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At least 19 recordsLinked to original sources
PubMed2026

Identification and full genome sequencing of previously unknown sandfly-borne phleboviruses using a newly established capture-based next-generation sequencing approach.

Sandfly-borne phleboviruses cause febrile illness and neuroinvasive disease in humans. While infections are reported in the Mediterranean region, the discovery of previously unknown phleboviruses in sandflies from Kenya suggests a wider geographic distribution. Detection and characterization of novel phleboviruses are often hindered by low-quality and low-viral-load samples. We developed a capture-based target enrichment next-generation sequencing approach that showed a 99%-100% fold enrichment of viral genomes from primary material and provides a robust tool for generating complete genomes of both known and previously unknown viruses. From a collection of 15,652 sandflies in Kenya, we recovered seven complete coding sequences of Embossos, Bogoria, and Kiborgoch viruses, and of two previously unknown phleboviruses, which were named Sosoik and Shable viruses. Sosoik virus shared 83% amino acid identity in its RdRp gene with that of Bogoria virus, while Shable virus shared ca. 88% amino acid identity with viruses of the Salehabad serocomplex. Additionally, a reassortant of Shable virus was detected that possessed an M segment from an undescribed Ponticelli-like virus. DNA barcoding of blood-fed sandflies revealed several potentially novel Sergentomyia species and evidence of host-feeding on humans, livestock, and reptiles, suggesting possibilities for zoonotic transmission. Overall, our findings increase the known genetic diversity of Old World sandfly-borne phlebovirus species from 18 to 25 (by 38.9%), including the detection of viruses from all pathogenic sandfly-borne phlebovirus serocomplexes in East Africa, opening new horizons in disease ecology research.IMPORTANCEKnowledge of the genetic diversity of circulating pathogens is crucial for providing appropriate diagnostics and disease management. This study established a novel capture-based target enrichment next-generation sequencing approach that enabled the near-complete viral genome recovery from primary samples, while native NGS yielded negative or poor-quality results. In addition to the five recently discovered sandfly-borne phleboviruses in Kenya, two previously unknown phleboviruses were detected in sandflies from the same region. The viruses were detected in several sandfly species, which showed diverse host-feeding behaviors, including mixed feeding on humans and chickens. The study significantly advances the understanding of sandfly-borne phleboviruses by uncovering their broader geographic distribution and genetic diversity, particularly in East Africa, highlighting the importance of expanding surveillance efforts beyond traditionally studied regions.

Phlebovirus↗
PubMed2024

Molecular epidemiology of enteroviruses from Guatemalan wastewater isolated from human lung fibroblasts.

The Global Specialized Polio Laboratory at CDC supports the Global Poliovirus Laboratory Network with environmental surveillance (ES) to detect the presence of vaccine strain polioviruses, vaccine-derived polioviruses, and wild polioviruses in high-risk countries. Environmental sampling provides valuable supplementary information, particularly in areas with gaps in surveillance of acute flaccid paralysis (AFP) mainly in children less than 15 years. In collaboration with Guatemala's National Health Laboratory (Laboratorio Nacional de Salud Guatemala), monthly sewage collections allowed screening enterovirus (EV) presence without incurring additional costs for sample collection, transport, or concentration. Murine recombinant fibroblast L-cells (L20B) and human rhabdomyosarcoma (RD) cells are used for the isolation of polioviruses following a standard detection algorithm. Though non-polio-Enteroviruses (NPEV) can be isolated, the algorithm is optimized for the detection of polioviruses. To explore if other EV's are present in sewage not found through standard methods, five additional cell lines were piloted in a small-scale experiment, and next-generation sequencing (NGS) was used for the identification of any EV types. Human lung fibroblast cells (HLF) were selected based on their ability to isolate EV-A genus. Sewage concentrates collected between 2020-2021 were isolated in HLF cells and any cytopathic effect positive isolates used for NGS. A large variety of EVs, including echoviruses 1, 3, 6, 7, 11, 13, 18, 19, 25, 29; coxsackievirus A13, B2, and B5, EV-C99, EVB, and polioviruses (Sabin 1 and 3) were identified through genomic typing in NGS. When the EV genotypes were compared by phylogenetic analysis, it showed many EV's were genomically like viruses previously isolated from ES collected in Haiti. Enterovirus occurrence did not follow a seasonality, but more diverse EV types were found in ES collection sites with lower populations. Using the additional cell line in the existing poliovirus ES algorithm may add value by providing data about EV circulation, without additional sample collection or processing. Next-generation sequencing closed gaps in knowledge providing molecular epidemiological information on multiple EV types and full genome sequences of EVs present in wastewater in Guatemala.

Humans↗
PubMed1987

Polyadenylation of Chinese hamster dihydrofolate reductase genomic genes and minigenes after gene transfer.

The major alternative polyadenylation sites in the Chinese hamster dihydrofolate reductase (dhfr) gene have been identified by DNA sequencing and RNase protection experiments. Comparison of the 3' gene sequence and polyadenylation sites with those of the mouse reveals that, despite an overall sequence homology, the major sites are different in the two species. A series of minigenes was constructed containing the dhfr promoter and the first intron but lacking the four large introns of the genomic sequence. These minigenes contained either all three polyadenylation sites, no polyadenylation sites, or just the first site. All of these minigenes, as well as a cosmid clone containing the full genomic sequence, could transform DHFR-deficient Chinese hamster ovary cell mutants to a DHFR-positive phenotype with approximately equal efficiencies. A minigene lacking the first intron was markedly less efficient. Analysis of dhfr mRNA from transfectant clones derived from minigenes showed that the dhfr polyadenylation sites were used when included, but novel sites were often used in addition. When endogenous polyadenylation sites were absent, new sites in flanking carrier or host DNA were recruited. Transfectants produced by the full genomic dhfr gene yielded mRNA species that were identical in size and relative abundance to the endogenous dhfr gene. The results indicate that the minimal signals for polyadenylation are not complex and can be easily acquired from foreign sequences.

Animals↗
PubMed2026

An enhanced multisegment RT-PCR method for influenza A virus sequencing: Improved performance and reduced preparation time over traditional methods.

Influenza A viruses (IAVs) remain a major global health threat, affecting both human and animal populations. Whole-genome sequencing is essential for monitoring viral evolution, zoonotic transmission, and emerging variants. However, conventional RT-PCR methods often result in incomplete gene coverage, amplification biases, and reduced sequencing accuracy, particularly in clinical samples. We developed a robust In-house method for IAV full-genome sequencing using the Oxford Nanopore Technologies (ONT) long-read sequencing platform. This method integrates an in-house multisegment Reverse Transcription PCR (RT-PCR) method with a streamlined 2-pool primer design targeting all eight IAV gene segments. RNA extracted from clinical and stock virus samples was reverse-transcribed and amplified using Superscript IV-based chemistry, followed by magnetic bead purification to ensure high-quality amplicons. Sequencing libraries were prepared with the Native Barcoding Kit 24 (SQK-NBD114.24) and sequenced on R10.4.1 flow cells on the MinION MK1C device. Data analysis using the Iterative Refinement Meta-Assembler (IRMA) confirmed improved read depth, uniform coverage, and complete genome recovery. Compared to conventional methods, our In-House Multisegment 2-Pool (IH-MS2P) RT-PCR method generated higher numbers of matched read counts, minimized chimeric artifacts, and delivered superior genome coverage across human, swine, and avian isolates. This optimized RT-PCR method provides a high-performance, time-efficient, and portable solution for influenza genomics, demonstrating robust applicability even with clinical samples of low RNA yield.

Influenza A virus↗
PubMed2026

Optimization of a Semi-nested PCR Protocol for Amplifying the Entire Spike Protein Region to Identify SARS-CoV-2 Variants in Wastewater.

Wastewater-based monitoring of SARS-CoV-2 and other pathogens is a widely adopted tool for assessing epidemic dynamics. While quantitative assays are commonly used to estimate infection levels in catchment populations, phylogenetic information-such as identifying circulating variants-is also crucial for public health. However, applying the widely used ARTIC protocol for full-genome sequencing to wastewater samples has proven challenging, likely due to the limited specificity and sensitivity of multiplex RT-PCR in such complex matrices. In this study, we developed and optimized a semi-nested RT-PCR assay targeting the full S-protein coding region (~4000 bases) for phylogenetic characterization of SARS-CoV-2 in wastewater. By reducing multiplexing and using single-plex reactions for both RT and PCR steps, we successfully amplified ~2000&#xa0;bp fragments. Amplicons were sequenced using the Flongle Flow Cell platform. The optimized method-consisting of reverse transcription with specific primers followed by three parallel single-plex semi-nested PCRs-yielded over 1,000 SARS-CoV-2-like reads per primer set in 30 out of 39 wastewater samples in treatment plants in Japan, including those with <10 copies per analyte. Variant proportions were estimated using a newly developed approach based on single-nucleotide variant pattern matrix, revealing the presence of multiple co-circulating variants, including XBB lineages, JN.1, and notably BA.2.75, which was undetected in domestic clinical surveillance. These results highlight the effectiveness of our approach for detecting temporal shifts in SARS-CoV-2 variants, even at low RNA concentrations.

Polymerase Chain Reaction↗
PubMed2026

Classification and sequencing of hepatitis D virus from a large cohort of chronically infected individuals paired with co-infecting hepatitis B virus sequencing: a genomic characterisation study.

BACKGROUND: The most severe form of viral hepatitis is caused by co-infection of hepatitis D virus (HDV) and hepatitis B virus (HBV). Phylogenetic analyses classify HBV and HDV into eight major genotypes: HBV GTA to GTH and HDV GT1 to GT8. Paired HBV and HDV sequencing data from participants with chronic hepatitis delta are scarce. We aimed to sequence and genotype HDV and HBV from a large cohort of participants from clinical studies and diverse countries of origin. METHODS: 407 participants with chronic hepatitis D from 24 countries were characterised (124 participants from MYR301 clinical trial, 93 from MYR204, 114 from MYR202, and an additional 76 participants from diverse geographical locations). HBV and HDV from participants were analysed using sequencing, enzyme immunoassay, or both to determine HBV and HDV genotypes. BLAST analysis and phylogenetics were used to determine HBV and HDV genotypes with reference sequence libraries. Bulevirtide treatment response (measured by HDV RNA decline and normalisation of alanine aminotransferase) was compared by genotype for MYR trial participants. FINDINGS: HDV sequencing assays were successful for 386 (95%) of 407 participants and HBV sequencing or serology-based HBV genotyping assays were successful for genotyping 395 (97%) participants. For individual genotypes, HBV GTD (336 [83%] participants) and HDV GT1 (364 [89%]) were the most prevalent. For paired HBV-HDV genotypes, HBV-HDV D/1 was most common (320 [79%] of 407) followed by A/1 (30 [7%]). Phylogenetic analyses of HDV full-genome sequences showed distinct clusters of sequences within HDV GT1, and four novel provisional HDV GT1 subgenotypes, HDV GT1fp to HDVGT1ip, were identified. For 218 MYR clinical trial participants, bulevirtide treatment response was similar across HDV GT1 subgenotypes (both established and newly identified). INTERPRETATION: Novel HDV subgenotypes identified in this study indicate a greater genetic diversity of HDV GT1 than previously recognised. This knowledge will be important for developing better diagnostics, and in understanding HDV genotype-specific biology and response to treatment. More extensive HDV sequencing from under-sampled regions, such as Africa, is needed to determine the true breadth of HDV sequence and genotype diversity. FUNDING: Gilead Sciences.

Hepatitis Delta Virus↗
PubMed2025

Reversible phenotypic resistance to phage infection via capsule downregulation in Klebsiella pneumoniae.

Capsule loss is a major mechanism by which bacteria evade phage infection. This has traditionally been attributed to mutations in capsule biosynthesis genes. Here, we investigated phage resistance in Klebsiella pneumoniae, a medically relevant encapsulated bacterium. Phage infection rapidly selected for resistant acapsular cells. As expected, transcriptomic analysis revealed a marked downregulation of capsule biosynthesis genes. However, full genome sequencing showed that capsule loss occurred without evidence of mutations, and acapsular phage-resistant cells were able to rapidly restore their capsule once phage pressure was removed. These findings highlight that phage-driven selective pressure can act on non-heritable variation in gene expression, providing a faster and more flexible resistance mechanism compared to the traditional mutation-selection process. This reversible resistance may complicate predictability and limit the long-term efficacy of phage therapy.

Cell biology↗
PubMed1990

Structure and expression of the human polymorphic epithelial mucin gene: an expressed VNTR unit.

The human polymorphic epithelial mucin (PEM) is expressed apically by glandular epithelium and by the carcinomas that develop from these tissues. Previously isolated cDNA clones revealed that the core protein contained a large domain consisting of variable numbers of 60 bp tandem repeats (TR), making it an expressed minisatellite. We now report the full genomic sequence of the PEM gene, including 803 bp of 5' flanking sequence. The gene is composed of 7 exons and varies in size from approximately 4 to approximately 7 kb, depending on the number of tandem repeats in exon 2. Expression of PEM was obtained from a genomic clone in an Epstein-Barr virus based vector, after transfection into a human epithelial cell line, indicating the presence of effective regulatory sequences in this clone.

Amino Acid Sequence↗
PubMed2019

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis.

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity of these viruses. Because of high mutation and recombination rates, genome replication by viral RNA-dependent RNA polymerases leads to populations of closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited to reconstruct large numbers of full-length haplotypes of (1) RNA virus genomes and (2) subgenome-length (sg) RNAs composed of noncontiguous genome regions. Here, we used a full-length, direct RNA sequencing (DRS) approach based on nanopores to characterize viral RNAs produced in cells infected with a human coronavirus. By using DRS, we were able to map the longest (&#x223c;26-kb) contiguous read to the viral reference genome. By combining Illumina and Oxford Nanopore sequencing, we reconstructed a highly accurate consensus sequence of the human coronavirus (HCoV)-229E genome (27.3 kb). Furthermore, by using long reads that did not require an assembly step, we were able to identify, in infected cells, diverse and novel HCoV-229E sg RNAs that remain to be characterized. Also, the DRS approach, which circumvents reverse transcription and amplification of RNA, allowed us to detect methylation sites in viral RNAs. Our work paves the way for haplotype-based analyses of viral quasispecies by showing the feasibility of intra-sample haplotype separation. Even though several technical challenges remain to be addressed to exploit the potential of the nanopore technology fully, our work illustrates that DRS may significantly advance genomic studies of complex virus populations, including predictions on long-range interactions in individual full-length viral RNA haplotypes.

Cell Line↗