Search Results (288)

Schmallenberg virus (SBV) is a transboundary animal pathogen that causes reproductive disorders in ruminants, necessitating standardized molecular surveillance. Adhering to the Chinese national standard GB/T 43159—2023, we developed armored RNA quality control materials using MS2 bacteriophage technology targeting the conserved SBV S segment and comprehensively characterized their physicochemical properties. Transmission electron microscopy (TEM) showed icosahedral symmetric virus-like particles (VLPs, approximately 25 nm), with the stock concentration determined to be 3.48 × 10¹⁰ copies/mL via digital PCR (dPCR). The armored RNA control effectively withstood RNase A degradation and remained stable at 37 °C for over 30 days. In bovine serum matrix simulation assays at identical medium concentrations, the serum armored RNA group (Ct 26.57) was detected 4.89 cycles earlier than the degraded serum naked RNA group (Ct 31.46). This confirms the physical protective efficacy of the MS2 capsid. Concurrently, the serum armored RNA group showed a delay of only 2.17 cycles compared to the aqueous armored RNA group (Ct 24.40), exhibiting a typical matrix effect. Overall, this armored RNA enables full-process quality control encompassing extraction, reverse transcription, and amplification, providing a safe and stable technical reference to support molecular surveillance and diagnostic preparedness for cross-border SBV. Full article

The high mortality associated with largemouth bass ranavirus (LMBRaV) disease requires rapid and sensitive diagnosis methods to prevent virus spread and subsequent outbreak. The recombinase polymerase amplification combined with the CRISPR/Cas12a system (RPA-CRISPR/Cas12a) assay and RPA-CRISPR/Cas12a, coupled with lateral flow dipstick (RPA-CRISPR/Cas12a-LFD) assay for LMBRaV, were established in this study for low viral load surveillance. The major capsid protein (mcp) gene is highly conserved and is widely used as the target sequence in LMBRaV detection. First, based on the mcp gene sequence of LMBRaV, three candidate crRNAs were designed. Among them, the crRNA-2, which exhibited the highest cleavage activity, was selected through dual evaluation of fluorescence signal intensity and LFD color development. Then, the key reaction conditions for both assays were optimized as follows: LbCas12a protein concentration of 150 nM, crRNA-2 concentration of 200 nM, and ssDNA reporters’ concentration of 200 nM. Moreover, specificity evaluation showed that both combined assays specifically recognized LMBRaV, with no cross-reactivity detected against other aquatic pathogens, such as CyHV-2, GSIV, ISKNV, WSSV, GCRV II or CrERV. The detection limit was 1 copy/μL of DNA sample for both RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-LFD assays. Finally, validation using 24 clinical samples (16 positive, eight negative) showed that both LMBRaV RPA-CRISPR/Cas12a assays achieved 100% detection rate, whereas conventional PCR detected 13 positive samples (detection rate 81.26%). ddPCR served as the reference method for clinical validation. Therefore, the LMBRaV RPA-CRISPR/Cas12a assay and the RPA-CRISPR/Cas12a-LFD assay provide sensitive, specific, and easy-to-operate methods for the rapid detection of LMBRaV. Full article

SARS-CoV-2 relies on a virus-encoded RNA capping pathway to produce 5′ cap structures that are essential for mRNA stability, efficient translation, and evasion of host innate immune surveillance. Within this pathway, nonstructural protein 14 (nsp14) catalyzes N7 methylation of the guanine cap, a key step that converts the cap core into a functional Cap-0 structure and enables subsequent maturation. Owing to its essential role in viral replication and its high conservation across coronaviruses, nsp14 has emerged as an attractive antiviral target. Recent structural and biochemical studies have elucidated the architecture of the nsp14 N7-methyltransferase domain, revealing an S-adenosyl-L-methionine (SAM)-dependent fold with a defined cofactor-binding site and an adjacent cap-binding pocket that orients the RNA substrate for methyl transfer. These insights have guided the development of diverse inhibitor classes, including SAM-competitive analogs, bisubstrate-like compounds, and non-nucleoside inhibitors identified through screening approaches. While early SAM-like inhibitors demonstrated target tractability, their therapeutic potential has been limited by challenges in selectivity and cellular permeability. More recent inhibitors that target the cap-binding pocket or exploit product-assisted ternary complex mechanisms highlight alternative strategies for achieving improved potency and specificity. Despite these advances, current structural models rely on truncated RNA substrates and isolated protein constructs, which may not fully capture the native catalytic environment. Future efforts to resolve nsp14 within the replication–transcription complex and develop novel inhibition strategies will be critical for advancing mechanistic understanding and antiviral development. Full article

Seasonal influenza remains a major global public health challenge despite decades of vaccine development, underscoring the need for more effective and adaptable immunization strategies. The recently approved mRNA-1010 vaccine represents a promising advance by applying messenger RNA technology to a pathogen characterized by continuous antigenic drift. Recent Phase 3 clinical trials conducted in support of regulatory review have demonstrated robust immunogenicity and superior protective efficacy against seasonal influenza compared with standard influenza vaccines in adults aged 50 years and older. However, increased reactogenicity, particularly among younger adults, together with the need for continued post-marketing safety surveillance, highlights the balance between enhanced immune responses and tolerability. Beyond seasonal influenza, mRNA-1010 provides a foundation for next-generation respiratory vaccines, including combination formulations targeting influenza, SARS-CoV-2, and respiratory syncytial virus (RSV), while illustrating the broader potential of mRNA vaccine technology. At the same time, challenges related to manufacturing scalability, cost, cold-chain requirements, and global accessibility remain important considerations for widespread implementation. This perspective examines whether mRNA-1010 represents an incremental advance in influenza vaccination or a potential transformative milestone in respiratory immunization, while considering the continuing roles of alternative vaccine platforms in achieving optimal effectiveness, safety, and equitable global access. Full article

To establish a rapid nucleic acid detection method for field screening of Brucella spp., a visual assay based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a was developed. Four pairs of RPA primers and two crRNAs were designed according to the conserved Brucella bcsp31 sequence. The optimal RPA primer–crRNA combination was screened using agarose gel electrophoresis and fluorescence readout, and the concentrations of Cas12a protein, crRNA, and ssDNA fluorescent reporter were optimized. RPA primer pair 2 showed the best amplification performance, and RPA2-crRNA1 was selected as the optimal combination for the assay. The final reaction conditions were 200 nM Cas12a protein, 200 nM crRNA, and 250 nM ssDNA fluorescent reporter. The limit of detection of the established method was 2.47 × 10⁰ copies/μL. No cross-reactivity was observed with Mycoplasma capricolum subsp. capripneumoniae, Chlamydia abortus, Chlamydia psittaci, or Salmonella enterica serovar Typhimurium. Among 56 clinical sheep nasal swab samples collected during routine surveillance, qPCR identified 22 positive and 34 negative samples, while the RPA-CRISPR/Cas12a assay identified 20 positive and 36 negative samples. The positive agreement, negative agreement, overall agreement, positive predictive value, negative predictive value, and Cohen’s kappa value were 90.91%, 100.00%, 96.43%, 100.00%, 94.44%, and 0.924, respectively. These findings provide preliminary evidence that the developed bcsp31-targeted RPA-CRISPR/Cas12a assay is rapid and visually interpretable and may be useful for preliminary Brucella spp. screening in routine veterinary surveillance settings. Larger-scale validation using different sample types and samples from different regions is still required. Full article

The identification of a pathogenic variant (PV) in one of the mismatch repair (MMR) genes confirms the diagnosis of Lynch syndrome (LS). Hence, the correct classification of MMR gene variants is of utmost importance for appropriate counselling, surveillance, and treatment of LS patients and their families. In 7/200 unrelated Tyrolean-suspected LS patients, we identified the rare variant MLH1:c.836T>G. Clinical and tumor data strongly indicate that this founder variant is associated with an increased risk for early-onset LS-associated tumors. We also demonstrate that the variant leads to aberrant mRNA splicing. However, the splice effect’s leakiness together with the small effect of the amino acid change p.(Val297Gly) encoded by the residual full-length transcripts in a functional assay preclude its formal classification as (likely) PV according to internationally accepted variant interpretation guidelines. The family histories of the carriers suggest that the obstacles to classify the variant as (likely) PV may be related with a reduced penetrance. Nonetheless, and despite the formal classification of MLH1:c.836T>G as a variant of uncertain significance, we show that carriers should undergo cancer surveillance and predictive testing should be offered to relatives. This variant illustrates the need for an improved classification framework for appropriate categorization of lower-penetrance alleles. Full article

Background/Objectives: Tenofovir disoproxil fumarate (TDF)-containing regimens remain the cornerstone of first-line antiretroviral therapy (ART) in many low- and middle-income countries. However, concerns regarding treatment failure and antiretroviral resistance highlight the need to evaluate the effectiveness of treatments under routine clinical conditions. This study evaluated the virologic outcomes of treatment-naïve individuals living with HIV who received predominantly TDF-based first-line ART in Davao City, Philippines. Methods: A retrospective observational study was conducted among treatment-naïve HIV patients who initiated ART between 2016 and 2020. Demographic and clinical characteristics, ART regimens, HIV surveillance stage classifications, and viral load results were extracted from routinely collected medical records. Virologic suppression was defined as an HIV RNA viral load of <1000 copies/mL and virologic failure as ≥1000 copies/mL. The availability of viral load monitoring and the interval between ART initiation and the latest viral load measurement were also evaluated. Results: A total of 494 treatment-naïve patients with HIV were included. Most patients were male (97.8%) and received TDF-containing regimens (99.0%), predominantly TDF + 3TC + EFV (95.1%). Viral load results eligible for outcompe analysis were available for 174 (35.2%) patients. Among these patients, 165 achieved virologic suppression, corresponding to a suppression rate of 94.8% (95% CI: 90.5–97.3), whereas virologic failure was observed in nine patients (5.2%; 95p% CI: 2.7–9.5). Viral load availability declined substantially among patients initiating ART in recent years, reflecting shorter follow-up durations and fewer opportunities for routine viral load monitoring. High levels of virologic suppression were observed across the demographic and clinical subgroups. Conclusions: Predominantly TDF-based first-line ART demonstrated high virologic effectiveness among treatment-naïve HIV patients with evaluable viral load measurements, with nearly 95% of patients achieving virologic suppression. However, incomplete viral load monitoring, particularly among patients initiating ART in later years, limits the evaluation of treatment outcomes at the program level. Strengthening routine viral load monitoring and long-term follow-up will improve the future real-world evaluation of ART effectiveness. Full article

Acute hepatopancreatic necrosis disease (AHPND) is one of the most devastating bacterial diseases affecting Pacific white shrimp (Litopenaeus vannamei) aquaculture worldwide. Although Vibrio parahaemolyticus has been recognized as the principal causative agent of AHPND, increasing evidence indicates that Vibrio campbellii also plays an important role in disease outbreaks. The present study investigated the pathogenicity and temporal dynamics of pirA transcription in V. campbellii isolated from cultured L. vannamei. The isolate was identified by 16S rRNA sequencing, and pirA expression was quantified during bacterial growth under in vitro conditions and throughout experimental infection in vivo using quantitative real-time PCR. In vitro, pirA transcription increased rapidly during the exponential growth phase and reached its highest level at 6 h after incubation, followed by a gradual decline despite continued bacterial growth. Immersion challenge experiments using juvenile shrimp (1 × 10⁶ CFU/mL) resulted in cumulative mortality exceeding 90% within 48 h post-infection. In vivo, pirA transcription remained relatively low during the early stage of infection but increased markedly at 36 h post-infection, coinciding with rapid mortality and severe hepatopancreatic damage. Histopathological examination revealed progressive epithelial cell sloughing, degeneration of B- and F-cells, tubular collapse, and extensive tissue necrosis, which are characteristic lesions of AHPND. These findings demonstrate that V. campbellii possesses strong pathogenic potential. Temporal induction of pirA transcription was closely associated with severe hepatopancreatic lesions and mortality during experimental infection. Collectively, these findings provide new insights into the temporal dynamics of pirA transcription during AHPND progression and its association with tissue pathology and mortality, supporting the development of early molecular diagnostics, disease surveillance, and biosecurity strategies for sustainable shrimp aquaculture. Full article

The RNA exosome plays a critical role in surveilling nuclear mRNA biogenesis and regulating co-translational mRNA decay in the cytoplasm. Unlike canonical translation, repeat-associated non-AUG (RAN) translation of a GGGGCC hexanucleotide repeat expansion (HRE) within an intron of the C9orf72 locus leads to the synthesis of neurotoxic dipeptide-repeat (DPR) proteins, contributing to the pathogenesis of frontotemporal dementia and amyotrophic lateral sclerosis (C9-ALS/FTD). However, it remains unclear whether aberrant RAN translation is monitored and regulated co-translationally or how C9orf72 HRE (C9-HRE) mRNA is degraded during this process. Here, we demonstrate that RAN translation triggers the rapid decay of C9-HRE mRNA. During this process, the RNA exosome engages the translating ribosome-C9-HRE mRNA complex to mediate RAN translation-coupled mRNA decay. Moreover, overexpression of EXOSC3, a key subunit of the RNA exosome cap, promotes RAN translation-coupled decay of C9-HRE mRNA and suppresses DPR production. In iPSC-derived neurons, a reduction in EXOSC3 levels blocks C9-HRE mRNA decay in a translation-dependent manner, further confirming its role in RAN translation surveillance. These findings highlight the essential function of the RNA exosome, particularly EXOSC3, in mitigating RAN translation-associated toxicity and preventing pathological DPR production. This work provides insights into potential therapeutic strategies for C9-ALS/FTD and may have broader implications for other disorders involving RAN translation. Full article

Integrated proteomics and untargeted metabolomics were employed to systematically characterize molecular differences between Acipenser schrenckii embryos with normal versus abnormal 24 h post-fertilization (24 hpf), which were classified as high-quality (HQ) and poor-quality (PQ) groups. Among 1636 proteins and 1102 metabolites, 220 differentially expressed proteins (DEPs) and 365 differential metabolites (DMs) were identified. Functional enrichment demonstrated that HQ samples were predominantly enriched in pathways associated with amino acid biosynthesis, glycolysis/tricarboxylic acid cycle, nucleotide metabolism, and mRNA surveillance, which collectively supported material accumulation, energy supply, and embryonic developmental competence. In contrast, PQ samples were mainly enriched in oxidative phosphorylation, mitochondrial stress response, arachidonic acid metabolism, and immune and inflammatory signaling pathways, indicating severe lipid metabolic disorders and excessive oxidative stress. Spearman correlation analysis identified L-pyroglutamic acid and ascorbic acid as core correlated metabolic hubs associated with high-quality eggs, while natamycin and tetrahydrocorticosterone were correlated characteristic metabolic signatures enriched in deteriorated eggs. Key protein nodes showing strong correlations with 24 hpf developmental phenotypes included GAPDH, glutathione S-transferase, and CYP450 2E1. Collectively, this study reveals divergent correlated molecular profiles linked to normal versus abnormal 24 hpf development and screens correlative multi-omics candidate signatures for distinguishing well-developed and deteriorated oocytes. Full article

In this study, one H9N2 subtype avian influenza virus (AIV) was isolated in Jiangsu Province, China, in March 2025. According to phylogenetic analysis, the HA gene fragment of the H9N2 isolate was classified as the B4.7 lineage, with the remaining seven segments falling into the Eurasian lineage. This H9N2 virus underwent complex genetic reassortment across multiple regions in China during its evolution, and key amino acid substitutions were identified, which may enhance its viral polymerase activity and replication efficiency in mice. Critically, a mouse experiment demonstrated that the H9N2 virus replicated effectively in the lungs and nasal turbinates of BALB/c mice without requiring prior adaptation. In addition, poultry infection studies showed that the H9N2 virus could replicate and spread more efficiently in chickens than in ducks. Moreover, the virus could be transmitted to contacted ducks through infected chickens with greater efficiency than from ducks to ducks. Furthermore, in the lungs of inoculated chickens and ducks, the mRNA expression levels of pattern-recognition receptors (PRRs) and cytokines were upregulated, while the overall fold increase in ducks was lower than that in chickens. In conclusion, these results highlighted the potential public health risk presented by chicken-derived H9N2 AIV, emphasizing the critical need for enhanced routine surveillance of H9N2 AIVs from poultry. Full article

Purpose: To characterize the messenger RNA (mRNA) and microRNA (miRNA) expression profiles of the normal human cornea and conjunctiva using impression cytology (IC) samples and to investigate their molecular characteristics and regulatory networks. Methods: Corneal and conjunctival IC samples were collected from healthy subjects. Whole-transcriptome and miRNA sequencing were performed, followed by differential expression and bioinformatics analyses. Regulatory networks, protein interaction networks, and functional enrichment analyses were constructed. Selected genes and miRNAs were validated by RT-qPCR. Results: A total of 1676 differentially expressed genes and 175 differentially expressed miRNAs were identified between the cornea and conjunctiva. Functional analyses revealed that genes showing higher expression in the cornea were mainly associated with epithelial structure, barrier function, and antiviral immune responses. In contrast, genes showing higher expression in the conjunctiva were primarily involved in immune regulation, secretion, metabolic detoxification, and tissue remodeling. PPI network analysis showed that hub genes in the cornea were predominantly interferon-stimulated genes related to antiviral responses, while those in the conjunctiva were mainly involved in cell cycle regulation and metabolic detoxification. GO and KEGG analyses further supported these functional distinctions. RT-qPCR validation generally supported the expression patterns identified by RNA sequencing. Conclusions: Our findings reveal that the cornea is characterized by gene expression programs supporting epithelial homeostasis, barrier function, and antiviral immunity, whereas the conjunctiva exhibits transcriptional signatures related to immune surveillance, secretion, metabolic processing, and tissue remodeling. The miRNA–mRNA regulatory networks constructed in this study provide new insights into the molecular regulatory mechanisms of the ocular surface and offer a theoretical basis for future research into disease mechanisms and targeted therapeutic strategies. Full article

HPV DNA-positive women with ASC-US/LSIL cytology constitute a heterogeneous triage group. We evaluated genotype-specific HPV E6/E7 mRNA testing with PreTect HPV-Proofer’7 in a population-based cohort from Tromsø and compared the findings descriptively with a previously published cohort from Bodø. This retrospective quality-assurance study included 1006 HPV DNA-positive women with ASC-US/LSIL cytology screened between 2019 and 2024, with linkage to regional pathology records through 31 December 2025. The assay detects E6/E7 mRNA from HPV16, 18, 31, 33, 45, 52, and 58. ASC-US/LSIL accounted for 42.5% of linked HPV DNA-positive women in Tromsø compared with 22.3% in Bodø. Among the mRNA-tested ASC-US/LSIL cohorts, mRNA positivity was similar (40.8% vs. 44.6%). In Tromsø, CIN2+ was recorded in 26.1% of mRNA-positive and 7.9% of mRNA-negative women (RR 3.31, 95% CI 2.41–4.55), whereas CIN3+ was recorded in 3.9% and 1.0%, respectively (RR 3.88, 95% CI 1.53–9.82). For CIN2+, sensitivity was 69.5%, specificity 64.4%, PPV 26.1%, and NPV 92.1%. Among HPV16/18 DNA-positive women, CIN2+ risk was 42.7% in mRNA-positive and 13.4% in mRNA-negative women. Simulated mRNA-guided referral reduced immediate colposcopy referrals by 59.2%. Genotype-specific mRNA testing provided clinically relevant risk stratification, with directionally similar patterns being observed in the Tromsø and Bodø cohorts; however, mRNA-negative women require structured surveillance. Full article

Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine (Phe) metabolism caused by pathogenic variants in the phenylalanine hydroxylase (PAH) gene, resulting in toxic phenylalanine accumulation that, if untreated, causes profound intellectual disability and neurodevelopmental impairment. PKU is especially significant in the Kingdom of Saudi Arabia (KSA), where high consanguinity rates substantially elevate disease prevalence relative to Western populations, and the country’s expanding newborn screening programs have highlighted PKU as a persistent public health concern. This review provides a translational synthesis of the Saudi PKU literature, covering epidemiology, molecular pathophysiology, genetics, clinical presentation, diagnosis, treatment, prognosis, and future directions. We summarize global and regional incidence data and show that Saudi Arabia, driven by consanguinity, is among the countries with the highest reported PKU burden worldwide. We review the metabolic basis of phenylalanine neurotoxicity and the allelic heterogeneity of the PAH gene, with attention to variants enriched in Saudi and Arab cohorts, including the founder allele p.R252W. Diagnostic pathways anchored in newborn screening and tandem mass spectrometry are discussed alongside neurodevelopmental outcomes and gaps in Saudi PKU surveillance. We outline established and emerging therapies, including dietary management, sapropterin (BH4), pegvaliase, large neutral amino acids, and investigational gene and mRNA therapies. Throughout, we identify where genuine Saudi-specific evidence exists and where general PKU knowledge is extrapolated to the Saudi context because of limited local data, most notably the absence of a national PKU registry. This review is intended to serve as a translational reference for clinicians, metabolic dietitians, geneticists, and policymakers engaged in PKU care in Saudi Arabia and the wider Arab region. Full article

Two chikungunya (CHIKV) vaccines have now been licensed, but the human clinical evidence base remains fragmented across vaccine platforms, populations, follow-up periods, and safety settings, complicating product-specific interpretation of durability and benefit–risk. We conducted a PRISMA-ScR–guided scoping review of human CHIKV vaccine evidence indexed in PubMed, Embase, and Web of Science from January 2000 to June 2026. After screening 890 records, we included 77 sources of evidence and mapped them at both the record level and the candidate/product level. The included evidence clustered around a limited number of vaccine programs, including TSI-GSD-218, VRC-CHKVLP059-00-VP/PXVX0317/Vimkunya, MV-CHIK/V184, VLA1553/IXCHIQ, ChAdOx1 Chik, and mRNA-1388/VAL-181388. Late-stage and post-authorization evidence was concentrated mainly in VLA1553/IXCHIQ and PXVX0317/Vimkunya, whereas viral-vector and mRNA candidates remained largely restricted to early-phase adult studies. Evidence has expanded to adolescents and adults aged ≥65 years for selected products but remains limited or product-specific for children, pregnant individuals, immunocompromised populations, and medically complex older adults. Short-term trial safety data were characterized primarily by mild or moderate local and systemic reactogenicity, while post-authorization safety evidence remains recent and concentrated in licensed products. This scoping review provides a structured evidence map for CHIKV vaccine development and highlights priorities for standardized immunogenicity assessment, longer-term durability data, broader population representation, endemic-region effectiveness studies, and continued post-marketing surveillance. Full article