Search Results (243)

Background: Wastewater-based surveillance (WBS) has emerged as a valuable tool for population-level monitoring of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) transmission, yet the interplay between sampling strategies and disease prevalence in shaping detection performance remains ambiguous. We investigated how grab and composite sampling influence SARS-CoV-2 ribonucleic acid (RNA) detection dynamics and predictive lag times across high- and low-prevalence communities in Selangor, Malaysia. Methods: A 28-week longitudinal study was conducted in Selangor, Malaysia, comparing grab and composite wastewater sampling in communities with high and low Coronavirus disease 2019 (COVID-19) prevalence. SARS-CoV-2 RNA in 348 samples was quantified using digital Reverse Transcription Polymerase Chain Reaction (RT-dPCR), and viral lineages were characterized by Nanopore sequencing. Detection sensitivity and lead times relative to reported cases were evaluated. Results: In low-prevalence settings, grab sampling showed higher detection sensitivity than composite sampling (92.0% vs. 70.0%), whereas both methods achieved similarly high detection in high-prevalence areas (>97.0%). Lag-time analysis indicated that grab sampling in high-prevalence settings was significantly associated with case trends at potential two-week lead (p = 0.024), while composite sampling in low-prevalence settings showed the strongest association at a potential one-week lead (p = 0.0022). Overall, lag structures varied by both sampling strategy and prevalence context. Both sampling approaches captured the replacement of Omicron sublineages (XBB.1.5, XBB.1.9.1, XBB.1.16) and identified additional circulating variants, including EG.5, that were not captured in the available clinical sequencing dataset during the same period. Conclusions: These findings reveal that local transmission intensity is associated with the utility of different sampling designs. Context-specific optimization of WBS sampling strategies enhances sensitivity, reduces detection lag, and strengthens early warning and genomic-tracking capacity in public health surveillance frameworks. Full article

Highly mutated Omicron sub-lineages JN.1 and NB.1.8.1 harbor extensive spike changes, but their impact in preterm infants is poorly documented. We report a preterm male infant with three hospitalizations in seven weeks: severe SARS-CoV-2 ARDS at 40 days of life (DOL 40) requiring ventilation caused by JN.1.16, HCoV-OC43 infection at DOL 65, and a mild SARS-CoV-2 reinfection at DOL 87 due to NB.1.8.1, the first detection of this variant in Tunisia. Spike analysis showed a shared JN.1 backbone but distinct N-terminal and receptor-binding domain changes, supporting intra-Omicron reinfection driven by antigenic divergence and immature immunity and underscoring the value of pediatric genomic surveillance, including phylogenetic placement of case genomes within local Omicron diversity. Full article

Streptococcus dysgalactiae subsp. equisimilis (SDSE) is an emerging cause of invasive disease, yet contemporary genomic data from Canada remain scarce. We investigated 56 cases of invasive SDSE infection identified between 2018 and 2022 in two major tertiary care teaching hospitals in Toronto, Ontario, and characterized 49 corresponding isolates by whole-genome sequencing. Nearly three-quarters of infections were caused by the globally expanding ST20 emm type stG62647 lineage. Patients infected with this lineage were significantly older than those infected with non-ST20 lineages across both bloodstream and non-blood infections. Core-genome phylogenetic analysis revealed a highly clonal ST20 cluster, although two isolates had divergent emm types suggesting recombination at the emm locus. Non-ST20 lineages were numerically smaller and genetically more heterogeneous, including distinct sublineages within ST3 and ST34. All isolates were susceptible to β-lactams and vancomycin. Resistance to tetracycline, erythromycin, and clindamycin was detected in a subset of isolates and was associated with genes tetM, tetO, ermA, ermB, and msrD. Several antimicrobial resistance determinants were located on mobile genetic elements, including integrative and conjugative elements. Our findings provide a contemporary genomic view of invasive SDSE in Toronto, highlighting the dominance of the ST20 stG62647 lineage in agreement with recent global observations. Full article

Background/Objectives: Influenza, RSV, and SARS-CoV-2 co-circulate and evolve under immune and therapeutic pressures, complicating decision-making for both vaccine formulation and antiviral use. Fragmented, pathogen-specific sequencing approaches limit cross-virus comparability. Methods: We applied a standardized, multiplexed, amplicon-based next-generation sequencing (NGS) workflow to 34 diagnostic specimens (Ct < 35) positive for influenza A/B, RSV-A/B, or SARS-CoV-2. Sequencing libraries were generated and run on an Illumina MiSeq platform (2 × 250 bp). Although the wet-lab workflow is standardized across pathogens, consensus generation and annotation utilized two different analysis environments: Geneious Prime for influenza and MicrobioChek for RSV and SARS-CoV-2. Quality metrics included genome breadth and depth of coverage. Results: Near-complete genomes (mean coverage ≥98%) were recovered for all samples. Influenza A(H1N1)pdm09 sequences clustered in clade 6B.1A; A(H3N2) clustered in subclade 3C.2a1b.2a.2; and influenza B belonged to the Victoria lineage V1A.3a.2. RSV sequences were assigned to Nextclade clades A.D.5.1, A.D.1.10, A.D.2.1, and A.D.3 (RSV-A) and to B.D.4.1.3 and B.D.E.1 (RSV-B), consistent with the ON1 (RSV-A) and BA (RSV-B) genotypes prevalent in recent seasons. Clinically relevant mutations included changes in the influenza HA site and neuraminidase substitutions, RSV F-protein polymorphisms, and spike protein substitutions associated with recent Omicron sublineages (L455F/S, F456L) in SARS-CoV-2. Conclusions: A unified amplicon–NGS approach yields harmonized genomic data across respiratory viruses, enabling timely detection of antigenic drift and resistance markers while supporting integrated, cross-pathogen surveillance. Full article

PRRSV-2 represents a major threat to the swine industry. Canada is one of the world’s leading pork producers and a major trading partner of live pigs with the United States, yet PRRSV-2 evolutionary dynamics in these two countries are often studied independently, partly due to limited publicly available sequence data from Canada. We analyzed more than 3000 PRRSV-2 ORF5 sequences collected between 2000 and 2024 from five Canadian provinces. Thirteen previously described sub-lineages were detected in Canada, while approximately one-third of the sequences could not be assigned to any known sub-lineage. Phylogenetic analyses incorporating global reference sequences revealed that most unclassified sequences clustered into four distinct monophyletic clades, exhibiting genetic distances greater than 9.5% from recognized sub-lineages. We propose two new sub-lineages, 1K and 1L, corresponding to clades that were prevalent and persistent over time, whereas the remaining two clades were rare and last detected in 2021. We reconstructed cross-border transmission histories and found that sub-lineages 1C, 1H, 1I, 1K, and 1L originated in Canada, whereas 1A, 1B, 1E, and 1F originated in the United States. Transmission patterns varied across sub-lineages, ranging from unidirectional to bidirectional movement. Our findings refine PRRSV-2 classification and provide insights to inform targeted surveillance, particularly at national borders. Full article

Recent reports have advocated the global resurgence of human metapneumovirus (hMPV) infections in the post-COVID-19 era. Considering the absence of relevant data from Greece, the present study aimed to explore the molecular epidemiology and possible resurgence of significant genotypic variants. Whole genome and F gene-specific sequencing were implemented in order to obtain complete information on the genotype and lineage distribution of circulating hMPV strains in Greece. The results showed a statistically significant increase in monthly positivity rates in 2025, especially from February to April, compared to the respective period in 2024. Overall, 21 strains were classified as genotype A2.2.2 (42.9%) and 27 as genotype B2 (55.1%), whereas only one strain belonged to genotype B1. The G gene of most completely sequenced A2.2.2 strains harbored a 111nt duplication sequence and a genotype-specific pattern of N-glycosylation sites. Maximum likelihood and time-scaled, Bayesian phylogenetic analyses demonstrated the dominance of specific sub-lineages at the regional level and international transmission events. This complex epidemic pattern in conjunction with the differential evolutionary pressure exerted on the hMPV genes, advocates continuous surveillance of hMPV epidemiology with multiple genes, or complete genome sequencing methodologies. Full article

Highly pathogenic influenza A viruses of the H5 subtype continue to diversify worldwide through mutation and genetic reassortment, generating novel variants with unpredictable consequences under the One Health approach. Between 2024 and 2025, five outbreaks of avian influenza A viruses were detected in backyard poultry across Michoacán, Estado de México, and Ciudad de México. We conducted molecular and genetic characterization of five highly pathogenic H5N2 viruses isolated from these events. All cases tested positive for influenza A virus and the H5 hemagglutinin, exhibiting high pathogenicity with intravenous pathogenicity index values ranging from 2.88 to 3.0. Whole-genome sequencing revealed novel reassortants containing hemagglutinin from Eurasian H5N1 clade 2.3.4.4b and neuraminidase from the endemic Mexican H5N2 lineage. The viral genome of the isolate from Michoacán contained six segments derived from Eurasian H5N1 viruses introduced into North America in 2021–2022, while nucleoprotein and neuraminidase originated from Mexican H5N2 viruses. In contrast, viruses from Estado de México and Ciudad de México contained five H5N1-derived segments and incorporated polymerase basic protein 1, nucleoprotein, and neuraminidase from low-pathogenic H5N2 viruses circulating in 2024. Phylogenetic analyses confirmed the emergence of a distinct H5N2 Mexican sublineage, providing evidence of active viral reassortment and local evolutionary processes in Mexico. Full article

Objective: The aim of this retrospective study was to evaluate clinical and laboratory characteristics in adult patients with neuroinvasive West Nile virus (WNDD). We also studied the phylogeny and molecular characteristics of some of the WNV strains. Methods: A retrospective analysis was conducted at “Annunziata” Hub Hospital, a secondary referral facility in Calabria region, in Southern Italy. Sample pre-processing, sequencing and bioinformatic analyses were carried out at IRCCS Sacro Cuore Don Calabria Hospital in Negrar di Valpolicella, Verona, Veneto region in North-East Italy. Results: Nine cases of WNDD were analyzed, involving eight males and one female, with a mean age of 70.33 years (range 60–85). The overall average hospital stay was 20.6 days (range 6–46). Six patients made a full recovery after a mean of 35.3 days of acute care. Thirty-day mortality rate was 23%. VNDD in some of our patients manifested itself in the form of cerebral hemorrhage (ICH) in three patients, causing lethality in two patients and other unusual manifestations, such as Guillain–Barré syndrome with fatal outcome and severe facial palsy. Phylogenetic analysis shows that our sequences are closely related to other southern-Italian and cluster with Central–Southern–Eastern European sequences, while being evidently separated from northern Italian and Central–Western European ones, belonging to the sub-lineage 2a of the WNV-2, clustering with sequences from the Central–South–Eastern clade, mainly to Hungary. Conclusions: Cerebrovascular complications of WNE may be an important clinical manifestation of WNV neuroinvasive infection. Preliminary data do not allow us to determine whether our strains, closely related to other southern-Italian and cluster with Central–Southern–Eastern European sequences, really presented an increased neurovirulence. Full article

Background/Objectives: Methicillin-resistant Staphylococcus aureus (MRSA) is a major causative pathogen in Republic of Korea. While numerous variants exist, the long-term evolutionary history of indigenous lineages remains unclear. Therefore, this study aimed to reconstruct the high-resolution population structure of Korean MRSA. Methods: A total of 191 MRSA clinical isolates collected between 1999 and 2025 were obtained from four Korean biobanks. Whole-genome sequencing was conducted and international MRSA genomes from the National Center for Bioinformatics were used as a control group. A genome-wide association study, including single-nucleotide polymorphism (SNP)-based phylogenomic analysis, principal component analysis (PCA), and ADMIXTURE, was performed for distribution analysis. A time-scale epidemiological analysis was conducted using SNP-based phylogenetic data. Additional profiling was performed via core genome multilocus sequence typing (cgMLST) for comparison with the SNP-based phylogenomic results. Finally, antimicrobial resistance and virulence factor genes were annotated using the ResFinder and VirulenceFinder databases. Results: Phylogenetic analysis identified five major clades: 1 (ST5), 2 (ST6), 3 (ST72), 4 (ST1/ST188), and 5 (ST8/ST239/ST254). Time-scaled analysis estimated that these major clades began to diverge in the early 20th century (e.g., Clade 1 around 1918). Notably, Korean ST5 isolates formed a sublineage distinct from North American strains, characterized by unique AMR profiles and divergence in the 1960s. ST72 formed an independent clade that was phylogenetically closer to clade 4 (ST1/ST188) than to the canonical CC8 group (clade 5). Furthermore, the ST1 isolates showed a temporal split into an older lineage and a recent sublineage, with expanded AMR pro-files. Conclusions: By integrating time-scale phylogenetics with cgMLST, we elucidated the evolutionary history and transmission dynamics of Korean MRSA. Full article

South China’s freshwater biodiversity has been shaped by Quaternary climatic oscillations and persistent geological barriers. We investigated the phylogeography and conservation implications of the primary freshwater fish Microphysogobio kachekensis across mainland China and Hainan Island using mitochondrial (cyt b and control region) and nuclear (RAG2 and rpS7-1) markers from 200 individuals. Mitochondrial analyses recovered two major lineages and multiple sublineages largely structured by drainage basins, whereas nuclear data resolved four geographically concordant lineages. Population differentiation was strong (high F_ST), and SAMOVA/AMOVA supported major barriers restricting gene flow, including the Qiongzhou Strait, Gulf of Tonkin, Yunkai Mountains, and Nanling Mountains. Ancestral-area reconstruction inferred the Pearl River region as the most likely source area, followed by dispersal to northern Hainan and subsequent expansion to southern Hainan and the Red River, with additional northward expansion to the Zhejiang–Fujian region. Despite high haplotype diversity, within-population nucleotide diversity was low, consistent with long-term river isolation and complex demographic history. We propose six ESUs and four MUs for evolutionarily informed conservation and to guide stock enhancement in southern China. Full article

West Nile virus lineage 2 (WNV-2) is a growing public health concern in Europe causing West Nile fever or West Nile neuroinvasive disease (WNND) with substantial morbidity and mortality; however, genomic data from southern Italy are limited despite recent expansion of autochthonous transmission. The aim of the study was to characterize the phylogenetic and molecular features of the WNV-2 strain responsible for the first autochthonous human infection reported in Calabria (2023), and two more additional WNND cases detected in 2024. Full WNV-2 genomes were generated from the three cases. Phylogenetic analysis was performed using all publicly available WNV sequences up to September 2025. Amino acid changes in the polyprotein were compared with known WNV-2 lineage and sub-lineage signatures. The three sequences formed a monophyletic group within sub-lineage WNV-2a, clustering with strains circulating in Central-South-Eastern Europe and showing closest affinity to Hungarian sequences. Non-synonymous substitutions characteristic of the Hungary 578/10 strain (NS2B-119I, NS4B-14G, NS4B-49A, and NS5-298A) were identified and were absent from Central-Northern-Western European and previously reported Italian sequences. Additional substitutions (E-159T, E-399R, and NS3-249P) corresponded to signatures from a fatal WNV-2 infection in a Great Grey Owl in Slovakia. Our study provides the first report of Central-South-Eastern European WNV-2 circulation outside Eastern Europe, supporting its likely spread through the Balkans into Italy by 2022. These findings underscore the rapid spread of WNV-2 in newly affected areas and highlight the critical need for sustained molecular surveillance. Full article

The global rise of multidrug resistance (MDR) across bacterial pathogens poses a severe threat to public health, with the food chain serving as a critical reservoir and transmission route for resistant clones. This study investigated the genomic epidemiology of Staphylococcus aureus in retail pork from Beijing, China, and Copenhagen, Denmark, with a focus on MDR patterns and associated genetic elements. Among 134 isolates, the livestock-associated clonal complex CC398 was the dominant lineage (24.63%) and exhibited a high burden of MDR (48.48%), carrying resistance genes to β-lactams (blaZ and mecA), tetracyclines (tetM and tetK), and aminoglycosides. Notably, MRSA isolates displayed a significantly higher MDR prevalence (73.53%) compared to MSSA isolates (18.00%), underscoring methicillin resistance as a key marker for broader resistance phenotypes. Phylogenetic analysis revealed the segregation of CC398 into distinct sub-lineages, with the livestock-associated branch consistently linked to a characteristic tetracycline–β-lactam MDR profile. Furthermore, high frequencies of mobile genetic elements, such as the rep16 plasmid, were associated with MDR dissemination in CC398. These findings highlight retail meat as an important reservoir for MDR S. aureus and illustrate how livestock-adapted clones contribute to the environmental burden of antimicrobial resistance. This study underscores the need for integrated One Health surveillance that connects veterinary, food safety, and human health sectors to monitor and contain the spread of MDR bacteria across ecological niches. Full article

Hypera postica (Gyllenhal) is a major pest of alfalfa. We combined mitochondrial COI and CytB gene sequences to characterize the genetic diversity of 20 geographic populations of H. postica across Xinjiang, China, and to elucidate their lineage relationships at both regional and global scales. We found that Nucleotide diversity (Pi) was markedly higher in western Xinjiang populations (Pi > 0.016), specifically Wusu (0.023), Tekes (0.023), Jinghe (0.023), Wenquan (0.021), Bole (0.021), Habahe (0.020), Nilka (0.020), Tacheng (0.019), Toli (0.018), Altay (0.017), Emin (0.016), Xinyuan (0.016), and Zhaosu (0.016), whereas central Xinjiang populations exhibited substantially lower diversity (Pi < 0.014), including Shawan (0.014), Qitai (0.011), Jimsar (0.007), Urumqi (0.004), Hutubi (0.003), Fukang (0.001), and Manas (0.001). Pairwise F_ST analysis revealed pronounced genetic divergence between the western Xinjiang group (Altay, Bole, Wenquan, Tacheng, Emin, Toli, Nilka, Xinyuan, Tekes, Zhaosu) and the central Xinjiang group (Qitai, Urumqi, Fukang, Habahe, Hutubi, Jimsar, Shawan, Manas). At the global level, H. postica can be divided into two major phylogroups: the Western and Eastern lineages. All Xinjiang populations belong to the Eastern lineage. Haplotype network analysis identified two distinct sublineages, western and central Xinjiang, with H2 and H26 as their respective dominant shared haplotypes; both are unique to China. Both maximum likelihood (ML) and Bayesian phylogenetic trees robustly support the central Xinjiang lineage as a distinct clade. Neutrality tests provided strong evidence of recent demographic expansion across the Xinjiang H. postica population as a whole (Fu’s Fs = −21.987, p < 0.05), with particularly pronounced signals in Hutubi (HTB: Tajima’s D = −1.966, Fu’s Fs = −0.781, p < 0.05), Jimsar (JMSE: Tajima’s D = −2.176, Fu’s Fs = −0.962, p < 0.01), and Wenquan (WQ: Fu’s Fs = −11.159, p < 0.01). Our results reveal a clear phylogeographic split within Xinjiang H. postica populations, comprising western and central sub-lineages, with the western sub-lineage likely representing ancestral lineage. The western Xinjiang sub-lineage appears to be shaped primarily by mountainous topography, whereas the central Xinjiang sub-lineage likely results from the combined effects of piedmont plain geography and infection with the endosymbiont Wolbachia strain wHypera4. Full article

Pseudomonas aeruginosa is an opportunistic pathogen commonly associated with nosocomial infections and environmental dissemination. Among its high-risk clones, ST244 is notable for its global distribution and distinctive genomic traits. This study reports whole-genome sequencing of ten ST244 isolates from hospitalized patients and wastewater in a healthcare complex in Southeastern Brazil. Genomic comparisons revealed a highly conserved clonal group, with nine isolates forming a tight monophyletic cluster based on rMLST, SNP phylogeny, and average nucleotide identity (>99.5%). One isolate showed close phylogenetic proximity to strains from Asia and North America, suggesting international dissemination. Serotype analysis revealed both O5 and O12 variants, indicating intra-lineage antigenic diversity. Resistance profiling identified multidrug-resistant phenotypes carrying carbapenemase genes (bla_OXA-494, bla_OXA-396) and diverse insertion sequences (ISPa1, ISPa6, ISPa22, ISPa32, and ISPa37), facilitating horizontal gene transfer. Virulence gene analysis showed conserved elements related to adhesion, iron uptake, secretion systems, and quorum sensing, while the cytotoxin gene exoU was absent. These results highlight clonal persistence, possible intra-hospital transmission, and links to globally circulating ST244 sublineages. Our findings underscore the importance of genomic surveillance to track high-risk P. aeruginosa clones at the clinical–environmental interface. Full article

We describe RNA-Seq analyses conducted on nasopharyngeal swabs collected from 37 patients admitted to an Australian intensive care unit from October 2022 to August 2023. During this time, the dominant omicron sublineage infections broadly progressed from BA.5 to BA.2-like, to XBB-like, then XBC, consistent with global trends. Viral load and patient metadata correlations indicated this cohort was broadly representative of severe COVID-19 patients. Human gene expression analyses were complicated by the large range (>5 log) and variability in viral reads. Nevertheless, the comparison of XBC and BA.5 samples that had comparable viral read counts, revealed differentially expressed genes and a cellular deconvolution signature that indicated increased targeting of ciliated epithelial cells by XBC. To obtain more evidence for increased targeting of ciliated epithelial cells by the later omicron sublineage viruses, a series of mouse strains were infected with a BA.5 or a XBB isolate. Increased infection of the nasal turbinates and ciliated epithelial cells by XBB was demonstrated by viral titrations and immunohistochemistry, respectively. Compared with previous lineages, the omicron lineage showed increased targeting of ciliated epithelia in the upper respiratory tract, with the data presented herein suggesting this trend continued for the omicron sublineages. Full article