Whole Genome Sequencing Research Articles

Whole genome sequencing completes the molecular genetic testing workflow of patients with Lynch syndrome

Multigene panel tests (MGPTs) revolutionized the diagnosis of Lynch syndrome (LS), however noncoding pathogenic variants (PVs) can only be detected by complementary methods including whole genome sequencing (WGS). Here we present a DNA-, RNA- and tumor tissue-based WGS prioritization workflow for patients with a suspicion of LS where MGPT detected no LS-related PV. Among the 100 enrolled patients, MGPT detected 28 simple PVs and an additional 3 complex PVs. Among the 69 MGPT-negative patients, the lack of somatic MLH1 promoter methylation in a patient with a distinguished MLH1 allelic imbalance selected this sample for WGS. This returned a germline deep intronic MLH1 variant, with further functional studies confirming its’ pathogenicity. Interestingly, all three complex PVs and the MLH1 deep intronic PV were found to be recurrent at our center. Our straightforward and cost-effective prioritization workflow can optimally include WGS in the genetic diagnosis of LS.

Comparative Analysis of the Probiotic Features of Lysinibacillus and Enterobacter Strains Isolated from Gut Tract of Triploid Cyprinid Fish.

Gut mucosal immunity of teleost is mainly governed by mucosa-associated lymphoid tissues (MALT) and indigenous microbiota on mucosal surfaces of gut tract, which can confer protection against pathogenic invasion. However, the probiotic features of bacterial isolates from gut tract of triploid cyprinid fish (TCF) were largely unclear. In this study, Lysinibacillus and Enterobacter strains were isolated for probiotic identification. Whole genome sequencing (WGS) analysis indicated that Lysinibacillus and Enterobacter isolates possessed a variety of functional genes associated with probiotic features. Biofilm-forming activity (BFA) were one of the most important probiotic features, which can enable probiotic strains to communicate with indigenous microbiota by forming sessile community and then confer protection against stressors and invading pathogens. In this study, Lysinibacillus and Enterobacter isolates displayed high levels of BFA, hydrophobicity as well as aggregating potentials. Moreover, supernatants of probiotic isolates not only decreased pathogenic BFA and growth activity, but also showed high decomposing activity to macronutrients. These results indicated that probiotic isolates from gut tract of TCFs may pose protective roles in health of farmed fish.

Antimicrobial resistance profiles and molecular epidemiology of Klebsiella pneumoniae isolates from Scottish bovine mastitis cases.

Klebsiella pneumoniae are opportunistic pathogens which can cause mastitis in dairy cattle. K. pneumoniae mastitis often has a poor cure rate and can lead to the development of chronic infection, which has an impact on both health and production. However, there are few studies which aim to fully characterize K. pneumoniae by whole-genome sequencing from bovine mastitis cases. Here, K. pneumoniae isolates associated with mastitis in dairy cattle were identified using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and whole-genome sequencing. Furthermore, whole-genome sequence data were used for phylogenetic analyses and both virulence and antimicrobial resistance (AMR) prediction, in parallel with phenotypic AMR testing. Forty-two isolates identified as K. pneumoniae were subject to whole-genome sequencing, with 31 multi-locus sequence types being observed, suggesting the source of these isolates was likely environmental. Isolates were examined for key virulence determinants encoding acquired siderophores, colibactin, and hypermucoidy. The majority of these were absent, except for ybST (encoding yersiniabactin) which was present in six isolates. Across the dataset, there were notable levels of phenotypic AMR against streptomycin (26.2%) and tetracycline (19%), and intermediate susceptibility to cephalexin (26.2%) and neomycin (21.4%). Of importance was the detection of two ESBL-producing isolates, which demonstrated multi-drug resistance to amoxicillin-clavulanic acid, streptomycin, tetracycline, cefotaxime, cephalexin, and cefquinome.

Identification of Potential Intervention Targets Involved in Prior Exercise that Attenuates Peripheral Neuropathic Pain by Integrating Transcriptome and Whole-genome Bisulfite Sequencing Analyses.

Changes in DNA methylation and subsequent alterations in gene expression have opened a new direction in research related to the pathogenesis of peripheral neuropathic pain (PNP). This study aimed to reveal epigenetic perturbations underlying DNA methylation in the dorsal root ganglion (DRG) of rats with peripheral nerve injury in response to prior exercise and identify potential target genes involved. Male Sprague-Dawley rats were divided into three groups, namely, chronic constriction injury (CCI) of the sciatic nerve, CCI with prior 6-week swimming training (CCI_Ex), and sham operated (Sham). Mechanical withdrawal threshold (MWT) and thermal withdrawal latency (TWL) were used as the main observation indicators to evaluate behavioral changes associated with pain. In this study, 6-week swimming training before CCI prevented later chronic pain. In particular, CCI rats with prior exercise showed a significant increase in the MWT and TWL of the injured lateral hind paw compared with CCI rats without exercise on days 14, 21, and 28 after CCI. Whole-genome bisulfite sequencing from the injured lumbar (L4-L6) DRGs on the 28th day after surgery was detected. We also generated DNA methylation maps of the two comparisons (sham group vs. CCI and CCI groups vs. CCI_Ex group), and 396 overlapping differentially methylated region-related genes were found between the two comparisons. Moreover, we integrated RNA sequencing to understand the mechanism by which differential DNA methylation after CCI may influence gene expression. Finally, Ryr1 and Xirp2 were identified through association analysis of two omics and quantitative reverse-transcription polymerase chain reaction, respectively. The methylation levels of Ryr1 and Xirp2 were upregulated with a corresponding increase in their mRNA expression in the DRGs of CCI rats, whereas prior exercise downregulated Ryr1 methylation and restore its expression level. Functional enrichment analysis of both omics found that the calcium signaling pathway was significantly enriched. Therefore, the potential intervention targets (Ryr1 and Xirp2) in L4-L6 DRGs may be involved in prior exercise that attenuates PNP induced by CCI. This study provides crucial insights into the epigenetic regulation of PNP responses to prior exercise.

Complete genome sequence of Bifidobacterium animalis subsp. lactis BI040, a probiotic strain with multiple health benefits from the NORDBIOTIC collection.

The complete genome of Bifidobacterium animalis subsp. lactis BI040-a human stool isolate, was sequenced using Illumina and Oxford Nanopore technologies. The BI040 genome is composed of a circular 1,944,141-bp chromosome which carries genes potentially involved in vitamin synthesis and gut health.

Familial Oculoauriculovertebral Spectrum: A Genomic Investigation of Autosomal Dominant Inheritance.

Oculoauriculovertebral spectrum (OAVS) encompasses abnormalities on derivatives from the first and second pharyngeal arches including macrostomia, hemifacial microsomia, micrognathia, preauricular tags, ocular, and vertebral anomalies. We present genetic findings on a 3-generation family affected with macrostomia, preauricular tags and ptosis following an autosomal dominant pattern. We generated whole-genome sequencing data for the proband, affected father, and unaffected paternal grandmother followed by Sanger sequencing on 23 family members for the top candidate gene mutations. We performed parent and sibling-based transmission disequilibrium tests (TDTs) and burden analysis via a penalized linear mixed model, for segregation and mutation burden, respectively. Next, via bioinformatic tools we predicted protein function, mutation pathogenicity, and pathway enrichment to investigate the biological relevance of mutations identified. Rare missense mutations in SIX1, KDR/VEGFR2, and PDGFRA showed the best segregation with the OAVS phenotypes in this family. When considering any of the 3 OAVS phenotypes as an outcome, SIX1 had the strongest associations in parent-TDTs and sib-TDTs (P = 0.025, P = 0.052) (unadjusted P-values). Burden analysis identified SIX1 (RC = 0.87) and PDGFRA (RC = 0.98) strongly associated with OAVS severity. Using phenotype-specific outcomes, sib-TDTs identified SIX1 with uni- or bilateral ptosis (P = 0.049) and ear tags (P = 0.01), and PDGFRA and KDR/VEGFR2 with ear tags (both P < 0.01). SIX1, PDGFRA, and KDR/VEGFR2 are strongly associated to OAVS phenotypes. SIX1 has been previously associated with OAVS ear malformations and is co-expressed with EYA1 during ear development. Efforts to strengthen the genotype-phenotype co-relation underlying the OAVS are key to discover etiology, family counseling, and prevention.

Resolving Rapid Radiation of Voles and Lemmings (Arvicolinae: Cricetinae, Rodentia) with QuaddRAD Sequencing and Transcriptome Analysis

Voles and lemmings (subfamily Arvicolinae) remain some of the most difficult groups for disentangling phylogenetic relations owing to recent and very fast explosive radiation. The rapid radiation events are challenging for phylogenetic analysis and produce bushes of various shapes that are impossible to resolve with a straightforward approach using individual loci. Here using the quaddRAD-seq technique and transcriptomes, we tested whether data from the nuclear genome are consistent with trees inferred earlier from individual loci and from mitogenomes in topology and divergence dating. Both the transcriptome and quaddRAD-seq data convincingly place Arvicola as the earliest derivative within Arvicolini. This result is in agreement with the fossil record and conventional taxonomy. For the first time, whole-genome sequencing data resolved relations within the third radiation wave of the subfamily’s taxa, namely the tribes Arvicolini, Lagurini, and Ellobiusini, which have formed polytomy in mitochondrial trees in earlier articles. This study indicates that divergence dating is highly dependent on the number and position of calibration points in a tree and on taxa sampling. In terms of phylogenetic inference, the position of nodes with insufficient taxa representation is the most susceptible to errors.

Sherlock-Genome: an R Shiny application for genomic analysis and visualization

MotivationNext-generation sequencing technologies, such as whole genome sequencing (WGS), have become prominent in cancer genomics. However, managing, visualizing, and integratively analyzing WGS results across various bioinformatic pipelines remains challenging, particularly for non-bioinformaticians, hindering the usability of WGS data for biological discovery.ResultsWe developed Sherlock-Genome, an R Shiny app for data harmonization, visualization, and integrative analysis of WGS-based cancer genomics studies. Following FAIR principles, Sherlock-Genome provides a platform and guidelines for managing and sharing finalized sample-level WGS analysis results, enabling users to upload results, inspect analyses locally, and perform integrative analyses. It includes modules for major cancer genomic analyses, allowing interactive data visualizations and integrative analyses with other data types. Sherlock-Genome supports both local and cloud deployment, facilitating the sharing of results for related publications. This tool has the potential to be widely adopted in cancer genomics, significantly enhancing the accessibility and usability of sample-level WGS analysis results for comprehensive biological discovery and research advancements.Availability and implementationThe source code and installation instructions for Sherlock-Genome can be accessed via Github https://github.com/xtmgah/Sherlock-Genome. Documentation and data requirements for user project data can also be found on the same GitHub page.

Characterization of Streptococcus pyogenes Strains from Tonsillopharyngitis and Scarlet Fever Resurgence, 2023—FIRST Detection of M1UK in Bulgaria

Recently a resurgence of Streptococcus pyogenes infections has arisen, with concerns around the highly virulent M1UK lineage. Our aim was to characterize S. pyogenes, the immune responses it causes, and to determine the presence of the M1UK lineage in Sofia, Bulgaria. In our study, the infections were confirmed by culture testing or rapid antigen test. Identification was performed by MALDI-TOF and was followed up by antibiotic susceptibility testing (EUCAST). Virulence factors were identified using multiplex PCR and whole genome sequencing (WGS). Immune responses were measured through detection of serum complement levels, lymphocyte subsets, and cytokine profiling. Out of 82 children, 38 had scarlet fever and the rest had streptococcal pharyngitis. Strains were susceptible to penicillin (β-lactams), macrolides, clindamycin, tetracyclines, co-trimoxazole, fluoroquinolones, and linezolid. Superantigen profiles were identified: SpeA + SpeJ (45%), SpeC, and SpeI + SpeH (27.5% each). A novel Multilocus sequence typing (MLST) haplotype in the mutS gene (d90b) was found in four strains. The M1UK lineage was detected for the first time in Bulgaria. We observed an increase in complement fractions C3 and C4 and a decrease in T lymphocytes. A significant increase in the levels of IFN-γ, IL-6, and IL-10 with corresponding reduction in IL-17A were revealed. In conclusion, the studied S. pyogenes strains were characterized by their susceptibility to antibiotics and the predominance of SpeA superantigen; for the first time in Bulgaria the presence of M1UK and a novel SNP variation in the mutS gene (d90b) were found. A mixed pattern of pro- and anti-inflammatory immune responses in patients was observed.

On the ability to extract MLVA profiles of Vibrio cholerae isolates from WGS data generated with Oxford Nanopore Technologies

ObjectiveMultiple-Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) is widely used to subtype pathogens causing foodborne and waterborne disease outbreaks. The MLVAType shiny application was previously designed to extract MLVA profiles of Vibrio cholerae isolates from whole-genome sequencing (WGS) data, and provide backward compatibility with traditional MLVA typing methods. The previous development and validation work was conducted using short (pair-end 300 and 150 nt long) reads from Illumina MiSeq and Hiseq sequencing. In this study, the MLVAType application was validated using long reads generated by Oxford Nanopore Technologies (ONT) sequencing platforms. In silico MLVA profiles of V. cholerae isolates (n = 9) from the Democratic Republic of the Congo were generated using the MLVAType application on Nanopore WGS data. The WGS-derived in silico MLVA profiles were extracted from Canu (v.2.2) assemblies obtained through MinION and GridION sequencing by ONT. The results were compared to those obtained from SPAdes assemblies (v3.13.0; k-mer 175) generated from short-read (pair-end 300-bp) reference data obtained by MiSeq sequencing, Illumina.ResultsFor each isolate, the in silico MLVA profiles were concordant across all three sequencing methods, demonstrating that the MLVAType application can accurately predict the MLVA profiles from assembled genomes generated by long-reads ONT sequencers.

From Traditional Typing to Genomic Precision: Whole-Genome Sequencing of Listeria monocytogenes Isolated from Refrigerated Foods in Chile

Ready-to-eat (RTE) foods are the most common sources of Listeria monocytogenes transmission. Whole-genome sequencing (WGS) enhances the investigation of foodborne outbreaks by enabling the tracking of pathogen sources and the prediction of genetic traits related to virulence, stress, and antimicrobial resistance, which benefit food safety management. The aim of this study was to evaluate the efficacy of WGS in the typing of 16 L. monocytogenes strains isolated from refrigerated foods in Chile, highlighting its advantages in pathogen identification and the improvement of epidemiological surveillance and food safety. Using cgMLST, a cluster was identified comprising 2 strains with zero allele differences among the 16 strains evaluated. Ninety-four percent of the isolates (15/16) were serotype 1/2b, and 88% of them (14/16) were ST5. All strains shared identical virulence genes related to adhesion (ami, iap, lapB), stress resistance (clpCEP), invasion (aut, iapcwhA, inlAB, lpeA), toxin production (hly), and intracellular regulation (prfA), with only 13 strains exhibiting the bcrBC and qacJ gene, which confer resistance to quaternary ammonium. The pCFSAN010068_01 plasmids were prevalent, and insertion sequences (ISLs) and composite transposons (cns) were detected in 87.5% of the strains. The presence of various antibiotic resistance genes, along with resistance to thermal shocks and disinfectants, may provide L. monocytogenes ST5 strains with enhanced environmental resistance to the hygiene treatments used in the studied food production plant.

Targeted sputum sequencing for rapid and broad drug resistance of Mycobacterium tuberculosis.

Rapid detection of drug resistance in Mycobacterium tuberculosis (Mtb) from clinical samples facilitates the timely provision of optimal treatment regimens for tuberculosis (TB) patients. In November, 2023, the WHO released its second catalogue of resistance-conferring mutations in Mtb. Utilizing this information, we developed a single 17-plex PCR assay covering 16 key resistance genes and modified thermo-protection buffer to amplify 30 kbp DNA directly from sputum samples for nanopore sequencing. We implemented our protocol using rapid barcoding for sequencing with both a Flongle and a MinION flow cell. The single multiplex PCR assay was successfully validated on clinical sputum samples using the thermo-protection buffer. The protocol was applied to both Flongle and MinION flow cells, analyzing 12 and 40 samples, respectively. Data analysis suggested that optimal performance could be achieved by processing 6 and 12 samples with similar microscope staining scores on these two platforms. This approach facilitated rapid antimicrobial resistance (AMR) predictions directly from sputum on the day of collection or the following day, with a cost of less than $35 per sample. Compared to AMR predictions based on whole-genome sequencing (WGS) using Mykrobe and TBProfiler, our amplicon-based analysis tool, ARapidTb, demonstrated superior resistance detection capabilities. When analyzing publicly available nanopore WGS datasets for 442 isolates, ARapidTb achieved agreement rates of 95.8% and 98.0%, outperforming Mykrobe (89.4% and 98.3%) and TBProfiler (75.6% and 89.8%). Our study significantly reduces the time required for drug resistance detection, enabling quicker initiation of appropriate treatments and potentially improving patient outcomes and TB management.

Whole-genome sequencing-based characterization of Salmonella enterica Serovar Enteritidis and Kentucky isolated from laying hens in northwest of Iran, 2022–2023

BackgroundThe transmission of Salmonella spp. to human through the consumption of contaminated food products of animal origin, mainly poultry is a significant global public health concern. The emerging multidrug resistant (MDR) clones of non-typhoidal Salmonella (NTS) serovars, have spread rapidly worldwide both in humans and in the food chain. In this study NTS strains were isolated from diseased laying hens in Iran and were further studied by whole-genome sequencing (WGS) to investigate the prevalent serovars, multilocus sequence types, antimicrobial resistance and virulence genes.ResultsOut of eight isolated Salmonella spp. six were identified as S. Enteritidis serovar ST11 (n = 5) or ST5824 (n = 1), and two isolates were recognized as S. Kentucky serotype ST198 lineages. The aminoglycoside resistance gene aac(6′)-Iaa was the most frequently detected gene being present in all serovars, but it did not confer phenotypic resistance to corresponding agents (tobramycin and amikacin). All S. Enteritidis isolates carried a single GyrA D87N/Y substitution. Other identified antimicrobial resistance genes (ARGs) including tetA, floR, sul1, dfrA1, aph(3′)-Ia and double gyrA and parC mutations conferring high-level ciprofloxacin resistance (CIPR) (MIC ≥ 16mg/L) were only found in S. Kentucky isolates. The comparison of phenotypic and genotypic antimicrobial resistance (AMR) profiles revealed inconsistent results for some antibiotics. A total of 11 different Salmonella Pathogenicity Islands (SPIs) including SPIs-1, to 5, 9, 10, 13, 14, C63PI, CS54 and several virulence genes related to type III secretion system, adhesins, iron and magnesium uptake, serum and antimicrobial peptide resistance were detected among the isolates.ConclusionsOur study reports emergence of a highly MDR- CIPRS. Kentucky ST198 clone form poultry associated sources in Iran. The presence of numerous virulence determinants, SPIs and ARGs in the examined NTS isolates poses a significant risk for food safety. The inconsistencies between the genotypic and phenotypic AMR profiles indicate that WGS data alone may not be always sufficient for guiding therapeutic strategies.

Complete genome sequence of bacteriophage Godfather isolated from Microbacterium foliorum.

Microbacteriophage Godfather was collected from a soil sample in Stephenville, Texas. The 17,452-bp double-stranded genome contains 24 protein-coding genes. The genome shares >99% nucleotide sequence identity with cluster EE microbacteriophages Scamander, Danno, Kojax4, and Burgy.

Genome-Guided Identification and Characterisation of Broad-Spectrum Antimicrobial Compounds of Bacillus velezensis Strain PD9 Isolated from Stingless Bee Propolis.

The emergence of multidrug-resistant pathogens presents a significant global health challenge, which is primarily fuelled by overuse and misuse of antibiotics. Bacteria-derived antimicrobial metabolites offer a promising alternative strategy for combating antimicrobial resistance issues. Bacillus velezensis PD9 (BvPD9), isolated from stingless bee propolis, has been reported to have antibacterial activities against methicillin-resistant Staphylococcus aureus (MRSA). This study aimed to characterise and identify the antimicrobial compounds (AMCs) synthesised by BvPD9 through integration of genome mining and liquid chromatography-mass spectrometry (LC-MS) analysis. The whole-genome sequence of BvPD9 contained 4,263,351 base pairs and 4101 protein-coding sequences, with 12 potential AMC biosynthetic gene clusters. Comparative genomic analysis highlighted the unique profile of BvPD9 that possesses the largest number of unknown proteins, indicating significant potential for further exploration. The combined genomics-metabolic profiling uncovered five AMCs in BvPD9 extract, including bacillibactin, bacilysin, surfactin A, fengycin A, and bacillomycin D. The extract exhibited a broad antibacterial spectrum against 25 pathogenic bacteria, including both Gram-positive and Gram-negative bacteria, with the lowest minimum inhibitory concentration (MIC, 0.032mg/ml) against S. epidermidis ATCC 12228, and the lowest minimum bactericidal concentration (MBC; 0.128mg/ml) against MRSA ATCC 700699 and Aeromonas hydrophilia. The robust stability of BvPD9 extract was demonstrated at high temperatures, over a wide range of pH conditions (6 to 12) and in the presence of various hydrolytic enzymes. Additionally, the extract showed 50% haemolytic and cytotoxicity activity at 0.158 and 0.250mg/ml, respectively. These characteristics suggest potential applications of BvPD9 metabolites for tackling antimicrobial resistance and its applicability across diverse industries.

Locally Specific Genome-Wide Signatures of Adaptation to Environmental Variation at High Resolution in an Alpine Plant.

Microevolutionary processes shape adaptive responses to heterogeneous environments, where these effects vary both among and within species. However, it remains largely unknown to which degree signatures of adaptation to environmental drivers can be detected based on the choice of spatial scale and genomic marker. We studied signatures of local adaptation across two levels of spatial extents, investigating complementary types of genomic variants-single-nucleotide polymorphisms (SNPs) and polymorphic transposable elements (TEs)-in populations of the alpine model plant species Arabis alpina . We coupled environmental factors, derived from remote sensed digital elevation models (DEMs) at very high resolution (0.5 m), with whole-genome sequencing data of 304 individuals across four populations. By comparing putatively adaptive loci detected between each local population versus a regional assessment including all populations simultaneously, we demonstrate that responses of A. alpina to similar amounts of abiotic variation are largely governed by local evolutionary processes. Furthermore, we find minimally overlapping signatures of local adaptation between SNPs and polymorphic TEs. Notably, functional annotations of candidate genes for adaptation revealed several symbiosis-related genes associated with the abiotic factors studied, which could represent selective pressures from biotic agents. Our results highlight the importance of considering different spatial extents and types of genomic polymorphisms when searching for signatures of adaptation to environmental variation. Such insights provide key information on microevolutionary processes and could guide management decisions to mitigate negative impacts of climate change on alpine plant populations.

Gallibacterium faecale sp. nov., Isolated from Dairy Cow Feces.

A facultative anaerobic, Gram-stain-negative, non-motile, rod-shaped bacterial strain AGMB14963T was isolated from the feces of a dairy cow. A 16S rRNA gene sequence-based phylogenetic analysis revealed that strain AGMB14963T belongs to the genus Gallibacterium, with Gallibacterium salpingitidis F150T being the closest species (95.8% 16S rRNA gene sequence similarity). Whole-genome sequence analysis revealed that strain AGMB14963T had a 37.0% G + C genomic DNA content and a genome size of 2.58Mb. In addition, the strain contained 53 tRNA and 2 rRNA genes. The average nucleotide identity and digital DNA-DNA hybridization values between strains AGMB14963T and G. salpingitidis F150T were 82.3 and 24.9%, respectively. Further, strain AGMB14963T was positive for oxidase and catalase, but negative for urease. Optimal growth of strain AGMB14963T occurred at 37 ℃, pH 8, and in 0.5% NaCl-containing medium. The predominant cellular fatty acids (> 10%) in strain AGMB14963T were C14:0, C16:0, and summed feature 3. Strain AGMB14963T produced lactic acid and isobutyric acid as the major end products of glucose fermentation. Polar lipids consisted of one phospholipid and one phosphoaminolipid. According to the genomic, physiologic, and chemotaxonomic characteristics, strain AGMB14963T represents a novel species of the genus Gallibacterium, for which the name Gallibacterium faecale sp. nov. is proposed. The type strain is AGMB14963T (= KCTC 25487T = NBRC 116419T).

Ongoing measles outbreak in Romania: Clinical investigation and molecular epidemiology performed on whole genome sequences.

Romania is currently facing a prolonged measles outbreak. The aim of the study was to analyse the circulating human measles virus (HMV) strains by combining whole genome sequencing (WGS) with phylogenetic analysis, with a focus on the haemagglutinin gene. We conducted an observational study in the first five months of 2024, in which 168 patients diagnosed with measles were randomly included. We have evaluated the clinical and epidemiological differences between children and adults. Screening for samples to be sequenced was performed with a commercial kit (PrimerDesign). WGS was done on Illumina MiSeq platform and phylogenetic analysis was performed with ML FastTree. No significant epidemiological and clinical differences between patients in the two age groups were identified. WGS was successfully performed for a number of 124 HMV strains. Genotype analysis indicated all the sequences as D8, except one that was B3. Phylogenetic analysis identified two well supported clusters, suggestive for at least two local transmission networks in Romania. One large transmission network (n = 108) consisted of sequences both from adults and children. Only one sequence from outside Romania (reported in Russia in 2023) clustered within this group. Another small transmission cluster was identified (14 sequences of which 11 from patients infected in the spring of 2024 and three in 2022). A few differences between the two co-circulating viral variants/clusters were observed. The median duration of hospitalisation was 2 days longer for patients in smaller cluster compared to those in the larger one (p = 0.019). Furthermore, these two clusters presented different mutation profiles in the hemagglutinin (HA) and neuraminidase (N) genes with implications for molecular surveillance. The current measles epidemic in Romania is driven mainly by two D8 genotype variants with different mutation profiles and slightly different severity of the disease, highlighting the usefulness of sustained molecular surveillance.

Comparison of whole genome sequencing performance from fish swabs and fin clips

ObjectiveFin clipping is the standard DNA sampling technique for whole genome sequencing (WGS) of small fish. The collection of fin clips requires anaesthesia or even euthanisation of the individual. Swabbing may be a less invasive, non-lethal alternative to fin-clipping. Whether skin and gill swabs are comparable to fin clips in terms of DNA extraction quality and sequence read mapping performance from WGS was tested here on Eurasian minnows (Phoxinus phoxinus).ResultsOf 49 fin clips, all met the DNA concentration threshold of 20 ng/μl, whereas 43 of 88 swabs met this requirement. Preserving swabs in ATL buffer and treatment with Proteinase K during DNA extraction consistently raised skin swab DNA concentrations above the cut-off. All samples passed the A260/A280 absorbance ratio cut-off of 1.3. Ultimately, 93.88% of the fin clips, 30.61% of the skin, and 7.69% of the gill swabs were suitable for sequencing. Mapping performances of all three tissues were comparable in reads passing quality filtering, percentage of reads mapping to the P. phoxinus reference genome, and coverage. Overall, skin swabs treated with Proteinase K during extraction, can match fin clips in WGS performance and represent a viable non-invasive DNA sampling alternative.

Clinical Features of Human Parvovirus B19-Associated Encephalitis Identified in the Dakar Region, Senegal, and Viral Genome Characterization

Neurological manifestations associated with human parvovirus B19 (B19V) infections are rare and varied. Acute encephalitis and encephalopathy are the most common, accounting for 38.8% of all neurological manifestations associated with human B19V. Herein, we report on the clinical features of 13 laboratory-confirmed human cases of B19V-associated encephalitis in Senegal in the framework of a hospital-based surveillance of acute viral encephalitis conducted from 2021 to 2023. Overall, B19V was detected from 13 cerebrospinal fluid samples using specific real time PCR. The mean age was 16.7 years among B19V-positive patients, with a higher prevalence in 0–5-year-old children and the sex ratio (male/female) was 2.25. The B19V-positive patients mainly exhibited hypoleukocytosis, normal glycorrhachia, and normal proteinorrachia in the cerebrospinal fluid. While the main neurological symptoms included meningeal and infectious syndromes. Furthermore, three complete B19V genome sequences were successfully characterized using next-generation sequencing. The newly characterized sequences belonged to the genotype 1a and represent, to date, the first complete B19V genome sequences from Senegal. These sequences could be useful not only in future phylodynamic studies of B19V but also in the development of prevention or treatment countermeasures. Our study is noteworthy for the identification of acute B19V-associated encephalitis in Senegal More investigations on the risk factors associated with B19V transmission in Africa are warranted.