARS{square}CoV{square}2-positive individuals and 47 healthy controls from central India. Fungal diversity and community structure were compared using alpha- and beta-diversity analyses, while differential taxa were identified using prevalence-based testing and LEfSe. SARS{square}CoV{square}2-positive samples showed significantly higher fungal alpha diversity than controls, with increased Shannon diversity (p = 0.000319) and Simpson diversity (p = 0.017). Beta-diversity analysis showed significant separation between groups for both Bray-Curtis and Jaccard distances (PERMANOVA p = 0.001), with significant dispersion effects as well (PERMDISP p = 0.001). Differential analysis identified more SARS{square}CoV{square}2-enriched than control-enriched taxa, including Candida orthopsilosis, Malassezia furfur, M. sympodialis, M. globosa, Aspergillus niger, A. terreus, and A. nidulans. Aspergillus sydowii was the main control-enriched taxon. LEfSe and concordant multi-test analysis supported these findings, and sensitivity analysis confirmed robustness across thresholds. Certain SARS{square}CoV{square}2-enriched taxa were linked to confirmed or probable COVID{square}19-associated fungal infections, whereas no such pathogens were detected in controls. These findings indicate that SARS{square}CoV{square}2 infection is associated with URT mycobiome dysbiosis and enrichment of clinically relevant opportunistic fungi in community cases.

The ubiquitin-conjugating enzyme UBE2J1 functions in the proteasomal degradation of proteins at the ER. Existing evidence suggests that it plays a role during viral infection, with elevated UBE2J1 levels generally associated with increased infection. This is particularly relevant for some RNA viruses; however, the regulation of UBE2J1 during infection has not been well studied. Here, we used a BHK21 cell model to demonstrate that UBE2J1 overexpression promotes the replication of Vesicular Stomatitis Virus (VSV), as indicated by a significant increase in viral titres. To better understand the underlying molecular processes, cells were co-transfected to express the VSV-G protein and wild-type UBE2J1 protein, and we observed a significant increase in the syncytial fusion area. This effect was not observed when catalytically inactive (C91S) or phospho-deficient (S184A) forms of the protein were used. Interestingly, overexpression of a truncated, non-ER localized form of UBE2J1 ({Delta}TM) led to the largest increase in the syncytial fusion area. This arose as a result of increased syncytia size, and may indicate an enhanced cellular role if soluble forms of UBE2J1 are not anchored to the ER. Additional studies using truncated, mutated and wild-type proteins confirmed that UBE2J1 increases VSV viral replication and is associated with an increase in the number of infection plaques. Considering the emerging evidence for UBE2J1 involvement in viral infection, our finding should help in understanding the role of this protein in viral pathogenesis and cellular processes linked to syncytialization.