Search Results (1,204)

SARS-CoV, MERS-CoV, and SARS-CoV-2 have posed tremendous threats to human health, highlighting the necessity of monitoring cross-species transmission of animal coronaviruses to humans. Hedgehogs infected with coronaviruses have been reported in several countries across Europe and Asia, raising concerns about the potential transmission of coronaviruses from hedgehogs to humans. In this study, we investigated coronavirus infections in hedgehogs inhabiting greenspaces in metropolitan Beijing and identified a Merbecovirus subgenus coronavirus with a prevalence rate of 30% (95% CI: 25–35%) among 317 hedgehogs. Phylogenetic analysis of 23 complete viral genome sequences revealed a monophyletic origin, showing close relatedness to Erinaceus hedgehog coronavirus HKU31 (Ea-HedCoV HKU31) with genome-wide nucleotide identities of 93.24–96.42%, and evidence of recombination with Tylonycteris bat coronavirus HKU4. These findings suggest that the increase in wildlife populations associated with urban greenspace development may pose a potential threat to human health that should not be overlooked. Full article

The continuing spread of SARS-CoV-2 and the associated morbidity and mortality, especially in vulnerable populations, highlight the need for the development of antiviral therapeutics. Reverse genetics systems and reporter viruses are valuable for antiviral screening by simplifying methods to detect and quantify virus infections. This study aimed to generate wild-type and Nluc reporter full-length SARS-CoV-2 molecular clones and viruses as tools for high-throughput antiviral assays. The large SARS-CoV-2 genome (~30 kb) makes cDNA cloning and virus rescue technically challenging, so we opted to use cDNA chemical synthesis services to generate full-length wild-type and reporter Delta and Omicron clones. Clone-derived Delta and Omicron wild-type and reporter viruses were successfully rescued and showed replication kinetics comparable to patient-derived isolates. Nluc reporter viruses displayed stable luciferase expression that correlated with viral titres, supporting their reliability as replication substitutes. Antiviral assays measuring replication inhibition by Remdesivir, Molnupiravir, and Nirmatrelvir, based on Nluc expression, yielded IC₅₀ values and selectivity indices consistent with published ranges. Finally, Delta Nluc viruses replicated in primary human bronchial epithelial cells, demonstrating the application of clone-derived viruses in physiologically relevant models. The SARS-CoV-2 cDNA clones and Nluc reporter viruses derived from DNA synthesis services provide a rapid, scalable reverse genetics platform for generating new viruses and developing assays to rapidly assess antiviral compounds against current and emerging SARS-CoV-2 variants or coronaviruses that may emerge in the future. Full article

Affordable, flexible surveillance tools are needed to detect SARS-CoV-2 and other pathogens in wildlife. Standard nucleic acid amplification tests (NAATs) are reliable but restricted to predefined targets, limiting their ability to detect co-infections or emerging pathogens. To address this, we adapted a targeted next-generation sequencing (tNGS) panel for mesocarnivores to the Oxford Nanopore Technologies (ONT) MinION platform and combined it with a SARS-CoV-2 whole-genome sequencing assay. Merging both assays before library preparation enables simultaneous SARS-CoV-2 detection, variant identification, and broader pathogen screening. The MinION platform also improves turnaround time because sequencing can begin immediately on small numbers of samples, reducing costs in low-volume workflows. We converted our validated carnivore tNGS panel from the Ion Torrent system to MinION, optimizing amplification conditions, primer pools, and barcoding for multiplexing. Analytical sensitivity was measured using contrived wildlife samples spiked with serial dilutions of SARS-CoV-2 and tested in parallel with a commercial NAAT. Diagnostic sensitivity was assessed using contrived positives, and specificity was evaluated using NAAT-negative wildlife samples and in silico analyses. All 161 wildlife samples were NAAT-negative. MinION tNGS detected SARS-CoV-2 down to Ct 34 and produced ≥ 99% genome coverage for Ct ≤ 24 while simultaneously identifying additional pathogens. Diagnostic sensitivity and specificity were 96.7% and 100%. This workflow offers a low-cost, scalable approach for comprehensive wildlife pathogen surveillance. Full article

Influenza and other respiratory viruses pose significant public health threats among SARI patients, yet comprehensive surveillance data remain limited in Pakistan. This prospective, multi-center study characterized the burden, distribution, and molecular evolution of respiratory viruses among hospitalized SARI patients across seven tertiary hospitals from November 2022 to June 2025. Specimens were tested using RT-PCR for influenza, SARS-CoV-2, and RSV, with 375 samples sequenced via Oxford Nanopore Technology. Among 11,451 specimens, 2818 (24.6%) tested positive: RSV (1648, 14.4%), influenza (855, 7.5%; 45% H1N1pdm09, 35% H3N2, 20% influenza B), and SARS-CoV-2 (315, 2.8%). RSV predominantly affected children under 2 years (63%), while influenza and SARS-CoV-2 primarily impacted adults aged 15–40 years. Male predominance (65–79%) reflected healthcare access barriers. Strong winter seasonality (December–February) was observed for influenza and RSV. ICU admission rates were 17% for influenza, 16% for RSV, and 4% for SARS-CoV-2. Shortness of breath was associated with influenza (OR = 1.62) and RSV (OR = 1.27), while malaise (OR = 2.24) and myalgia (OR = 3.87) was associated with SARS-CoV-2. Phylogenetic analysis revealed vaccine-matched influenza clades and rapid SARS-CoV-2 variant succession (3–4 months). RSV is the primary SARI pathogen in young children, necessitating maternal vaccines and nirsevimab implementation. Sustained genomic surveillance remains essential for pandemic preparedness. Full article

RNA viruses, such as SARS-CoV-2 and influenza, pose a persistent threat to global public health. Their high mutation rates undermine the effectiveness of conventional direct-acting antivirals (DAAs) and facilitate drug resistance. As obligate intracellular parasites, RNA viruses rely extensively on host cellular machinery and metabolic pathways throughout their life cycle. This dependency has prompted a strategic shift in antiviral research—from targeting the mutable virus to targeting relatively conserved host dependency factors (HDFs). In this review, we systematically analyze how RNA viruses exploit HDFs at each stage of infection: utilizing host receptors for entry; remodeling endomembrane systems to establish replication organelles; hijacking transcriptional, translational, and metabolic systems for genome replication and protein synthesis; and co-opting trafficking and budding machinery for assembly and egress. By comparing strategies across diverse RNA viruses, we highlight the broad-spectrum potential of HDF-targeting approaches, which offer a higher genetic barrier to resistance, providing a rational framework for developing host-targeting antiviral therapies. Full article

Background: The continuous emergence of SARS-CoV-2 variants represents a major public health concern. Next-generation sequencing (NGS) enables genomic surveillance, facilitating the detection and monitoring of mutations that impact viral evolution. Methods: In this study, full-length SARS-CoV-2 genomes were analyzed between February 2022 and March 2024 as part of routine genomic surveillance conducted in Verona, Italy. Mutations in the envelope (E), membrane (M), and nucleocapsid (N) structural proteins were investigated. Only substitutions with a total prevalence of greater than 1% in the study dataset were considered. Results: A total of 178 mutations were identified across the three proteins (E: 16; M: 33; N: 129), of which 18 met the inclusion threshold (E: 3; M: 5; N: 10). Mutations were classified according to temporal dynamics as fixed, emerging, or transient. Throughout the study period, fixed mutations were consistently prevalent, emerging mutations appeared later but persisted with an ascending trend, while transient mutations displayed a single frequency peak before disappearing. Several mutations were reported with potential structural or functional relevance based on the existing literature, while others remain of unknown significance. Conclusions: The mutational patterns detected in this study broadly reflect global evolutionary trends of SARS-CoV-2. These findings emphasize the importance of continued genomic surveillance and underline the need for integrated experimental approaches to clarify the biological and epidemiological impact of poorly characterized mutations. Full article

GC-rich sequences affect DNA replication, recombination and repair, as well as RNA transcription in vivo. Such sequences may also impede site-directed mutagenesis in vitro. P3a site-directed mutagenesis is a highly efficient method, but it has not been tested with plasmids possessing GC-rich sequences. Here we report that it is very efficient with a BRPF3 expression vector but unsuccessful with that for KAT2B. Because two GC-rich regions located within the synthetic CAG promoter and the KAT2B coding region may form guanine (G)-quadruplexes and hinder plasmid denaturation during PCR, we developed P3b site-specific mutagenesis, achieving an average efficiency of 97.5% in engineering ten KAT2B mutants. Importantly, deletion mutagenesis revealed that either of the two GC-rich regions is sufficient for rendering the plasmid incompatible with P3a mutagenesis. Consistent with this, only P3b mutagenesis worked efficiently with several widely used sgRNA/Cas9 expression vectors, which contain the CAG promoter, and with an expression vector for CDK13, which possesses an intrinsically disordered domain encoded by a GC-rich DNA fragment. Thus, this study highlights serious challenges posed by GC-rich sequences to site-directed mutagenesis and provides an effective remedy to address such challenges. The findings support that G-quadruplex formation is one mechanism whereby such sequences impede regular PCR-based mutagenesis methods. Full article

The Indo-Pacific region hosts several annual military exercises that involve the deployment of thousands of U.S. and partner-nation military personnel. Respiratory and diarrheal diseases pose a significant health risk to exercise participants and represent a substantial portion of medical encounters and lost duty days. We conducted surveillance for respiratory and diarrheal illness at the Cobra Gold and Balikatan military exercises in Thailand and the Philippines from 2023–2025. Through coordination with health providers in the field, military personnel that reported acute symptoms were asked to provide a nasopharyngeal swab or stool sample. These samples were transported to a field lab and tested by PCR for common respiratory and diarrheal pathogens. Follow-up analyses included bacterial culture, antimicrobial susceptibility testing, and viral whole-genome sequencing. From 84 respiratory and 61 diarrheal samples analyzed, we found that respiratory illness was primarily attributed to rhinoviruses/enteroviruses (23%), common coronaviruses (21%), and SARS-CoV-2 (11%) while diarrheal disease was attributed to a high rate of diarrheagenic E. coli (73%) and norovirus (20%), followed by Salmonella spp. (18%) and Campylobacter spp. (13%). Our findings highlight the distinct etiologies of respiratory and diarrheal disease in military field settings and demonstrate the feasibility of conducting real-time infectious disease surveillance in operational environments. Full article

Importance: The coronavirus disease 2019 (COVID-19) was the third leading cause of mortality in the United States for three years in a row. The genetic contributions to disease severity remain unclear and many previously identified single nucleotide polymorphisms (SNPs) have not been replicated nor linked with functional significance. Objective: To identify SNPs associated with mortality among hospitalized COVID-19 patients supplemented by expression quantitative trait loci (eQTL) evidence to infer plausible functional mechanisms related to COVID-19 severity. Design: A quality-controlled genome-wide association study (GWAS) supported by robust gene-level omnibus kernel association tests (SKAT-O), functional prediction, and eQTL analyses of the top GWAS signal. Setting: Massachusetts General Hospital (MGH). Participants: 370 adult ICU patients with SARS-CoV-2 infection and acute hypoxemic respiratory failure and floor patients with mild hypoxemia managed with supplemental oxygen consecutively admitted to MGH between March and June 2020 (Surge 1), and January and March 2021 (Surge 2) with baseline clinical characteristics and demographics collected. Exposures: Low-pass genotyped SNPs from whole blood and aggregated SNP-sets of potential disease susceptibility loci with $\pm$500 kb flanking regions. Main Outcomes & Measures: Genome-wide individual SNP associations and SNP-set associations with mortality outcomes from 370 severe COVID-19 cases. Results: After LD pruning (<0.8) and false discovery rate adjustment (<0.05), we identified rs7420371 G>A of the receptor transporter protein 5 (RTP5) gene as the top independent signal significantly associated with 30- and 60-day mortality among severe COVID-19 patients (OR, 2.32; 95% CI, 1.59–3.39; p = 4.92 × 10⁻⁹ and OR, 2.06; 95% CI, 1.43–2.97; p = 5.43 × 10⁻⁸, respectively). SKAT-O analyses on the RTP5 SNP-set showed associations with both mortality outcomes (p = 5.90 × 10⁻⁵ and 6.17 × 10⁻⁵, respectively). eQTL analysis showed rs7420371 A allele significantly upregulated the mRNA expression of RTP5 in 266 cerebellum tissues, in 277 cerebellar hemisphere tissues, and in 270 cerebral cortex samples. Conclusions & Relevance: We discovered a novel, independent, and potentially functional SNP RTP5 rs7420371 G>A to be significantly associated with COVID-19 mortality. The A allele is significantly associated with elevated mRNA expression of RTP5 in the brain, an important protein coding gene that modulates olfactory binding and taste perceptions in response to SARS-CoV-2 infection. Full article

Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host–virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, enabling cloning, manipulation, and the rescue of recombinant SARS-CoV-2 (rSARS-CoV-2) with high fidelity and high viral titers after a single passage. Using this system, we generated and characterized spike protein mutants Y453F and N501Y, as well as a U76G mutation in the 5′-UTR. Y453F showed reduced replication kinetics, lower cell binding, and diminished fitness, while N501Y exhibited comparable replication and fitness, highlighting the distinct effects of these spike protein mutations. The U76G mutation is located within a novel NSP9 binding site in the 5′-UTR and leads to impaired RNA synthesis and reduced viral replication efficiency, suggesting an important role in transcription and replication. Our findings highlight the robustness and adaptability of this reverse genetics system, providing a versatile, cost-effective tool for studying SARS-CoV-2 mutations and their effects on replication and fitness, with potential applications in vaccine and therapeutic development. Full article

Face masks are widely recognized as a key intervention to limit SARS-CoV-2 transmission, yet the distribution and persistence of viral RNA across different mask regions and layers remain poorly understood. To address this, we analyzed 185 masks collected from 60 SARS-CoV-2-positive individuals in Rio de Janeiro between December 2020 and September 2022. Masks were sectioned into anatomical regions (nose, mouth, sides) and structural layers (inner, middle, outer), and viral RNA was quantified using RT-qPCR. Samples with the highest viral loads were selected for partial sequencing of the spike gene, and paired analyses with swab samples were performed. Statistical comparisons included non-parametric tests and a linear mixed-effects model. Our results showed that the inner layer and nose region consistently harbored the highest viral RNA levels, with no significant differences between surgical and fabric masks. Viral load decreased by an estimated 39% per day, consistent with exponential decay. Sequencing confirmed identical viral genomes in masks and swabs and allowed identification of circulating variants, including Gamma and Omicron. These findings indicate that masks serve not only as effective physical barriers but also as non-invasive sources for genomic surveillance, providing insights into viral shedding patterns and informing strategies for monitoring and controlling SARS-CoV-2 transmission. Full article

This study aimed to unravel the genomic uniqueness of Streptomyces chrestomyceticus ADP4 using whole-genome sequence analysis, with a focus on identifying strain-specific genes/proteins associated with a novel therapeutic source. The genome of the strain ADP4 was sequenced and assembled to a total size of 9.64 MB. A total of 8378 coding regions were identified. Strain ADP4 was found to be clustered into a clade of the species S. chrestomyceticus. Fifty-one biosynthetic gene clusters were predicted in the genome of the strain ADP4, and three of them were common to all the strains of S. chrestomyceticus. A comparative metabolic profile of S. chrestomyceticus revealed a unique metabolic protein, supporting strain-level variations. Comparative genome analysis led to the identification of the genomic sequences that were specific to the strain ADP4. These strain-specific unique sequences of ADP4 were identified across the available data, underscoring their distinct genetic identity. Among these eight functionally uncharacterized hypothetical proteins (HPs), only two could be assigned with functional attributes, wherein one of them, HP2, was ascertained to be a peptide with possible antiviral activity, underscoring its potential as a novel drug candidate for aantiviraltherapy. The structural validation and peptide–protein molecular docking have evidently demonstrated anantiviralctivity of HP2 against significant human viral pathogens, for example, HIV, SARS-CoV2, HCV, ZIKV, JEV, and DENV. Full article

The ability of a virus to adapt is key to its survival, and this is achieved through mutation, which allows the virus to change and adapt to new environments. To capture the full extent of SARS-CoV-2 diversity in Asturias, samples obtained from nasopharyngeal swabs were characterised using whole-genome sequencing. Between 2020 and July 2024, a total of 4001 sequences were analysed and 5302 mutations were identified. An increase in the positivity rate was observed between 2022 and 2024 in children under 1 year of age. During this period, 55 new circulating variants belonging to 41 pangolin lineages were detected: 24 originated throughout the world and 31 in Asturias (10 detected only in the region, 8 in the rest of Spain, and 13 around the world). A total of 31 new non-synonymous mutations were fixed in the viral population 250 ± 46 (93–620) days after their appearance. During seasonal SARS-CoV-2 circulation, surveillance systems developed during the pandemic continue to detect new indigenous and imported variants without indicating an increase in severity. Full article

The coronavirus disease 2019 (COVID-19) pandemic in Tocantins, Brazil, exhibited distinct phases between 2020 and 2025, with high mortality concentrated in 2020–2021 and subsequent stabilization at residual levels. Using epidemiological data, statistical modeling, and genomic surveillance, we show that the crisis peaked in 2021, coinciding with the circulation of Gamma and Delta, when health system capacity was severely strained. From 2022 onwards, the spread of Omicron led to record incidence but proportionally low mortality, reflecting accumulated immunity, vaccination, and improved clinical management. Vaccination represented the turning point, reducing hospitalizations and deaths by over 90% and driving a clear decoupling between incidence and severity. Interrupted time-series and generalized additive model (GAM) analyses confirmed sustained reductions in transmission and severity associated with mass immunization. Genomic sequencing of 3941 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes identified 166 lineages and successive variant replacements, culminating in the predominance of LP.8.1.4 in 2025. To our knowledge, this is one of the few integrated, long-term analyses (2020–2025) combining epidemiological and genomic data, capturing the full succession of variants up to LP.8.1.4 and highlighting Tocantins as a strategic “variant corridor” linking Brazil’s North and Central-West regions. These findings underscore the dual role of vaccination and genomic surveillance in shaping the epidemic trajectory and the importance of sustaining both strategies to mitigate future health crises. Full article

The emergence of high-threat pathogens—such as Ebola, Lassa fever, and most recently SARS-CoV-2—has highlighted critical weaknesses in global surveillance systems, particularly in resource-limited settings where many zoonotic spillovers originate. Despite the World Health Organization’s (WHO) prioritization of these diseases for research and development (R&D), the current surveillance infrastructures in these regions remain under-resourced, fragmented, and often reactive rather than anticipatory. This narrative review explored the literature and structured relevant findings in three key dimensions: (i) the structural and operational limitations of existing surveillance systems for the WHO priority diseases in resource-limited settings including challenges in data integration, laboratory capacity, workforce, and community engagement; (ii) how these surveillance gaps could delay detection and hinder the response to future emerging threats, particularly a hypothetical but inevitable Disease X; and (iii) innovative and context-adapted strategies to strengthen epidemic intelligence including integrated One Health surveillance, digital and genomic tools, participatory approaches, and regional data-sharing mechanisms. We argue that building agile, equity-centered, and decentralized surveillance systems is not only essential for managing known threats, but also foundational to the early detection and rapid containment of the next public health emergency in resource-limited settings. This review uniquely frames surveillance limitations in resource-limited settings as a global security concern and outlines context-adapted, equity-centered innovations to strengthen epidemic intelligence in preparation for Disease X. Full article