Search Results (182)

Background: Influenza A viruses (IAV) cause seasonal flu and occasional pandemics. In addition, the potential for the emergence of new strains presents unknown challenges for public health. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of these viruses. Metal ions embedded into PPE have been demonstrated to inactivate respiratory viruses, but the underlying mechanism of inactivation and potential for resistance is presently not well understood. Methods: In this study, we used hemagglutination assays to quantify the effect of zinc ions on IAV sialic acid receptor binding. We varied the zinc concentration, incubation time, incubation temperature, and passaged IAV in the presence of zinc ions to investigate if resistance to zinc ions could evolve. Results: We found that zinc ions impact the ability of IAV particles to hemagglutinate and observed inhibition within 1 min of exposure. Maximum inhibition was achieved within 1 h and sustained for at least 24 h in a concentration-dependent manner. Inhibition was also temperature-dependent, and optimal above room temperature. Serial passaging of IAV in the presence of zinc ions did not result in resistance. Conclusions: e conclude that zinc ions prevent IAV hemagglutination in a concentration and temperature-dependent manner for at least 24 h. Overall, these findings are in line with previous observations indicating that zinc-embedded materials can inactivate the IAV hemagglutinin and SARS-CoV-2 spike proteins, and they support work toward developing robust, passive, self-cleaning antiviral barriers in PPE. Full article

Bacterial persisters are dormant cells that survive antibiotic treatment, serving as a reservoir for the emergence of resistant mutations. The evolution of antibiotic resistance poses a significant challenge to public health. In this study, we investigated the development of resistance in Pseudomonas aeruginosa persister cells by exposing the reference strain PA14 to meropenem and tracked the emergence of resistance mutations over serial passages. Whole-genome sequencing of the populations or individual resistant strains revealed evolutionary trajectories. In the initial passages, low-level meropenem-resistant mutants harbored various mutations, accompanied by increasing population survival. Then, mutations in the oprD gene appeared, followed by mutation in the mexR gene in most of the cells, leading to high-level meropenem resistance and collateral resistance to ciprofloxacin. Our study provides insights into the evolutionary pathways of P. aeruginosa under lethal antibiotic pressure, highlighting the dynamic interplay between persister cells and the emergence of resistance mutations. Full article

Pseudomonas aeruginosa is a major opportunistic pathogen with high levels of antibiotic resistance. Phage therapy represents a promising alternative for the treatment of difficult infections both alone and in combination with antibiotics. Here, we isolated and characterized three novel lytic myoviruses, Cisa, Nello, and Moonstruck. Genomic analysis revealed that Cisa and Nello belong to the Pbunavirus genus, while Moonstruck is a novel Pakpunavirus species. All lacked lysogeny, virulence, or resistance-associated genes, supporting their therapeutic suitability. Phage Nello and Moonstruck were active against P. aeruginosa Pa3GrPv, isolated from a patient with lung infection candidate for phage therapy. Moonstruck exhibited superior lytic activity with ciprofloxacin sub-MIC value (0.125 µg/mL), achieving bacterial suppression for 48 h. However, to improve the lytic efficacy of the phages on the clinical isolate, phage adaptation via serial passage was investigated. The killing efficacy of Nello was enhanced, whereas Moonstruck showed a less consistent improvement, suggesting phage-specific differences in evolutionary dynamics. Sequencing of the evolved phages revealed point mutations in tail-associated genes, potentially linked to a better phage–host interaction. These results support the use of phage–antibiotic combinations and directed evolution as strategies to enhance phage efficacy against drug-resistant infections. Overall, these findings support the therapeutic potential of the newly isolated phages in treating P. aeruginosa lung infections. Full article

This study elucidates potential genetic determinants and mechanisms involved in the synergistic effects of daptomycin (DAP) + fosfomycin (FOF) combination therapy. Among 33 clinically derived DAP-susceptible (S)/DAP-resistant (R) isogenic strain pairs, mutations in the mprF gene occurred in 30/33 DAP-R strains, including polymorphisms of L826F (33%) or T345A/L/I (15%). Strain variants of DAP-S CB1483 serially passaged in vitro for 10 days in DAP +/− FOF identified a key non-synonymous mutation in mprF (L826F) only in the DAP monotherapy arm. Interestingly, passage in FOF alone or DAP + FOF prevented the emergence of this mprF mutation following 10-day passage. This L826F mprF polymorphism, associated with a “gain-in-function” phenotype, exhibited increased amounts of lysyl-phosphatidylglycerol (L-PG) in the cell membrane (CM). Transcriptomics revealed a relatively modest number (~10) of distinct genes that were significantly up- or downregulated (≥2 log fold) in both the DAP-S and DAP-R strain pairs upon DAP + FOF exposures (vs. DAP or FOF alone). Of note, DAP + FOF decreased expression of lrgAB and sdrE and increased the expression level of fosB. In a rabbit infective endocarditis (IE) model, the DAP-R CB185 strain treated with DAP +/− FOF showed significantly reduced lrgB expression in vegetations compared with DAP treatment alone. Overall, these findings indicate that DAP + FOF therapy impacts MRSA through multiple specific mechanisms, enhancing bacterial clearance. Full article

Bovine alphaherpesvirus 1 (BoAHV-1) is a promising oncolytic virus that can infect the human lung carcinoma cell line A549. In an effort to adapt the virus to grow more rapidly in these cells through the serial passaging of viral progeny, we were unsuccessful. Here, we found that extracellular vesicles (EVs) secreted by BoAHV-1-infected A549 cells (referred to as EDVs) contain 59 viral proteins, including both viral structure proteins (such as gC and gD) and viral regulatory proteins (such as bICP4 and bICP22), as identified via a proteomic analysis. These EDVs can bind to and enter target cells, inhibit viral particles binding to cells, and stimulate the production of IFN-α and IFN-β in A549 cells. When EDVs are inoculated into rabbits via either the conjunctival sacs or intravenously, they can be readily detected in neurons within the trigeminal ganglia (TG), where they reduce viral replication and promote the transcription of IFN-γ. Furthermore, incorporation of the known anti-herpesvirus drug Acyclovir (ACY) into the EDVs leads to synergistically enhanced antiviral efficacy. Collectively, the EDVs exhibit antiviral effects by blocking viral binding to target cells and stimulating the innate immune response, thereby leading to the failure of the serial passaging of viral progeny in these cells, and these EDVs may serve as a promising vector for delivering drugs targeting TG tissues for antiviral purposes. Full article

In the baculovirus expression system, recombinant viruses generated via bacmids often exhibit reduced expression and genetic stability of target genes during serial passages. This instability is thought to arise from the proximity of non-essential exogenous genes to the target gene insertion site. This study investigated the impact of the target gene insertion locus on its expression and stability within the recombinant viral genome. In addition to the conventional polyhedrin (polh) locus, we evaluated four additional loci located distal to the non-essential exogenous genes. Among them, the ODV-e56 and ChiA/v-cath loci maintained target gene expression and genetic stability more effectively than the polh and p10 loci, even after ten consecutive undiluted viral passages. Notably, essential or functionally important viral genes were located near the ODV-e56 and ChiA/v-cath loci, whereas such genes were absent near the p10 locus. These findings suggest that enhanced stability and expression are associated with the proximity to essential viral genes. Therefore, the strategic selection of target gene insertion sites in close proximity to essential viral elements, rather than simply avoiding non-essential exogenous regions, represents a promising strategy to enhance recombinant protein production in baculovirus expression systems. Full article

Background: African Swine Fever (ASF) is a viral hemorrhagic disease characterized by diverse clinical and pathological manifestations depending on the virulence of isolates/strains and the immunological status of pigs. The use of modified live viruses (MLVs) is currently the most common approach in developing vaccines against ASF. However, despite the availability of dozens of MLV candidates that meet basic safety and efficacy criteria—such as the absence of severe clinical signs and survival after challenge with a virulent strain—no broadly accepted vaccine has yet been developed. Here, we propose viremia testing as an essential criterion for evaluating candidate ASF vaccines, with levels exceeding 10⁴ HAD₅₀/TCID₅₀ and lasting longer than 21–28 days post vaccination considered unfavorable indicators. Methods: We analyzed ASF MLV vaccines obtained through the deletion of one, two, or more genes, focusing on viremia kinetics after vaccination and challenge with virulent ASFV strains. Post mortem data were used to assess viral persistence in organs. Results: Most MLV candidates, especially those with single-gene deletions, demonstrated relatively high viremia levels after vaccination and challenge. Viral persistence was frequently detected in organs upon necropsy. MLVs with an additional EP402R gene deletion showed low viremia after vaccination but high levels after challenge. Nevertheless, several candidates with favorable viremia profiles were identified, including those obtained via targeted deletions or serial passaging in cell cultures. Conclusions: Incorporating viremia assessment as a primary screening criterion can significantly narrow down the selection of promising MLV candidates and help accelerate the development of effective emergency vaccines for use in ASF-affected regions. Full article

Influenza A virus (IAV) is a highly diverse pathogen with genetic variability primarily driven by mutation and reassortment. Using next-generation sequencing (NGS), we characterised defective viral genomes (DVGs) generated during the serial passaging of influenza A/Puerto Rico/8/1934 (H1N1) virus in embryonated chicken eggs. Deletions were the most abundant DVG type, predominantly accumulating in the polymerase-encoding segments. Notably, we identified and validated a novel class of multisegment DVGs arising from intersegment recombination events, providing evidence that the IAV RNA polymerase can detach from one genomic template and resume synthesis on another. Multisegment recombination primarily involved segments 1–3 but also occurred between other segment pairings. In specific lineages, certain multisegment DVGs reached high frequencies and persisted through multiple passages, suggesting they are not transient by-products of recombination but may possess features that support stable maintenance. Furthermore, multisegment DVGs were shown to be encapsidated within virions, similar to deletion DVGs. The observation of recombination between segments with limited sequence homology underscores the potential for complex recombination to expand IAV genetic diversity. These findings suggest recombination-driven DVGs represent a previously underappreciated mechanism in influenza virus evolution. Full article

Bacterial infections, particularly those caused by multidrug-resistant strains, remain a significant global public health challenge. The growing resistance to traditional antibiotics highlights the urgent need for novel antibacterial strategies. Herein, we successfully synthesized three types of nitrogen-doped carbon dots (tBuCz-CDs, HAH-CDs, and EC-CDs) via hydrothermal method using tert-butyl carbazate, hydroxyacetic acid hydrazide, and ethyl carbazate as precursors. tBuCz-CDs, HAH-CDs, and EC-CDs exhibited potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA), with minimum inhibitory concentrations (MICs) of 100, 100, and 150 µg/mL, respectively. Their antibacterial effect on MRSA was comparable to that of the widely used antibiotic vancomycin hydrochloride, as shown by the zone of inhibition assay. Furthermore, the carbon dots exhibited low cytotoxicity and hemolytic activity showing their excellent biocompatibility both in vitro and in vivo. They also significantly promoted wound healing compared to untreated controls. Notably, the serial passaging of MRSA exposed to these carbon dots did not result in the bacterial resistance. Mechanistic studies revealed that the carbon dots exerted antibacterial effects through multiple mechanisms, including the disruption of bacterial membranes, inhibition and eradication of biofilm formation, generation of reactive oxygen species, and DNA damage. This work highlights the potential of nitrogen-doped CDs as a promising material for combating drug-resistant bacterial infections and underscores their potential for further biomedical development. Full article

Background/Objectives: Equine rhinopneumonia, caused by equine herpesvirus types 1 and 4 (EHV-1 and EHV-4), continues to be a significant health and economic concern in the global equine industry, particularly in Kazakhstan. While vaccines targeting EHV-1 are available, there is currently no licensed monovalent vaccine for EHV-4, and existing formulations offer limited protection against this serotype. This study aimed to develop and evaluate a freeze-dried, live-attenuated EHV-4 vaccine with improved safety, stability, and immunogenicity. Methods: A field isolate of EHV-4 was attenuated through serial passaging in primary lamb testicle (LT-KK49) cell cultures. Viral biomass was concentrated and formulated with various stabilizers before freeze-drying. The most effective stabilizer composition—sucrose, gelatin, and lactalbumin hydrolysate—was selected based on viral titer retention. Safety and immunogenicity were assessed in mice, guinea pigs, rabbits, donkeys, and horses. A guinea pig reproductive challenge model was used to evaluate protective efficacy. Results: The optimized lyophilized vaccine retained infectivity (>6.0 log₁₀ TCID_50/cm³) for at least six months at 4 °C. No adverse clinical signs were observed in any test species. Immunization induced robust neutralizing antibody responses in both small animals and equines. In the guinea pig model, vaccinated females demonstrated 100% pregnancy retention and fetal viability following challenge with a virulent EHV-4 strain. Conclusions: This freeze-dried, live-attenuated EHV-4 vaccine candidate is safe, immunogenic, and thermostable. It offers a promising platform for the targeted prevention of EHV-4 infection, particularly in young horses and in regions with limited cold-chain infrastructure. Full article

Background/Objectives: Eurasian avian-like (EA) H1N1 swine influenza viruses, with their persistent evolution and zoonotic potential, seriously threaten both swine and human health. The objective was to develop an effective vaccine against these viruses. Methods: A cold-adapted, temperature-sensitive live-attenuated influenza vaccine (LAIV) candidate, GX18ca, was developed. It was derived from the wild-type EA H1N1 strain A/swine/Guangxi/18/2011 (GX18) through serial passaging in embryonated eggs at temperatures decreasing from 33 °C to 25 °C. Its characteristics were studied in mice, including attenuation, immune responses (mucosal IgA, serum IgG, IFN-γ+ CD4⁺/CD8⁺ T-cell responses), and protective efficacy against homologous (GX18), heterologous EA H1N1 (LN972), and human 2009/H1N1 (SC1) viruses. Results: GX18ca showed cold-adapted and temperature-sensitive phenotypes. In mice, it was attenuated, with viral titers in the nasal turbinates and lungs reduced 1000–10,000-fold compared to the wild-type strain, and it cleared by day 5 post infection. Intranasal immunization elicited strong cross-reactive immune responses. Mucosal IgA had broad reactivity, and serum IgG titers reached high levels. IFN-γ+ CD4^+/CD8⁺ T-cell responses were detected against all the tested viruses. A single dose of GX18ca fully protected against GX18 and LN972 challenges, and two doses significantly reduced SC1 lung viral loads, preventing mortality and weight loss. Conclusions: GX18ca is a promising LAIV candidate. It can induce broad immunity, addressing the cross-protection gaps against evolving EA H1N1 SIVs and zoonotic H1N1 variants, which is crucial for swine influenza control and pandemic preparedness. Full article

Humanized mice generated by hematopoietic stem cell (HSC) transplantation are limited by the immune system developed being allogeneic to the tumor. We have innovated a platform to reconstitute an autologous human immune system (HIS) in immunodeficient NOG-EXL mice from mobilized peripheral blood (MPB)-CD34 cells, along with PDX generated from the same patient’s tumor tissue. Patients consented under an IRB-approved protocol for tumor biopsy and HSC apheresis at Emory University. HSC collection included mobilization with G-CSF and plerixafor, immunomagnetic bead isolation with CliniMACS, and cryopreservation of CD34⁺ cells. PDX were established from biopsies or surgical specimens by passaging into immunodeficient mice. Irradiated NOG-EXL mice were engrafted with HSCs by intravenous transplantation of CD34⁺ HSC. Engraftment of human T cells, B cells, and myeloid cells in peripheral blood was assessed by serial flow cytometry of blood samples, with final assessment of immune components in spleen and bone marrow at 30 weeks. Twenty-eight PDX models were generated from 43 patients with HNSCC; 1 patient underwent apheresis. HSC engraftment in blood was observed in 100% of NOG-EXL mice at 8 weeks post-transplant, with 5–20% hCD45⁺ cells present in the periphery. B-cell development was predominant at early time points and declined over time. Human T-cell and subset development of CD4⁺ and CD8⁺ T cells were observed in blood from 15 weeks post-transplant. Strong development of the myeloid lineage (CD33⁺) was observed starting at 8 weeks and persisted throughout the study. These data demonstrate that mobilization and apheresis of HNSCC patients is technically and clinically feasible and may allow the establishment of autologous HIS-PDX mice. Full article

Background: Stenotrophomonas maltophilia is one of the common causative agents of hospital-acquired infections worldwide. The major concern regarding S. maltophilia infections is its extreme resistance to multiple antibiotics. Methods: Enrofloxacin-resistant mutants of S. maltophilia K279a were selected using a serial passage technique. Results: In this study, we showed that one of the mutant strains, KE507, which was selected from S. maltophilia K279a for its resistance to the veterinary drug enrofloxacin, conferred resistance to trimethoprim/sulfamethoxazole (co-trimoxazole), levofloxacin, and minocycline as per the Clinical and Laboratory Standards Institute guideline. These antibiotics are the first-line drugs routinely used to treat S. maltophilia infections. The KE507 mutant also showed increased resistance to all tested quinolones, azithromycin, and neomycin. Molecular characterization using whole genome sequencing, antibiotic resistance gene expression profiles, and mutational analysis indicated that inactivation of SmeRv (Q208insHSPRFTW), a transcriptional regulator of the SmeVWX multidrug efflux pump, contributes to resistance to quinolones (including levofloxacin), co-trimoxazole, and partially to neomycin, but not to azithromycin or minocycline. These data, together with in silico structural analysis, suggest that the mutation of SmeRv causes a conformational change in the SmeRv structure, which leads to the activation of SmeVWX efflux transporter expression and subsequent resistance to co-trimoxazole and quinolone antibiotics. Conclusion:S. maltophilia can thus acquire resistance to the antibiotics primarily used to treat S. maltophilia infections through the mutation of SmeRv. Full article

Porcine deltacoronavirus (PDCoV) is a newly discovered enteropathogenic coronavirus primarily responsible for diarrhea and mortality in piglets, with the potential to infect humans, thereby posing a significant threat to both human health and the global pig industry. Currently, there is no commercially available live-attenuated vaccine for PDCoV. In this study, an isolated virulent PDCoV strain, DHeB1, was continuously passaged in LLC-PK1 cells for up to 110 passages. The virus growth kinetics in cell culture and complete genome sequences of various passages (F11, F40, F70, F90, and F110) were determined. The results indicated significant increases in virus titers at passages F40 and F90. Sequence analysis revealed that only a few single-nucleotide mutations (some of which resulted in amino acid changes) and one nucleotide insertion were observed throughout successive passages. Notably, the eight and seven amino acid mutations that emerged in F40 and F70, respectively, remained stable in subsequent passages and were predominantly located in the S glycoprotein. The pathogenicity of F11, F40, F70, and F90 was assessed in 5-day-old piglets, revealing markedly reduced clinical symptoms, histopathological lesions, and intestinal PDCoV antigen distributions in piglets inoculated with F70 or F90. Importantly, F90 exhibited little to no virulence in piglets. The immunogenicity of F70, F90, and F110 was further evaluated in weaned piglets, with results indicating that the neutralizing antibody titers induced by F70 and F90 were comparable and significantly higher than those induced by F110. Collectively, these findings suggest that the PDCoV strain DHeB1 has been attenuated and can be used to develop a live-attenuated vaccine against PDCoV. Full article

Nasal carriage of Staphylococcus aureus and its antibiotic-resistant derivative, methicillin-resistant S. aureus (MRSA), is a risk factor for nosocomial S. aureus infections. Mupirocin is a topical antibiotic and a key in the decolonization of both methicillin-susceptible S. aureus (MSSA) and MRSA carriage in patients and health care personnel. Recent observations have shown a global increase in the prevalence of mupirocin-resistant MSSA and MRSA, reducing the efficacy of mupirocin in decolonization regimens. LTX-109 is a peptidomimetic synthetic compound that has shown broad-spectrum bactericidal antimicrobial activity in vitro and in animal experiments. However, the development of resistance against LTX-109 in clinical isolates of MRSA and MSSA has not been systematically examined. Background/Objectives: Here, we assess the development of spontaneous and adaptive resistance against LTX-109 in genomically diverse MRSA (n = 3) and MSSA (n = 4) strains. Methods: Adaptive and mutational resistance were examined by serial passaging strains over 60 cycles in a range of LTX-109 and mupirocin concentrations. Spontaneous resistance was examined in high-inoculum agar plates with 2–8 times the concentration above MIC. Results: Throughout serial passage, LTX-109 MICs varied less than 4-fold compared to the initial MIC of 4–8 mg/L, while mupirocin MICs increased in all susceptible strains (n = 5) from 0.25 mg/L to 16–512 mg/L. The spontaneous resistance assay demonstrated no resistance development at 4–8× MIC LTX-109 and an inoculum effect at 2× MIC. Conclusions: Our results demonstrate the novelty of LTX-109 as an antimicrobial agent with no detectable in vitro resistance development in selected clinical strains of MRSA and MSSA. Full article