Pathogens

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.

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.

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.

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.

Burkholderia cepacia (B. cepacia) is an opportunistic pathogen with versatile virulence mechanisms. The pathogenesis of B.cepacia in the immunocompetent host following intranasal exposure largely remains ambiguous. Male BALB/c mice were intranasally inoculated with B. cepacia strain 20209 (1x10{square} CFU) and evaluated on days 3, 7, 14, and 21 post-infection. Histopathology of lung, liver, spleen, and kidney tissues were performed using H&E and PAS staining. Plasma cytokines were quantified using commercial multiplex assays and ELISA. Matrix metalloproteinase-2 (MMP-2) activity was assessed via gelatin zymography and metabolomic profiling by high-resolution mass spectrometry (HRMS). Histopathological analysis revealed organ-specific pathological indices such as interstitial pneumonitis, bronchitis, leukocyte infiltration, hepatic inflammation, as well as splenic hyperplasia. Similarly, MMP-2 activity revealed time-dependent modulation, reflecting dynamic proteolytic responses. Plasma and tissue IL-18 and IL-1{beta} levels demonstrated a temporal regulation, with IL-18 peaking on day 7 post-infection, while IL-1{beta} showed a biphasic expression peaking on day 3 and 14. Untargeted metabolomics revealed differential expression of lipid metabolism, and energy pathways, with higher expression of phospholipids and sphingolipids. Together, our study portrayed a physiologically relevant intranasal BALB/c model that captures both localized and systemic inflammatory responses to B. cepacia. Our findings highlight organ-specific pathologic progression and sustained inflammation providing key insights into host-pathogen interactions.

Respiratory viral-bacterial co-infections cause severe disease across species, yet the molecular mechanisms underlying enhanced pathogenesis remain poorly understood. This study characterised H3N8 equine influenza A virus (IAV) and Streptococcus equi subspecies zooepidemicus (SEZ) co-infections using complementary ultrastructural and transcriptomic approaches. Transmission electron microscopy demonstrated direct physical binding between spherical (A/equine/Miami/63) and filamentous (A/equine/Sussex/89 and A/equine/Newmarket/5/2003) IAV isolates and SEZ, including when SEZ was heat-inactivated ({theta}SEZ). Lectin staining revealed that SEZ expresses predominantly 2,3-linked sialic acids, the receptor for equine IAV. However, virus-bacteria binding persisted despite neuraminidase treatment. Scanning electron microscopy quantification demonstrated that viral pre-infection significantly enhanced bacterial adherence to cells of the DH82 canine macrophage-like cell line (2-fold increase, p<0.01) but not ExtEqFL (equine lung-derived) cells, revealing cell-type-specific enhancement. RNA-sequencing analysis showed that bacterial infection drove most transcriptional changes during co-infection with little difference in the number of differentially expressed genes (DEGs) between infection with SEZ alone (146 DEGS) or after pre-infection with either A/equine/Sussex/89 (166 DEGS) or A/equine/Newmarket/5/2003 (149 DEGS). Validation of upregulation of selected cytokines by RT-qPCR and ELISA demonstrated that SEZ infection drives dramatic cytokine upregulation compared to mock or {theta}SEZ controls. Viral pre-infection did not alter the SEZ-induced pro-inflammatory cytokine responses (IL-6, IL-8, TNF-) but significantly reduced IFN-{beta} expression compared to SEZ infection alone. These findings suggest that direct virus-bacteria physical interactions may drive cell-type-specific enhancement of bacterial colonisation, fundamentally advancing our understanding of respiratory co-infection pathogenesis.

BackgroundThe gut-kidney axis plays a direct role in gastrointestinal and kidney health. Gut-derived metabolites like uremic toxins are associated with the pathophysiology of feline chronic kidney disease (CKD). The aim of the study was to identify novel fecal biomarkers and investigate the roles of gastrointestinal metabolites in feline CKD. ResultsFecal samples from 41 healthy non-CKD (control) and 67 CKD cats, including 5 IRIS stage 1 (CKD1), 37 stage 2a (CKD2a), 18 stage 2b (CKD2b), and 7 stage 3 (CKD3), were subject to fecal untargeted metabolomics and targeted short-chain fatty acid (SCFA) analyses. Multiple linear regression, adjusted for sex, age, body weight and study site, identified 64 differential metabolites between control and across CKD groups (P<0.0001 and FDR<0.10). Approximately 65% of the metabolites were lipids, including polyunsaturated long-chain fatty acids, acylcarnitines, and ceramides. Random Forest algorithm selected N1-methyl-2-pyridone-5-carboxamide (2PY), a uremic toxin from nicotinamide catabolism, as the top fecal marker for classifying feline CKD. Fecal 2PY was increased in CKD1 (P = 0.03), CKD2a, CKD2b, and CKD3 (all P<0.0001) compared to the controls. Data mining revealed serum concentration of 2PY was significantly increased with severity of CKD in cats, possibly due to impaired renal excretion. Cholesterol and arachidonic acid, markers for enterocyte shedding and inflammation, were increased in CKD3 versus control (both P<0.05). In healthy non-CKD cats, evident suggested fecal lipids increased with age (P<0.0001), and were higher in females versus males (P<0.0001). While fecal indole and p-cresol were increased in CKD3 versus control (both P<0.05), no change was observed in indoxyl sulfate (IS) or p-cresol sulfate (PCS). Fecal indole-3-acetic acid (IAA) was decreased in several CKD groups compared to the controls (all P<0.05). Finally, two branched SCFAs, isobutyrate and isovalerate, were increased in CKD3 versus control (both P<0.05). ConclusionsThe study revealed 2PY as a novel marker and unveiled profound alterations in intestinal lipid compositions with a potential link to gut barrier integrity and inflammation in CKD.

Molecular diagnostics to detect Vibrio cholerae (Vc) may be negatively impacted by pathogen-specific lytic bacteriophage (phage) predation. To address this problem, phage detection as a proxy for pathogen detection has been proposed. However, efforts to modernize cholera diagnostics with molecular tools require addressing knowledge gaps on best practices to detect Vc and associated bacteriophages. We conducted polymerase chain reaction (PCR), quantitative PCR (qPCR), and nano-liter (nl) qPCR targeting Vc and known phages (ICP1/2/3) on stool samples collected from patients admitted at hospitals across Bangladesh. Of 4,975 patients enrolled, 2,574 diarrheal samples were collected and over 65,000 reactions were conducted, including replicates. We analyzed the results for target-specific assay alignment and then used machine learning to determine the effect of phage predation on Vc-assay alignment. Standard curve analyses were used to set qPCR-positivity thresholds at 7.3x105 CFU/mL for Vc and 1.7x103, 9.3x103, and 3.0x105 PFU/mL for ICP1, ICP2, and ICP3, respectively. Among 2,462 samples assayed by qPCR, target detection was 25.3% (623), 7.8% (193), 0.5% (13), and 5.8% (144) for Vc, ICP1, ICP2, and ICP3, respectively. There was strong alignment between assays for Vc detection ({kappa}=0.785) and moderate alignment for phage detection ({kappa}=0.609, 0.593, and 0.533 for ICP1/2/3, respectively). Phages were ranked as the first (ICP1) and third (ICP3) effectors of Vc diagnostic alignment. These findings provide insights on how to prioritize molecular methods in the cholera field as well as related less tractable diseases facing similar diagnostic challenges. IMPORTANCEThis paper presents a comprehensive comparison of molecular methods to detect Vibrio cholerae (Vc) and associated bacteriophage (phage) which can be used as a proxy for pathogen detection. This initiative is an important step towards modernizing cholera diagnostics with molecular tools. In this study, we found that quantitative polymerase chain reaction (qPCR) represents a reasonable approach to detect Vc and associated phages balancing assay performance, cost, and accessibility. A key additional finding was that phage predation was found to be a leading factor that impacts the alignment of molecular methods to detect Vc. While we recommend qPCR be added to the cholera diagnostic toolkit, the effects of phage predation need to be accounted for in the development and evaluation of cholera diagnostics. These findings have applicability to less tractable disease where diagnostics share similar vulnerabilities.

Successive dengue virus (DENV) outbreaks can progressively reshape population immunity influencing disease expression and diagnostic performance. Objectives The aim was to evaluate the impact of secondary infections across sequential outbreaks on clinical severity, serotype dynamics and diagnostic concordance. Methods This retrospective study analyzed 976 febrile-stage samples from three sequential outbreaks in Misiones, Argentina. For serotyping and clinical analyses, 869 viremic samples confirmed by at least one direct method were included (2016: n=512; 2019: n=148; 2024: n=209). Additionally, 318 samples, including 107 non-viremic cases, were used to compare NS1 rapid diagnostic tests (NS1 Ag) and RT-PCR. Viral serotyping and clinical and laboratory markers of disease severity were evaluated. Results Secondary infections increased from 31.05% (2016) to 43.24% (2019) and 53.87% (2024) (p<0.0010). Serotype distribution shifted from DENV-1 predominance in 2016 (95.12%), DENV-1/DENV-4 co-circulation in 2019 (60.71%/39.29%), and DENV-2 predominance in 2024 (97.60%). Secondary infections were associated with more severe disease manifestations, particularly in 2024, with higher hematocrit (p=0.0120) and hemoglobin (p=0.0080), lower white blood cells (p=0.020) and platelet counts (p=0.0030), and elevated AST (p=0.0007) and ALT (p=0.0130). Concordance between NS1 Ag and RT-PCR was lower in secondary infections (k=0.457 vs k=0.759, p=0.0013). Conclusions The rising frequency of secondary infections may affect both clinical severity and diagnostic performance during outbreaks. The clinical impact was more evident in 2024, likely associated with the introduction of a new serotype. These findings highlight the need for optimized surveillance and diagnostic strategies to improve case detection and patient management during epidemics.

Citrus yellow vein clearing virus (CYVCV) is the causal agent of an emerging disease representing a potentially high-impact threat for citrus production. Despite remaining outside Europe for decades, CYVCV has now expanded towards two important European citrus producers, Italy and, more recently, Spain. The presence of this virus in the EPPO region represents a current threat with unpredictable and potentially devastating consequences for European citriculture. Therefore, urgent protective measures need to be taken to prevent CYVCV spread and minimize its impact. Diagnostics is a key measure in the management of viral diseases, highlighting the need for harmonized methods suitable for reliable routine detection of the currently known CYVCV diversity. In this study, an inclusive, efficient and highly sensitive real-time RT-qPCR for the detection of CYVCV in plant material and transmission vectors has been developed and validated according to EPPO standards. Moreover, the validated method has been successfully adapted to both PCR digital platforms, that allow high-sensitive absolute quantitative detection, essential in the diagnostics at low viral concentrations; and PCR portable tools, that can be applied in a real diagnostic context for on-site detection. This versatility combines standard validated performance, absolute sensitive quantitation and real on-site detection. The study has also addressed sampling strategies to support reliable molecular diagnostic performance. Our results represent an improvement in the detection of CYVCV to be applied in epidemiological studies and different real diagnostic contexts for the containment of this important citrus pathogen.

Close to 50% of all bird species are reservoirs of potentially pathogenic fungi, including those listed as priority by the World Health Organization. In Malawi, data on diversity, pathogenic potential, and ecological avian sources of medically important yeast are scarce. A cross-sectional study using a descriptive approach was conducted in Blantyre, Southern Malawi, to characterise medically important yeasts recovered from environments contaminated with excreta/guano from synanthropic pigeons. A total of 20 samples were collected from 4 peri-urban areas, which yielded 71 yeast isolates. To assess the pathogenic potential of the environmental isolates, we compared their phenotypic virulence traits with those of 21 clinical yeast isolates collected from referral hospital laboratories. Pichia kudriavzevii (39%) and Candida orthopsilosis (30%) were the commonly isolated species in the pigeon-guano-contaminated environments. Candida parapsilosis sensu stricto (29%) and Candida albicans (24%) constituted most of the clinical yeast isolates. Half of the species isolated in the pigeon-guano-contaminated environments were also identified among the clinical isolates. A majority of the environmental isolates showed virulence traits similar to or stronger than clinical isolates. The findings underscore the critical need for integrated surveillance under the One Health framework, especially in bird-inhabited spaces close to human settlements.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

Human metapneumovirus (HMPV) causes acute lower respiratory infections, primarily affecting young children and older adults, with seasonal outbreaks peaking annually in March or April in the United States and other temperate regions in the Northern hemisphere. However, the factors driving HMPV seasonality in the United States remain poorly understood. We analyzed laboratory-confirmed HMPV cases and age-specific emergency department visits across 10 US regions, fitting an age-stratified dynamic transmission model to assess spatiotemporal patterns and investigate the influence of environmental variables and viral interference from RSV on HMPV transmission rates. We found that models incorporating climate variables into the transmission rate, including vapor pressure, precipitation, potential evapotranspiration, and minimum temperature, could not capture the timing of HMPV activity across all regions. Instead, HMPV timing was associated with RSV activity, with the HMPV transmission rate reduced in the presence of RSV. We showed that, unlike RSV, only models incorporating viral interference could reproduce the biennial pattern of HMPV observed in some regions, characterized by alternating late-small and early-large epidemics. Furthermore, our model successfully reproduced post-COVID-19 HMPV and RSV epidemics and predicted that RSV interventions are not likely to lead to a substantial increase in HMPV activity despite decreasing competition from RSV. Our work unravels the spatiotemporal dynamics of HMPV and its interaction with RSV, informing future seasonal forecasting and intervention strategies for HMPV.

Nocardiosis is a human infectious disease caused by several species of Nocardia and primarily affecting the skin, lungs and central nervous system. The first line treatment is based on cotrimoxazole, combining trimethoprim and sulfamethoxazole. These two drugs target respectively the dihydrofolate synthase (DHFR) and the dihydropteroate synthase (DHPS) involved in the essential folate synthesis pathway. The occurrence of drug resistance to these two drugs is however frequent. While the molecular mechanisms of trimethoprim resistance are well documented in other bacteria, they remain poorly explored and documented in Nocardia. This is partly because few biochemical structural or genetic studies have been conducted on DHFR from this genus. In this study, we report the biochemical and structural characterization of DHFR from Nocardia asteroides (DHFRNad). We show that overexpression of DHFRNad in N. asteroides confers strong resistance to trimethoprim. We recombinantly expressed and purified active DHFRNad and determined its inhibition constant for trimethoprim. We solved the crystal structure of DHFRNad bound to trimethoprim at high resolution. Further, biochemical studies of mutant DHFR variants pinpointed the role of important residues for trimethoprim binding and drug-resistance. HighlightsFirst biochemical and structural characterization of Nocardia asteroides DHFR. Overexpression of DHFRNad induces high-level trimethoprim resistance in N. asteroides. Crystal structure of DHFRNad reveals key residues for trimethoprim binding. Mutagenesis confirms residues critical for trimethoprim susceptibility. IC50 data confirm strong DHFRNad inhibition by trimethoprim and methotrexate

Cryptosporidium parvum is a protozoan parasite responsible for cryptosporidiosis, significantly threatening immunocompromised individuals, particularly HIV/AIDS patients, by causing severe diarrhea and potential mortality. Current treatments are largely ineffective, prompting investigations into new therapeutic options. This study evaluated two antiparasitic drugs: Mebendazole, used for helminth infections, and Artemisinin, used for malaria. The SKSR gene family encodes virulence factors in C. parvum, and Calcium-dependent protein kinase1 (CpCDPK1) regulates the life cycle of C. parvum; targeting these proteins may reduce growth and infection in hosts. In the current study, molecular docking was conducted taking Mebendazole and Artemisinin drugs as ligands, SKSR gene family and CpCDPK1 proteins as drug targets. Results with SKSR showed binding energy of -4.9 kcal/mol, -6.72 kcal/mol for Mebendazole and Artemisinin, respectively. Whereas, with CpCDPK1, the binding energies were -6.44 kcal/mol, -9.18 kcal/mol for Mebendazole and Artemisinin, respectively. Docking of Nitazoxanide (an in-use drug for C. parvum) with SKSR and CpCDPK1 revealed binding energies -4.2 kcal/mol, -4.81 kcal/mol, respectively. The stability of the proteins (targets) upon binding to the ligands was assessed by performing all-atom MD simulations for 100ns using the GROMACS package. No major variations were observed upon binding of Artemisinin and Mebendazole to SKSR and CpCDPK1. The findings of MD simulations imply that both proteins maintain their stability upon binding of Artemisinin and Mebendazole. Molecular Docking and MD simulation studies suggest that Artemisinin and Mebendazole are potential candidates for repurposing in the treatment of C. parvum infections, with recommendations for in vitro studies to validate these findings.

Background Oropouche virus (OROV) is an emerging vector-borne virus with rapidly expanding geographic range, increasing case counts, and growing evidence of severe outcomes including neuroinvasive disease and vertical transmission. Because OROV infection presents with nonspecific febrile illness that overlaps clinically with other viruses including dengue, zika, and chikungunya, accurate molecular diagnostics are essential for patient care and surveillance. Yet existing assays rely on single genomic targets and are vulnerable to detection failure as the virus evolves and reassorts. Methodology/Principal Findings To support diagnostic capacity, we developed and clinically validated a multiplexed qPCR assay targeting three regions of the OROV S segment, incorporating redundancy to preserve sensitivity across viral diversity while enabling robust clinical interpretation. The multiplex also includes an assay targeting RNaseP as an internal sample control to ensure adequate sample processing. We evaluated assay performance using both historical and contemporary OROV strains and validated the assay on contrived serum, plasma, and cerebrospinal fluid samples, assessing linearity, limit of detection (LOD), accuracy, specificity, precision, and sample stability. The assay met or exceeded all predefined acceptance criteria for clinical testing and achieved an LOD as low as 6 copies per reaction for contemporary outbreak strains. We further implemented a logic-based interpretation matrix that reduced false-positive risk while maintaining sensitivity near the analytical LOD. Conclusions/Significance Our assay sensitively and specifically detects OROV RNA in serum, plasma, and cerebrospinal fluid while incorporating safeguards against viral evolution and reassortment. The assay has been approved for use by CLIA at Nexus Medical Labs in 49 U.S. states, expanding access to timely OROV diagnostics in the United States and providing a durable framework for molecular detection of reassorting, rapidly evolving viruses as OROV continues to spread into new regions.

Currently circulating swine influenza viruses (SIVs) mainly include H1N1, H1N2, and H3N2 subtypes. In this study, two G4 genotype Eurasian avian-like (EA) H1N1 SIVs were isolated from 556 samples collected between 2023 and 2026. A systematic analysis was conducted on the two EA H1N1 isolates (FYD30 and YZF69) to assess their pandemic potential. The hemagglutinin (HA) proteins of both H1N1 viruses possessed residues 225E and 228S, indicating enhanced affinity for human-like -2,6-linked sialic acid receptors, which was confirmed by receptor-binding assays. Polymerase activity tests demonstrated that the two SIVs exhibited significantly higher activity in mammalian cells, relative to avian cells, which is consistent with the efficient replication in mammalian cells. Challenge experiments revealed that both H1N1 caused significant pathogenicity in mice and pigs, with YZF69 exhibited higher virulence than FYD30. The higher virulence of YZF69 may be attributed to its molecular features, including the NP Q357K mutation, and an additional glycosylation site in HA. In conclusion, currently circulating EA H1N1 SIVs have acquired key molecular signatures of mammalian adaptation, exhibit enhanced virulence in mammals, and continue to undergo extensive reassortment driven by international swine trade. These findings highlight the potential pandemic risk of SIVs and underscore the urgent need for strengthened surveillance.

Colibacillosis, caused by Avian Pathogenic Escherichia coli (APEC), result in substantial economic losses in global poultry production. The emergence of multidrug-resistant (MDR) APEC poses zoonotic risks through horizontal transfer of antimicrobial resistance (AMR) genes. Bacteriophage therapy emerges as a safe alternative to antibiotherapy; however, comprehensive characterization of phages targeting MDR-APEC from diverse geographical regions remains limited. We isolated five lytic bacteriophages from poultry fecal samples collected from five Indian states and characterized them through morphological analysis, physiological stability testing, whole-genome sequencing, and in vivo efficacy assessment. Host range was determined against APEC isolates, and therapeutic potential was validated in Galleria mellonella infection model. All five phages showed Myovirus-like morphology and stability across physiologically relevant temperatures (up to 55-70{degrees}C) and pH conditions (3-11). Their genome size ranges from 170 to 356 kb, belonging to three distinct genera; Dhakavirus, Gaprivervirus, and Asteriusvirus. Genomic analysis confirmed absence of antimicrobial resistance, virulence, toxin, or lysogeny genes. 51 APEC strains were isolated, of which 23 (45.1%) were MDR. Individual phages lysed 37-51% of tested APEC and 17-39% of MDR strains. Three Escherichia phages (fBSZT1, fUAMT1, fPKPT2) significantly improved larval survival to 60-80% at MOI 10 in G. mellonella infection models compared to untreated controls. This study establishes a well-characterized phage bank targeting MDR-APEC strains, providing foundation for developing phage-based interventions to reduce antibiotic dependency and mitigate AMR transmission risks under One Health framework.

The neutralizing activity present in human serum is considered a correlate of protection against SARS-CoV-2 infection and disease but the mechanisms by which serum antibodies are transported into the lumen of the respiratory tract, where they are required to interact with virus particles and infected cells remain incompletely understood. The transcytosis and neutralizing activity of serum-derived IgG and IgA antibodies was investigated using an in vitro SARS-CoV-2 infection model with primary differentiated human nasal and basal epithelial cells (hNECs and hBECs) cultures. Expression of the antibody transport receptors neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) in hNECs cultures was confirmed by qPCR, immunofluorescence microscopy, and flow cytometry. Both receptors were expressed throughout the epithelial cultures, with enriched expression observed in ciliated cells compared with goblet and basal cells. Purified IgG and IgA isolated from convalescent plasma demonstrated specificity for SARS-CoV-2 spike protein and inhibited ACE2-Spike interactions, although activity was reduced against later variants. Purified IgG contained higher anti-spike antibody titers than purified IgA. Functional neutralization assays showed that transcytosed IgG and IgA significantly reduced SARS-CoV-2 infection compared with untreated controls. However, serial dilution studies demonstrated that IgG-mediated neutralization was more potent than IgA-mediated neutralization. Similar results were determined for influenza A virus H3N2 subtype. The transcytosis of IgG was more efficient in hBEC cultures while IgA transcytosis was higher in hNEC cultures, reflecting the levels of the corresponding transport proteins. Together, these findings demonstrate that serum-derived IgG and IgA can undergo transepithelial transport across human nasal epithelium while retaining SARS-CoV-2 or influenza A virus neutralizing activity in vitro. These results suggest that FcRn- and pIgR-mediated antibody transport may contribute to mucosal protection following vaccination or infection and may help identify antibody responses associated with protection against SARS-CoV-2. ImportanceSerum antibody levels are considered correlates of protection for SARS-CoV-2 and Influenza A virus but its unclear how those antibodies are transported to the apical surface of respiratory epithelial cells, where they must be present for optimal activity. We show that IgG and IgA specific for SARS-CoV-2 or influenza A virus is transcytosed across respiratory epithelial cell cultures, the efficiency of which reflects the level of FcRn or pIgR expression levels, suggesting that cells of the upper and lower respiratory tract transport different antibodies from the blood.

Powassan virus (POWV) is an emerging tick-borne flavivirus that can cause severe encephalitis in humans. Currently no vaccines or therapeutics are approved to treat POWV. POWV is spread by the deer tick, Ixodes scapularis, which is ubiquitous across the Northeastern United States. To better understand POWV prevalence in high-risk populations, we examined POWV seroprevalence in Cadets at United States Military Academy (USMA) in West Point, New York. Cadets at USMA, located in a heavily wooded area, are at high risk for tick exposure during outdoor military training. 1,051 serum samples from the Cadet class of 2017 were screened for POWV seropositivity using a POWV Envelope (E) DIII ELISA. A seropositivity rate of 1.3% was determined. Several ELISA-positive samples were also able to neutralize both reporter virus particles bearing the POWV E protein and authentic POWV. This study demonstrates populations at risk for tick exposure may have significant seroprevalence of POWV.