Search Results (403)

Human herpesvirus 8 (HHV-8) is the etiologic agent of Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), multicentric Castleman disease (MCD), and KS-associated immune reconstitution inflammatory syndrome (IRIS-KS). Quantifying HHV-8 DNA in whole blood is clinically relevant, yet laboratory practices remain heterogeneous. Here, we developed and validated an in-house quantitative PCR (qPCR) assay targeting ORF26, optimized for whole blood. Assay calibration used plasmid, BCBL-1 cell–derived, and commercial HHV-8 DNA standards. Analytical validation was performed following the Clinical and Laboratory Standards Institute (CLSI) guidelines and the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines and showed a 95% limit of detection of 65.7 copies/reaction, efficiencies of 90–101% (R² > 0.99), and intra/inter-assay coefficients of variation < 6.5%. Strong correlations were observed among the three calibrators (R² > 0.97).Clinical validation against a composite reference yielded 100% sensitivity, specificity, PPV, and NPV. Viral loads (log₁₀ copies/mL) varied by clinical condition: classic KS and transplant-associated KS showed the lowest medians (2.30–2.23), MCD HIV− and PEL intermediate values (2.83–3.72), and epidemic KS, MCD HIV+, and IRIS-KS the highest (4.12, 4.86, and 5.03, respectively). Viremia > 5 log₁₀ copies/mL was associated with uncontrolled E-KS, MCD HIV+, and IRIS-KS. Longitudinal follow-up revealed viral load decline paralleled clinical improvement. This validated assay provides a robust, affordable tool for HHV-8 quantification in whole blood and supports its integration into diagnostic workflows and patient monitoring. Full article

Background/Objectives: Glomerular diseases (GD) possess strong polygenic susceptibility, yet exact causal genes remain unclear because most variants identified by genome-wide association studies (GWAS) reside in non-coding regions. While transcriptome-wide association studies (TWAS) effectively decode complex traits, cross-tissue profiling for GD remains largely unexplored. Therefore, this study employs an integrative cross-tissue TWAS and Mendelian randomization framework to systematically identify and validate novel GD susceptibility genes. Methods: We conducted a systematic cross-tissue TWAS integrating Genotype-Tissue Expression (GTEx) v8 eQTL data across 49 tissues. Candidate genes were nominated using five complementary frameworks (sparse canonical correlation analysis (sCCA), functional summary-based imputation (FUSION), fine-mapping of causal gene sets (FOCUS), summary-data-based Mendelian randomization (SMR), and multi-marker analysis of genomic annotation (MAGMA)). Findings were refined via Mendelian randomization (MR), pathway enrichment, protein interaction networks, and druggability profiling. Results: We identified 21 candidate susceptibility genes for GD, with 10 genes (AGER, C6orf48, CSNK2B, CYP21A2, HLA-DRB1, HSD17B8, LST1, MICB, PRRT1, TCF19) strongly supported by MR analysis. Notably, five of these MR-prioritized genes (C6orf48, CSNK2B, HSD17B8, LST1, and PRRT1) were previously unreported. Functionally, these prioritized genes are primarily involved in immune modulation, inflammation, and steroid metabolism. Furthermore, five genes (AGER, CSNK2B, CYP21A2, HLA-DRB1 and MICB) were identified as potentially druggable targets. Conclusions: This first systematic cross-tissue TWAS of GD prioritizes a set of genetically supported susceptibility genes. By uncovering novel drivers and druggable proteins, this study advances the mechanistic understanding of GD and provides a foundation for future therapeutic development and precision nephrology. Full article

Background/Objectives: Opioid use disorder (OUD) is caused by a complex interplay between genetic and non-genetic factors. DNA methylation is an epigenetic mechanism that modulates gene expression. Data on DNA methylation and opioid addiction and treatment are limited. This association study was designed to assess the difference in genome-wide methylation patterns between individuals with OUD in methadone maintenance treatment (MMT) (n = 114) and those with OUD who achieved long-term abstinence (>10 years) without mu opioid receptor agonist treatment (n = 136). Methods: Differential DNA methylation analysis was performed in whole blood using the Illumina EPIC array. Results: A total of 135 differentially methylated probes (DMPs) reached epigenome-wide significance (p < 1 × 10⁻⁷), controlling for sex, age, estimates of blood cell proportions, and the first two principal components based on genome-wide SNP genotypes. The methylation sites were annotated to 157 genes, including 32% long non-coding RNAs. These genes are related to several systems, including cell adhesion (e.g., SAXO4), immune system and inflammation (e.g., UBTF, USP39, C10orf90, PRKCA), stress response (e.g., CRHR1, GPR19), and spermatogenesis (e.g., SPATA16, COX7B2). DMP cg11641410 is located in lncRNA ENSG00000254687, an antisense to OPRK1. Six of the DMPs were also identified in a related longitudinal study of MMT. Conclusions: At this point, it is not possible to determine whether the minor methylation differences observed in this study cause clinically relevant changes in gene expression. However, these findings have the potential to identify biomarkers and to provide new targets for treatment optimization. Full article

Hepatitis B virus (HBV) is the smallest partially double-stranded, reverse-transcribing DNA virus, with four open reading frames (ORFs) encoding viral proteins. It is classified into nine geographically distributed genotypes (A–I). In Kenya, the molecular characterization of HBV among patients seeking medical care remains poorly defined. This observational study aimed to characterize HBV among patients seeking medical care in Kenya’s endemic region, focusing on circulating genotypes and ORF mutations. Serum samples were collected from the outpatient departments of selected health facilities, with demographic and clinical information extracted from patients’ medical records. Hepatitis B surface antigen (HBsAg) was tested at the facilities, and 85 HBsAg-positive samples were collected for molecular analysis. The basal core promoter and pre-core (BCP/PC), polymerase, and surface regions of the viral genome were amplified and sequenced to determine genotypes and to profile their mutations. Out of 85 HBsAg-positive samples, 38 samples tested positive for HBV DNA, and 26 samples were successfully sequenced. HBV genotype A was prevalent at 73.1% (19/26), followed by genotype D at 23.1% (6/26), and genotype E at 3.8% (1/26). Genotype A sequences clustered with both A1 Asian and African subgenotypes, whereas genotype D clustered with subgenotypes D6 and D1. All HBV genotype A, D, and E sequences were serotypes adw2, ayw2, and ayw4, respectively. HBV core promoter mutations (A1762T/G1764A) were detected in both genotype D and genotype A isolates. The pre-core G1896A mutation was highly prevalent in genotype D samples (5/6; 83.3%) but was not observed in genotypes A or E. Analysis of mutations within the “a” determinant region revealed genotype-specific patterns: genotype A predominantly harbored V14A, P46H, S58C, and P67Q substitutions; genotype E showed N59S; and genotype D exhibited V14A, C69stop, S104T, and W182stop mutations. Two drug resistance mutations (V191I and A194T) were present in two chronic patients, one with genotype A and the other with genotype D. In conclusion, HBV genotypes A and D are the most prevalent among Kenyan patients with chronic HBV infection. The presence of point mutations in the ORFs among patients seeking medical care highlights the need for molecular surveillance to better understand the viral diversity and its potential clinical and public health implications. Full article

TEA domain transcription factors are critical regulators of tissue development and regeneration in mammals, yet their roles in aquatic invertebrate regeneration remain poorly understood. Here, a full-length cDNA encoding a putative transcriptional enhanced associate domain protein 1 (TEAD1) ortholog in Apostichopus japonicus (AjTEAD1) was cloned and characterized. The open reading frame (ORF) of AjTEAD1 is 1344 bp, encoding a polypeptide of 447 amino acids with a conserved TEA domain (Asp⁴⁰–Leu¹¹¹) and a protein-binding domain (Gly²³¹–Asp⁴⁴⁶). Function analysis demonstrates that AjTEAD1 is essential for intestinal regeneration. AjTEAD1 expression was significantly upregulated during the regeneration process. Functional impairment of AjTEAD1 suppressed intestinal regeneration and attenuated cell proliferation. At the molecular level, we identified the cell cycle gene in A. japonicus (AjCyclin E), whose expression pattern coincided with that of AjTEAD1 and was downregulated following AjTEAD1 knockdown. Dual-luciferase reporter assays further confirmed that AjTEAD1 binds to specific sites in the AjCyclin E promoter and transcriptionally activates its expression. In summary, our study reveals that AjTEAD1 promotes cell proliferation and drives intestinal regeneration in A. japonicus by directly upregulating AjCyclin E transcription. These findings identify the TEAD–Cyclin E axis as a key regulator of echinoderm regeneration, shedding new light on the regenerative processes and cytological mechanisms in economically important species. Full article

Euproctis pseudoconspersa is a devastating pest in Camellia oleifera plantations, necessitating the development of sustainable molecular intervention strategies. This study targeted the chitin synthase A gene (EpCHSA) to evaluate and compare the RNA interference (RNAi) efficacy of dsRNA synthesized via the T7 in vitro transcription system and the Escherichia coli HT115 (DE3) expression system. The EpCHSA gene (2199 bp ORF) was cloned and characterized, exhibiting peak expression during the fourth-instar stage, and predominantly in the head tissues of fifth-instar larvae. Bioassays demonstrated that larvae fed with 500 ng/μL in vitro synthesized dsRNA exhibited continuous gene silencing for five days, reaching a maximum efficiency of 68.1%. Conversely, treatment with 100× concentrated bacterial broth (5000 ng/μL) elicited a superior silencing effect of 79.3% within 24 h. Furthermore, the bacterial treatment group reached a 14-day mortality rate of 46.66%, significantly higher than the in vitro group (38.33%). Both methods induced severe phenotypic abnormalities, including molting failure and pupal malformation. These findings conclude that the E. coli expression system offers a cost-effective and highly potent platform for dsRNA production. This research provides a critical technical foundation and promising application prospects for the field-scale management of E. pseudoconspersa utilizing RNAi-based biopesticides. Full article

Human adenovirus (HAdV), a double-stranded DNA virus, targets terminally differentiated cells in the upper respiratory tract. As a key platform for gene therapy vectors, elucidating HAdV’s virulence factors is vital for optimizing therapeutic applications and mitigating risks. To achieve productive replication, HAdV strategically neutralizes host immune defenses and induces S-phase pathways essential for viral propagation. This review synthesizes the latest insights into the key pathways through which HAdVs harness these early proteins to enhance virulence, skilfully evading and counteracting host defense mechanisms while propelling viral replication. As foundational platforms for gene therapy vectors (e.g., in oncology and rare disease treatments) and vaccine backbones (e.g., COVID-19 vaccines like ChAdOx1), understanding HAdV’s immunoshadowing—the multifaceted strategies used to cloak innate and adaptive immunity—is crucial for enhancing vector safety and efficacy. Recent insights unveil how early viral proteins—including E1A, E1B-55K, E4orf1, E4orf3, E4orf6, and the E3 complex—participate in these processes. This review critically synthesizes these pathways, evaluating study limitations such as reliance on immortalized cell lines that underestimate the role of these proteins in immunological competent cells, and addresses unresolved controversies, including differential immunoshadowing efficacy across HAdV species that impacts vaccine design. Full article

Background: Hepatitis E virus (HEV) is an increasingly recognized cause of acute viral hepatitis in Thailand as the burden of hepatitis A, B, and C has declined. HEV is a positive-sense RNA virus in the family Hepeviridae with three major open reading frames encoding replication proteins (ORF1), the capsid protein (ORF2), and an accessory protein involved in viral egress (ORF3). Unlike highly endemic regions where genotypes 1 and 2 are linked to waterborne outbreaks, infections in Thailand are reported mainly as sporadic cases associated with zoonotic transmission, most commonly genotype 3. Objectives: This review summarizes the epidemiology, transmission routes, and public health implications of HEV infection in Thailand. Methods: Peer-reviewed studies on HEV seroprevalence, molecular epidemiology, and transmission in Thailand were identified through PubMed using combinations of the keywords “HEV” and “Thailand”. Two investigators independently screened titles, abstracts, and full texts. Eligible studies were synthesized qualitatively. Results: Earlier studies suggested low population exposure, but more recent evidence indicates substantial cumulative risk. A nationwide survey among blood donors reported anti-HEV IgG seroprevalence of about 30%, with geographic variation and increasing prevalence with age. Detection of HEV RNA in pigs, slaughterhouse environments, and retail pork products, together with links to raw or undercooked pork consumption, supports pigs as the principal reservoir and foodborne exposure as an important route. Transfusion-associated infection has also been documented. Conclusions: In Thailand, HEV infection is linked mainly to zoonotic and foodborne transmission involving genotype 3. Stronger surveillance, food safety measures, and risk-based blood safety policies are needed. Full article

Background: The ORF3 protein of swine hepatitis E virus (HEV-4) is a key virulence factor involved in viral assembly, egress, and host signaling regulation. The mammalian target of rapamycin (mTOR) pathway plays a pivotal role in autophagy, metabolism, and immunity, and is often modulated by viruses to promote replication. However, it remains unknown whether HEV-4 ORF3 modulates the mTOR pathway via circular RNAs (circRNAs). Methods: Using an adenovirus-mediated ORF3 overexpression system in HepG2 cells, we integrated circRNA and transcriptome high-throughput sequencing data, followed by KEGG enrichment analysis to identify mTOR-associated differentially expressed genes. A circRNA–miRNA regulatory network was constructed using bioinformatics tools, and the expression changes of m6A-related genes, including YTHDF3, were evaluated. Results: ORF3 overexpression significantly activated the mTOR pathway (p < 0.05) and led to the identification of 20 mTOR-related circRNAs (e.g., circRNA5142). These circRNAs regulated downstream autophagy and lipid metabolism genes by sponging miRNAs such as hsa-let-7d-5p and hsa-miR-132-3p. Altered YTHDF3 expression indicated possible m6A-dependent epitranscriptomic regulation of the mTOR pathway. Conclusions: Our integrated analysis suggests that HEV-4 ORF3 may modulate the mTOR pathway through a circRNA–miRNA network, perturbing host autophagy and metabolic balance, which may contribute to viral immune evasion. Targeting the ORF3-mediated circRNA-mTOR regulatory axis represents a promising therapeutic approach and provides a theoretical basis for novel anti-HEV-4 strategies. Full article

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis worldwide, with zoonotic genotypes detected in humans and animals. In Africa, limited data exist on environmental HEV circulation. Here, we report the first detection of Rocahepevirus ratti (RHEV) in urban wastewater from Conakry, Guinea. From December 2024 to April 2025, Rocahepevirus ratti (RHEV) has been detected in 35 out of 180 urban untreated wastewater samples in Conakry, Guinea. The phylogenetic analysis of partial HEV ORF1 genome segments reveals clustering with African rodent RHEV strains, highlighting environmental contamination and potential zoonotic risk for human populations in proximity. This finding underscores the need for integrated One Health surveillance to monitor HEV transmission at the human–animal–environment interface in West Africa, particularly in Guinea. Full article

Salt, drought and freezing stress were major abiotic factors limiting plant growth, development and yield. Halostachys caspica (Amaranthaceae), a halophyte native to saline-arid desert regions, tolerated multiple abiotic stresses, but its molecular mechanisms of stress tolerance remain unclear. By integrating the small RNA library and transcriptome data of H. caspica under high salinity, HcmiR172e was identified as a differentially expressed miRNA and selected for the study of multiple abiotic stress responses. Using its mature sequence (20 nt) to align with upregulated genes from the transcriptome, HcTOE3 (AP2 subfamily transcription factor belonging to the AP2/ERF family) was preliminarily predicted as its target gene through bioinformatic analysis. Our previous work demonstrated that HcTOE3 was strongly upregulated by multiple abiotic stresses, including salinity, drought, heat and low temperature. Furthermore, overexpression of HcTOE3 conferred freezing tolerance to Arabidopsis throughout the entire growth period. In this study, miRNA expression analyses showed that HcmiR172e was significantly downregulated in the assimilating branches of H. caspica under low temperature, heat, salt, drought, oxidative stress and abscisic acid (ABA) application. Tobacco transient expression assays and 5′RLM-RACE confirmed that HcmiR172e directly cleaved HcTOE3 transcripts in the region close to the 5′end of the ORF. HcmiR172e-overexpressing Arabidopsis displayed increased sensitivity to salt, drought, freezing stresses and ABA treatment, along with enhanced growth inhibition, elevated reactive oxygen species (ROS) accumulation, decreased osmolyte content and downregulation of stress-responsive genes. In contrast, HcTOE3-overexpressing Arabidopsis exhibited the opposite phenotypes, physiological responses and corresponding gene expression patterns under multiple stress treatments. These findings collectively elucidated the antagonistic regulatory roles of HcmiR172e and HcTOE3 in plant abiotic stress responses, providing novel molecular targets for engineering stress-tolerant crops for saline, arid, freezing environments. Full article

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) poses a serious threat to the swine industry in China. As a major pig-producing province, Shandong requires continuous epidemiological monitoring of PRRSV. To elucidate the molecular epidemiology of the virus, 1621 clinical samples were collected from suspected cases across different regions of Shandong Province between 2023 and 2025, primarily from Tai’an, Linyi, Jining, and Liaocheng. RT-qPCR detection showed that the positive rate for PRRSV-2 was 20.05% (325/1621). Genetic analysis based on ORF5 and NSP2 genes indicated that Sublineage L1C (NADC30-like) was the dominant strain for 38.38% of ORF5 gene and 72.73% of NSP2 sequencing results. This was followed by Sublineage L8E and L1A and L5A strains. Key virulence-related mutations were identified at residues R13 and R151 in the GP5 protein, which are associated with enhanced pathogenicity. Additionally, variations in neutralizing epitope and the number of N-glycosylation sites (ranging from 2 to 5 per strain) suggested potential immune evasion. Notably, 26.79% (15/56) of sequenced samples showed discordant ORF5 and NSP2 genotyping results, indicating widespread recombination among PRRSV strains in Shandong Province. These finding demonstrated that the genetic diversity, high recombination frequency, and key amino acid variations in circulating PRRSV strains collectively undermine vaccine effectiveness. This study highlights the need to optimize vaccination strategies, enhance biosecurity measures, and implement effective disease control and elimination programs to reduce the impact of PRRSV in Shandong Province. Full article

PRRSV-2 represents a major threat to the swine industry. Canada is one of the world’s leading pork producers and a major trading partner of live pigs with the United States, yet PRRSV-2 evolutionary dynamics in these two countries are often studied independently, partly due to limited publicly available sequence data from Canada. We analyzed more than 3000 PRRSV-2 ORF5 sequences collected between 2000 and 2024 from five Canadian provinces. Thirteen previously described sub-lineages were detected in Canada, while approximately one-third of the sequences could not be assigned to any known sub-lineage. Phylogenetic analyses incorporating global reference sequences revealed that most unclassified sequences clustered into four distinct monophyletic clades, exhibiting genetic distances greater than 9.5% from recognized sub-lineages. We propose two new sub-lineages, 1K and 1L, corresponding to clades that were prevalent and persistent over time, whereas the remaining two clades were rare and last detected in 2021. We reconstructed cross-border transmission histories and found that sub-lineages 1C, 1H, 1I, 1K, and 1L originated in Canada, whereas 1A, 1B, 1E, and 1F originated in the United States. Transmission patterns varied across sub-lineages, ranging from unidirectional to bidirectional movement. Our findings refine PRRSV-2 classification and provide insights to inform targeted surveillance, particularly at national borders. Full article

Apostichopus japonicus undergoes evisceration in response to adverse environmental stimuli, and its coelomocytes undergo rapid regeneration within 6–24 h to restore innate immune function. FOS, an immediate early gene, regulates cell proliferation and cycle, but its role in A. japonicus coelomocyte regeneration after evisceration is unclear. In this study, AjFOSL from A. japonicus was cloned, which harbors a 609 bp open reading frame (ORF) encoding 202 amino acids (aa) with a conserved bZIP domain and is localized on chromosome 14. It shares 58% homology with FOS from Holothuria leucospilota and Lytechinus pictus. Phylogenetic analysis revealed that AjFOSL clusters closely with FOS from Magallana gigas and Mytilus edulis. Tissue distribution analysis showed that AjFOSL was widely expressed in various tissues, with the highest expression level detected in the tentacles. Temporal expression profiling demonstrated that AjFOSL was significantly upregulated by 1.75-fold at 6 h after evisceration. After AjFOSL knockdown, the peak expression of Cyclin A, Cyclin B, and E2F was delayed and the coelomocyte number was consistently reduced compared with that in the evisceration-only group. The AjFOSL acted as an immediate early response gene and was associated with the regulation of coelomocyte regeneration by modulating the expression of cell cycle-related genes. This study provides novel insights into the molecular associations underlying coelomocyte regeneration and the evolutionary adaptation of FOS genes in echinoderms. Full article

(1) Background: Swine hepatitis E (SHE) is an emerging zoonotic disease caused by the swine hepatitis E virus (SHEV). The open reading frame 3 (ORF3) protein is a recognized virulence factor of SHEV. Jaundice, the typical clinical sign of SHE, primarily results from disruptions in bile production, secretion, and excretion. However, the mechanism by which SHEV ORF3 influences bile metabolism remains unclear. (2) Methods: Building on our previous work involving adenovirus-mediated overexpression of genotype IV SHEV ORF3 in HepG2 cells and subsequent high-throughput lncRNA/transcriptome sequencing, this study performed KEGG enrichment analysis on differentially expressed lncRNAs. Candidate lncRNAs were validated via qRT-PCR. Cis-regulated target genes were predicted by integrating differentially expressed mRNA data. Furthermore, AlphaFold 3.0 was employed to analyze the molecular binding sites between lncRNA UBC (MSTRG.6881.4) and its target, UBC protein. (3) Results: We identified three lncRNAs associated with the bile secretion pathway (ko 04976) in HepG2 cells expressing genotype IV SHEV ORF3, which were further confirmed by qRT-PCR: lncRNA UBC (MSTRG.6881.4), lncRNA UBC (MSTRG.6881.9), and lncRNA UBC (MSTRG.6881.12). Bioinformatics prediction suggested six lncRNA-mRNA regulatory networks involved these lncRNAs and two downregulated UBC mRNA transcripts (ENST00000540700 and ENST00000536769). Molecular docking indicated that nucleotides 395U and 41C of lncRNA UBC (MSTRG.6881.4) could potentially bind to residues 82Lys, 88Thr, and 90Thr of the UBC protein, with predicted binding energies ranging from −4.73 to −0.75 kcal/mol. (4) Conclusions: The successful identification of bile secretion-related lncRNAs, coupled with the prediction of their regulatory networks and molecular interaction sites, has advanced our understanding of SHEV ORF3 function and the pathogenesis of SHEV infection. Full article