Search Results (1,500)

Liver fibrosis is a significant consequence of severe liver injury resulting from viral hepatitis, alcohol, and metabolic dysfunction. Progressive fibrosis and ultimate cirrhosis are leading causes of morbidity and mortality worldwide, generally irreversible and poorly targeted by current therapies. Traditional in vitro models and animal models mostly fail to fully recapitulate human multicellular crosstalk, extracellular matrix (ECM) remodeling, and the chronic, immune modulated nature of the disease. Recent advances in three-dimensional (3D) cell culture models including organoids, spheroids, bioprinted constructs, and organ-on-a-chip systems are advantageous for reconstructing cellular diversity and mechanical microenvironments to understand pathophysiology and aid in drug discovery. Emerging multi-organ models are capable of incorporating microbiome derived cues and using multi-omics readouts and imaging-enabled mechanistic dissection for more predictive anti-fibrotic screening. These technologies align well with the recent Modernization 3.0 regulation and New Approach Methodologies by the Food and Drug Administration (FDA) and recent EU Pharmaceutical Reform. This review summarizes the pathophysiology of liver fibrosis, the current landscape of 3D human liver models, and examines how microbiome interfaces modulate fibrogenesis. Full article

Hepatitis B virus (HBV) remains a major global health threat because covalently closed circular DNA (cccDNA) persists in hepatocytes and limits the efficacy of current antiviral therapies. Effective HBV research and drug screening require culture models that recapitulate the complete viral life cycle and allow for quantitative monitoring of replication. In this study, an 11-amino acid luminescent reporter, HiBiT, was inserted at multiple sites within the preS1 region of a genotype D HBV genome, and the C terminus of preS1 was identified as optimal for maintaining robust replication. We then established HepG2-B4 cells stably replicating HiBiT-HBV with HiBiT at the preS1 C terminus. Extracellular HiBiT activity and supernatant levels of HBV-DNA, HBsAg, and HBcAg increased continuously until day 42 and were reduced by nucleos(t)ide analog treatment, and cccDNA was confirmed by Southern blot analysis. Supernatants from HepG2-B4 cells infected naïve HepG2-NTCP cells and primary human hepatocytes, as shown by extracellular HiBiT activity. Transcriptome analysis revealed distinct gene expression changes in HepG2-B4 cells compared with parental HepG2 cells. These findings indicate that the HepG2-B4 system provides a rapid, quantitative, and scalable platform for HBV replication and infection studies and is suitable for mechanistic investigations and high-throughput antiviral screening. Full article

Background: Rhinovirus C (RV-C) is one of three species of rhinoviruses (RVs), which cause the common cold, preschool wheezing illnesses and exacerbations of asthma. RV-C types are more virulent, especially in children, but progress in developing treatments is limited by difficulties in generating high-titer virus preparations. The goals of this study were to optimize methods for large-scale production and purification of RV-C to facilitate structure and immune response studies. Methods: We optimized protocols for the propagation and purification of RV-C15a, a clinical isolate adapted to HeLa-E8 cells stably expressing virus receptor CDHR3. We compared virus yields in adherent and suspension cultures, evaluated the effects of calcium supplementation and infection timing, and tested multiple purification strategies, including ultracentrifugation, dialysis, and lipase treatment. Results: RV-C15a yields were significantly lower in suspension vs. adherent cultures despite comparable virus binding and entry, suggesting post-entry replication limitations in suspended cells. In adherent cultures, infecting soon after cell seeding and calcium supplementation reduced the time of virus production and modestly improved virus progeny yields. Surface CDHR3 expression declined over time, potentially restricting viral spread. Among purification methods, lipase treatment of infected cell lysates followed by ultracentrifugation produced highly pure and concentrated virus preparations suitable for structural and immunological applications, with high yields. Conclusions: We present a robust system for large-scale RV-C15a production in adherent HeLa-E8 cells and recommend a lipase-based purification method as a rapid and effective approach for producing high-quality viral preparations. These advances will support structural studies and accelerate the development of RV-C-targeted therapeutics and vaccines. Full article

Background/Objectives: SARS-CoV-2, the causative agent of COVID-19, has been responsible for more than seven million deaths worldwide since its emergence. The Bacillus Calmette–Guérin (BCG) vaccine, used for over 100 years to prevent tuberculosis, induces a Th1-prominent immune response that is important for protection against Mycobacterium tuberculosis, other mycobacteria, and intracellular pathogens. BCG has also been shown to induce innate immune memory and heterologous protection against non-related infections. Additionally, BCG has been used as a vector to express heterologous proteins, showing protective effects against various diseases, particularly respiratory viral infections, including SARS-CoV-2. In this report, we constructed two recombinant BCG strains as potential vaccine candidates based on the receptor-binding domain (RBD) of the Spike antigen: one expressing only the RBD protein (rBCG-RBD) and another expressing the RBD protein in fusion with the LTB (Escherichia coli Labile Toxin subunit B) adjuvant (rBCG-LTB-RBD). Methods: We evaluated the induction of SARS-CoV-2-specific humoral and cellular immune responses using these vaccine candidates in a prime–boost strategy with a booster dose using the rRBD protein (produced in cell culture) and the Alum adjuvant. Antisera were evaluated for neutralization of the Wuhan and Omicron SARS-CoV-2 pseudotyped virus. Results: Either immunization scheme (rBCG-RBD/rRBD or rBCG-LTB-RBD/rRBD) induced high IgG antibody titers, with antibody neutralization against a Wuhan SARS-CoV-2 pseudotyped virus after 10 weeks. The antibody levels induced by rBCG-RBD/rRBD were maintained for up to 9 months. Interestingly, only the sera from mice receiving the prime–boost with rBCG-LTB-RBD/rRBD showed cross-reactive neutralization against the Omicron SARS-CoV-2 pseudotyped virus. Immunization with rBCG-RBD or rBCG-LTB-RBD and a rRBD booster dose promoted the induction of specific CD4+ and CD8+ T cells producing Th1/Th2 cytokines (IL-4, TNF-α and IFN-γ). Conclusions: Our study highlights the potential of the prime–boost immunization strategy using rBCG-RBD/rRBD to induce long-term immunity and rBCG-LTB-RBD/rRBD to induce cross-protection against different variants, both of which could serve as promising vaccine candidates. Full article

Coxsackieviruses B are single-stranded RNA viruses belonging to the Enterovirus genus and are associated with various clinical outcomes, ranging from acute infections to chronic diseases, such as type 1 diabetes (T1D). It was previously shown that inoculation of Swiss albino mice with CVB4 by the intraperitoneal route induced both anti-CVB4 neutralizing and enhancing activities of serum. This study aimed to investigate the humoral immune response of mice inoculated with CVB4 by the mucosal route. Mice were inoculated orally or intranasally with CVB4, and the anti-CVB4 neutralizing activity of serum and saliva was assessed by a cell culture neutralization assay. Anti-enterovirus (EV) IgG and IgA antibodies were detected in serum and saliva, respectively, by ELISA. The serum-dependent enhancement of CVB4 infection in cultures of murine splenocytes was evaluated by detecting intracellular viral RNA using RT-qPCR. At day 45 post-inoculation, an anti-CVB4 neutralizing activity, the extent of which depends on the amount of inoculated infectious particles, was detected in the serum of mice exposed orally or intranasally. An increase in anti-CVB4 neutralizing activity was observed in the saliva of mice inoculated orally or intranasally during the follow-up. Oral or intranasal inoculation of CVB4 induced a systemic IgG and mucosal IgA response. In addition, serum from these mice harbored an anti-CVB4 enhancing activity in vitro. These data indicate that Swiss albino mice exposed to CVB4 via the mucosal route constitute a potentially useful model for testing strategies to promote the production of protective mucosal and systemic anti-CVB4 antibodies and for verifying whether or not enhanced antibodies are produced. Full article

Advances in diagnostic techniques, along with environmental changes driven by human activity, have intensified the surveillance and monitoring of virus and arbovirus circulation on the Amazon. These efforts have increased the detection of insect-specific viruses in field-collected hematophagous arthropods. This study reports the first isolation of the Aguas Brancas virus from mosquitoes collected in the Brazilian Amazon and in a rural area of Brasília, Federal District, Brazil. Arthropods of the family Culicidae, genus Limatus durhamii, were collected at ground level in forest fragments. Sample BEAR812610 originated from Ananindeua, Pará, within the Evandro Chagas Institute’s grounds, and sample BEAR839941 from a forest fragment in Brasília (Ceilândia—Núcleo Rural Boa Esperança, Site B4). Specimens were identified to the species/genus level, macerated, and the supernatant inoculated into C6/36 and Vero cell cultures for viral isolation. The presence of arboviruses was determined by indirect immunofluorescence using antibodies against major arbovirus groups. Positive samples were sequenced for nucleotide and amino acid identification, and phylogenetic analysis confirmed the virus as belonging to the genus Orthoflavivirus. This represents the first report of the isolation and characterization of the insect-specific Aguas Brancas virus. Full article

The flavivirus NS1 protein is a component of the viral replication complex and plays diverse, yet poorly understood, roles in the viral life cycle. To enable real-time visualization of the developing replication organelle and biochemical analysis of tagged NS1 and its interacting partners, we engineered a replication-competent yellow fever virus (YFV) replicon encoding a C-terminal fusion of NS1 with green fluorescent protein (NS1–GFP). The initial variant was non-viable in the absence of trans-complementation with wild-type NS1; however, viability was partially restored through the introduction of co-adaptive mutations in GFP (Q204R/A206V) and NS4A (M108L). Subsequent cell culture adaptation generated a 17-nucleotide frameshift within the NS1–GFP linker, resulting in a more flexible and less hydrophobic linker sequence. The optimized genome, in the form of a replicon, replicates in packaging cells that produce YFV structural proteins, as well as in naive BHK-21 cells. In the packaging cells, the adapted NS1–GFP replicon produces titers of infectious particles of approximately 10^6 FFU/mL and is genetically stable over five passages. The expressed NS1–GFP fusion protein localizes to the endoplasmic reticulum and co-fractionates with detergent-resistant heavy membranes, a hallmark of flavivirus replication organelles. This NS1–GFP replicon provides a novel platform for studying NS1 functions and can be further adapted for proximity-labeling strategies aimed at identifying the still-unknown protease responsible for NS1–NS2A cleavage. Full article

The concentration of extracellular vesicles (EVs) in the peripheral blood of COVID-19 patients is increased. Nevertheless, their potential role in the transmission of infection remains unclear. This study was performed to determine whether EVs produced by the sub-genomic replicon system developed in Baby Hamster Kidney (BHK-21) cells could transfer SARS-CoV-2 replicon RNA, leading to the establishment of a viral replication system in human cells. Purified EVs from the SARS-CoV-2 sub-genomic replicon cell line BHK-21 were cultured with a naive human cell line. The success of EV-mediated transfer of SARS-CoV-2 replicon RNA and its productive replication was assessed using G-418 selection, a luciferase assay, immunostaining, and Western blot. We found that the A549 cell line cultured with EVs isolated from SARS-CoV-2 BHK-21 replicon cells developed G-418-resistant cell colonies. SARS-COV-2 RNA replication in A549 cells was confirmed by nano luciferase, Nsp1 protein. SARS-CoV-2 RNA replication causes massive morphological changes. Treatment of cells with the FDA-approved Paxlovid demonstrated a dose-dependent inhibition of viral replication. We isolated two human epithelial cell lines (gastrointestinal and neuroblastoma) and one vascular endothelial cell line that stably support high-level replication of SARS-CoV-2 sub-genomic RNA. Viral elimination did not revert the abnormal cellular shape, vesicle accumulation, syncytia formation, or EV release. Our study’s findings highlight the potential implications of EV-mediated transfer of replicon RNA to permissive cells. The replicon model is a valuable tool for studying virus-induced reversible and irreversible cellular reprogramming, as well as for testing novel therapeutic strategies for SARS-CoV-2. Full article

The family Arenaviridae encompasses zoonotic, rodent-borne pathogens (e.g., Lassa, Machupo, and Junín viruses) that cause severe viral hemorrhagic fevers with high case fatality rates. The current therapeutic landscape is severely limited, underscoring the urgent need for novel antiviral strategies. A promising approach involves combining directly acting antivirals with host-targeted antivirals. A compelling host-targeted antiviral target is the aryl hydrocarbon receptor (AHR). This ubiquitous ligand-activated transcription factor is a recognized pro-viral host factor across multiple viral families. Building on prior work with Junín and Tacaribe viruses, we investigated whether the AHR inhibitor CH223191 could enhance the virus-directed antiviral activity of favipiravir against these viruses. First, we evaluated the toxicity and antiviral potential of CH223191 against a lethal Junín virus infection in male and female hTfR1 mice. After demonstrating substantial protection, we conducted preliminary assays to study the antiviral effects of combining CH223191 and favipiravir on Tacaribe virus (TCRV) infections in the Vero cell culture model. We observed synergistic interaction with all four models (ZIP, Loewe, Bliss, and HSA). We next determined the sub-optimal dose of favipiravir and conducted an antiviral combination study in the AG129 mouse model infected with TCRV. The combination effectively protected mice from a lethal TCRV infection and showed cooperative effects, reducing weight loss and viral loads. Overall, these results show that the AHR is a promising pharmacological target for the development of novel antivirals. Furthermore, we discovered a cooperative interaction between the activities of favipiravir and CH223191. Full article

Background/Objectives: Syndromic multiplex PCR assays such as BIOFIRE FilmArray^® Pneumonia (PN) panel enable rapid and simultaneous detection of bacterial and viral pathogens in respiratory specimens, improving diagnostic accuracy and patient management in lower respiratory tract infections (LRTIs). Methods: In this retrospective observational study, PN panel results in 410 bronchoalveolar lavage (BAL) samples from hospitalized patients with suspected pneumonia were analyzed and compared with those obtained using the conventional culture (CC) method. Results: The PN panel showed an overall positivity rate of 54%, detecting bacteria in 39.0% of samples, viruses in 7.1%, and atypical bacteria in 2.2%. Using the conventional culture (CC) method, 33.9% of samples tested positive. Overall, 83 (20.2%) samples that were positive by the PN panel were negative by CC, whereas only 14 specimens (3.4%) were positive by CC and negative by PN panel. The most frequently detected pathogen by both the PN panel and CC was Staphylococcus aureus (n = 67, 16.34% for PN; n = 40, 9.76% for CC). Regarding diagnostic performance, the PN panel demonstrated a sensitivity of 89.02%, a specificity of 97.86%, and an overall accuracy of 97.63%. Lower sensitivity values were observed only for the Enterobacter cloacae complex (57.14%) and the Klebsiella pneumoniae group (75%). Specificity exceeded 92% for all bacterial targets. Conclusions: The PN panel confirms enhanced pathogen detection and a shortened time-to-result. It serves as a valuable adjunct for the timely diagnosis of LRTIs, supporting antimicrobial stewardship through more precise and appropriate antibiotic selection. Full article

Background: Survivin is an anti-apoptotic protein highly expressed during embryonic development and, in adults, mainly in the gastrointestinal epithelium. Its levels decrease in human gastric tissue and cultured cells upon exposure to Helicobacter pylori gamma-glutamyl transpeptidase (GGT), though the underlying mechanism remains unclear. Objective: We aimed to investigate the role of cap-independent translation driven by the Survivin 5′ untranslated region (5′UTR) in response to H. pylori infection in vitro. Methodology: Human cell lines (AGS, GES-1, HeLa, HEK293T) were used alongside bicistronic and monocistronic (Firefly/Renilla luciferases) reporter assays to assess short and long variants of the Survivin 5′UTR and HIV-1 internal ribosome entry site (IRES) sequences. Additional methods included in vitro transcription/translation, RT-qPCR, agarose gel electrophoresis, Western blotting, coupled/uncoupled translation assays, and siRNA silencing. Results: The short variant of the Survivin 5′ UTR supported cap-independent translation, like the HIV-1 IRES. Notably, H. pylori infection suppressed this translation in a GGT-dependent manner in gastric cells, and a similar reduction was observed following treatment with ATO, a known prooxidant. Conclusion: The Survivin 5′UTR exhibits cap-independent translation activity that is inhibited by H. pylori in a GGT-dependent manner, likely via oxidative stress. This mechanism helps to explain the downregulation of Survivin during gastric infection and indicates that oxidative stress can negatively affect both cellular and viral IRES-mediated translation. Full article

Astroviruses are non-enveloped, positive-sense single-stranded RNA viruses known to infect various mammals and birds, including humans, often causing gastrointestinal disorders. In recent years, astroviruses have also been linked to neurological and respiratory diseases across several species, including ruminants, mink, deer, and other mammals. Notably, astrovirus infections in goats have been documented in countries such as Switzerland and China, where novel genotypes have been identified in fecal samples. However, their role in the context of disease remains unclear, and reports focusing solely on goat astrovirus in the United States have not been published. A necropsy case of a Boer goat kid with a history of diarrhea was submitted for investigation following death in January 2025. Fresh tissues were received and used for histopathology and enteric pathogen testing, including parasitic, bacterial, and viral workups. Metagenomic-based next-generation sequencing (mNGS) was also applied for this case. Histological examination revealed severe necrotizing enterocolitis. The small intestine exhibited epithelial ulcerations, villus atrophy, hyperplastic and dilated crypts with necrotic debris, few intraenterocytic coccidian parasites, and increased inflammatory cells in the lamina propria. The large intestine showed similar findings with pleomorphic crypt enterocytes. Standard enteric pathogen tests were negative except for aerobic culture that identified Escherichia.coli and Enterococcus hirae. mNGS and bioinformatic analysis identified a novel astrovirus in the intestinal content that showed the highest nucleotide identity (86%) to the sheep strain Mamastrovirus 13 sheep/HA3 from China based on BLAST analysis. Phylogenetic analysis indicated that the newly identified caprine astrovirus IL90175 clustered with astrovirus strains from small ruminants in Asia and Europe. This research reports the discovery, histopathologic features, and genetic characteristics of a gastrointestinal disease-causing astrovirus in a goat kid, which had not been previously described in the United States. Full article

Tobacco mosaic virus (TMV) threatens crop yield and quality, while chemical antivirals offer limited efficacy and potential environmental hazards. Marine fungal polysaccharides are promising eco-friendly alternatives due to their biocompatibility and biodegradability. Here, extracellular polysaccharides (EPSs) from the deep-sea fungus Beauveria bassiana T2-2 was isolated, characterized, and produced under optimized conditions (28 °C, 200 rpm, 9 days, pH 8, inoculum 4%) using an L9 (3⁴) orthogonal medium, yielding 3.42 g/L, which is a 48% increase over unoptimized culture. EPSs were glucose-rich, with a molecular weight of 3.56 × 10⁴ Da, containing 90.05% total sugar, 0.28% protein, 1.15% uronic acid, and 1.18% sulfate. In a Nicotiana benthamiana–TMV model, EPSs alleviated viral symptoms, maintained chlorophyll content, enhanced antioxidant enzymes (SOD, POD, CAT), reduced malondialdehyde, and upregulated defense genes in SA, ET, ROS, and phenylpropanoid pathways. EPSs, alone or combined with Ribavirin, activated multi-pathway antiviral immunity, highlighting its potential as a sustainable plant-protective agent. Full article

Background: Efficient nucleic acid extraction is essential for reliable viral load testing, yet performance can differ widely depending on the extraction system and sample type. We compared three automated platforms, QIAcube, EZ1 Advanced, and Maxwell RSC, for their ability to recover cytomegalovirus (CMV) DNA and West Nile virus (WNV) RNA from common clinical matrices. Methods: Mock specimens were prepared using whole blood, plasma, serum, and urine collected from two donors. Samples were spiked with CMV or WNV culture material and extracted in triplicate on each platform according to the manufacturers’ protocols. Viral loads were measured using ELITech ELITE MGB assays on the InGenius system. Whole blood samples were also tested after a 1:4 dilution. Matrix-specific standard curves were applied, and viral loads were compared using Wilcoxon rank-sum tests with false-discovery rate adjustment. Results: Extraction efficiency differed substantially by platform and specimen type. For CMV, QIAcube consistently produced the highest DNA recovery across all matrices, with particularly large differences in plasma and serum, where EZ1 and Maxwell RSC yielded significantly lower loads. The WNV results varied by matrix: EZ1 and QIAcube performed similarly in plasma, while Maxwell RSC achieved the highest RNA recovery in whole blood. While the QIAcube exhibited reduced WNV recovery in blood, it achieved the best performance in serum, as specified by the kit. No significant platform differences were observed for urine. Diluting whole blood reduced variability between platforms. Conclusions: Both sample matrix and extraction system strongly influence nucleic acid recovery. These results highlight the need for matrix-specific validation and standardized extraction approaches in molecular diagnostics. Full article

The Emergency Department (ED) represents an ideal location for antimicrobial stewardship (AMS) intervention, given the large volume of antibiotics seen prescribed to a wide variety of patients. This is particularly true in paediatrics, where most infectious presentations are viral in nature. A recent European Society of Clinical Microbiology and Infectious Diseases (ESCMID) position paper addressed four key areas affecting adult ED. This included: (1) the utility of biomarkers or rapid pathogen tests, (2) the impact of blood cultures on antibiotic prescribing, (3) the effect of watchful waiting on clinical outcomes, and (4) the potential for structured follow-up programmes within the ED to impact prescribing. Comparatively, the paediatric ED remains underrepresented in the literature with regard to AMS interventions. In this review article, we review the evidence surrounding the above four key areas as they relate to the paediatric population. Full article