Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomic surveillance is crucial for understanding viral evolution and guiding public health responses. However, many countries, particularly in Central America, have limited sequencing capacity, resulting in scarce and delayed data. This study addresses this gap by analysing 320 SARS-CoV-2 genomes sequenced from a major diagnostic centre in Guatemala City, Guatemala, between April and August 2021. Clade 21J (Delta) was predominant (46.2%), followed by 19B (29.4%) and 20J (Gamma, 6.6%). The most reported symptoms were cough, headache, malaise and myalgia/arthralgia. Among patients infected with the Delta variant, 39.9% reported being contacts from a confirmed case, less than reported by the patients infected with non-Delta variants (53.2%, P=0.017). The proportion of signs and symptoms was similar among these two groups, except for the history of fever, which was increased by ~twofold in the Delta group. This research contributes valuable genomic and epidemiological data to elucidate SARS-CoV-2 variant dynamics in Central America and emphasizes the importance of global genomic surveillance for pandemic preparedness and response.

The elasmobranch dermal microbiome may be important for buffering effects of environmental stress on host health and population viability via functional metabolic interactions. Dermal microbiomes among elasmobranch orders co-vary with host phylogeny (phylosymbiosis), indicating functional co-evolution with their hosts at deep phylogenetic splits. However, the extent of phylosymbiosis and potential for functional co-evolution within particularly species-rich elasmobranch families remains unknown. Here, we re-analyse Illumina amplicon sequence data from the 16S rRNA gene from eight Carcharhinid shark species (plus one Ginglymostomatid outgroup) across six independent studies and explicitly examine the extent of phylosymbiosis in dermal microbiomes within this family. We found extensive divergence in operational taxonomic unit (OTU) abundance and functional metabolic capacity between studies, driven by disparity in OTU sharing and probably reflecting geographical and seasonal factors. Total microbiome structure was incongruent with shark phylogeny, providing no evidence for phylosymbiosis when considering all species and OTUs. However, using bootstrapping and subsampling methods, we identified several subsets of OTUs where Bray–Curtis dissimilarity supported perfect topological congruence with shark phylogeny or strong associations with phylogenetic distances, but not both. Partial Mantel tests identified ten candidate OTUs that supported a moderately strong signal of phylosymbiosis across all shark species and included the immunostimulant skin symbiont genera Lactiplantibacillus and Alcaligenes. Overall, this provides provisional evidence for phylosymbiosis in a minority fraction of the elasmobranch dermal microbiome within the Carcharhinidae family and will necessitate coordinated large-scale studies to establish the generality of these findings.

Introduction. Alongshan virus (ALSV) is a tick-borne Flaviviridae. It has been detected in Ixodes ricinus and Ixodes persulcatus across China, Russia, Finland, Switzerland and Germany.Hypothesis/Gap Statement. However, the clinical relevance and the pathogenicity of ALSV in humans remain unclear. Sensitive and specific molecular tools are needed to support surveillance and to enable clinical investigations of ALSV in suspected cases of tick-borne meningoencephalitis.Aim. We aimed to develop, validate and integrate two ALSV-specific TaqMan real-time quantitative PCR (qPCR) assays on our open, high-throughput molecular diagnostic platform.Methodology. We designed assays targeting conserved regions of the NS3 (helicase-protease) and NS5 (RNA-dependent RNA polymerase) genes, incorporating degenerate bases and locked nucleic acid modifications where needed to accommodate documented viral diversity and to harmonize the annealing temperature with TaqMan probe-related technologies and our platform. Analytical sensitivity and reproducibility were assessed using synthetic plasmids carrying the targets; specificity was evaluated against 41 cerebrospinal fluid (CSF) pathogens and 30 winter CSF specimens from patients with suspected central nervous system infection. ALSV-positive Swiss tick extracts served as biological positives.Results. Detection frequencies for NS3 PCR were 100%, 100%, 92%, 72%, 20%, 28 and 0% at 1,000, 100, 10, 5, 2, 1 and 0.1 copies per reaction, respectively. For NS5, the detection frequencies were 100%, 100%, 92%, 88%, 40%, 20% and 0% at the same concentrations. Using a priori definition of limit of detection (LoD) as ≥95% positive replicates, LoD was 100 copies per reaction for both real-time qPCRs. However, as the PCRs are performed in triplicate in our platform, the LoD can be estimated at five copies per reaction for the NS3 real-time qPCR and two copies per reaction for the NS5 PCR. Intra- and inter-run reproducibility across five independent runs met diagnostic standards. Specificity was 100% (71/71). ALSV-positive tick samples were detected by both assays, with lower cycle thresholds for NS5.Conclusions. We validated two ALSV real-time qPCR assays suitable for integration into open molecular diagnostic platforms. These assays enable syndromic testing alongside other encephalitis-associated viruses (e.g., Tick-borne encephalitis virus and West Nile virus) and will facilitate timely clinical management of suspected cases, high-throughput tick surveillance and future clinical studies of potential ALSV pathogenic role.

Xanthomonas spp. are increasingly recognized as a global threat to agriculture, impacting a broad range of economically important crops. We report the whole-genome sequences of six Xanthomonas strains isolated from tomato and pepper plants in Turkey that were experiencing symptoms of bacterial spot disease. Phylogenomic analysis with representative Xanthomonas genomes from each species revealed that three of these strains belonged to Xanthomonas perforans, two to Xanthomonas euvesicatoria and one to Xanthomonas campestris. We then analysed the phylogenomic relatedness of these strains with other strains from these respective species and characterized their type III secreted effector content. These genomic data represent a valuable resource for understanding the genetic diversity and local epidemiology of bacterial spot disease in Turkey.

The Ralstonia solanacearum species complex (RSSC) is a globally distributed group of Gram-negative, soil-borne bacteria that cause wilt diseases on a broad range of hosts. Due to these pathogens’ impact on economically important plant species, there is a need for consolidated and visualized information on RSSC pathogen isolation data. We developed an interactive dashboard designed to allow users to explore the diversity and biogeography of the RSSC. The dashboard visualizes data in the form of maps, charts and tables, with a variety of user-interactive filters for taxonomic, geographic and host of isolation specifications. This Ralstonia Wilt Dashboard will aid in communicating knowledge to researchers, regulatory scientists and other stakeholders to improve disease control and regulation. This report highlights the deployment of the Ralstonia Wilt Dashboard and provides four case studies that address focused, scientific questions (https://ralstoniadashboard.shinyapps.io/RalstoniaWiltDashboard/).

Assessment of the pathogenic potential (virulence and toxicity) in non-pathogenic bacterial species is a challenge as it relies on methods developed for assessment of species known to be pathogenic. Here, we have applied and evaluated some of these methods on industrially relevant bacteria to differentiate between 'true' virulence factors applying only to pathogens and niche factors being defined as promoting colonization and survival rather than pathogenicity and as being present also in non-pathogenic bacteria. We examined the pathogenicity of 49 strains from 9 industrially relevant bacterial species (Lactobacillus gasseri, Lactobacillus jensenii, Lactobacillus delbrueckii, Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum, Latilactobacillus curvatus, Ligilactobacillus salivarius, Staphylococcus carnosus and Staphylococcus xylosus), including 14 clinical isolates of the same species, through genomic screening and phenotypically through assays established for pathogenic bacteria. The genomes were screened against the Virulence Factor Database (VFDB), and thresholds (>80% nucleotide or protein identity, >70% coverage) provided by the European Food Safety Authority (EFSA) were adopted to differentiate between genes of potential concern and genes of no concern. Core genome analysis was performed to determine whether the clinical isolates were phylogenetically related to the industrial isolates. The genotypic assessment did not reveal the presence of true virulence factors in the examined strains, and in the core genome analysis, the clinical isolates could not be distinguished from the industrial strains. Furthermore, cytotoxicity toward Vero cells, negative impact on Caco-2 cell viability and haemolytic activity on blood agar plates were examined, and none of the tested strains exhibited any activity in these assays. Overall, the results suggest that VFDB screening with the EFSA thresholds can be used to differentiate between true virulence factors and niche factors. Furthermore, the use of phenotypic assays supports the genotypic assessment, albeit expert knowledge is required to interpret the results.

Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disorder in preterm infants with a high mortality rate. The aetiology of NEC appears to be multifaceted; however, gut microbiota dysbiosis likely plays a significant role. This systematic review aimed to describe how the gut microbiota of preterm infants with NEC differs from infants without NEC (PROSPERO: CRD42022344126). Databases were searched from inception to 22 June 2022 to identify eligible studies that examined the gut microbiota composition of preterm infants with and without NEC using sequencing methods. Results were described narratively. We identified 28 eligible studies. Overall, findings were heterogeneous and no single gut microbiota signature was associated with NEC in all studies. Importantly, 3 studies reported no difference in the gut microbiota composition between NEC and healthy infants, while studies reported a difference using one or more analytical method (i.e. alpha diversity, beta diversity or differential abundance analysis). Of note, NEC (or development of NEC) was positively associated with increased detection and/or abundance of Enterobacteriaceae (n=11 studies), Clostridium (n=8) and Proteobacteria (n=2). The taxa most frequently associated with NEC (Enterobacteriaceae, Clostridium, and Proteobacteria) may play an important role in the pathogenesis of NEC and should be further explored.

Fresh produce-associated outbreaks of the foodborne pathogen Escherichia coli O157 are responsible for a number of disease cases, hospitalizations and deaths. In many cases, the source of contamination can be linked to the growing media of the food, although pathogen detection is problematic in these complex soil ecosystems. In this study, direct quantitative real-time PCR without pre-enrichment was used to detect 310 copies of the Tir gene, using a primer sequence specific to E. coli O157, in horticultural compost purchased from a commercial supplier. The pathogen could not be cultured on selective media but was visualized using peptide nucleic acid fluorescence in situ hybridization and cell elongation viability assay, confirming the viability. Enumeration of elongated E. coli O157 determined that there were 205 live cells per gram of compost. The nonculturability and confirmation of viability of the pathogen indicates its viable but nonculturable (VBNC) status. The detection of VBNC foodborne pathogens in environmental samples challenges current understanding of the nature of foodborne pathogen contamination.

Ticks are arthropod vectors that transmit pathogens important to human and animal health. The objective of this work was to identify Uukuvirus lihanense in the metatranscriptome of hard ticks. Between October 2022 and June 2023, ticks were collected from rural areas of the Colombian Caribbean area of the departments of Córdoba and Cesar. High-throughput sequencing (next-generation sequencing) was performed using MGI’s DNBSEQ-G50RS. Bioinformatics analyses were performed in Galaxy, diamond and IQ-TREE2. A total of 766 ticks were collected; 87.33% (669/766) were Rhipicephalus microplus, 5.4% (42/766) Dermacentor nitens, 4.2% (32/766) Rhipicephalus sanguineus and 3.0% (23/766) Amblyomma dissimile. Complete and partial L and S segments of Uukuvirus lihanense (LITV) were detected in the metatranscriptome of A. dissimile, D. nitens and R. microplus. The LITV sequences found are phylogenetically related to those detected in R. sanguineus and A. variegatum from the French Antilles, in R. microplus from Trinidad and Tobago and R. microplus from Brazil. LITV was identified in D. nitens and R. microplus; the first report was in A. dissimile. Although LITV is not considered necessary in public health, the virus belongs to the Phenuiviridae family, which includes viruses of public health importance, such as Dabie banda-virus and Bandavirus heartlandense.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection triggers complex host responses, including alterations in RNA transcription and modification. Understanding these changes is crucial for elucidating viral pathogenesis and identifying potential therapeutic targets. We used direct RNA sequencing to comprehensively profile the transcriptomic and epitranscriptomic landscapes of human HEK-AT cells infected with SARS-CoV-2 at 8 h post-infection, compared to mock controls. We analysed viral and host transcriptomes, focusing on gene and transcript expression, isoform usage and RNA m6A modifications. Viral RNA sequencing reads showed 3′ end-biassed coverage indicative of subgenomic RNA synthesis, with high expression of N gene subgenomic RNA reads. Sixteen m6A modification sites were consistently identified in the viral genome, primarily within the ORF1ab and S genes. In the human transcriptome, we found 254 positions with significantly altered m6A modification rates, with 119 showing decreased modification and 135 showing increased modification in infected cells. Genes with decreased m6A modifications were enriched in the neurotrophin signalling pathway. Transcript-level analysis identified 19 upregulated and 12 downregulated transcripts. Notably, transcript discovery and quantification revealed a novel isoform of the HIST1H2BK gene, which was significantly more expressed in infected cells compared to mock controls. Isoform switching analysis revealed 24 significant switches involving 21 genes, implicating mitochondrial reprogramming and immune-related pathways. In conclusion, this study provides a detailed, direct RNA sequencing-based characterization of host–virus RNA interactions, revealing key insights into SARS-CoV-2 infection mechanisms and potential therapeutic targets.