Oxford Nanopore Sequencing Research Articles

Detection of Nuclear RNA Decay Intermediates Using a Modified Oxford Nanopore RNA Sequencing Strategy.

The nuclear RNA exosome complex is crucial for noncoding RNA processing and RNA quality control in the nucleus. Identifying substrates and intermediates of RNA decay pathways, such as those mediated by the exosome complex using Oxford Nanopore sequencing can be difficult in part because a simple method to detect them has been lacking and also because some of these RNAs lack abundant poly(A) tails which are required for Oxford Nanopore-based sequencing. Here we describe an Oxford nanopore-based approach which can be used to identify long reads corresponding to precursors and products of nuclear exosome processing. We are able to observe accumulation of unprocessed snoRNAs, cleavage products of the yeast nuclear RNase III endonuclease Rnt1p when the nuclear exosome component Rrp6p is inactivated.

EPCO-23. LONGITUDINAL TRACKING AND LONG-READ CHROMATIN FIBER SEQUENCING OF HUMAN ASTROCYTES WITH TELOMERE DYSFUNCTION REVEALED ALTERED CHROMATIN ACCESSIBILITY AND INCREASED STRUCTURAL VARIATION

Abstract Glioma is an aggressive cancer of the central nervous system that is genetically characterized by aneuploidy and frequent structural variation (SV). We sought to investigate the role of telomere dysfunction (TD) in shaping the SV landscape in glioma. Normal human astrocytes (NHA) were engineered to express viral E6E7 oncoproteins which are known to spontaneously trigger TD by suppressing p53/pRb. We hypothesized that NHA-E6E7 cells gradually accumulate SVs and aneuploidy over time because of progressive TD. We therefore longitudinally tracked bulk and single-cell clones of NHA-E6E7 cells for a period of over 6 months since viral infection. Overall, NHA-E6E7 cells demonstrate a growth benefit over primary NHA cells. We obtained Illumina short-read sequencing for NHA-E6E7 cells at multiple time points. Three time points were additionally selected for both Oxford Nanopore and PacBio HiFi long-read sequencing using the single-molecule chromatin fiber sequencing (fiber-seq) protocol. Copy number variation (CNV) analysis of short-read data showed deletion events in chromosome 13 as early as population doubling level (PDL) 48. K-mer based CNV analysis using the long-read data confirmed this finding and also detected deletion events in chromosome 7 at PDL 50. From the long-read data, we found an elevated number of SVs in NHA-E6E7 cells compared to the primary NHA cells. In particular, we detected telomere junctions and found that telomere-associated SVs were more prevalent in NHA-E6E7 cells compared to primary cells. Next, chromatin accessibility and regulatory elements were predicted from fiber-seq data with exogenous N6-methyladenosine (m6A) stencils. We observed large-scale chromatin actuation changes in various regulatory elements across the genome that intensified in later passages of NHA-E6E7 cells. For example, we found a decrease in the accessibility in FBXO7 which is a gene that maintains chromosome stability. Our preliminary results indicated that TD is an important mediator of structural variation in glioma.

Microbiome shifts elicited by ornamental lighting of granite facades identified by MinION sequencing

Night-time outdoor illumination in combination with natural sunlight can influence the visible phototrophic colonizers (mainly algae) growing on stone facades; however, the effects on the microbiome (invisible to the naked eye) are not clear. The presence of stone-dwelling microbes, such as bacteria, diatoms, fungi, viruses and archaea, drives further biological colonization, which may exacerbate the biodeterioration of substrates. Considering the microbiome is therefore important for conservation of the built heritage. The impact of the following types of lighting on the relative abundance and diversity of the microbiome on granite ashlars was evaluated in a year-long outdoor pilot study: no lighting; lighting with a metal halide lamp (a traditional lighting system currently used to illuminate monuments); and lighting with a novel LED lamp (an environmentally sound prototype lamp with a biostatic effect, halting biological colonization by phototrophs, currently under trial). Culturable fractions of microbiome and whole-genome sequencing by metabarcoding with Oxford Nanopore Sequencing (MinION) was conducted for bacteria and fungi in order to complement both community characterization strategies. In addition, the possible biodeteriorative profiles of the isolated strains, relative to calcium carbonate precipitation/solubilisation and iron oxidation/reduction, were investigated by plate assays. Alpha and beta diversity indexes were also determined, along with the abundance of biocide and antibiotic resistance genes. Culture-dependent microbiological analysis failed to properly show changes in community composition, for which metagenomic approaches like MinION are better suited. Thus, MinION analysis identified shifts in the granite microbiome elicited by ornamental lighting. The novel LED lamp with the biostatic effect on phototrophs caused an increase in the diversity of bacteria and fungi. In this case, the microbiome was more similar to that in the unlit samples. In the samples illuminated by the metal halide lamp, dominance of bacteria was favoured and the presence of fungi was negligible.

Accurate bacterial outbreak tracing with Oxford Nanopore sequencing and reduction of methylation-induced errors.

Our study investigates the effectiveness of Oxford Nanopore Technologies for accurate outbreak tracing by resequencing 33 isolates of a 3-year-long Klebsiella pneumoniae outbreak with Illumina short-read sequencing data as the point of reference. We detect considerable base errors through cgMLST and phylogenetic analysis of genomes sequenced with Oxford Nanopore Technologies, leading to the false exclusion of some outbreak-related strains from the outbreak cluster. Nearby methylation sites cause these errors and can also be found in other species besides K. pneumoniae Based on these data, we explore PCR-based sequencing and a masking strategy, which both successfully address these inaccuracies and ensure accurate outbreak tracing. We offer our masking strategy as a bioinformatic workflow (MPOA) to identify and mask problematic genome positions in a reference-free manner. Our research highlights limitations in using Oxford Nanopore Technologies for sequencing prokaryotic organisms, especially for investigating outbreaks. For time-critical projects that cannot wait for further technological developments by Oxford Nanopore Technologies, our study recommends either using PCR-based sequencing or using our provided bioinformatic workflow. We advise that read mapping-based quality control of genomes should be provided when publishing results.

A cry for kelp: Evidence for polyphenolic inhibition of Oxford Nanopore sequencing of brown algae.

Genomic resources have yielded unprecedented insights into ecological and evolutionary processes, not to mention their importance in economic and conservation management of specific organisms. However, the field of macroalgal genomics is hampered by difficulties in the isolation of suitable DNA. Even when DNA that appears high quality by standard metrics has been isolated, such samples may not perform well during the sequencing process. We here have compared Oxford Nanopore long-read sequencing results for three species of macroalgae to those of nonmacroalgal species and determined that when using macroalgal samples, sequencing activity declined rapidly, resulting in reduced sequencing yield. Chemical analysis of macroalgal DNA that would be considered suitable for sequencing revealed that DNA derived from dried macroalgae was enriched for polyphenol-DNA adducts (DNA with large polyphenols chemically attached to it), which may have led to sequencing inhibition. Of note, we observed the strongest evidence of sequencing inhibition and reduced sequence output when using samples dried using silica gel-suggesting that such storage approaches may not be appropriate for samples destined for Oxford Nanopore sequencing. Our findings have wide-ranging implications for the generation of genomic resources from macroalgae and suggest a need to develop new storage methods that are more amenable to Oxford Nanopore sequencing or to use fresh flash-frozen tissue wherever possible for genome sequencing.

ONT sequencing identifies a high prevalence of crt sensitive, triple mutant dhfr and single mutant dhps parasites within an ANC population in Nigeria.

Malaria in pregnancy is a major public health issue, particularly among vulnerable populations in malaria-endemic sub-Saharan African countries. To mitigate its risks, WHO recommends sulphadoxine-pyrimethamine (SP) for chemoprevention and artemisinin-based combination therapy (ACT) to treat uncomplicated Plasmodium falciparum malaria. These interventions have helped to alleviate the risk associated with malaria in pregnancy; however, in the context of the emergence of SP- and ACT-resistant P. falciparum, maintained efficacy is under threat. Molecular surveillance is a reliable tool to monitor the emergence of resistance where molecular markers are known. Thus, the objective of the study was to use a multiplexed amplicon Oxford Nanopore sequencing approach to assess the molecular markers for antimalarial resistance among pregnant women in Nigeria. Dried blood spots (DBS) were collected from pregnant women who received IPTp-SP at the enrollment and follow-up visits. P. falciparum genomic DNA was extracted by the Chelex® method and Pf18S qPCR was used to detect parasite DNA in each sample. With nested PCR assays, fragments of Pfdhps, Pfdhfr, Pfmdr1, Pfcrt, Pfk13 and Pfama1 genes were amplified and multiplexed amplicon-based sequencing was conducted on the minION Oxford Nanopore Technology. In total, 251 pregnant women were enrolled in the study and 457 DBS samples were collected. P. falciparum genomic DNA was detected in 12% (56/457) of the samples, 31 at baseline and the remaining during the follow-up visits. Pfama1, pfk13, Pfdhps, Pfdhfr, Pfmdr1 and Pfcrt were successfully sequenced in a single run. Notably, k13 artemisinin resistance mutations were absent, the frequencies of Pfdhfr and Pfdhps SP resistance haplotypes, IRN for pyrimethamine resistance and ISGKA/IAGKA associated with sulphadoxine resistance were 82% (36/44) and 64% (27/42), respectively, and the Pfcrt CVIET resistant haplotype was at approximately 22% (7/32). Here a multiplexed amplicon-based ONT assay established that triple mutant Pfdfhr-IRN, double mutant Pfdhps-SG haplotypes and the chloroquine sensitive strain were prevalent among pregnant women in Nigeria.

Haplotype-resolved and near-T2T genome assembly of the African catfish (Clarias gariepinus)

Airbreathing catfish are stenohaline freshwater fish capable of withstanding various environmental conditions and farming practices, including breathing atmospheric oxygen. This unique ability has enabled them to thrive in semi-terrestrial habitats. However, the genomic mechanisms underlying their adaptation to adverse ecological environments remain largely unexplored, primarily due to the limited availability of high-quality genomic resources. Here, we present a haplotype-resolved and near telomere-to-telomere (T2T) genome assembly of the African catfish (Clarias gariepinus), utilizing Oxford Nanopore, PacBio HiFi, Illumina and Hi-C sequencing technologies. The primary assembly spans 969.62 Mb with only 47 contigs, achieving a contig N50 of 33.71 Mb. Terminal telomeric signals were detected in 22 of 47 contigs, suggesting T2T assembled chromosomes. BUSCO analysis confirmed gene space completeness of 99% against the Actinopterygii dataset, highlighting the high quality of the assembly. Genome annotation identified 25,655 protein-coding genes and estimated 43.94% genome-wide repetitive elements. This data provides valuable genomic resources to advance aquaculture practices and to explore the genomic underpinnings of the ecological resilience of airbreathing catfish and related teleosts.

NanoCore: core-genome-based bacterial genomic surveillance and outbreak detection in healthcare facilities from Nanopore and Illumina data.

Genomic surveillance involves sequencing the genomes and measuring the relatedness of bacteria from different patients or locations in the same healthcare facility, enabling an improved understanding of pathogen transmission pathways and the detection of "silent" outbreaks that would otherwise go undetected. It has become an indispensable tool for the detection and prevention of healthcare-associated infections and is routinely applied by many healthcare institutions. The earlier an outbreak or transmission chain is detected, the better; in this context, the Oxford Nanopore sequencing technology has important potential advantages over traditionally used short-read sequencing technologies, because it supports "real-time" data generation and the cost-effective "on demand" sequencing of small numbers of bacterial isolates. The analysis of Nanopore sequencing data, however, can be challenging. We present NanoCore, a user-friendly software for genomic surveillance that works directly based on Nanopore sequencing reads in FASTQ format, and demonstrate that its accuracy is equivalent to traditional gold standard short read-based analyses.

Analyzing Antibiotic Resistance in Bacteria from Wastewater in Pakistan Using Whole-Genome Sequencing.

Background: Wastewater is a major source of Antibiotic-Resistant Bacteria (ARB) and a hotspot for the exchange of Antibiotic-Resistant Genes (ARGs). The occurrence of Carbapenem-Resistant Bacteria (CRB) in wastewater samples is a major public health concern. Objectives: This study aimed to analyze Antibiotic resistance in bacteria from wastewater sources in Pakistan. Methods: We analyzed 32 bacterial isolates, including 18 Escherichia coli, 4 Klebsiella pneumoniae, and 10 other bacterial isolates using phenotypic antibiotic susceptibility assay and whole-genome sequencing. This study identified the ARGs, plasmid replicons, and integron genes cassettes in the sequenced isolates. One representative isolate was further sequenced using Illumina and Oxford nanopore sequencing technologies. Results: Our findings revealed high resistance to clinically important antibiotics: 91% of isolates were resistant to cefotaxime, 75% to ciprofloxacin, and 62.5% to imipenem, while 31% showed non-susceptibility to gentamicin. All E. coli isolates were resistant to cephalosporins, with 72% also resistant to carbapenems. Sequence analysis showed a diverse resistome, including carbapenamases (blaNDM-5, blaOXA-181), ESBLs (blaCTX-M-15, blaTEM), and AmpC-type β-lactamases (blaCMY). Key point mutations noticed in the isolates were pmrB_Y358N (colistin) and ftsI_N337NYRIN, ftsI_I336IKYRI (carbapenem). The E. coli isolates had 11 different STs, with ST410 predominating (28%). Notably, the E. coli phylogroup A isolate 45EC1, (ST10886) is reported for the first time from wastewater, carrying blaNDM-5, blaCMY-16, and pmrB_Y358N with class 1 integron gene cassette of dfrA12-aadA2-qacEΔ1 on a plasmid-borne contig. Other carbapenamase, blaNDM-1 and blaOXA-72, were detected in K. pneumoniae 22EB1 and Acinetobacter baumannii 51AC1, respectively. The integrons with the gene cassettes encoding antibiotic resistance, and the transport and bacterial mobilization protein, were identified in the sequenced isolates. Ten plasmid replicons were identified, with IncFIB prevalent in 53% of isolates. Combined Illumina and Oxford nanopore sequencing revealed blaNDM-5 on an IncFIA/IncFIC plasmid and is identical to those reported in the USA, Myanmar, and Tanzania. Conclusions: These findings highlight the environmental prevalence of high-risk and WHO-priority pathogens with clinically important ARGs, underscoring the need for a One Health approach to mitigate ARB isolates.

Nanopore sequencing enables novel detection of deuterium incorporation in DNA

Identifying active microbes is crucial to understand their role in ecosystem functions. Metabolic labeling with heavy, non-radioactive isotopes, i.e., stable isotope probing (SIP), can track active microbes by detecting heavy isotope incorporation in biomolecules such as DNA. However, the detection of heavy isotope-labeled nucleotides directly during sequencing has, to date, not been achieved.In this study, Oxford nanopore sequencing was utilized to detect heavy isotopes incorporation in DNA molecules. Two isotopes widely used in SIP experiments were employed to label a bacterial isolate: deuterium (D, as D2O) and carbon-13 (13C, as glucose). We hypothesize that labeled DNA is distinguishable from unlabeled DNA by changes in the nanopore signal. To verify this distinction, we employed a Bayesian classifier trained on signal distributions of short oligonucleotides (k-mers) from labeled and unlabeled sequencing reads.Our results show a clear distinction between D-labeled and unlabeled reads, based on changes in median and median absolute deviation (MAD) of the nanopore signals for different k-mers. In contrast, 13C-labeled DNA cannot be distinguished from unlabeled DNA. For D, the model employed correctly predicted more than 85% of the reads. Even when metabolic labeling was conducted with only 30% D2O, 80% of the obtained reads were correctly classified with a 5% false discovery rate.Our work demonstrates the feasibility of direct detection of deuterium incorporation in DNA molecules during Oxford nanopore sequencing. This finding represents a first step in establishing the combined use of nanopore sequencing and SIP for tracking active organisms in microbial ecology.

Performance of somatic structural variant calling in lung cancer using Oxford Nanopore sequencing technology

BackgroundLung cancer is a heterogeneous disease and the primary cause of cancer-related mortality worldwide. Somatic mutations, including large structural variants, are important biomarkers in lung cancer for selecting targeted therapy. Genomic studies in lung cancer have been conducted using short-read sequencing. Emerging long-read sequencing technologies are a promising alternative to study somatic structural variants, however there is no current consensus on how to process data and call somatic events. In this study, we preformed whole genome sequencing of lung cancer and matched non-tumour samples using long and short read sequencing to comprehensively benchmark three sequence aligners and seven structural variant callers comprised of generic callers (SVIM, Sniffles2, DELLY in generic mode and cuteSV) and somatic callers (Severus, SAVANA, nanomonsv and DELLY in somatic modes).ResultsDifferent combinations of aligners and variant callers influenced somatic structural variant detection. The choice of caller had a significant influence on somatic structural variant detection in terms of variant type, size, sensitivity, and accuracy. The performance of each variant caller was assessed by comparing to somatic structural variants identified by short-read sequencing. When compared to somatic structural variants detected with short-read sequencing, more events were detected with long-read sequencing. The mean recall of somatic variant events identified by long-read sequencing was higher for the somatic callers (72%) than generic callers (53%). Among the somatic callers when using the minimap2 aligner, SAVANA and Severus achieved the highest recall at 79.5% and 79.25% respectively, followed by nanomonsv with a recall of 72.5%.ConclusionLong-read sequencing can identify somatic structural variants in clincal samples. The longer reads have the potential to improve our understanding of cancer development and inform personalized cancer treatment.

Draft genome sequence of Listeria aquatica strain SG_BD1, isolated from a cow dung sample in Bangladesh.

Here, we present the genome of Listeria aquatica strain SG_BD1, isolated from cow dung in Dhaka, Bangladesh, and assembled after Oxford Nanopore sequencing. The genome is 2,690,148 bp with 2,855 predicted coding DNA sequences, G + C content of 39.6%, and displays a putative virulence gene clpP and 9 CRISPRs.

Characterizing a lethal CAG-ACE2 transgenic mouse model for SARS-CoV-2 infection using Cas9-enhanced nanopore sequencing.

The SARS-CoV-2 pandemic has underscored the necessity for functional transgenic animal models for testing. Mouse lines with overexpression of the human receptor ACE2 serve as the common animal model to study COVID-19 infection. Overexpression of ACE2 under a strong ubiquitous promoter facilitates convenient and sensitive testing of COVID-19 pathology. We performed pronuclear microinjections using a 5kb CAG-ACE2 linear transgene construct and identified three founder lines with 140, 72, and 73 copies, respectively. Two of these lines were further analyzed for ACE2 expression profiles and sensitivity to SARS-CoV-2 infection. Both lines expressed ACE2 in all organs analyzed. Embryonic fibroblast cell lines derived from transgenic embryos demonstrated severe cytopathic effects following infection, even at low doses of SARS-CoV-2 (0,1-1.0 TCID50). Infected mice from the two lines began to show COVID-19 clinical signs three days post-infection and succumbed between days 4 and 7. Histological examination of lung tissues from terminally ill mice revealed severe pathological alterations. To further characterize the integration site in one of the lines, we applied nanopore sequencing combined with Cas9 enrichment to examine the internal transgene concatemer structure. Oxford Nanopore sequencing (ONT) is becoming the gold standard for transgene insert characterization, but it is relatively inefficient without targeted region enrichment. We digested genomic DNA with Cas9 and gRNA against the ACE2 transgene to create ends suitable for ONT adapter ligation. ONT data analysis revealed that most of the transgene copies were arranged in a head-to-tail configuration, with palindromic junctions being rare. We also detected occasional plasmid backbone fragments within the concatemer, likely co-purified during transgene gel extraction, which is a common occurrence in pronuclear microinjections.

Antidepressant-like effects of the leaf extract of Mallotus oppositifolius (Geiseler) Müll. Arg. (Euphorbiaceae) in the chronic unpredictable mild stress model: A role of the gut-brain axis

The gut microbiota has been posited as a target for the treatment of major depressive disorder. Herein, we investigated the effect of the hydroethanolic leaf extract of Mallotus oppositifolius (MOE) on the gut microbiota of mice and how this contributes to its known antidepressant-like effect. A 6-week chronic unpredictable mild stress (CUMS) procedure was employed in 7 groups of mice to induce depression. From the third week, oral MOE treatments (10, 30, 100 mg/kg) and two reference drugs, fluoxetine (12 mg/kg) and minocycline (40 mg/kg), known to affect the gut microbiota, were administered. The sixth and seventh groups were the vehicle stressed (VEH-S) and non-stressed groups (VEH-NS). Changes in depressive-like behaviors were assessed using sucrose preference test while the forced swimming test (FST) was used to assess sustained antidepressant-effect after treatment discontinuation. Moreover, changes in prefrontal cortex (PFC) and hippocampal serotonin (5-HT) levels were evaluated using enzyme-linked immunosorbent assay (ELISA). The effect of treatment on the profile of the gut microbiota of the groups was elucidated using 16S rRNA Oxford Nanopore sequencing. MOE and reference drugs reversed the depression-associated reduction in sucrose preference when compared to VEH-S. MOE (with peak effect at 30 mg/kg) reduced immobility while increasing swimming and climbing behaviors. MOE reversed CUMS-induced reduction of 5-HT concentration in PFC and hippocampus. The behavioral effects of MOE were associated with shifts in the gut microbiota of CUMS-exposed mice. The study has provided seminal evidence that MOE ameliorates CUMS-induced depressive symptoms by modulating gut microbiota and increasing brain 5-HT levels.

Combined analysis of genome-wide DNA methylome and transcriptome reveals the first epigenetic-based antibiotic-resistance mechanism in Acinetobacter baumannii

The World Health Organization (WHO) has defined carbapenem-resistant Acinetobacter baumannii (A. baumannii) as a critical species for which new therapeutic strategies are needed. In this tragic context, the discovery of new antibiotics with innovative pharmacological targets is essential. Currently, limited information is available on the relationship between epigenetic changes of m5C/m6A DNA and drug resistance in A. baumannii. In this study, we used Oxford Nanopore sequencing to analyze m5C/m6A epigenetic marks and Illumina RNA-Seq to evaluate the transcriptome of 5 A. baumanni isolates with different resistance profiles. The data clearly demonstrated that the m5C pattern of resistant strains globally differs from the multi susceptible strain, with more than 600 hypo- and hypermethylated sites for each resistant strain. For m6A, less severe changes occurred, however, more than 40 hyper- and hypomethylated sites were still found between each resistant and susceptible strain. Combined whole-genome DNA methylome and transcriptome analysis revealed a greater involvement of m5C modification in drug resistance processes. This study provides a new understanding of the epigenetic regulation of antibiotic resistance in A. baumannii and has potential implications for future antibiotic resistance research.

Molecular detection of intestinal parasites in a rural community of Colombia: A one health approach to explore potential environmental-zoonotic transmission.

Protozoan and helminth parasitic infections pose significant public health challenges, especially in developing countries with rural populations marked by suboptimal hygiene practices and socio-economic constraints. The parasites are the etiological agents of these infections and have a notably elevated global prevalence. Therefore, this study focuses on estimating the frequency and transmission dynamics of several parasitic species, including Blastocystis, Giardia, Cryptosporidium spp., Entamoeba histolytica, Ascaris lumbricoides, Trichuris trichiura, Taenia spp. and hookworms, within a rural community in southwest Colombia with a particular emphasis on the One Health framework, considering environmental and zoonotic transmission potentials. This study involved the analysis of 125 samples, encompassing human participants (n = 99), their domestic pets (dogs) (n = 24) and water sources (n = 2). Parasite detection was carried out utilizing a combination of microscopy and molecular techniques. Furthermore, the characterization of Blastocystis subtypes (STs) was achieved through Oxford Nanopore sequencing of the rRNA-18S gene. The investigation also entailed the examination of potential associations between intestinal parasitism and various sociodemographic factors. Results revealed a high frequency of parasitic infections when employing molecular methods, with Blastocystis (n = 109/87%), Giardia (n = 20/16%), Ancylostoma duodenale (n = 28/22%), Ancylostoma ceylanicum (n = 7/5.6%), E. histolytica (n = 6/4.8%), Cryptosporidium spp. (n = 12/9.6%) and even Taenia (n = 1/0.8%) detected. Cryptosporidium spp. was also identified in water samples. Coinfections were prevalent, with 57% (n = 70) of samples exhibiting single-parasite infections and 43% (n = 53) showing various degrees of polyparasitism, emphasizing the complexity of transmission dynamics. Blastocystis subtyping, conducted via Oxford Nanopore sequencing, revealed a diversity of subtypes and coexistence patterns, with ST2 being the most prevalent. This research underscores the importance of using molecular techniques for frequency estimation, particularly emphasizing the relevance of zoonotic transmission in parasitic infections. It highlights the significance of the One Health approach in comprehending the circulation of parasites among animals, humans and environmental sources, thereby directly impacting public health and epidemiological surveillance.

Shedding Light on Viral Shedding: Novel Insights into Nuclear Assembly, Cytoplasmic Transformation and Extracellular Vesicle Release of the BK Virus.

Despite the high prevalence of BK polyomavirus (BKPyV) and the associated risk for BKPyV-associated nephropathy (BKPyVAN) in kidney transplant (KTX) recipients, many details on viral processes such as replication, maturation, assembly and virion release from host cells have not been fully elucidated. VP1 is a polyomavirus-specific protein that is expressed in the late phase of its replicative cycle with important functions in virion assembly and infectious particle release. This study investigated the localization and time-dependent changes in the distribution of VP1-positive viral particles and their association within the spectrum of differing cell morphologies that are observed in the urine of KTX patients upon active BKPyV infection. We found highly differing recognition patterns of two anti-VP1 antibodies with respect to intracellular and extracellular VP1 localization, pointing towards independent binding sites that were seemingly associated with differing stages of virion maturation. Cells originating from single clones were stably cultured out of the urine sediment of KTX recipients with suspected BKPyVAN. The cell morphology, polyploidy, virus replication and protein production were investigated by confocal microscopy using both a monoclonal (mAb 4942) and a polyclonal rabbit anti-VP1-specific antibody (RantiVP1 Ab). Immunoblotting was performed to investigate changes in the VP1 protein. Both antibodies visualized VP1 and the mAb 4942 recognized VP1 in cytoplasmic vesicles exhibiting idiomorphic sizes when released from the cells. In contrast, the polyclonal antibody detected VP1 within the nucleus and in cytoplasm in colocalization with the endoplasmic reticulum marker CNX. At the nuclear rim, VP1 was recognized by both antibodies. Immunoblotting revealed two smaller versions of VP1 in urinary decoy cell extracts, potentially from different translation start sites as evaluated by in silico analysis. Oxford Nanopore sequencing showed integration of BKPyV DNA in chromosomes 3, 4 and 7 in one of the five tested primary cell lines which produced high viral copies throughout four passages before transcending into senescence. The different staining with two VP1-specific antibodies emphasizes the modification of VP1 during the process of virus maturation and cellular exit. The integration of BKPyV into the human genome leads to high virus production; however, this alone does not transform the cell line into a permanently cycling and indefinitely replicating one.

Complete genome sequences of two new strains of the shipworm endosymbiont, Teredinibacter turnerae.

We present the complete genome sequences of two strains of Teredinibacter turnerae, SR01903 and SR02026, shipworm endosymbionts isolated from the gills of Lyrodus pedicellatus and Teredo bartschi, respectively, and derived from Oxford Nanopore sequencing. These sequences will aid in the comparative genomics of shipworm endosymbionts and understanding of host-symbiont selection.

Towards improved nepovirus detection and identification in Xiphinema nematodes

Several Xiphinema nematode species are vectors of regulated nepoviruses. The aim of this study was to develop an optimized method to detect virus-carrying Xiphinema specimens. The study compared various techniques for nematode extraction from soil (Automated Zonal Centrifuging and Flegg’s Modified Cobb method), nematode cuticle disruption (slicing, bead beating, and bead beating with collagenase), RNA extraction (RNeasy Plant Mini Kit, CTAB, and KingFisher MagMAX RNA Isolation Kit), and nepovirus detection (nepovirus generic subgroup and species-specific RT-(q)PCR assays, as well as shotgun sequencing with Oxford Nanopore Sequencing technology using a MinION device). Based on this comparison, a diagnostic procedure is proposed including the best performing methods: nematode extraction using automated zonal centrifugation, physical disruption of nematode cuticle using bead beating or slicing, and RNA extraction using either the KingFisher MagMAX or the RNeasy Plant Mini Kit method. While existing generic nepovirus subgroup RT-PCR assays lacked sensitivity, species-specific RT-(q)PCR assays successfully detected ArMV, GFLV, and ToRSV in X. diversicaudatum, X. index, and X. americanum s.s., respectively. The reliability of nepovirus detection was higher with adults compared to juveniles. The minimum nematode quantity required varied depending on the specific nepovirus and the detection method used.

Closing the genome of Teredinibacter turnerae T7902 by long-read nanopore sequencing.

We present the complete closed circular genome sequence derived from Oxford Nanopore sequencing of the shipworm endosymbiont Teredinibacter turnerae T7902 (DSM 15152, ATCC 39867), originally isolated from the shipworm Lyrodus pedicellatus (1). This sequence will aid in the comparative genomics of shipworm endosymbionts and the understanding of host-symbiont evolution.