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We report the genome of Vanta, a lytic bacteriophage isolated from wastewater using Pseudomonas aeruginosa PA14. Phage Vanta is a Yuavirus with a circularly permuted, 61,663-bp, 64.5% guanine-cytosine, dsDNA genome. Head width is 56 ± 2 nm, and tail length is 133 ± 5 nm. Vanta plaques on two drug-resistant clinical P. aeruginosa isolates.
We report the genome of Luminis, a lytic bacteriophage isolated from wastewater using Pseudomonas aeruginosa mPAO1Δpf4Δpf6. Phage Luminis is a Yuavirus with 61,617 bp of circularly permuted dsDNA and 64.4% GC content. In addition to reference strains, Luminis plaques on two drug-resistant clinical isolates of P. aeruginosa.
The health benefits of dietary soy isoflavones are largely mediated by specific microbial metabolites, such as O-desmethylangolensin (O-DMA). However, the diversity and application potential of O-DMA-producing strains remain poorly explored, primarily due to the limited availability of isolated strains, narrow ecological sources, and a lack of practical applications. In this study, an O-DMA-producing bacterium, designated strain FRJF5, was isolated from Chinese stinky tofu under anaerobic conditions and was identified as Clostridium beijerinckii. The biosynthesized O-DMA exhibited an enantiomeric excess (e.e.) of 78.6%. Based on phylogenetic and average nucleotide identity analyses against 235 public C. beijerinckii genomes, the clustering of FRJF5 with strains from diverse habitats-including industrial fermentation settings, animal feces, and soil-highlights the broad ecological diversity within this species. Functional gene mining and intra-species comparative genomics revealed a unique flavonoid metabolism gene cluster in FRJF5. Using apigenin as a representative flavonoid, we confirmed the successful conversion to 3-(4-hydroxyphenyl)-propionic acid. Moreover, the strain was predicted and verified to possess a substantial butyrate-producing capacity. Genomic screening for virulence or antibiotic resistance genes, combined with phenotypic tests (hemolysis, antibiotic susceptibility, and mouse gavage), revealed a favorable safety profile for strain FRJF5. Finally, intervention experiments in a mouse model of colitis supported its potential in alleviating the disease. Collectively, this study identifies C. beijerinckii FRJF5 as a strain capable of simultaneously producing O-DMA and butyrate, highlighting its potential for future applications in functional foods.IMPORTANCESoy isoflavones require gut bacterial conversion into bioactive metabolites-such as the anti-inflammatory compound O-desmethylangolensin (O-DMA)-to exert health benefits. Yet O-DMA-producing strains remain scarce, largely confined to fecal sources, and poorly characterized. Here, we isolated Clostridium beijerinckii FRJF5 from Chinese stinky tofu, an unexplored ecological niche. This strain not only produces enantiomerically enriched O-DMA but also co-produces butyrate, a metabolite known to strengthen gut barrier function. Genomic mining uncovered a unique flavonoid metabolism gene cluster responsible for this dual activity. Combined with favorable safety profiles, FRJF5 emerges as a strong candidate for functional food applications. This work expands the known diversity of O-DMA producers and bridges traditional fermented foods with next-generation probiotic development.
We report the complete genome sequence of the attenuated human cytomegalovirus strain TR-VAC, developed for subviral particle vaccine production. Oxford Nanopore duplex sequencing confirmed all engineered modifications, including UL130 repair, UL25 stop codons, ddFKBP insertion, GFP deletion, and retention of the bacterial artificial chromosome backbone, without large-scale structural rearrangements.
Aeromonas dhakensis has emerged as a significant pathogen affecting both aquatic animals and humans; however, genomic data for isolates from marine mammals remain scarce. In this study, we characterised the genome of A. dhakensis strain KDL-001, isolated from a fatal dolphin case, using whole-genome sequencing and comparative genomics. Taxonomic analyses, including MLST and average nucleotide identity (ANI), confirmed the isolate as A. dhakensis. Core-genome phylogeny further revealed that KDL-001 is closely related to strains derived from fish and aquatic environments. Notably, in silico screening of virulence-associated genes showed that the virulence-associated gene profile of the dolphin isolate was broadly comparable to those of other A. dhakensis strains, with no isolate-specific virulence-associated genes being identified within the limits of this analysis. These findings demonstrate that the dolphin-derived isolate is genomically comparable to previously described A. dhakensis strains and possesses conserved virulence-associated genes commonly found within the species.
Influenza B viruses contribute substantially to global morbidity and mortality, yet genomic data from the Middle East remain limited. We retrospectively performed whole-genome sequencing of six influenza B virus-positive residual nasopharyngeal specimens collected at King Abdulaziz Medical City, Riyadh, during the 2024-2025 season, including one fatal pediatric case, and described their genomic features alongside clinical outcomes. All six genomes belonged to the B/Victoria lineage and clustered within V1A.3a.2-derived subclades circulating globally during 2024-2025. Five genomes, including the fatal pediatric case, were assigned to subclade C.5.6, whereas one non-fatal case belonged to C.5.7. The Saudi sequences were interspersed among contemporaneous reference strains from Europe, Asia, and North America, without evidence of a distinct local lineage. The fatal isolate did not occupy a distinct phylogenetic position and contained none of the screened virulence-associated markers, including the neuraminidase N342K substitution. These genomes provide regional surveillance data from an underrepresented setting. Given the small sample size and inclusion of a single fatal case, the findings are descriptive and do not permit inference regarding genomic determinants of disease severity. Larger studies integrating viral genomic, clinical, and host data are needed.
Fusarium wilt of cotton, caused by Fusarium oxysporum f. sp. vasinfectum (Fov), remains a persistent threat to cotton production worldwide. Among the known races, Fov race 4 and its extra-virulent variants cause particularly severe losses in Upland cotton. Although several Fov genome assemblies have been assigned to races, the genomic diversity and evolutionary relationships among pathogenic and non-pathogenic isolates associated with cotton outbreaks remain poorly understood at the whole-genome level. This study addressed these gaps by generating and comparing high-quality genome assemblies of four Fusarium isolates collected from Texas cotton fields: two pathogenic (TX17-24 and TX18-9) and two non-pathogenic (TX17-6 and TX18-6). Draft assemblies were generated using Oxford Nanopore long reads and polished with Illumina reads. Comparative genomic analyses showed that pathogenic isolates possessed larger genomes and more conserved orthologous families, whereas non-pathogenic isolates contained more unique genes. Analyses of predicted secreted effectors, transposable elements, and carbohydrate-active enzymes further distinguished pathogenic and non-pathogenic lineages, suggesting roles in virulence adaptation and genome plasticity. Phylogenomic analyses using k-mer-based, assembly- and alignment-free methods incorporated all available long-read Fov genomes and revealed substantial genetic diversity within races 1 and 4, clustering isolates into multiple sublineages. These findings show that Fov race diversification is underestimated when based on traditional classification schemes and may be shaped by host specialization, geographic separation, or horizontal gene transfer. This work advances our understanding of the genomic diversity and evolutionary dynamics of Fov and establishes a foundation for improved race identification and characterization of Fusarium wilt pathogenesis in cotton.
Avian metapneumovirus (aMPV) subtypes A and B emerged in United States poultry in late 2023 and early 2024, prompting genome-scale surveillance from clinical samples. Here, we developed and optimized targeted amplicon sequencing (TAS) assays for both subtypes and applied them to 104 subtype-positive clinical samples collected from chicken and turkey farms across nine US states between early 2024 and early 2026. TAS recovered 91 genomes suitable for comparative analysis, including 44 aMPV-A and 47 aMPV-B sequences, with successful recovery extending to Ct values of 34.6 for aMPV-A and 31.2 for aMPV-B. Recovered genomes showed near-complete breadth and high mapping efficiency. Phylogenetic analyses of both G-gene and whole-genome datasets showed that US aMPV-A field strains formed a distinct monophyletic lineage within group IV and resolved into three closely related clusters. Cluster 2, first recognized in North Carolina and later detected in Ohio, spread across 30 turkey and chicken farms. Cluster 2 genomes were defined by a concentrated G-protein hotspot within residues 209-275, and most North Carolina Cluster 2 genomes carried 10-11 nonsynonymous substitutions in this region, including multiple proline substitutions suggestive of local structural change. Missouri Cluster 3 remained cohesive but distinct from both Cluster 1 and Cluster 2 in the G-gene and whole-genome trees. In contrast, US aMPV-B field strains remained highly homogeneous across hosts and states, with more than 99% nucleotide identity by both G-gene and WGS analyses. We also identified 12 vaccine-derived genomes on both vaccinated and nonvaccinated farms. These included six genomes related to aMPV-A vaccine and six related to aMPV-B vaccines (VCO3/50 and 1062), all of which retained vaccine-defining markers together with additional substitutions consistent with continued circulation after vaccine use in the field. Selection analyses showed that the G gene had the highest gene-wise dN/dS ratio in both subtypes. Additional elevated signal was observed in SH and M2, and candidate positively or episodically selected codons were concentrated in the subtype A Cluster 2 G-gene hotspot. These findings show that TAS supports direct-from-sample aMPV genomic surveillance and provides genomic context for field clusters, vaccine-derived lineages, and continued adaptive change in aMPV in US poultry.
As a key subunit of the SWItch/sucrose nonfermentable chromatin-remodeling complex, SMARCA4 plays a critical role as a tumor suppressor in various tumors. However, the clinicopathological and molecular features of SMARCA4-deficient carcinoma of the gallbladder (SMARCA4-dGBC) have not been well explored. In this study, a retrospective cohort of 926 nonsquamous cell gallbladder carcinomas (GBCs) was analyzed on tissue microarrays using immunohistochemistry for SMARCA4, comprising 813 adenocarcinomas, 53 adenosquamous carcinomas, 43 undifferentiated carcinomas, 7 sarcomatoid carcinomas, 6 small cell neuroendocrine carcinomas, and 4 large cell neuroendocrine carcinomas. Twenty-six (2.8%) SMARCA4-dGBCs were identified and further analyzed using immunohistochemistry, whole-exome sequencing, and clinicopathological data. SMARCA4-dGBCs are frequently identified in advanced stages and exhibit diverse patterns of differentiation. The majority were identified as monotonous diffuse sheets, nests, and cords, whereas a subset exhibited gland-forming and rhabdoid morphologies (11.5%). Tumors retained mismatch repair proficiency (100%) but showed variable HER2 expression (11.5% scored as 2+/3+) and limited PD-L1 positivity. Genomic profiling revealed SMARCA4 alterations in 88.5% (23/26) of patients, predominantly deletions (91.3%) and truncating mutations-p.K892∗ and p.R979∗-that disrupt the critical ATPase/helicase domains. Co-occurring TP53 mutations (56.5%) highlighted the presence of synergistic chromatin-remodeling defects. Enrichment of oncogenic signaling pathways, including the RTK-RAS (78.3%), TP53 (60.9%), NOTCH (47.8%), and HIPPO (39.1%) pathways, was observed. Patients with SMARCA4-dGBC exhibited significantly shorter progression-free survival (median, 6 vs 14 months) and overall survival (median, 11 vs 16 months) than those with SMARCA4-retained tumors. Overall, these findings revealed that SMARCA4-dGBC is a rare, distinct entity characterized by the destabilization of the SWItch/sucrose nonfermentable complex, genomic instability, and resistance to conventional therapies. The prevalence of targetable pathways, such as RTK-RAS and cell cycle dysregulation, highlights opportunities for precise therapeutic strategies involving EZH2, CDK4/6, or ATR inhibitors. SMARCA4 immunohistochemistry and molecular profiling are essential for accurate diagnosis, prognostic stratification, and therapeutic innovation of this GBC subtype.
Staphylococcus aureus is an important pathogen associated with bovine mastitis and dairy product contamination, posing economic and public health risks through the food chain. In this study, a temperate phage, vB_SauL_202595, was isolated from a dairy farm environmental sample using S. aureus SHZ-0127 as the host, and its biological characteristics, genomic features, and antibacterial activity in dairy matrices were evaluated. vB_SauL_202595 lysed 18 of 66 tested S. aureus strains, with a lysis susceptibility rate of 27.3%, including 5 highly susceptible strains, indicating a relatively limited host range. The optimal multiplicity of infection was 0.01, the latent period was approximately 30 min, and the burst size was approximately 316 PFU/cell. The phage remained stable at 4°C-37°C and pH 6-10. Genome analysis showed that vB_SauL_202595 belongs to the class Caudoviricetes, has a genome of 44,503 bp with 33.59% GC content, and encodes 63 predicted proteins. No typical antibiotic resistance genes or major virulence factors were detected; however, integrase and repressor genes were identified, supporting its temperate nature. vB_SauL_202595 inhibited S. aureus SHZ-0127 growth, reduced mature biofilm biomass, and decreased viable bacterial counts in milk and yogurt, with reductions of 1.23 and 1.42 log10 CFU/mL under representative conditions, respectively. From a One Health perspective, these findings provide foundational evidence for reducing S. aureus contamination and related antimicrobial resistance risks along the dairy chain. Overall, vB_SauL_202595 represents a candidate phage resource for dairy-associated S. aureus biocontrol research, but its limited host range and lysogeny-related genes require further safety assessment before food-related applications.IMPORTANCEStaphylococcus aureus is a major pathogen associated with bovine mastitis and a common contaminant in dairy products, causing economic losses and public health risks through the food chain. Although phage-based biocontrol has emerged as a promising strategy for controlling S. aureus contamination in dairy products, systematic evidence regarding phage activity in actual dairy matrices remains limited. In this study, we isolated and characterized a dairy farm environment-derived temperate phage, vB_SauL_202595, and evaluated its biological characteristics, genomic features, host range, stability, biofilm removal ability, and antibacterial performance in milk and yogurt. These findings provide foundational experimental evidence for phage-based dairy biocontrol against S. aureus. However, due to its limited host range and lysogeny-related genomic features, vB_SauL_202595 should be considered a candidate phage resource for further study. Broader validation, including phage-cocktail testing, long-term storage assays, product quality assessment, and regulatory safety evaluation, is needed before practical application.
BACKGROUND: Aggressive evolution of PitNETs is rare; metastatic spread is even more. Defining aggressiveness and malignancy is challenging, subsequently hard to predict, and to understand. The aim was to provide a molecular definition of aggressiveness using genomic approaches. METHODS: PitNETs from 206 patients were included. Associations between 9 clinicopathological features of aggressiveness and PitNETs' omics were explored. Omics included transcriptome, DNA methylation, chromosomal alterations, and mutations. Clonal tumor evolution was monitored in 7 patients. RESULTS: Among the 9 clinicopathological features of aggressiveness, only rapid progression, progression after radiotherapy, Ki67/MIB1 proliferation index ≥10%, temozolomide treatment, metastases, and specific death were associated with specific omics signatures, while tumour maximal diameter ≥40 mm, cavernous, and sphenoid invasion were not. The omic signatures associated with these features of aggressiveness overlapped but remained distinct between corticotroph and mammo-somato-thyrotroph lineages. For each lineage, a common signature of aggressiveness was identified, associating a proliferative transcriptome signature and DNA hypermethylation. Alterations in specific genes were associated with aggressive features, including a novel PitNET gene, LRP1B, and known cancer genes (TP53, CDKN2A), while USP8 and GNAS alterations were not. Integration of gene alterations with methylome and transcriptome signatures isolated a subset of molecularly aggressive PitNETs. Molecular signatures were stable during the course of the disease, despite evolution toward aggressiveness and potential clonal divergence. CONCLUSION: This systematic analysis of clinicopathological features of aggressiveness using an integrated multiomic approach establishes a histomolecular definition of aggressiveness in PitNETs. Prospective cohort studies are needed to validate these molecular signatures and establish their prognostic value.
The cocirculation of CRF01_AE and CRF07_BC among men who have sex with men (MSM) in China may facilitate the emergence of genetically complex HIV-1 recombinants. Here, we identified and characterized a novel second-generation HIV-1 CRF01_AE/CRF07_BC recombinant, designated GY0192, from a 25-year-old MSM individual in Guiyang, Guizhou Province, southwest China, using near full-length genome (NFLG) analysis. Recombination analyses identified four breakpoints at HXB2 positions 3,125, 5,687, 6,375, and 9,176, generating a distinct five-segment mosaic genome. Subregion phylogenetic analyses showed that the two CRF01_AE-derived fragments clustered with the CRF01_AE cluster 5 lineage, while all three CRF07_BC-derived fragments grouped with CRF07_BC lineages frequently circulating among MSM in China, indicating that both parental components of GY0192 were phylogenetically related to lineages frequently reported among MSM populations in China. The virus was also predicted to be CCR5-tropic, adding epidemiological relevance because CCR5-tropic viruses are commonly involved in transmission and early infection. Together, these findings identify GY0192 as a distinct CRF01_AE/CRF07_BC mosaic and suggest that cocirculating MSM-associated HIV-1 lineages may provide opportunities for interlineage recombination. This case expands the known spectrum of CRF01_AE/CRF07_BC recombinants in Guizhou and underscores the value of NFLG-based surveillance for detecting underrecognized HIV-1 genetic complexity in southwest China.
BACKGROUND: This study aimed to elucidate the molecular epidemiology and virulence characteristics of ST11 carbapenem-resistant Klebsiella pneumoniae (CRKP) colonizing the intestinal tract of elderly inpatients in the Chongzhou region, providing a basis for controlling the transmission of such resistant bacteria in high-risk populations. METHODS: CRKP strains isolated from the intestines of elderly inpatients in this region between January 2023 and June 2024 were collected. ST11 strains were identified via multilocus sequence typing (MLST). Whole-genome sequencing, antimicrobial susceptibility testing, and string test, serum killing, biofilm formation, capsular polysaccharide quantification were employed to characterize their resistance genes, virulence genes, and molecular typing profiles. RESULTS: Among 58 CRKP isolates, 17 (29.3%) were ST11. ST11-KL64 was the dominant clone (70.6%). All isolates carried the carbapenemase gene bla KPC-2 and exhibited extensive drug resistance, with tigecycline retaining the highest susceptibility (64.7%). The yersiniabactin system genes (ybtS, fyuA, entB) were universally present, whereas the aerobactin gene cluster (iucABCD-iutA) was detected in only 17.6% of isolates. The virulence regulator rmpA2 was incomplete in all carriers. The hypermucoviscosity phenotype was observed in 35.3% of isolates, which correlated with serum resistance in some strains. Biofilm formation was variable. The mortality rate among colonized patients was 35.3%. CONCLUSION: The ST11-KL64 clone is dominant among CRKP strains colonizing the intestinal tract of elderly patients in this region. This clone universally carries the bla KPC-2 gene conferring carbapenem resistance and exhibits a unique virulence gene profile characterized by a low carriage rate of classical hypervirulence markers and an incomplete rmpA2 regulator gene. This finding clarifies the local epidemic status of this clone and underscores the importance of implementing active surveillance and targeted prevention strategies for high-risk populations.
BACKGROUND: Gut microbiota are potential sources of probiotics and play an essential role in maintaining intestinal health. Limosilactobacillus reuteri PSC102 (L. reuteri PSC102), which was isolated from the feces of healthy pigs, exhibited health-beneficial properties. AIM: We aimed to conduct a whole-genome sequencing analysis of L. reuteri PSC102 to determine its molecular characteristics as a probiotic strain. METHODS: Limosilactobacillus reuteri PSC102 cells were cultured in De Man-Rogosa-Sharpe medium, followed by DNA extraction for genomic analysis using the PacBio-Illumina sequencing platform. The EzBioCloud software was used to perform gene assembly, and the genes were interpreted by the National Center for Biotechnology Information (NCBI) and the Glimmer program. Core and pan-genomic analyses were performed to assess the extent of functional conservation in the genomic sequence. Moreover, the NCBI database and the Basic Local Alignment Search Tool software were used to identify antimicrobial resistance genes and virulence factors. RESULTS: Limosilactobacillus reuteri PSC102 consists of a single circular chromosome with 2,048,626 bp, a guanine- cytosine of 38.9%, 18 rRNA genes, and 69 tRNA genes. Among the 1,846 protein-coding sequences, genes associated with probiotic characteristics were identified, including genes involved in host-microbe interactions, stress tolerance, biogenesis, and defense mechanisms. Furthermore, the genome of L. reuteri PSC102 comprises 2,446 pan-genome and 1,222 core-genome orthologous gene clusters. A total of 74 unique genes were identified in L. reuteri PSC102 genome. These genes mostly encode proteins potentially involved in the transport and metabolism of amino acids and carbohydrates. Moreover, antibacterial resistance genes and virulence factors were absent in L. reuteri PSC102. CONCLUSION: The results of the molecular insight into L. reuteri PSC102 corroborates its use as a probiotic in humans and other animals.
In September 2025, West Nile virus (WNV) RNA was detected for the first time in a sheep in Zaghouan Governorate, Tunisia. Following sheep abortions, fetal specimens were analyzed and one tested positive for WNV by RT-qPCR. The generated near-complete WNV genome sequence by Oxford Nanopore Technologies belonged to Lineage 1a, previously associated with locally reported human neuroinvasive cases in the same area. Amino acid analysis of the envelope (E) protein confirmed the presence of the conserved NYS glycosylation motif (E154-E156), a characteristic feature of Euro-Mediterranean Lineage 1 WNV strains. These findings further support the endemic circulation of WNV in Tunisia and underline its significance for both animal and public health.
Enterococci are common commensals of the human gut and important opportunistic pathogens, with Enterococcus faecium and Enterococcus faecalis being the most clinically prevalent species. A significant epidemiological shift has emerged with an increasing clinical burden of E. faecium. To compare genomic evolution of E. faecium and E. faecalis, we performed whole-genome sequencing on 93 E. faecium and 32 E. faecalis isolates causing bloodstream infections at a single hospital (2022-2024). Analysis of patient demographics revealed that E. faecium infections originated from fewer sources than E. faecalis, with a higher proportion deriving from intra-abdominal infections. Multilocus sequence typing identified ST78 and ST789 as the predominant sequence types for E. faecium, whereas ST16 and ST179 were most common for E. faecalis. E. faecium carried more antimicrobial resistance genes and putative virulence marker (PVM)-type virulence genes than E. faecalis, with vancomycin resistance predominantly mediated by vanHAX (33/93, 35.5%) and a single E. faecalis isolate also carrying vanHAX (1/32, 3.1%); the structurally incomplete vanHMX gene cluster was detected in 11 E. faecium isolates. Pan-genome analysis indicated a larger core genome in E. faecalis compared to E. faecium, consistent with greater plasmid replicon diversity in the latter. Intra-host comparisons showed that two E. faecalis pairs from the same patient were clonally related, with one isolate acquiring a vanHAX plasmid conferring vancomycin resistance. In contrast, E. faecium isolates exhibited marked genomic diversity even among clonally related pairs. These findings suggest that E. faecium possesses greater genomic plasticity and adaptive potential to the clinical environment.IMPORTANCEThis study provides a detailed comparison of clinical and genomic features between Enterococcus faecium and Enterococcus faecalis from the same hospital setting. We show that E. faecium isolates, mainly ST78/ST789, carry more antimicrobial resistance genes and a higher number of putative virulence marker (PVM) genes than E. faecalis, reflecting their hospital-adapted nature. E. faecium also exhibits a smaller core genome and greater diversity of plasmid replicon types, indicating higher genomic plasticity and capacity for horizontal gene transfer. By contrast, E. faecalis retains a larger core genome and a set of classical virulence factors, and its within-host isolates are clonally related. These distinct genomic profiles help to understand how the two species adapt to clinical environments and may inform more targeted infection control strategies and resistance surveillance.
Aeromonas veronii is a pathogen of multiple fish species, yet systematic understanding of its infection in Nile tilapia (Oreochromis niloticus) remains limited. A dominant strain, NN0115, was isolated from a natural outbreak and identified as A. veronii by 16S rRNA and whole-genome average nucleotide identity (ANI, 96.33%). Experimental infection revealed high virulence (LD50 = 3.41 × 106 CFU/mL, equivalent to 8.53 × 104 CFU/fish). The genome is 4.58 Mb (58.57% GC) and encodes 4216 proteins. Virulence factor analysis identified 1253 genes, dominated by motility-related (264) and immune modulation (208) factors. Genomic island GI2 harbors 7 virulence genes and two dual-function resistance-virulence genes. The strain is resistant to 9 of 25 agents tested but carries three RND efflux pump genes whose predicted resistance was not phenotypically observed. The head kidney transcriptome of tilapia at 24 h post-bacterial infection identified 773 differentially expressed genes; among them, 57 were immunoglobulin (Ig) genes, and 56 were down-regulated. Integration of published single-cell transcriptomic data showed that non-Ig B-cell marker genes were down-regulated by 32%, whereas Ig genes were reduced by 63%, indicating selective transcriptional suppression of Ig genes rather than a general decrease in B-cell transcriptional activity. Together, this study provides a comprehensive characterization of a highly virulent A. veronii from Nile tilapia and reveals that selective downregulation of B-cell Ig genes is the dominant transcriptional feature of the host head kidney response.
Respiratory syncytial virus (RSV), a ~15.2 kb negative-sense RNA virus, causes acute respiratory infections in infants and older adults. Its two subtypes, RSV-A and RSV-B, evolve rapidly, making ongoing monitoring of circulating strains essential. The Georgia Public Health Laboratory (GPHL) developed and evaluated an amplicon-based whole-genome sequencing (WGS) assay for RSV surveillance. A total of 214 de-identified remnant clinical specimens (102 RSV-A and 112 RSV-B) with RT-PCR Cq values <31 were included. RSV genomes were amplified using ARTIC-style and custom primer sets, with the ARTIC set showing superior performance. Libraries were prepared using a modified Illumina COVIDSeq protocol, sequenced on NextSeq 1000/2000 instruments, and analyzed using the GPHL-RSV-PIPE bioinformatics pipeline. Among genomes meeting validation criteria, sequencing depth was slightly higher for RSV-A (median 53,433×; mean 51,076×) than RSV-B (median 49,699×; mean 46,945×), whereas genomic coverage was slightly lower for RSV-A (median 97.5%; mean 96.6%) than RSV-B (median 98.3%; mean 97.6%). Predominant lineages were A.D.3.1 and A.D.5.2 for RSV-A and B.D.E.1 for RSV-B. For RSV-A, the assay showed 92.8% accuracy, 96.2% sensitivity, 87.2% specificity, 92.6% positive predictive value, and 93.2% negative predictive value. Intra- and inter-run precision assessed using 16 and 53-57 genomes, respectively, showed nearly 100% consensus genome identity with 0-5 nucleotide differences. Specificity testing of 31 non-RSV specimens produced no false-positive detections. Limits of detection were 4.4 TCID50/mL for RSV-A and 18.6 TCID50/mL for RSV-B. These results demonstrate that the ARTIC-based RSV WGS assay enables near real-time surveillance and strengthens data-driven public health responses to future outbreaks.IMPORTANCERSV, with two major subtypes, RSV-A and RSV-B, causes acute respiratory infections that can be severe in infants under 6 months and older adults. Current RSV surveillance at the GPHL relies on the Thermo Fisher TaqMan Gene Expression Capillary assay, which detects and subtypes RSV but lacks resolution for lineage classification and identification of emerging variants. To address this critical gap, GPHL developed and evaluated an amplicon-based WGS assay using 214 de-identified RSV clinical specimens. Genomes were amplified using ARTIC-style and custom-primer sets, with ARTIC primers showing superior performance. The assay demonstrated strong sequencing depth, genomic coverage, specificity, repeatability, reproducibility, and low limits of detection. RSV lineages were accurately determined based on genetic variation. These results establish that the ARTIC-based WGS assay enables near real-time genomic surveillance, supporting monitoring of circulating RSV strains and informing data-driven public health responses.