Search Results (92)

Oncolytic viruses represent a promising class of anticancer therapeutics, and rapid, accurate quantification of viral titers is critical for ensuring both efficacy and safety during clinical development. Conventional viral titering methods, such as 50% cell culture infectious dose (CCID₅₀), are time-consuming and limited in sensitivity, thereby restricting their application in real-time clinical monitoring. This study aimed to develop and validate a rapid titer assay for oHSV2-PD-L1/CD3-BsAb, an oncolytic herpes simplex virus expressing a PD-L1/CD3 bispecific antibody, to support preclinical and clinical monitoring. A dual-reporter cell system was established using Vero-PD-L1-GFP (Vero cells expressing PD-L1 and GFP) cells as target cells and Jurkat-NFAT-Fluc (Jurkat cells expressing NFAT and Fluc) cells as effector cells. Viral infection activates the NFAT signaling pathway, driving Fluc expression, thereby enabling rapid quantification of infectious virus. The assay was evaluated for specificity, limit of detection (LOD), and lower limit of quantification (LLOQ), and compared with the conventional CCID₅₀ method. Its applicability was further assessed using clinical simulation samples, including PBMCs and swabs. The rapid titer assay accurately quantified virus at 10³ CCID₅₀/mL after 8 h of incubation, consistent with CCID₅₀ results, while extending the incubation to 18 h improved the LLOQ to 10^2.5 CCID₅₀/mL, demonstrating enhanced sensitivity. The assay exhibited high reproducibility and stability in both PBMC and swab samples, enabling reliable quantification of low-titer virus in complex biological matrices. Compared with CCID₅₀, the method substantially reduced assay time (from 3–5 days to 8–18 h) while improving sensitivity and specificity. The developed rapid titer assay for oHSV2-PD-L1/CD3-BsAb provides a sensitive and specific platform for viral quantification. It offers a valuable tool for oncolytic virus development, production quality control, and clinical monitoring, facilitating efficient safety evaluation and risk management in ongoing and future clinical applications. Full article

Background: Viral vectors are indispensable tools in gene therapy and neural circuit mapping, offering promising therapeutic strategies for diverse genetic diseases and advancing neuroscience research. To achieve high transduction efficiency while mitigating impurity-induced immunogenicity, the development of viral vectors with improved purity and quality is essential. However, this critical requirement is often unmet by conventional purification methods such as ultracentrifugation, which are time-consuming and frequently result in limited product purity. The pseudorabies virus (PRV) is extensively employed as a viral tool for mapping neural circuits, where improved purity contributes to enhanced accuracy of neural tracing. PRV531 is a retrograde trans-synaptic tracer modified from the PRV Bartha strain, specifically designed to facilitate the precise visualization of hierarchical neural networks. Methods: In this study, we developed a method for the concentration and purification of PRV531 by integrating hollow fiber ultrafiltration (HF) with Capto^TM Core 700 (CC700) chromatography. Initially, to concentrate the viral supernatant, a 500 kDa HF membrane was employed, maintaining a feed flow rate of 80 mL/min, a shear rate ranging from 2000 to 6000 s⁻¹, and a transmembrane pressure (TMP) between 0.5 and 1 bar. Following concentration, the virus underwent purification through CC700 chromatography, operating at linear flow rates ranging from 100 to 300 cm/h. Results: Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) revealed distinct bands consistent with the expected sizes of major PRV structural proteins, each with molecular weights ranging from 25 kDa to 150 kDa, concurrently demonstrating a substantial reduction in host cell proteins (HCPs) contamination. The purified PRV531 achieved a high final infectious titer of 3.55 × 10⁹ PFU/mL, with an overall functional virus recovery of 8.88% from the crude supernatant to the final product. Conclusion: These data demonstrate that TFF combined with CC700 resin can efficiently purify retrograde trans-synaptic PRV tracer. Furthermore, this approach provides a promising strategy for purifying other viral-based tracers that traditionally rely on conventional centrifugation methods. Full article

Efficient in vitro production of rabies virus is essential for vaccine development and quality control applications. High-density cultivation systems offer practical advantages for rabies virus production but also create culture conditions in which nutrient depletion, waste accumulation, and progressive deterioration of host-cell condition may limit infectious virus output. In this study, we evaluated the effects of sodium pyruvate supplementation on rabies virus CVS-11 production in Vero and BSR cells cultivated in a high-density macrocarrier-based tide-motion culture system under serum-containing and serum-free conditions, with complementary comparative observations in conventional monolayer cultures of BHK cells. Cultures were infected at a multiplicity of infection of 0.01, and infectious virus production was assessed over time, together with cell density, glucose consumption, and pH dynamics. Sodium pyruvate supplementation was associated with significantly higher infectious virus titers, delayed culture deterioration, prolonged maintenance of viable cell populations, and higher peak infectious titers in both Vero and BSR cultures. The highest infectious titers were observed under serum-free pyruvate-supplemented conditions, reaching 7.5 log₁₀ FFU/mL in Vero cells and 7.2 log₁₀ FFU/mL in BSR cells. Across the tested conditions, serum-free cultivation and pyruvate supplementation were both associated with significantly higher peak infectious titers. In contrast, exploratory correlation analysis based on condition-level summary values indicated an inverse association between minimum culture pH and peak infectious titer. Together, these findings show that sodium pyruvate supplementation can improve infectious rabies virus yield and prolong the productive phase in high-density macrocarrier-based cultures, supporting its use as a practical culture-modulation strategy for CVS-11 production in adherent cell systems. Full article

Background: Modified Vaccinia Ankara (MVA) vectors are highly immunogenic vaccine platforms for the delivery of recombinant antigens. Efficient downstream processing is still challenging, particularly because substantial fractions of the virus remain intracellular. While chemical cell lysis that releases MVA particles into the supernatant before clarification can greatly enhance process efficiency and scalability, this step remains insufficiently characterized. Methods: This study assessed the compatibility of ionic, non-ionic, and zwitterionic detergents with the virus as purification target. Polysorbate 20 (Tween 20) was selected as a candidate detergent and evaluated across harvest times of 48–72 h post-infection (hpi) at concentrations of 0.01–0.5% (v/v). Results: The addition of 0.01% to 0.05% Tween 20 at 48 hpi resulted in a twofold increase in supernatant virus within one hour of application. Extended exposure to Tween 20, combined with a 650 mM mixture of NaCl, NaBr, and KCl, promoted virus particle release. However, Tween 20 concentrations above 0.1% reduced MVA infectivity. A filtration cascade using pore sizes of 5 µm and 1.2 µm achieved product yields of 77–83% at 48 hpi and 41–69% at 72 hpi, respectively. Host-cell DNA is an important contaminant during viral vector processing. However, the application of 0.05% (v/v) Tween 20 resulted in a 35% reduction of dsDNA released into the culture supernatant; the nuclei could not be preserved intact under high-salt conditions to avoid the release of cellular DNA. Conclusions: In summary, this comprehensive data demonstrated that non-ionic detergents can be used to induce cell lysis while maintaining infectious activity of enveloped MVA. Full article

Infectious bronchitis virus (IBV) is a highly infectious disease of chickens posing a major threat to poultry production worldwide. Due to genetic and antigenic variability, it is difficult to establish one universal protocol for vaccination against IB. The protectotype vaccination strategy, combining antigenically distinct vaccine strains, offers broader cross-protection than homologous IBV vaccination approaches. This field study aimed to establish serological baseline values in broiler chickens in a protectotype vaccination program using Avishield IB H-120 and Avishield IB GI-13, and to evaluate the program’s efficacy under field conditions on commercial farms where heterologous IBV genotypes were detected. The study was conducted on 25 broiler farms in 2023–2024. Day-old chicks were vaccinated via coarse spray. Blood samples were taken from them at six weeks of age and analyzed using IDEXX and BioChek ELISAs, and cecal tonsils were tested by real-time RT-PCR. Production performance was assessed using the European production efficiency factor (EPEF), with a cut-off value of 360. No IB-related clinical signs were reported, and the mean EPEF of the included farms was 408.7. The detected IBV genotypes were Mass + 793B, 793B, and 793B + VAR2. The presence of heterologous VAR2 did not negatively impact flock health or performance. Preliminary serological baseline values for vaccinated, non-VAR2 farms were established at 891–1332 (in chicks tested with the IDEXX ELISA) and 4193–5204 (in those tested with the BioChek assay). The H-120 + GI-13 program demonstrated high field efficacy, reliable serological profiles, and cross-protection in the presence of heterologous IBV. Continuous monitoring of IBV genotypes remains essential. Full article

Influenza viruses cause mild to severe lower respiratory infections, sometimes resulting in hospitalization and death. Vaccination remains the primary prophylactic strategy. Live attenuated influenza vaccines (LAIVs) efficiently induce antiviral immune responses and contain temperature-sensitive and cold-adapted mutations that render them safe. These mutations are principally located in the PB1 and PB2 subunits of the viral RNA polymerase, but the mechanism by which they attenuate the virus is unclear. We introduced the PB1 and PB2 mutations from two LAIV backbones, A/Ann Arbor/6/1960 H2N2 (AA) and A/Leningrad/134/17/1957 H2N2 (Len), into the model influenza strain A/Puerto Rico/8/1934 H1N1 (PR8). In contrast to the wild-type (WT) PR8 polymerase, the two “PR8-LAIV” polymerase complexes demonstrated maximal activity at cold temperatures (30–32 °C) and greatly reduced activity at elevated temperatures (>37 °C). To further understand the impact of the LAIV mutations, we infected MDCK cells with WT and mutated PR8 viruses that contain the Len and AA LAIV mutations in PB1 and PB2. The PR8-LAIV mutant viruses exhibited a selective, temperature-dependent defect in the replicase activity of the viral RNA polymerase relative to WT PR8, while also demonstrating a temperature-dependent enhancement in the transcriptional activity of the enzyme. In addition, the PR8-LAIV mutant viruses produced similar levels of viral proteins to WT PR8 at 37 °C, but greatly (2–3 log₁₀) reduced levels of infectious viral progeny. Collectively, these data show that LAIV mutations selectively alter influenza viral RNA polymerase function, favoring transcription over genome synthesis at 37 °C, thereby preserving viral antigen production while also contributing to viral attenuation. Full article

Coronaviruses, including avian infectious bronchitis virus (IBV), utilize host cellular pathways to evade the host immune response. The aryl hydrocarbon receptor (AhR), a key antiviral regulator exploited by mammalian coronaviruses like SARS-CoV-2, remains unclear in avian coronavirus pathogenesis. This study examined AhR’s involvement during IBV infection using H1299 and Vero cells with pharmacological modulation (AhR antagonist CH223191/agonist kynurenine) and shRNA-mediated silencing. Viral replication was quantified through plaque assays, qRT-PCR, and Western blot. The results reveal IBV-induced AhR activation, driving downstream CYP1A1 expression and pro-inflammatory cytokine production. CH223191 treatment reduced IBV titers, RNA loads, and N protein expression dose-dependently, while kynurenine showed no effect. AhR knockdown similarly reduced N protein expression, confirming its proviral role. An IBV-encoded noncoding RNA was identified as a modulator of AhR activation, suggesting viral balancing of immune evasion and replication efficiency. These results establish AhR as a conserved host factor co-opted by IBV, and highlight AhR antagonism as a promising therapeutic strategy. By bridging insights from avian and mammalian coronaviruses, this work informs strategies to address IBV’s genetic variability and supports development of broad-spectrum antiviral therapies. Full article

The dengue virus (DENV) exploits host cell exosome pathways to disseminate and evade immunity. However, the host factors enabling this process remain poorly defined. Here, we demonstrate that DENV infection robustly induces expression of the short isoform of Reticulon 3 (RTN3S) in hepatic (Huh7) and monocytic cells, and that RTN3S is a critical driver of infectious exosome biogenesis. RTN3S physically associates with double-stranded viral RNA and the DENV non-structural protein 3 (NS3) in infected cells, indicating its integration into the viral replication complex. Loss of RTN3 markedly reduced exosome production and the exosomal export of viral RNA and proteins, demonstrating that RTN3S is required for efficient exosome-mediated viral release. Conversely, overexpression of full-length RTN3S dramatically increased the release of infectious virus-containing exosomes; truncation of the RTN3S C-terminal domain abolished this enhancement, confirming the essential role of the C-terminus in RTN3S’s pro-viral exosomal function. In DENV-infected monocytes, we observed a shift toward a CD16-positive intermediate phenotype, accompanied by the upregulation of genes involved in vesicle biogenesis and stress response. These infected monocytes also secreted higher levels of inflammatory cytokines. Similarly, monocytes from Dengue patients exhibited high RTN3 expression, which correlated with an expansion of intermediate (CD16⁺) subsets and enriched expression of vesicle trafficking machinery genes. These findings reveal a previously unrecognized mechanism by which DENV hijacks RTN3S to promote the formation of infectious exosomes, thereby facilitating viral dissemination and immune evasion. RTN3S thus represents a novel element of the Dengue pathogenesis and a potential target for host-directed antiviral strategies. Full article

African swine fever (ASF) is a highly pathogenic and hemorrhagic swine infectious disease caused by the African swine fever virus (ASFV). It encodes over 150 proteins, among which the CD2v protein plays multiple roles throughout the infection process. Single B-cell antibody technology is a cutting-edge method for preparing monoclonal antibodies (mAbs), which has the advantages of rapid, efficient, and high yield in antibody production, while possessing natural conformations. In this study, by cloning and expressing antibody genes in vitro, 14 murine-derived mAbs were prepared using recombinant CD2v proteins as immunogenic sources, which brings sufficient enrichment and selectivity for the development of antibodies based on the single B-cell antibody technique. All 14 mAbs demonstrated reactivity with CD2v protein by indirect ELISA, whereas 8 mAbs successfully detected CD2v in ASFV-infected PAM cells by IFA, indicating the tested mAbs can effectively recognize and bind to ASFV CD2v. Finally, a blocking ELISA method for detecting CD2v antibodies using CD2v mAb C89 was established, which holds significant potential for broad application in the serological diagnosis of ASFV with determination of the CD2v-blocking ELISA specificity, sensitivity, reproducibility, and compliance rate. It could be used for the rapid clinical detection of ASFV CD2v protein to provide a powerful tool for the monitoring of epidemics. Full article

Background/Objectives: Glucan particles (GPs), derived from Saccharomyces cerevisiae yeast, possess unique biomedical properties. Nevertheless, it is imperative that a comprehensive risk assessment is conducted during pre-clinical development. GPs are primarily constituted of a naturally occurring polymer known as β-glucan. This study characterized GPs, focusing on physicochemical attributes, biocompatibility, and immunomodulatory potential. Methods: GPs were characterized for size, morphology, surface charge, and protein encapsulation efficiency using dynamic light scattering (DLS), electron microscopy, and encapsulation assays. Biocompatibility was assessed through cytotoxicity assays (MTT), hemolysis tests, and measurement of reactive oxygen (ROS) and nitric oxide (NO) production in immune cells. Immunomodulatory potential was evaluated by cytokine and chemokine secretion analysis in peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (moDCs) and through in vivo immunization studies in a murine model, focusing on cellular immune responses. Results: GPs demonstrated stable physicochemical properties and efficient protein encapsulation, highlighting their suitability as vaccine delivery systems. They exhibited biocompatibility by not inducing cytotoxicity, hemolysis, or excessive ROS and NO production. In PBMCs, GPs stimulated cytokine secretion, suggesting their adjuvant potential. GPs were efficiently internalized by monocytes and led to specific chemokine secretion in stimulated moDCs. In a murine model, GPs induced distinctive cellular immune responses, including TNF-α and IFN-γ production and effector memory T cell activation. Conclusions: These findings emphasize GPs’ biocompatibility and immunomodulatory effects, highlighting their potential in immunotherapy and vaccine development, particularly for targeting infectious agents like hepatitis B virus. Full article

Background: Vaccines represent one of the most affordable and efficient tools for controlling infectious diseases; however, the development of efficacious vaccines against complex pathogens remains a major challenge. Adjuvants play a relevant role in enhancing vaccine-induced immune responses. One such molecule is interferon-stimulated gene 15 (ISG15), a key modulator of antiviral immunity that acts both through ISGylation-dependent mechanisms and as a cytokine-like molecule. Methods: In this study, we assessed the immunostimulatory potential of ISG15 as an adjuvant in Modified Vaccinia virus Ankara (MVA)-based vaccine candidates targeting Zika virus (ZIKV) and Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Early innate responses and immune cell infiltration were analyzed in immunized mice by flow cytometry and cytokine profiling. To elucidate the underlying mechanism of action of ISG15, in vitro co-infection studies were performed in macrophages. Finally, we evaluated the magnitude and functional quality of the elicited antigen-specific cellular immune responses in vivo. Results: Analysis of early innate responses revealed both platform- and variant-specific effects. ISG15AA preferentially promoted natural killer (NK) cell recruitment at the injection site, whereas ISG15GG enhanced myeloid cell infiltration in draining lymph nodes (DLNs), particularly when delivered via MVA. Moreover, in vitro co-infection of macrophages with MVA-based vaccine vectors and the ISG15AA mutant led to a marked increase in proinflammatory cytokine production, highlighting a dominant role for the extracellular, ISGylation-independent functions of ISG15 in shaping vaccine-induced immunity. Notably, co-infection of ISG15 with MVA-ZIKV and MVA-SARS-CoV-2 vaccine candidates enhanced the magnitude of antigen-specific immune responses in both vaccine models. Conclusions: ISG15, particularly in its ISGylation-deficient form, acts as a promising immunomodulatory adjuvant for viral vaccines, enhancing both innate and adaptive immune responses. Consistent with previous findings in the context of Human Immunodeficiency virus type 1 (HIV-1) vaccines, this study further supports the potential of ISG15 as an effective adjuvant for vaccines targeting viral infections such as ZIKV and SARS-CoV-2. Full article

Polyomaviruses have the potential to cause significant morbidity not only in transplant medicine, but also in other forms of disease or variants of immunosuppression. In kidney transplant recipients or recipients of human stem cell transplants, the BK-Virus is the major proponent of manifestations such as BKPyV-associated nephropathy or hemorrhagic cystitis. As no polyomavirus-specific drug with proven in vivo effects has been developed so far, methods to screen for such drugs are important. This work describes the establishment of a virus-secreting cell line. By infecting a pre-established monkey kidney cell line (COS-1) with a non-rearranged human BK polyomavirus isolated from a kidney transplant patient suffering from BKPyV-associated nephropathy, a continuously replicating cell type with consistent virus secretion could be established and was termed COSSA. Measurements of BKPyV replication, virion production, and secretion were performed both intracellularly and in the cell supernatant. Viral proteins such as VP1 and LTAg were accurately tracked by confocal microscopy, as well as by immunoblot and qPCR. An intracellular flow cytometry (FACS) assay detecting VP1 protein was established and revealed an expanded range of positive intracellular signals. The viruses produced proved to be infectious in human tubular epithelial cell lines. Long-range sequencing of the COSSA genome using Oxford Nanopore Technology revealed a total of five distinct BKPyV integration events. One integration of a partial BKPyV genome was located upstream of the epidermal growth factor receptor gene. The second and third, both truncated forms of integration, were close to histocompatibility gene locuses, while the fourth was characterized by a ninefold and the fifth by a fourfold tandem repeat of the BKPyV genome. From both of the repeat forms, virus replicates were derived showing deletions/duplications on early and late genes and inversions within the non-coding control region (NCCR). This pattern of repetitive viral genome integration is a potential key driver of enhanced viral replication and increased virion assembly, ultimately supporting efficient virus egress. Quantitative PCR analysis confirmed the release of approximately 10⁸/mL viral units per 48 h from 2 × 10⁵ COSSA cells into the culture supernatant. Notably, the NCCR region of the most frequent copies of circular virus and the integrated tetrameric tandem repeat exhibited a rearranged configuration, which may contribute to the observed high replication dynamics. The establishment of a consistent methodology to generate and secrete BKPyV from a cell line is expected to significantly facilitate antiviral drug development. Full article

Infectious laryngotracheitis virus (ILT) is the pathogen responsible for a respiratory ailment that has resulted in significant economic losses in the poultry industry, primarily due to high morbidity and mortality rates, as well as diminished egg production. However, for small producers living near the coast, where backyard flocks are located in rural areas, respiratory illness may pose a public health risk rather than an economic one, which must be considered in the differential diagnosis of ILT. Therefore, in this research, we focused on developing an efficient tool to detect, quantify, and classify infectious laryngotracheitis virus (ILTV) field variants. Our results demonstrates that qPCR detected positive samples collected between 2019 and 2023 in flocks exhibiting clinical signs of respiratory illness in Valdivia, in the Los Ríos Region of Southern Chile. Furthermore, the molecular characterization of positive samples using dideoxynucleotide sequencing revealed that the detected viruses were similar to tissue culture origin (TCO) vaccines, even though the birds had never been vaccinated. To the best of our knowledge, this is the first report characterizing ILTV variants in Chile using a molecular approach. Our findings indicate that the tool is useful for detecting ILTV and can also be used to quantify viral particles using a standard curve, making it a valuable tool for the differential diagnosis of respiratory pathogens in poultry. Full article

T lymphocytes infiltrate the CNS in response to murine cytomegalovirus (MCMV) infection and form a pool of long-lived brain tissue-resident memory T-cells (bT_RMs), which display markers of residency (i.e., CD103, CD69, CD49a). However, the functional role of these bT_RMs is still unknown. By 30 days postinfection, a latent viral brain infection was established, as indicated by absence of viral transcripts (IE1, E1, and gB) produced during productive infection. Following intracerebroventricular injection of either depleting α-CD8 Ab (clone YTS169.4) or α-CD103-sap (clone IT50) into the brain, 90–95% T-cell depletion was achieved. Using luciferase-expressing mice, we observed recommenced imaging signals indicative of de novo MCMV IE promoter activity in depleted animals. Surprisingly, using an explant assay, we efficiently recovered reactivatable, infectious virus from untreated, latent animals, but not from those depleted of bT_RMs (viral recovery in explants was reduced from 100% to 50% by day 21). We identified Lgals3 (galectin 3), Gpnmb (glycoprotein nonmetastatic melanoma protein B) and Hmox1 (heme oxygenase 1) as genes that were most upregulated in bT_RM-depleted groups. When bT_RMs were depleted, there was transient expression of viral IE genes which resulted in antiviral microglia with a phagocytic, disease-associated (DAM) or neurodegenerative (MGnD) phenotype. These data provide new insights into the role of bT_RMs in controlling both CNS reactivation and driving microglial phenotypes. Full article

Perioperative blood management strategies include evidence-based guidelines to efficiently manage blood products and transfusions while minimizing blood loss and improving patient outcomes. Perioperative Medicine has made evident that anemia is often under-recognized and not appropriately addressed prior to surgery. Early recognition and correction of anemia is imperative for better surgical optimization, fewer transfusions perioperatively, and improved outcomes. Patient blood management utilize evidence-based guidelines for the establishment of a framework to promote treatment of the causes of anemia, reduce blood loss and coagulopathy as well as to improve patient safety and outcomes by efficiently managing blood products, decrease complications associated with blood transfusions and reduce overall costs. Both liberal and restrictive strategies for blood transfusions established thresholds for hemoglobin: restrictive transfusion threshold of hemoglobin 7–8 g/dL in stable patients, and a higher transfusion threshold of hemoglobin > 8 g/dL may be considered in patients with cardiac disease. Intraoperatively, tests such as viscoelastic testing, including rotational thromboelastometry and thrombelastography, offer real-time analysis of a patient’s clotting ability, allowing for targeted transfusions of fresh frozen plasma, platelets, cryoprecipitate or antifibrinolytic drugs. Complications associated with blood transfusions include allergic reactions, delayed hemolytic reactions, transfusion related acute lung injury, transfusion-associated circulatory overload, and the transmission of infectious diseases such as Hepatitis B, Hepatitis C, and Human-immunodeficiency virus. This review will discuss the management of blood products for surgical patients in the entire perioperative setting, with specific considerations for the peri-, intra- and post-operative stages. Full article