Search Results (946)

Chilli veinal mottle virus (ChiVMV) is an important potyvirus that poses a serious threat to crop production. In this study, small RNA sequencing and molecular cloning were used to obtain the complete genome sequence of a ChiVMV isolate identified in pepper plants in Sichuan (SC1 isolate). Molecular evolutionary and phylogenetic analysis of SC1 and 35 ChiVMV isolates revealed four clades of ChiVMV isolates. Recombination analysis found 23 recombinant events and 28 recombinants, with the SC1 isolate arising from the recombination of the PK isolate from Pakistan and the YNpe isolate from Yunnan, China. A full-length infectious cDNA clone of ChiVMV was constructed and demonstrated to be infectious in both Nicotiana benthamiana and pepper plants. Moreover, a Myc-tag was inserted after NIb, and the derived infectious clone of ChiVMV remained infectious, and NIb-Myc was readily expressed in infected host plants. These reverse genetic tools will promote the study of the function of ChiVMV-encoded proteins, especially the NIb protein, and facilitate basic and translational studies of ChiVMV. Full article

AIG1 (avrRpt2-induced gene 1)-like proteins are a class of GTPases that play crucial roles in plants, functioning both in chloroplast protein import and disease resistance. However, their evolutionary history and the mechanisms driving this functional diversification remain poorly understood. Here, we performed a comprehensive genomic and evolutionary analysis of this gene family across the plant kingdom. We identified 90 AIG1-like genes from 11 sequenced plant species, representing major lineages from green algae to angiosperms. Phylogenetic analysis revealed that plant AIG1-like proteins form three monophyletic lineages corresponding to the Toc34, Toc159, and IAN subfamilies, which originated via two ancient duplications predating the divergence of green algae and land plants. These lineages exhibit dramatically divergent evolutionary patterns. The Toc34 subfamily is evolutionarily conserved, maintaining stable copy numbers and gene structure, indicative of strong functional constraints in its core role in plastid import. In contrast, the Toc159 and IAN subfamilies have undergone dynamic expansion via lineage-specific duplication mechanisms, including segmental duplication and prolific tandem duplication, respectively. Notably, we uncovered a novel mechanism for generating head-to-head tandem duplicates in the IAN subfamily, mediated by recombination between inverted repeats. Our analysis of ancestral gene numbers and gene gain/loss dynamics further highlights that functional diversification was driven by both the acquisition of distinct C-terminal targeting domains (M and TM domains) and profound differences in evolutionary rates and duplication modes among subfamilies. This study provides the first full-scale evolutionary framework for plant AIG1-like genes, establishing that functional specialization is rooted in distinct modes of sequence and genomic evolution. Full article

Background/Objectives: Whole exome sequencing (WES) is an effective method for detecting disease-causing variants. However, copy number variation (CNV) detection using WES data often has limited sensitivity and high false-positive rates. Methods: In this study, we constructed a reference CNV set using chromosomal microarray analysis (CMA) data from 44 of 180 individuals who underwent WES and CMA and evaluated four WES-based CNV callers (CNVkit, CoNIFER, ExomeDepth, and cn.MOPS) against this benchmark. For each tool, we first defined software-specific problematic genomic regions across the full WES cohort and filtered out the CNVs that overlapped these regions. Results: The four algorithms showed low mutual concordance and distinct distributions in the problematic regions. On average, 2210 sequencing target baits (1.23%) were classified as problematic; these baits had lower mappability scores and higher coefficients of variation in RPKM than the remaining probes. After the supplementary filtration step, all tools demonstrated improved performance. Notably, ExomeDepth achieved gains of 14.4% in sensitivity and 7.9% in positive predictive value. Conclusions: We delineated software-specific problematic regions and demonstrated that targeted filtration markedly reduced false positives in WES-based CNV detection. Full article

In recent years, the development of new diagnostic technologies, such as tandem mass spectrometry (MS/MS) and next-generation sequencing (NGS), has caused a veritable revolution in the diagnosis of genetic diseases, reducing time, cost, and invasiveness associated with prior diagnostic techniques. While MS/MS laid the foundation for the development of numerous, usually institutionally based, neonatal screening programs, NGS has gained traction in newborn screening (NBS), primarily through pilot projects and private funding across different countries. As a result, the traditional Wilson and Jungner criteria have been supplemented by additional criteria, including considerations of equity and access, in response to emerging technologies. This review aims to provide an up-to-date overview of the global landscape of metabolic screening panels, highlight the major ongoing genomic screening projects, and outline the current models for integrating these two screening systems. Substantial differences exist across countries in the numbers and types of diseases included in national NBS programmes. In this context, Italy represents a prominent case, as its neonatal screening framework has seen significant expansion and development in recent years, reaching a particularly comprehensive metabolic screening panel. Nonetheless, a number of initiatives to incorporate genomic technologies into the NBS pathway are currently underway, primarily involving high-income countries. Nonetheless, unlike metabolomic-based NBS programs, no country has a government-mandated NGS program as first-tier testing for newborns. New evidence is emerging from ongoing models of integration of multi-omics approaches into NBS, including the use of AI and machine learning. Identifying the most appropriate system for this integration to reduce the false-positive and false-negative rates associated with both screening types, ensure more equitable access to screening, and facilitate faster access to treatment may represent a useful and foresightful way to conceptualize NBS in the future. This transitional phase should promote rigorous improvements before full-scale adoption. Full article

Background: The enhanced sensitivity of next-generation sequencing (NGS) for assessing hepatitis B virus (HBV) quasispecies heterogeneity over clone-based sequencing (CBS) is well documented. However, its comparative reliability for genotype determination remains an open question. Objective: This study aimed to directly compare the performance of NGS and CBS for genotyping HBV using the entire viral genome. Methods: We selected five challenging clinical samples that previously could not be subgenotyped or showed conflicting results when using direct sequencing of the S open reading frame (ORF). The full HBV genome from these subjects was amplified and then analyzed in parallel by both NGS and CBS. Phylogenetic analysis was subsequently used to assign genotypes. Results: Both methods identified a range of genotypes, including B, C, and I, as well as aberrant and recombinant forms. For three of the five subjects, genotyping results were identical between the two platforms. In the remaining two cases, however, CBS revealed greater complexity, identifying additional subgenotypes and recombinant/aberrant strains not detected by NGS. Notably, for three individuals, the genotypes determined by both modern methods contradicted earlier results from 2011 based on direct S ORF sequencing. Furthermore, the specific mutations detected were incongruent between the platforms, with CBS identifying a higher number of variants than NGS. Conclusions: Our findings indicate that genotyping results from NGS and CBS can be discordant. Contrary to expectations, CBS may uncover more genetic diversity, including a greater number of subgenotypes and mutations, than NGS in certain contexts. The study also confirms that genotyping based solely on direct sequencing of the S ORF can be unreliable and lead to misclassification. Full article

The historic Rockborn strain of the canine distemper virus was widely used as a vaccine, but its use was discontinued due to safety concerns. Yet, Rockborn-like canine distemper virus strains are still used in some vaccine formulations. Genetic analysis of this strain was previously limited to the H gene, leaving its full evolutionary and pathogenic potential unclear. This study aimed to determine the complete genome sequence of the Rockborn strain to reconstruct its origin, understand its evolution, and provide a reference for improving diagnostics and future research on virulence markers. An amplicon-based sequencing protocol using MinION nanopore technology was employed to determine the complete genome of the Rockborn-46th laboratory strain. The genome was assembled, annotated, and analyzed in comparison with 223 genomes. The complete genome of the Rockborn strain was 15,690 nucleotides in length. Phylogenetic analysis revealed that Rockborn forms a unique lineage with field isolates from a masked civet in China and a dog in the United States. Crucially, a significant recombination event was identified, showing that the Rockborn strain acted as a parental strain, contributing its F and H genes to create mosaic viruses. The full-genome characterization of the Rockborn strain confirms that Rockborn-like viruses persist and actively contribute to the evolution of canine distemper virus through recombination. This finding highlights the inadequacy of single-gene analysis for diagnostics and surveillance, and underscores the necessity of whole-genome sequencing to accurately track the virus epidemiology and evolution. Full article

Fruit development and ripening in peach (Prunus persica) involve complex transcriptional and post-transcriptional regulation. While short-read sequencing has advanced transcriptome studies, it often fails to accurately resolve complex transcript isoforms. This study employed Oxford Nanopore Technologies’ (ONT) full-length RNA-Seq to comprehensively characterize the transcriptomic landscape of peach fruits across three key developmental stages: the first exponential stage, the second exponential stage, and the ripening stage. Our analysis identified 44,042 non-redundant isoforms, including 1109 novel genes and 32,289 novel isoforms, significantly expanding the peach genome annotation. We further investigated alternative splicing (AS) events, revealing that intron retention (IR) and alternative 3′ splice site (A3′S) were the most prevalent types, with AS abundance peaking at the S1 stage. A total of 10,236 differentially expressed transcripts (DETs) were identified, highlighting dynamic expression patterns during fruit development. Functional characterization focused on a MADS-box gene, PpMADS6, which produced two isoforms via alternative splicing. Dual luciferase assays in tobacco leaves demonstrated that the full-length isoform, PpMADS6a, specifically activated the promoter of the fruit-softening gene PpPG1, while the truncated isoform, PpMADS6b, lost this transactivation ability. This study provides a valuable resource of full-length transcriptomes for peach and underscores the critical role of alternative splicing in generating functional diversity to fine-tune fruit development and ripening processes. Full article

Pathogenic germline variants in the ATM gene are associated with a 20–30% lifetime risk of breast cancer. Crucially, a relevant fraction of loss-of-function variants in breast cancer susceptibility genes disrupts pre-mRNA splicing. We aimed to perform splicing analysis of ATM splice-site variants identified in the large-scale sequencing project BRIDGES (Breast Cancer After Diagnostic Gene Sequencing). To this end, we bioinformatically selected 47 splice-site variants across 17 exons that were genetically engineered into three minigenes and assayed in MCF-7 cells. Aberrant splicing was observed in 38 variants. Of these, 30 variants, including 7 missense, yielded no or negligible expression of the minigene full-length (mgFL) transcript. A total of 69 different transcripts were characterized, 48 of which harboured a premature termination codon. Some variants, such as c.2922-1G>A, generated complex patterns with up to 10 different transcripts. Alternative 3′ or 5′ splice-site usage was the predominant event. Integration of ATM minigene read-outs into the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular Pathology)-based specifications for the ATM gene enabled the classification of 30 ATM variants as pathogenic or likely pathogenic and 9 as likely benign. Overall, splicing assays provide key information for variant interpretation and the clinical management of patients. Full article

The overlapping circulation of influenza (Flu), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2; SC2), and respiratory syncytial virus (RSV) continues to challenge clinical laboratories, particularly in settings with limited automation and fragmented healthcare coverage. This study expanded the CDC Flu-SC2 assay by incorporating a laboratory-developed test (LDT) for RSV A/B detection into a fully automated quadruplex RT-qPCR (LDRA) on the Panther Fusion^® Open Access™ system. The design, based on more than 8000 RSV genomic sequences targeting the conserved M gene, achieved optimal amplification efficiencies (97–105%) and full multiplex compatibility. Analytical assessment established limits of detection between 9.6 and 37.8 copies per reaction, absence of cross-reactivity with 30 respiratory pathogens, and inclusivity for 32 viral variants. Commutability and diagnostic performance among the LDRA, CE IVD-marked Allplex™ SARS-CoV-2/FluA/FluB/RSV, and US IVD-marked Panther Fusion^® SARS-CoV-2/Flu A/B/RSV Assays were evaluated using 405 nasopharyngeal UTM-preserved swabs. The LDRA demonstrated excellent concordance (overall agreement ≥ 98%, κ > 0.95), strong diagnostic accuracy, and reliable detection of mixed infections. This quadruplex provides a fully automated, rapid, and accurate solution for the simultaneous detection of influenza A, influenza B, SARS-CoV-2, and RSV viruses, enhancing molecular diagnostic capacity and supporting equitable, timely clinical decision-making in middle-income healthcare systems such as that of the Dominican Republic. Full article

Circulating recombinant forms (CRFs) are important components of the HIV-1 pandemic. Previous studies have reported the propagation of diverse HIV-1 CRFs of South American origin in Europe. Here, through protease-reverse transcriptase (Pr-RT) and integrase sequence analyses, we identify a Spanish cluster (BC3) branching with a Brazilian virus (10BR_RJ009) previously classified as CRF146_BC. In Pr-RT, BC3 comprised 14 viruses and was nested within a larger cluster, comprising 22 Brazilian viruses and 1 Spanish virus branching outside of BC3. Near full-length genome analyses of five BC3 viruses revealed mosaic structures identical to 10BR_RJ009, with two breakpoints delimiting a ~0.3 kb subtype B fragment within an otherwise subtype C genome. Two other Brazilian viruses previously classified as CRF146_BC (10BR_RJ039 and 01_BR_RGS69) had one and two additional short subtype B fragments, respectively, and failed to group with the 10BR_RJ009/BC3 cluster in subtype C fragments. Based on these results, we contend that 10BR_RJ009 and BC3 viruses, but not 10BR_RJ039 and 01_BR_RGS69, should be classified as CRF146_BC. Bayesian analyses estimated the CRF146_BC emergence in Brazil to be around 1999 and its introduction in Europe around 2011. CRF146_BC is the 10th CRF of South American origin reported to circulate in Europe, reflecting the relationship between South American and European HIV-1 epidemics. Full article

Influenza D virus (IDV), an emerging orthomyxovirus with zoonotic potential, infects diverse hosts, causes respiratory disease, and remains poorly characterized in China despite its global expansion. From October 2023 to January 2025, we collected 563 nasal swabs from cattle across 28 farms in Jilin Province, Northeast China, and identified seven IDV-positive samples (1.2%), recovering two viable isolates (JL/YB2024 and JL/CC2024). Full-genome sequencing revealed complete, stable seven-segment genomes with high nucleotide identity (up to 99.9%) to contemporary Chinese D/Yamagata/2019 strains and no evidence of reassortment. Maximum-likelihood and time-resolved Bayesian phylogenies of 231 global hemagglutinin-esterase-fusion (HEF) sequences placed the Jilin isolates within the East Asian D/Yamagata/2019 clade and traced their most recent common ancestor to approximately 2017 (95% highest posterior density: 2016–2018), suggesting a cross-border introduction likely associated with regional cattle movement. No IDV was detected in parallel surveillance of swine, underscoring cattle as the principal reservoir and amplifying host. Bayesian skyline analysis demonstrated a marked decline in global IDV genetic diversity during 2020–2022, coinciding with livestock-movement restrictions imposed during the COVID-19 pandemic. Collectively, these findings indicate that IDV circulation in China is sporadic and geographically localized, dominated by the D/Yamagata/2019 lineage, and shaped by multiple independent incursions rather than a single emergence. Both the incorporation of IDV diagnostics into routine bovine respiratory disease surveillance and cattle-import quarantine programs, and the adoption of a One Health framework to monitor potential human spillover and future viral evolution, were recommend. Full article

In recent years, the quality of genome assemblies has notably improved, primarily due to advances in third-generation sequencing technologies and bioinformatics tools. In the present study, we obtained genome assemblies for two flax (Linum usitatissimum L.) varieties, K-3018 and Svyatogor, using Oxford Nanopore Technologies (ONT) simplex R10.4.1 data and the Hifiasm algorithm optimized for ONT reads. The K-3018 genome assembly was 491.1 Mb and consisted of thirteen full-length chromosomes and two one-gap chromosomes. The Svyatogor genome assembly was 497.8 Mb and consisted of twelve full-length chromosomes and three one-gap chromosomes. All chromosomes had telomeric repeats at their ends for both varieties. Hi-C contact maps and Illumina genomic data supported the accuracy of the obtained assemblies. The K-3018 and Svyatogor genome assemblies surpassed the quality of the best currently available flax genome assembly of variety T397, which serves as a reference for L. usitatissimum in the NCBI Genome database. Comparative analysis revealed that the flax genomes are generally quite similar at the chromosome level, with only a few large-scale differences. Thus, two near-T2T (telomere-to-telomere) flax genomes were assembled from the ONT simplex R10.4.1 reads using Hifiasm ONT without involving Pacific Biosciences (PacBio) HiFi or ultra-long ONT reads as well as optical maps. High-quality flax genomes are essential for improving the efficiency of genetic research, evaluating genetic diversity at the whole-genome level, and developing breeding and genome editing approaches of this valuable multipurpose crop. Full article

Bacterial cellulose hydrogels (BCHs) are characterized as exopolysaccharides of glucose polymers consisting of β–1–4–glycosidic linkage with various degrees of polymerization which are synthesized by bacteria. There is a paucity of information on the isolation and characterization of a BCH producer isolate from Nigeria. The study, therefore, aimed to characterize a new Acetobacter species that had previously been confirmed to produce BCH. The BCH-producing isolate was characterized by PCR amplification of the full-length 16S rRNA gene, as well as whole-genome sequencing analysis. The whole-genome sequence of the isolate was determined using the Illumina next-generation sequencing (NGS) platform, with downstream analysis of genomic reads through the metaWRAP pipeline. The BCH producer isolate was identified to be Acetobacter orientalis strain Zaria-B1, based on sequence identity with the reference Acetobacter orientalis strain VVS. Based on its annotated genome, the isolate had an approximate genomic size of 3.1 Mbp, 45 total RNAs, a GC content of 52.5%, 3046 total protein-encoding genes, an N50 of 253,774 bp, and an L50 of 4, as well as 30 contigs. The nucleotide BLAST of the cellulose synthase gene sequence confirmed the bin to be Acetobacter orientalis. The whole-genome characterization alongside the 16S rRNA genotyping confirmed the BCH-producing isolate to be Acetobacter orientalis. Full article

Background/Objectives: The vaginal microbiota constitutes a highly dynamic microbial ecosystem shaped by the distinct mucosal, hormonal, and immunological environment of the female genital tract. Accumulating evidence suggests that shifts in cervical microbial composition and function may influence host–microbe interactions and contribute to gynecological disease risk. Within this framework, the present study aimed to perform an in-depth genomic characterization of the cervical microbiota in a well-defined cohort of Greek women. The primary objective was to explore the functional microbial landscape by identifying dominant bacterial taxa, taxon-specific signatures, and potential microbial pathways implicated in cervical epithelial homeostasis, immune modulation, and disease susceptibility. Methods: Microbial genomic DNA was isolated from 60 cervical samples using the Magcore Bacterial Automated Kit and analyzed through full-length 16S rRNA gene sequencing using the Nanopore MinION™ platform, allowing high-resolution taxonomic assignment and enhanced functional inference. In parallel, cervical samples were screened for 14 HPV genotypes using a real-time PCR-based assay. Results: The cervical microbial communities were dominated by Lactobacillus iners, Lactobacillus crispatus, and Aerococcus christensenii, collectively representing over 75% of total microbial abundance and suggesting a functionally protective microbiota profile. A diverse set of low-abundance taxa—including Stenotrophomonas maltophilia, Stenotrophomonas pavanii, Acinetobacter septicus, Rhizobium spp. (Rhizobium rhizogenes, Rhizobium tropici, Rhizobium jaguaris), Prevotella amnii, Prevotella disiens, Brevibacterium casei, Fannyhessea vaginae, and Gemelliphila asaccharolytica—was also detected, potentially reflecting niche-specific metabolic functions or environmental microbial inputs. No HPV genotypes were detected in any of the cervical samples. Conclusions: This genomic profiling study underscores the functional dominance of Lactobacillus spp. within the cervical microbiota and highlights the contribution of low-abundance taxa that may participate in metabolic cross-feeding, immune signaling, or epithelial barrier modulation. Future large-scale, multi-omics studies integrating metagenomics and host transcriptomic data are warranted to validate microbial functional signatures as biomarkers or therapeutic targets for cervical health optimization. Full article

Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) is an RNA-binding protein known to play critical roles in metabolism, cell proliferation, and tumorigenesis. Although its involvement in muscle development has been documented in several species, the function of goose IGF2BP2 remains largely unexplored. In this study, we cloned and characterized the full-length cDNA and genomic DNA sequences of goose IGF2BP2. The cDNA is 2957 bp in length and contains a 1662 bp open reading frame encoding a 553-amino acid protein with five conserved RNA-binding domains. The genomic sequence spans 12,183 bp and consists of 12 exons and 11 introns. A total of 60 genetic variants were identified, including a deletion of a G base at position 2299 (g.2299delG) that results in a frameshift mutation. Expression analysis revealed high levels of IGF2BP2 mRNA in the liver, heart, and muscle tissues of female geese across embryonic (E25d), growing (A70d), and laying (L270d) stages, consistent with a potential role in muscle development (p < 0.05). Functionally, overexpression of IGF2BP2 in skeletal muscle satellite cells (SMSCs) was associated with significant changes in the expression of several genes linked to muscle development and signaling pathways, including upregulation of IGF1, EGFR, FGF19, BMP6, BMP2, ACVR1C and WNT5A and downregulation of MYBPC3, NODAL, HOXD13, TNXB, and ADD2 (Padj < 0.01). Furthermore, protein–protein interaction (PPI) network analysis of these genes suggests that IGF2BP2 may coordinate key genes, contributing to its potential role in skeletal muscle development in geese. Full article