Search Results (441)

Infectious Bronchitis Virus is one of several major viral infections in poultry, affecting the respiratory, reproductive, and renal systems and causing significant economic losses worldwide. Current vaccines, including the H120 strain, provide limited cross-protection against emerging variants, underscoring the need for improved vaccine strategies. In this study, the complete genome of IBV H120 was divided into 12 fragments, synthesized, and assembled using the Golden Gate Assembly (GGA) method. The recombinant virus (rH120) was successfully rescued in chicken fibroblast cells and propagated in embryonated specific-pathogen-free (SPF) chicken eggs. Growth kinetics in embryonated SPF chicken eggs revealed similar replication patterns between rH120 and the original H120 strain. In broiler chickens, rH120 replicated efficiently, as confirmed by viral RNA detection in throat and cloacal swabs, and induced a stronger antibody response by 14 days post-infection. The rH120 virus proved to be genetically stable, infectious, and immunogenic, indicating that GGA-based reverse genetics is an effective system for IBV vaccine development. Full article

This study aimed to evaluate in situ ruminal nutrient degradation, fractional disappearance rate, and effective ruminal degradation of hydroponically sprouted barley, wheat, and triticale. Two ruminally canulated lactating cows were used in a complete randomized block design with four treatments and nine incubation times (0, 2, 4, 8, 12, 24, 48, 72, and 240 h). Treatments were corn silage (control), and sprouted barley, triticale, and wheat. Quadruplicate samples (5 g each) were placed in Dacron bags and incubated in the rumen. Then, bags were rinsed and spun, dried (48 h × 55 °C; 3 h × 105 °C), and weighed to determine residual dry matter (DM). Data were analyzed using mixed models (MIXED, SAS 9.4) with treatment, time, and their interaction as fixed effects, and cow and replicate (cow) as random effects. Denominator degrees of freedom were adjusted using the Kenward–Roger method, and means were separated by Tukey–Kramer. Significance was declared at p ≤ 0.05 and tendencies at 0.05 < p ≤ 0.10. Sprouted triticale and wheat treatments had a greater rapidly soluble fraction for DM (p < 0.01), the greatest fractional disappearance rate for DM (p < 0.01) and neutral detergent fiber (NDF; p < 0.01), and greater effective ruminal degradability (ERD) for DM (p < 0.01) and crude protein (CP; p < 0.01). Sprouted wheat also had the greatest ERD for NDF (p < 0.01). In contrast, sprouted barley had the lowest rapidly soluble fractions for DM (p < 0.01), NDF (p < 0.01), and CP (p < 0.01), lower fractional disappearance rate for DM (p < 0.01) and NDF (p < 0.01) than sprouted triticale and wheat, and the lowest ERD for DM (p < 0.01) and CP (p < 0.01). Overall, sprouted triticale and wheat had greater in situ ruminal nutrient degradation, effective ruminal degradation, and nutrient degradation kinetics, indicating their potential for inclusion in dairy cattle diets to improve nutrient degradability. Full article

Background: Mini-puberty is a transient but critical postnatal activation of the hypothalamic–pituitary–gonadal axis, essential for male gonadal maturation, penile and testicular growth, and future reproductive potential: this physiological hormonal surge is absent or blunted in congenital hypogonadotropic hypogonadism (CHH), often manifesting as micropenis, cryptorchidism, and impaired Sertoli cell proliferation. Objective: The aim of this review is to summarize current evidence on the impact of early gonadotropin therapy in male infants with CHH. Methods: We conducted a comprehensive literature review using PubMed, including studies reporting on male infants with confirmed or suspected CHH receiving gonadotropin therapy. Keywords included “mini-puberty and hypogonadism”, “gonadotropins and infancy,” and “gonadotropin therapy in CHH.” Eligible studies reported biochemical outcomes (luteinizing hormone, follicle-stimulating hormone, testosterone, inhibin B, anti-Müllerian hormone) and clinical measures (penile length, testicular volume, testicular descent). Data extraction focused on endocrine responses, genital growth, and safety. Results: Twelve studies including 95 infants were analyzed. Early gonadotropin therapy effectively restored postnatal hormonal levels, with consistent increases in testosterone, inhibin B, and anti-Müllerian hormone. Clinically, treatment induced significant penile growth, increased testicular volume and partial or complete testicular descent in the majority of cases. Both continuous infusion and intermittent injection regimens were effective, though hormone kinetics and growth responses varied. No serious adverse events were reported, and therapy was generally well tolerated. Conclusions: Early gonadotropin therapy during mini-puberty represents a safe and effective intervention to replicate the physiological postnatal hormonal surge in male infants with CHH. Prospective longitudinal studies are warranted to evaluate sustained effects on puberty, fertility, and adult reproductive function. Full article

The SARS-CoV-2 nucleocapsid protein (Np) is essential for viral RNA replication and genomic RNA packaging. Phosphorylation of Np within its central Ser-Arg-rich (SRR) linker is proposed to modulate these functions. To gain mechanistic insights into these distinct roles, we performed in vitro biophysical and biochemical studies using recombinantly expressed ancestral Np and phosphomimetic SRR variants. Limited-proteolysis showed minor cleavage differences between wild-type (WT) and phosphomimetic Np, but no major structure or stability changes in the N- and C-terminal domains were observed by circular dichroism spectroscopy and differential scanning fluorimetry, respectively. Mass photometry (MP) revealed that WT Np dimerized more readily than phosphomimetic variants. Crosslinking-MP showed that WT Np formed discrete complexes on viral 5′ UTR stem-loop (SL) 5 RNA, whereas phosphomimetic Np assembled preferentially on SL1–4. WT Np bound non-specifically to all RNAs tested primarily via hydrophobic interactions, whereas phosphomimetic Np showed selectivity for SARS-CoV-2-derived RNAs despite binding more electrostatically. A major difference was observed in the binding kinetics; WT Np compacted and irreversibly bound single-stranded DNA, whereas phosphomimetic Np displayed reduced compaction and fast on/off binding kinetics. These mechanistic insights support a model where phosphorylated Np functions in RNA replication and chaperoning, while non-phosphorylated Np facilitates genomic RNA packaging. The findings also help to explain infectivity differences and clinical outcomes associated with SRR linker variants. Full article

Background: Parvovirus B19 (B19V; Erythroparvovirus primate 1) is now the most commonly detected virus in human endomyocardial biopsies from patients with myocarditis or dilated cardiomyopathy; however, its true causal role remains uncertain. By contrast, Protoparvovirus carnivoran 1, also known as canine parvovirus type 2 (CPV-2), is an apparent cause of myocarditis in neonatal puppies, where it replicates in cardiomyocytes, induces extensive cell death, and often leaves fibrotic scars in survivors. Conclusions: This review compares B19V and CPV-2 from basic biology to clinical expression. Divergent tropism and replication kinetics produce distinct injury patterns: predominantly endothelial and microvascular dysfunction with immune-mediated damage in adult human B19V infection versus direct, age-restricted cardiomyocyte lysis in neonatal CPV-2 infection, often followed by fibrosis. Because parvoviral DNA can persist in cardiac tissue, detection alone does not prove causality. We advocate an “evidence bundle” integrating viral load by quantitative polymerase chain reaction (qPCR), detection of viral transcripts and/or proteins when feasible, spatial co-localization with histological injury, and concordant clinical markers (cardiac troponins and advanced imaging, including cardiac magnetic resonance imaging [CMR]) to support etiologic attribution and guide management in human and veterinary cardiology. Full article

The continuing spread of SARS-CoV-2 and the associated morbidity and mortality, especially in vulnerable populations, highlight the need for the development of antiviral therapeutics. Reverse genetics systems and reporter viruses are valuable for antiviral screening by simplifying methods to detect and quantify virus infections. This study aimed to generate wild-type and Nluc reporter full-length SARS-CoV-2 molecular clones and viruses as tools for high-throughput antiviral assays. The large SARS-CoV-2 genome (~30 kb) makes cDNA cloning and virus rescue technically challenging, so we opted to use cDNA chemical synthesis services to generate full-length wild-type and reporter Delta and Omicron clones. Clone-derived Delta and Omicron wild-type and reporter viruses were successfully rescued and showed replication kinetics comparable to patient-derived isolates. Nluc reporter viruses displayed stable luciferase expression that correlated with viral titres, supporting their reliability as replication substitutes. Antiviral assays measuring replication inhibition by Remdesivir, Molnupiravir, and Nirmatrelvir, based on Nluc expression, yielded IC₅₀ values and selectivity indices consistent with published ranges. Finally, Delta Nluc viruses replicated in primary human bronchial epithelial cells, demonstrating the application of clone-derived viruses in physiologically relevant models. The SARS-CoV-2 cDNA clones and Nluc reporter viruses derived from DNA synthesis services provide a rapid, scalable reverse genetics platform for generating new viruses and developing assays to rapidly assess antiviral compounds against current and emerging SARS-CoV-2 variants or coronaviruses that may emerge in the future. Full article

Low-melting bioabsorbable polymers, such as poly(caprolactone-co-lactide) (PCLA), hold significant promise for biomedical applications. However, achieving high-precision micro- and nanotopographical functionalization remains a formidable challenge due to the material’s susceptibility to thermal deformation during conventional thermal molding processes. In this study, functional microstructured PCLA coatings were engineered via low-temperature nanoimprint lithography utilizing a TiO₂–SiO₂ gas-permeable mold. These molds were synthesized via a sol–gel method utilizing titanium dioxide and silicon precursors. The gas-permeable nature of the mold facilitated the efficient evacuation of trapped air and volatiles during the imprinting process, enabling the high-fidelity replication of microstructures (1.3 μm height, 3 μm pitch) and nanostructured PCLA coatings featuring linewidths as narrow as 600 nm. The resultant microstructured PCLA coatings demonstrated modulated surface wettability, evidenced by an increase in water contact angles from 70.1° to 91.4°, and exhibited enhanced FD4 elution kinetics. These results confirm morphology-driven functionalities, specifically hydrophobicity and controlled release capabilities. Collectively, these findings underscore the efficacy of this microfabrication approach for polycaprolactone-based materials and highlight its potential to catalyze the development of high-value-added biomaterials for advanced medical and life science applications. This study establishes a foundational framework for the practical deployment of next-generation bioabsorbable materials and is anticipated to drive innovation in precision medical manufacturing. Full article

RNA-based vaccination has been broadly applied in the COVID-19 pandemic. A characteristic of the immunization was fast-waning immunity. However, the time scale of this process varied considerably for virus subtypes and among individuals. Understanding the origin of this variability is crucial in order to improve future vaccination strategies. Here, we introduce a mathematical model of RNA-based vaccination and the kinetics of the induced immune response. In the model, antigens produced following vaccination give rise to an immune response leading to germinal center reactions and accordingly B-cell differentiation into memory B-cells and plasma cells. In a negative feedback loop, the antibodies synthesized by newly specified plasma cells shut down the germinal center reaction as well as antigen-induced differentiation of memory B-cell into plasma cells. This limits the build-up of long-lasting immunity and thus is accompanied by fast-waning immunity. The detailed data available on infection with and vaccination against SARS-CoV-2 enabled computational simulation of essential processes of the immune response. Through simulation, we analyzed to what extent a single- or double-dose vaccination provides protection against infection. We find that variability in the immune response in individuals, originating, e.g., in different immune-cell densities, results in a broad log-normal-like distribution of the vaccine-induced protection times that peaks around 100 days. Protection times decrease for virus variants with mutated antibody-binding sites or increased replication rates. Independent of these virus specifics, our simulations suggest optimal timing of a second dose about 5 weeks after the first in agreement with clinical trials. Full article

Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host–virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, enabling cloning, manipulation, and the rescue of recombinant SARS-CoV-2 (rSARS-CoV-2) with high fidelity and high viral titers after a single passage. Using this system, we generated and characterized spike protein mutants Y453F and N501Y, as well as a U76G mutation in the 5′-UTR. Y453F showed reduced replication kinetics, lower cell binding, and diminished fitness, while N501Y exhibited comparable replication and fitness, highlighting the distinct effects of these spike protein mutations. The U76G mutation is located within a novel NSP9 binding site in the 5′-UTR and leads to impaired RNA synthesis and reduced viral replication efficiency, suggesting an important role in transcription and replication. Our findings highlight the robustness and adaptability of this reverse genetics system, providing a versatile, cost-effective tool for studying SARS-CoV-2 mutations and their effects on replication and fitness, with potential applications in vaccine and therapeutic development. Full article

Feline herpesvirus-1 (FHV-1) is taxonomically classified within the family Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus, and species Felid alphaherpesvirus 1. The genome of FHV-1 is 135,797 bp in length and encodes 74 proteins. Among these proteins, serine/threonine protein kinase (pK) and thymidine kinase (TK) have been identified as potential virulence factors in alphaherpesviruses, although these kinases are dispensable for viral replication. As kinases, regulating phosphorylation modification is one of their functions, while the mechanism by which phosphorylation modification affects cell physiological functions and thereby influences viral replication remains unclear. In this study, we generated pK- and TK-deficient FHV-1 mutants by CRISPR/Cas9-mediated homologous recombination. The pK-deficient virus produced significantly smaller plaques than the TK-deficient virus. The replication kinetics of the pK-deficient virus were attenuated in multistep growth compared to the TK-deficient virus. These results indicate that deletion of the pK gene markedly reduces the replicative capacity of FHV-1. We applied data-independent acquisition (DIA) quantitative proteomics to profile changes in global protein expression and phosphorylation in F81 cells upon infection with TK⁻, pK^−, and wild-type FHV-1 strain. The pK-deficient virus exhibited 3632 differentially phosphorylated proteins containing 11,936 modification sites; the TK-deficient virus showed 4529 differentially phosphorylated proteins with 19,225 phosphorylation sites. Functional characterization through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses identified significant involvement of phosphoproteins in spliceosome pathways in pK-deficient virus and ATP-dependent chromatin remodeling pathway in TK-deficient virus. Notably, several splicing regulators—including Ess-2 and CDK13, which modulate host spliceosomal function—displayed significantly reduced phosphorylation levels in pK-deficient viruses. A significant enrichment of ATP-dependent factors, such as SMARCA5 and RSF1, was observed in the TK-deficient virus. To our knowledge, this is the first investigation into the effects of FHV-1 infection on the host cell phosphoproteome. These data offer new insights into the phosphoregulatory circuits and signaling networks triggered by FHV-1 and may enhance our understanding of the FHV-1 replication mechanism. Full article

The operational integrity of L415 pipeline steel, a critical component of China’s energy network, is severely threatened by the unique acidic red soil environments prevalent in Southern China. A significant knowledge gap exists regarding its specific failure mechanisms, particularly the interplay between Anodic Dissolution (AD) and Hydrogen Embrittlement (HE) in driving Stress Corrosion Cracking (SCC). This study systematically investigates the corrosion and SCC behavior of L415 steel in a simulated environment that replicates the typical soil chemistry of the Gannan region in Southern China. Results revealed that corrosion kinetics are highly dependent on environmental chemistry, with corrosion rates escalating nearly four-fold from 0.0505 mm/a to a severe 0.1949 mm/a, driven by the synergy of low pH and high SO₄²⁻ concentration. This behavior is governed by the integrity of the corrosion product film, where aggressive environments form porous, unprotective layers with low charge transfer resistance. Slow strain rate tensile (SSRT) tests confirmed that the steel’s susceptibility to SCC is strongly promoted by acidity. Critically, the dominant SCC mechanism was environment-dependent, transitioning from Hydrogen Embrittlement (HE) to intergranular cracking in the most acidic environment, and a mixed AD-HE mechanism causing transgranular cracking in high-chloride conditions. These findings provide a direct mechanistic link between soil chemistry and failure mode, offering a crucial scientific basis for developing environment-specific integrity management strategies for pipelines in these challenging terrains. Full article

Plant protease inhibitors (PPI) play a significant role against microbes, insects, and, to a considerable extent, human pathogens. PPIs inactivate hydrolase enzymes or depolarize the plasma membrane of the pathogens, thereby inhibiting their growth, replication, and invasion. Here, an active serine protease inhibitor was isolated and purified from the seeds of Cleome viscosa. The purified inhibitor was homogenous and exhibited a molecular weight of around 12 kDa as a monomer. The secondary structure analysis indicated that the inhibitor was predominantly composed of α-helical content. The kinetics experiments demonstrated a noncompetitive mode of inhibition towards serine protease when casein was used as the enzyme substrate. The inhibitor formed a stable complex with serine protease, having a likely 1:1 stoichiometry, as inferred from ITC, and the dissociation constant was examined to be K_d = 1.9 × 10⁻⁶ M with a Gibbs free energy of ΔG = −8.079 (kcal/mol). The inhibitor exhibits stable protease inhibition up to 90 °C. Further, in vitro preliminary studies revealed its inhibitory effects against HSV-2 function, evidence that it may have a role in the treatment of viral infections. Full article

This study investigates the production of volatile organic compounds (VOCs) in RK-13 cells infected with three equine viruses representing different families: equine arteritis virus (EAV) (Arteriviridae), equine herpesvirus 1 (EHV-1) (Herpesviridae), and equine rhinitis B virus (ERBV) (Picornaviridae). VOCs, which are byproducts of cellular metabolism and potential non-invasive diagnostic markers, were analyzed using headspace solid-phase microextraction (HS-SPME) and gas chromatography–mass spectrometry (GC-MS). Since viruses do not possess intrinsic metabolic activity, the observed changes in the VOC profiles were attributed to host responses, such as metabolic reprogramming, oxidative stress, and apoptosis. We hypothesized that each viral infection induces distinct metabolic changes, resulting in characteristic VOC signatures that mirror the virus type, replication kinetics, and cytopathic effects. Notably, viruses with rapid cytopathic effects (e.g., EHV-1) were anticipated to trigger more pronounced VOC alterations. In our experimental design, RK-13 cells were infected at a multiplicity of infection of 1 and incubated for 24 h, 48 h, or 72 h. Distinct VOC profiles emerged, with significant elevations in compounds like 2-ethyl-1-hexanol, particularly in EHV-1 infections, and selective increases in acetophenone and benzaldehyde. Principal component analysis (PCA) of the VOC concentration data showed the clear separation of data from viruses from different families. These findings support the potential of VOC profiling as a rapid diagnostic tool for viral infections. Full article

Transcription–replication conflicts (TRCs) often occur in cells and cause DNA replication fork stalling. In this study, we investigated the interplay of RNA polymerase (RNAP), DNA polymerase, and RNase H1 (RH1) during head-on TRC in vitro with precise control over the reaction conditions. We show that it is a catalytically competent transcription elongation complex (TEC) that interferes with the action of both the Klenow fragment and full-length DNA Pol I. An incompetent RNAP complex with an R-loop stimulates the 3′→5′ exonuclease activity and pauses the DNA polymerase during head-on TRC. As RNAP advances along the DNA template, elongating the RNA, the head-on TRC is slowly overcome in our model system, likely through the reassociation of the displaced DNA polymerase with the nontemplate DNA strand upstream of RNAP. An isolated R-loop containing an 11-nt heteroduplex (R-loop-11) does not interfere with DNA replication by the Klenow fragment. For DNA Pol I, such an R-loop also does not stall replication but stimulates its 3′→5′ exonuclease activity. We demonstrate that a stalled Klenow fragment does not interfere with transcription, whereas a Klenow fragment moving along the TRC substrate towards RNAP alters the kinetics of RNAP. Stalled DNA Pol I does not stop RNAP but stimulates its endonuclease activity. We find that RH1 alone does not displace stalled RNAP from a competent TEC containing R-loop-11 and does not resolve the head-on TRC. On the other hand, RH1 displaces RNAP from the incompetent complex with the TRC substrate. This eliminates the stimulation of the 3′→5′ exonuclease activity of DNA polymerase during head-on TRC. Full article

ZIKA virus (ZIKV) infections in human neonates and adults are associated with deleterious effects on brain cognition and neurological disorders. The mechanism(s) of ZIKV infection in neurons and associated neuronal antiviral responses are not fully understood. In this study, we determined the effects of ZIKV infectivity in human neuronal (SH-SY5Y) cells and mouse N2a cells/primary cultures of murine cortical neurons at early and late tested timepoints of infection. The human neuronal cells had higher ZIKV loads compared to the mouse N2a cells, but the viral loads in the murine cortical neurons were between the loads in these two in vitro cell lines. The murine cortical neurons were thought to be more permissive to ZIKV infection, but viral infection kinetics showed a declining trend like that observed in the mouse N2a cells. We noted that infectious extracellular vesicle (EV)-mediated ZIKV infection showed higher viral loads in the SH-SY5Y cells compared to direct infection with laboratory virus stocks. Similar results were obtained with ZIKV infectious EVs in the mouse N2a cells and cortical neurons. In addition, we noted that ZIKV infection significantly induced EV secretion from all three neuronal cells. Also, we found that ZIKV infection modulates the expression of type 1 interferons (IFNs) and entry receptors such as Tyro3, Axl, and MER-TK (TAM). Alongside the increased ZIKV loads in the SH-SY5Y cells, IFN-beta transcript levels and receptors Tyro3/MER-TK were upregulated at early timepoints of infection. Overall, the reduced ZIKV loads and decreasing IFN expression in the mouse neuronal cells suggested a unique murine cellular ability to restrict and limit viral replication. This could be one of the reasons for the unavailability of wild-type mouse models for ZIKV infection. Our data further shows that ZIKV may preferentially infect human rather than murine neuronal cells, and this could be the potential reason for microcephaly in newborns. Full article