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Betanodavirus is a causative agent of viral nervous necrosis (VNN) and a major threat to marine and brackish-water aquaculture globally. This virus causes epizootic outbreaks with particularly high morbidity and mortality in larval and juvenile stages and causes significant economic losses in aquaculture. Here, we developed a rapid and highly sensitive single-tube Reverse Transcription–Loop-Mediated Isothermal Amplification (RT-LAMP) assay for the detection of red-spotted grouper nervous necrosis virus (RGNNV) genotype infection in fish tissue samples. Six primers targeting eight conserved regions of the RNA2 coat protein gene of RGNNV were designed with conservation regions across RGNNV genotypes. RT-LAMP assay was completed within 60 min at 65 °C using a single-tube format that combined reverse transcription and isothermal amplification, and results were directly visualized by the addition of SYBR Green I dye, producing a colour change from orange (negative) to green (positive), observable with the naked eye or under UV illumination. The developed RT-LAMP assay was able to detect five copies of betanodavirus from infected samples, which was 20-fold more sensitive than conventional reverse transcription-PCR. The assay demonstrated diagnostic sensitivity and specificity in two fish hosts (Asian seabass and cobia) and showed no cross reactivity with other fish viruses such as tilapia lake virus and cyprinid herpesvirus-2. The developed assay is simple, cost-effective, specific, and enables rapid detection of betanodavirus in fish tissues. This single-tube RT-LAMP assay can be applied to screening broodstock facilities, fingerlings, aquaculture farms, quarantine facilities, and juveniles before stocking in ponds or cages, helping prevent disease outbreaks and the transmission of RGNNV in aquaculture systems. Full article

Background and objective: Natural killer (NK) cells are a subset of CD3^-CD56⁺ lymphocytes that can recognize and destroy virus-infected and cancerous cells. Infection is the main factor contributing to morbidity and mortality in individuals with multiple myeloma (MM). Therefore, this study was conducted to develop a framework for assessing the association between NK cell subsets (CD56bright and CD56dim) and infections in patients with newly diagnosed multiple myeloma (NDMM). Methods: Peripheral blood samples were obtained from 47 NDMM patients, and NK cell immunophenotype analysis was performed using flow cytometry. Screening for infection was based on the pathogens and combined clinical symptoms. Results: NDMM patients exhibited a significant increase in the overall frequency of circulating NK cells (p = 0.001) when compared to healthy controls. Clinically, infectious complications were observed in 31% of the NDMM patients, with viral infections accounting for 19.1% and bacterial infections for 12.7%. Interestingly, phenotypic analysis revealed a marked shift among NK cell subsets, characterized by an expansion of the CD56bright subset (p = 0.01) and a concurrent reduction in the CD56dim subset (p = 0.01). Additionally, NDMM patients who developed infections had significantly lower NK cell percentages, whereas those without infections showed higher levels, clustering at the upper end. Conclusions: These findings suggest a skewing of the NK cell population toward the CD56bright phenotype. According to the results, NDMM patients should be closely monitored for infection. Full article

Coconut (Cocos nucifera L.) is an important tropical horticultural crop valued for its edible endosperm with high oil content. However, the molecular mechanisms governing carbon partitioning and the regulation of glycolytic genes during endosperm development remain largely unclear. Fructose-1,6-bisphosphate aldolase (FBA), a key enzyme in glycolysis, plays a central role in carbohydrate metabolism, yet its transcriptional regulatory mechanisms in coconut have not been elucidated. In this study, the CoFBA promoter (proFBA) was isolated and used for yeast one-hybrid screening, leading to the identification of a GATA transcription factor, CoGATA2. Subcellular localization analysis confirmed that CoGATA2 is localized in the nucleus. Yeast one-hybrid assays, electrophoretic mobility shift assays (EMSA), and transient expression in coconut protoplasts demonstrated that CoGATA2 directly binds to a proFBA fragment containing the predicted GATA motif and represses CoFBA expression. Moreover, virus-induced gene silencing (VIGS) of CoGATA2 in coconut callus resulted in significant upregulation of CoFBA expression and increased fructose-1,6-bisphosphate (FBP) levels. Collectively, these findings demonstrate that CoGATA2 functions as a transcriptional repressor of CoFBA and that this regulation correlates with altered FBP levels in coconut callus. However, the precise mechanism by which FBP accumulation occurs and its relationship to glycolytic flux require further investigation. This study provides new insights into the transcriptional regulation of a key glycolytic gene in coconut and offers a foundation for future efforts to manipulate carbon partitioning in this important horticultural crop. Full article

In 2025, a severe mass mortality outbreak struck captive Geochelone sulcata at a zoo in Guangdong Province, China. To trace the causative agent, identify the viral strain and characterize associated pathological lesions, respiratory tract samples collected from diseased tortoises were subjected to a combined technical workflow. Metagenomic high-throughput sequencing was first applied to screen for potential pathogens, followed by virus isolation via cell culture. Transmission electron microscopy (TEM) was used to observe viral morphology. The major capsid protein (MCP) gene was amplified by PCR for molecular identification, and whole-genome sequencing together with phylogenetic analysis was performed to clarify the genetic features of the isolate. The results verified that ranavirus was the primary pathogen responsible for the mortality. Pathological examination demonstrated acute necrosis, hemorrhage and inflammatory infiltration in multiple organs including the liver, spleen, lung, and pancreas. TEM observation revealed typical iridovirus-like particles with an average diameter of approximately 70 nm. Molecular and genomic analyses confirmed the pathogen as Tiger Frog Virus (TFV) of the genus Ranavirus, designated TFV-CN2025, which shared 99.8% nucleotide sequence homology with known TFV reference strains. To our knowledge, this is the first report of lethal TFV infection in G. sulcata, which provides detailed pathological evidence for this cross-species transmission event from amphibian hosts to terrestrial chelonians. Our findings indicate that TFV poses considerable risks to the tortoise breeding industry and ecological security in China. We therefore suggest incorporating TFV detection into routine quarantine and disease surveillance programs for captive tortoises. Full article

Background: Respiratory syncytial virus (RSV) RNA-dependent RNA polymerase (RdRp) complex is an essential molecular machine for viral genome replication. The L protein, the catalytic subunit of this complex (L-RdRp), is well-characterized structurally and represents a highly promising target for the development of novel small-molecule drugs against RSV. Methods: To address the limitations of current QSAR-based virtual screening strategies for RSV L-RdRp inhibitor development, we established a multi-dimensional computer-aided drug screening framework integrating activity, toxicity, drug-likeness, and stability. Results: Two OECD-compliant 2D-QSAR models were developed and rigorously validated to predict inhibitory activity and cytotoxicity, respectively. The optimal inhibitory activity model exhibited strong statistical performance, with R² = 0.8281, $Q_{\mathrm{L}\mathrm{O}\mathrm{O}}^2=$ 0.7653, $R_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}^2=$ 0.8713, $Q_{\mathrm{F}\mathrm{n}}^2=$ 0.8594 ∼ 0.8837, CCC_test = 0.9301, MAE_test = 0.1966. Similarly, the best cytotoxicity model achieved R²= 0.8263, $Q_{\mathrm{L}\mathrm{O}\mathrm{O}}^2$ = 0.7422, $R_{\mathrm{t}\mathrm{e}\mathrm{s}\mathrm{t}}^2$ = 0.8951, $Q_{\mathrm{F}\mathrm{n}}^2$ = 0.8108~0.8530, CCC_test = 0.9081, MAE_test = 0.1685. Based on these models, a four-step screening workflow—QSAR-based filtering and molecular docking (15,758 → 2446 → 162 → 19 compounds), ADMET evaluation (19 → 5), and molecular dynamics simulations (MDSs)—was implemented to identify promising L-RdRp inhibitors. Conclusions: Ultimately, five candidate compounds were selected, all of which demonstrated predicted higher inhibitory activity, lower predicted cytotoxicity, a stable predicted binding mode, and favorable oral bioavailability compared with the reference drug remdesivir. These findings provide valuable in silico-derived lead candidates and a reliable computational workflow for identifying experimental L-RdRp inhibitors targeting RSV. Full article

In recent years, nanopore technology has enhanced analyte detection, enabled higher resolution and achieved single-molecule sensing capability. Aptamer-conjugated nanopore sensing technology combines the high specificity of aptamers with the single-molecule resolution of nanopores. By anchoring aptamers to biological, solid-state or hybrid nanopores, target binding events produce distinct electrical signatures that allow sensitive and label-free detection. This approach enables real-time monitoring of small molecules, proteins, and even pathogens, with promising applications in diagnostics, drug screening, environmental monitoring, etc. Hybrid biological/solid state devices produce robust signals and are suitable for PoC applications. The aptamers “magic bullets” have also been exploited to develop single-molecule antigen detection using nanopores, which offers a promising alternative for accurate virus testing to contain their transmission. Chemical conjugation of aptamers to nanopore interfaces improves selectivity for peptides/amino acids and expands robustness for practical samples. Aptamer-based nanopipettes offer high analytical precision by enabling label-free, real-time detection of target molecules in ultra-small sample volumes. This review maps aptamer–nanopore integration across biological, solid-state, and hybrid platforms. It also explores various types of aptamers integrated into nanopore platforms that cater to precise, single-molecule recognition, paving the way for highly sensitive, portable diagnostics and next-generation therapeutic monitoring tools. Full article

African swine fever (ASF), caused by African swine fever virus (ASFV), is a highly contagious disease that has brought severe economic losses to the global swine industry. ASFV dUTPase is a key enzyme modulating viral nucleotide metabolism and genome stability, serving as a specific structural target for anti-ASF lead discovery. Based on the crystal structure of ASFV dUTPase (Georgia 2008/1 strain), we established a molecular docking model to virtually screen a Traditional Chinese Medicine (TCM) compound library following ADME-based filtration. We further conducted 100 ns molecular dynamics (MD) simulations via GROMACS and calculated binding free energies using the MM/PBSA method, followed by in vitro enzymatic inhibition assays for verification. Four candidate compounds, namely Salvianolic acid B, Kukoamine B, Ligustroflavone and 9‴-Methyl salvianolic acid B, showed favorable structural accommodation and binding potential. In vitro tests indicated that Ligustroflavone and 9‴-Methyl salvianolic acid B exhibited preliminary inhibitory activity against ASFV dUTPase, with IC₅₀ values of 1.87 mM and 0.92 mM, respectively, while Kukoamine B showed no inhibitory effect. This study identified potential TCM-derived lead molecules targeting ASFV dUTPase, providing new references and candidate compounds for the development of anti-ASF therapeutics. Full article

Bovine viral diarrhea virus (BVDV) is a major cause of economic losses in the global cattle industry. This study investigated the clinical involvement and genetic diversity of BVDV in clinically affected cattle from the Thrace region in Türkiye, a critical border area with the European Union. A total of 533 nasal and rectal swabs were collected from 26 farms exhibiting clinical respiratory disease or diarrhea and analyzed using real-time RT-PCR. Positive samples were further characterized by sequencing and phylogenetic analysis to determine viral genotypes and subgenotypes. BVDV RNA was detected in 9 out of 26 farms (34.6%), with an overall positivity rate of 17.8% (95/533). Statistical analysis revealed significant associations between PCR positivity and both sampling year (p < 0.05) and animal age (p < 0.05). Genetic analysis of partial 5′-UTR sequences identified nine Pestivirus bovis subgenotype 1a strains, one Pestivirus bovis subgenotype 1f, and one Pestivirus brazilense. The Pestivirus bovis 1a strains clustered distinctly from previously reported Turkish BVDV-1 isolates, showing 97.32–99.11% nucleotide similarity with strains from Türkiye, Germany, China, Japan, the USA, Iran, and Argentina. Among the 533 samples tested, one Pestivirus brazilense isolate was identified, indicating its rare occurrence in the studied population despite targeted screening. This study represents the first report of Pestivirus brazilense in the Thrace region of Türkiye, expanding its known geographic range. The epidemiological data and genetic diversity analysis of BVDV strains characterized in this study offer critical insights for refining BVDV control programs, informing regional risk assessment, and supporting vaccine development. Full article

Background/Objectives: Hepatitis B virus (HBV) infection remains a major global health challenge because of its association with cirrhosis, hepatic failure, and hepatocellular carcinoma. Patients receiving rituximab-containing regimens are at particularly high risk for HBV reactivation due to profound B-cell depletion and prolonged immunosuppression. This study aimed to evaluate HBV serologic testing practices before rituximab initiation and to assess the incidence and outcomes of HBV reactivation among patients treated with rituximab at a tertiary care center in Jeddah, Saudi Arabia. Methods: A retrospective observational study was conducted on patients who received rituximab between 2010 and 2020 at a tertiary university hospital in Jeddah, Saudi Arabia. Demographic, clinical, laboratory, and treatment-related data were collected from electronic medical records. HBV reactivation was defined according to the American Association for the Study of Liver Diseases (AASLD) criteria. Results: A total of 611 patients were included. HBV serologic testing prior to rituximab initiation was documented in 60.8% of patients with hematological malignancies and 71.7% of those with non-hematological diseases. HBV reactivation occurred in 7 patients (3.4%) in the hematological malignancy cohort, all with lymphoma. Nine patients received antiviral prophylaxis. HBV reactivation was associated with significant morbidity, and liver-related mortality occurred in 28% of reactivation cases. Conclusions: HBV serologic testing was not universally documented prior to rituximab initiation, and antiviral prophylaxis was received by few patients. HBV reactivation was associated with significant morbidity and mortality. These findings support the need for systematic HBV evaluation and prophylaxis protocols in patients receiving rituximab. Full article

Hepatitis B surface antigen (HBsAg) signal-to-cutoff (S/CO) values and isolated anti-HBc positivity represent two significant diagnostic challenges in hepatitis B virus (HBV) serology. This retrospective study, conducted in Karabuk between 2021 and 2025, aimed to evaluate the correlation between these profiles and HBV DNA positivity to determine the optimal S/CO thresholds for confirmatory testing and clarify the clinical relevance of isolated anti-HBc reactivity. Among 17,356 patients screened for anti-HBc, isolated anti-HBc positivity was identified in 506 patients (2.9%), of whom 3.2% had detectable HBV DNA. Positivity was significantly higher in patients older than 50 years and in the Gastroenterology department. Separately, 213 specimens with HBsAg S/CO values between 1.0 and 10.0 underwent HBV DNA confirmation testing. ROC analysis yielded an excellent area under the curve of 0.937, with an S/CO threshold of ≥3.04, achieving 100% sensitivity for HBV DNA detection. All specimens with S/CO values between 1.0 and 3.0 were HBV DNA-negative. In this study population, no serum HBV DNA positivity was detected among samples with S/CO values below 3.04, suggesting that HBV DNA testing may help avoid unnecessary confirmatory work-up in this range. Anti-HBc screening should be prioritized in older patients. S/CO-based confirmatory algorithm using HBV DNA testing may improve diagnostic accuracy and reduce unnecessary clinical interventions in routine hepatitis B screening programs. Full article

Over the past decade, the incidence of infectious syphilis has been on the rise in the general Chinese population. Consequently, Treponema Pallidum (TP) testing has been proposed as a surrogate marker for sexually transmitted pathogens and for monitoring risky sexual behaviors among blood donors globally. In addition, sexual contact with individuals chronically infected with hepatitis B virus (HBV) is recognized as one of the primary routes of HBV transmission. Blood donors may acquire HBV infection through sexual contact with chronically infected partners, particularly with occult hepatitis B infections (OBIs), which are characterized by intermittent and extremely low viral loads. Therefore, the prevalence of OBIs among syphilis-positive blood donations and the corresponding risks to blood safety require further investigation. This study aimed to investigate the prevalence of OBIs among syphilis-positive blood donors and assess the surrogate value of TP testing for evaluating OBI-related risks to blood supply. After routine screening using serological assays and nucleic acid testing (NAT), blood donation samples with positive anti-TP enzyme-linked immunosorbent assay (ELISA) results were collected and further confirmed by the Treponema Pallidum Particle Agglutination Assay (TPPA). For blood donations confirmed positive for syphilis, further tests were performed to characterize whether the donations had HBV co-infection, including electrochemiluminescence immunoassay (ECLI) for the detection of hepatitis B surface antigen (HBsAg), anti-hepatitis B surface antibody (anti-HBs), hepatitis B e antigen (HBeAg), anti-hepatitis B e antibody (anti-HBe), and anti-hepatitis B core antibody (anti-HBc). Additionally, quantitative real-time polymerase chain reaction (qPCR) was used for HBV DNA quantification, and nested PCRs for the S and basal core promoter/precore (BCP/PC) region were conducted in combination with high-volume nucleic acid extraction. Subsequently, molecular characterization of HBV DNA in these co-infected samples was carried out by DNA sequencing to analyze the viral genetic features. Of 252 anti-TP ELISA+ donations screened from 64,871 blood samples, 138 (138/250, 55.2%) donations were confirmed syphilis-positive but NAT−, among which 78 (78/138, 56.5%) were anti-HBc-positive, and 88 (88/138, 63.7%) had anti-HBs. Notably, seven donations (7/138, 5.1%) were diagnosed as OBI co-infections, and available sequence analysis revealed that three cases were genotype B and one case was genotype C. In addition, several mutations in the S region of the HBV genome were identified, including Q101R, K122R, Q129H, T131N, M133T, G145R, and Y161F mutations. Furthermore, nucleotide mutations such as T1719G, A1752T, G1896A, and A1762T/G1764A in the BCP/PC regions were also detected in these OBI donations. These mutations may contribute to the extremely low HBV viral loads and/or failure in HBsAg detection, collectively leading to OBIs. These data indicate that syphilis screening of blood donors has potential to serve as an additional safeguard measure for excluding donations co-infected with OBIs. The high prevalence of undetected OBIs in syphilis-positive blood donors further supports that syphilis screening has the potential to serve as a surrogate marker for HBV-related risks in the blood supply. Full article

Viral infections remain a major threat to poultry health and production worldwide, highlighting the need for novel antiviral strategies. This study evaluated the in vitro antiviral and virucidal activities of six Thai medicinal plant extracts against herpesvirus of turkey (HVT), a non-oncogenic avian herpesvirus widely used as a live vaccine vector and employed here as a preliminary in vitro screening model. Cytotoxicity, total phenolic content (TPC), and antioxidant capacity were assessed using the MTT, Folin–Ciocalteu, DPPH, ABTS, and FRAP assays. Among the tested extracts, Caesalpinia sappan (CS) and Persicaria odorata (PO) exhibited the highest TPC and antioxidant activities and were selected for further antiviral evaluation. Both extracts significantly reduced HVT infectivity, infectious viral titers, and intracellular viral DNA levels in DF-1 cells (p < 0.01). CS produced lower infectious viral titers (1.995 × 10⁴ TCID₅₀/mL) than PO (8.24 × 10⁴ TCID₅₀/mL); however, both extracts significantly reduced viral titers compared with the untreated control (1.78 × 10⁶ TCID₅₀/mL). In the virucidal assay, pre-incubation of HVT with CS and PO significantly reduced infectious virus titers to 4.64 × 10⁴ and 2.15 × 10⁴ TCID₅₀/mL, respectively, compared with the untreated control (1.47 × 10⁶ TCID₅₀/mL) (p < 0.01). These findings identify C. sappan and P. odorata as promising sources of plant-derived compounds for future investigation as antiviral agents against avian herpesviruses. However, additional studies using virulent MDV-1, independent biological replicates, and in vivo models are required before their potential application to Marek’s disease control can be established. Full article

Cotton verticillium wilt caused by Verticillium dahliae severely restricts global cotton production. Mycovirus–induced hypovirulence provides a promising strategy for sustainable disease management. In this study, V. dahliae isolates from Xinjiang were screened by virome sequencing and RT–PCR, and strain 121–11C–1 co–infected with Verticillium dahliae partitivirus 1 (VdPV1) and Verticillium dahliae magoulivirus 1 (VdMoV1) was identified. Virus–free isogenic strains were obtained via single–conidium purification, verifying that both viruses can be vertically transmitted through fungal conidia. Dual–culture assays uncovered a novel, previously unreported horizontal transmission dependency between the two viruses: VdPV1 achieves horizontal transmission only with the assistance of VdMoV1, whereas VdMoV1 can transmit independently or co–transmit with VdPV1. Biological and pathogenicity assays demonstrated that both viruses significantly inhibit mycelial growth, reduce conidial production and attenuate fungal virulence. Notably, dual infection induced a significantly stronger hypovirulent phenotype than single infection. These findings elucidate the transmission dynamics of VdPV1 and VdMoV1, expand the repertoire of hypovirulent mycoviruses in V. dahliae, and provide evidence that viral co–infection enhances fungal attenuation, offering potential applications for biological control of cotton Verticillium wilt. Full article

Tomato yellow leaf curl virus (TYLCV) is a major threat to tomato production, creating a need for sensitive, low-cost detection methods that can be applied to early symptomatic or low-viral-load samples. Recombinant antigen configuration may influence serological assay development, although the specific contribution of multiple-cloning-site (MCS)-derived intermediate sequences remains uncertain. In this study, a recombinant Trx-His-coat protein (CP) fusion antigen was produced using an MCS-free direct-fusion construct that retained the Trx-His tag while removing the MCS-derived intermediate sequence, followed by gradient refolding. No direct comparison with linker-containing, tag-cleaved, or tag-free antigen constructs was performed. The purified antigen was used to immunize rabbits and generate a high-titre polyclonal antibody (pAb). The resulting indirect enzyme-linked immunosorbent assay (ELISA) achieved a theoretical limit of detection of 1.8 ng/mL and an estimated pre-dilution equivalent the limit of detection (LOD) of 72 ng/mL after sample dilution. The assay showed favourable tolerance to crude tomato leaf matrices, with spike-recovery rates of 95.45–100.40%. In a preliminary evaluation using a balanced panel of 32 field-collected samples, ELISA absorbance correlated with droplet digital PCR quantification (R² = 0.9819) and plant disease index values (R² = 0.9774). Liquid chromatography–tandem mass spectrometry (LC-MS/MS) peptide mapping and AlphaFold2-based modelling were used only to provide preliminary computational context for antigen interpretation. The assay showed cross-recognition toward Tobacco curly shoot virus (TbCSV), indicating that it should not be considered strictly TYLCV species-specific. Therefore, this assay may support preliminary serological screening under the tested conditions, whereas molecular confirmation remains necessary when species-level identification is required. Full article

Background: Adeno-associated virus (AAV) vector-mediated gene transfer is an emerging treatment strategy for severe cardiac disorders with genetic etiology. We refined the AAV toolkit to achieve efficient and selective expression of the therapeutic transgene in mouse hearts. Methods: Using vectors with a reporter transgene, we evaluated AAV administration routes, AAV serotype tropism to the myocardium, and cardiospecific promoters. Results: We showed that systemic AAV administration provides potent delivery and uniform transduction of cardiac tissue, outperforming localized injection techniques. The MyoAAV 2A capsid variant enabled an improved heart-to-liver transduction ratio compared to the parental AAV9 serotype. Screening a panel of cardiac and pan-muscular promoters in vitro and in vivo verified the superiority of the cardiac troponin T (cTnT) promoter for robust heart-specific transgene expression. Finally, we demonstrated that the cumulative properties of systemic AAV delivery, the MyoAAV 2A serotype, and the cTnT promoter allowed for efficient cardiac synthesis of the therapeutic transgene—an artificial miRNA designed for the gene suppression strategy of FLNC-related cardiomyopathy. Conclusions: Our findings establish an effective AAV approach for transgene transfer into the mouse heart and promote the development of gene therapy for cardiac disorders. Full article