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Antiviral innate immunity is the first line of defence against viruses. The interferon (IFN) signaling pathway, the DNA damage response (DDR), apoptosis, endoplasmic reticulum (ER) stress, and autophagy are involved in antiviral innate immunity. Viruses abrogate the antiviral immune response of cells to replication in various ways. Viral genes/proteins play a key role in evading antiviral innate immunity. Here, we will discuss the interference of viruses with antiviral innate immunity and the strategy for identifying viral gene/protein immune evasion.
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Inmunidad Innata , Humanos , Proteínas Virales/inmunología , Proteínas Virales/genética , Virus/inmunología , Virus/genética , Evasión Inmune , Virosis/inmunología , Virosis/virología , Animales , Genes Virales , Autofagia/inmunología , Interacciones Huésped-Patógeno/inmunología , Transducción de Señal/inmunologíaRESUMEN
Background: When individuals infected with human immunodeficiency virus (HIV) experience pulmonary infections, they often exhibit severe symptoms and face a grim prognosis. Consequently, early, rapid, and accurate pathogen diagnosis is vital for informing effective treatment strategies. This study aimed to use metagenomic next-generation sequencing (mNGS) and targeted mNGS (tNGS) to elucidate the characteristics of pulmonary infections in HIV and non-HIV individuals. Methods: This study enrolled 90 patients with pulmonary infection at the Department of Infectious Diseases of The First Hospital of Jilin University from June 2022 to May 2023, and they were divided into HIV (n=46) and non-HIV (n=44) infection groups. Their bronchoalveolar lavage fluid (BALF) was collected for mNGS analysis to evaluate the differences in pulmonary infection pathogens, and tNGS detection was performed on BALF samples from 15 HIV-infected patients. Results: A total of 37 pathogens were identified in this study, including 21 bacteria, 5 fungi, 5 viruses, 5 mycobacteria, and 1 mycoplasma. The sensitivity of mNGS was 78.9% (71/90), which is significantly higher than that of conventional methods (CTM) (39/90, P=1.5E-8). The combination of mNGS with CTM can greatly enhance the sensitivity of pathogen detection. The prevalence of Pneumocystis jirovecii (82.6% vs. 9.1%), cytomegalovirus (CMV) (58.7% vs. 0%), and Epstein-Barr virus (EBV) (17.4% vs. 2.3%) was significantly higher in the HIV infection group than in the non-HIV infection group (P<0.05). Although no statistically significant difference was observed, the detection rate of Mycobacteria was higher in HIV-infected patients (17.4%) than in the non-HIV group (6.8%). Furthermore, the tNGS results of BALF from 15 HIV-infected patients were not entirely consistent with the mNGS results., and the concordance rate of tNGS for the detection of main pathogens reached 86.7% (13/15). Conclusion: Next-generation sequencing (NGS) can accurately detect pathogens in the BALF of patients with pulmonary infection. The sensitivity of tNGS is comparable to that of mNGS. Therefore, this technique should be promoted in the clinic for better patient outcomes.
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Líquido del Lavado Bronquioalveolar , Infecciones por VIH , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Infecciones por VIH/complicaciones , Infecciones por VIH/virología , Masculino , Femenino , Metagenómica/métodos , Líquido del Lavado Bronquioalveolar/microbiología , Líquido del Lavado Bronquioalveolar/virología , Persona de Mediana Edad , Adulto , Bacterias/genética , Bacterias/aislamiento & purificación , Bacterias/clasificación , Anciano , Sensibilidad y Especificidad , Virus/genética , Virus/aislamiento & purificación , Virus/clasificación , Metagenoma , Infecciones del Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/diagnósticoRESUMEN
Human enteric and some respiratory viruses are identified to be involved with acute gastroenteritis that can be shed in feces of infected persons into the environment. Understanding the abundance of these viruses in wastewater is significant when assessing the public health impacts associated with discharge of wastewater into the environment. This study aimed to investigate the prevalence and seasonal variation of human enteric adenovirus (HAdV), Aichi virus (AiV-1), bocavirus (HBoV), and respiratory syndrome coronavirus 2 virus (SARS-CoV-2) in wastewater as well as their prevalence among hospitalized children with acute gastroenteritis. The viruses were detected and quantified with real-time PCR. HAdV was the most detected virus in raw sewage (88.5%), treated sewage (28%), and stool gastroenteritis (74%), followed by HBoV (45.8% for raw sewage, 14.6% for treated sewage, and 55.3% for stool samples). The detection rate of AiV-1 was 59.4%, 19.8%, and 62.6% in raw sewage, treated sewage, and stool samples, respectively. The rate of SARS-CoV-2 detection in raw sewage, treated sewage, and stool samples was 33.3%, 7.3%, and 20.6%, respectively. The viral concentrations ranged between 4.50 × 101 and 8.75 × 107 GC/ml in raw sewage samples, 1.20 × 101 and 5.43 × 106 GC/ml in treated sewage samples, and 4.80 × 101 and 9.88 × 108 GC/gram in stool samples. The overall log means of virus reduction during the wastewater treatment process ranged from 1.68 log10 (HAdV) to 3.31 log10 (AiV-1). The peak prevalence of the four viruses in raw sewage samples occurred during the winter season. This study showed the high detection rates of the four targeted viruses in wastewater and demonstrated that virological surveillance of wastewater in local wastewater treatment plants is a suitable model for epidemiological monitoring of diarrheal and respiratory diseases in middle- and low-resource countries.
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Heces , Gastroenteritis , Estaciones del Año , Aguas del Alcantarillado , Aguas Residuales , Humanos , Gastroenteritis/virología , Gastroenteritis/epidemiología , Aguas Residuales/virología , Prevalencia , Aguas del Alcantarillado/virología , Niño , Heces/virología , Preescolar , Niño Hospitalizado , Lactante , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Virus/aislamiento & purificación , Virus/clasificación , Virus/genética , Kobuvirus/aislamiento & purificación , Kobuvirus/genética , Bocavirus Humano/aislamiento & purificación , Bocavirus Humano/genética , MasculinoRESUMEN
Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system possess a broad range of applications for genetic modification, diagnosis and treatment of infectious as well as non-infectious disease. The CRISPR-Cas system is found in bacteria and archaea that possess the Cas protein and guide RNA (gRNA). Cas9 and gRNA forms a complex to target and cleave the desired gene, providing defense against viral infections. Human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpesviruses, human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cause major life threatening diseases which cannot cure completely by drugs. This chapter describes the present strategy of CRISPR-Cas systems for altering the genomes of viruses, mostly human ones, in order to control infections.
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Sistemas CRISPR-Cas , Edición Génica , Humanos , Sistemas CRISPR-Cas/genética , Virosis/genética , Virosis/terapia , Virosis/virología , Virus/genética , Genoma Viral/genéticaRESUMEN
Background: Although the emerging NGS-based assays, metagenomic next-generation sequencing (mNGS) and targeted next-generation sequencing (tNGS), have been extensively utilized for the identification of pathogens in pulmonary infections, there have been limited studies systematically evaluating differences in the efficacy of mNGS and multiplex PCR-based tNGS in bronchoalveolar lavage fluid (BALF) specimens. Methods: In this study, 85 suspected infectious BALF specimens were collected. Parallel mNGS and tNGS workflows to each sample were performed; then, we comparatively compared their consistency in detecting pathogens. The differential results for clinically key pathogens were confirmed using PCR. Results: The microbial detection rates of BALF specimens by the mNGS and tNGS workflows were 95.18% (79/83) and 92.77% (77/83), respectively, with no significant difference. mNGS identified 55 different microorganisms, whereas tNGS detected 49 pathogens. The comparative analysis of mNGS and tNGS revealed that 86.75% (72/83) of the specimens were complete or partial concordance. Particularly, mNGS and tNGS differed significantly in detection rates for some of the human herpesviruses only, including Human gammaherpesvirus 4 (P<0.001), Human betaherpesvirus 7 (P<0.001), Human betaherpesvirus 5 (P<0.05) and Human betaherpesvirus 6 (P<0.01), in which tNGS always had higher detection rates. Orthogonal testing of clinically critical pathogens showed a total coincidence rate of 50% for mNGS and PCR, as well as for tNGS and PCR. Conclusions: Overall, the performance of mNGS and multiplex PCR-based tNGS assays was similar for bacteria and fungi, and tNGS may be superior to mNGS for the detection of DNA viruses. No significant differences were seen between the two NGS assays compared to PCR.
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Líquido del Lavado Bronquioalveolar , Secuenciación de Nucleótidos de Alto Rendimiento , Metagenómica , Líquido del Lavado Bronquioalveolar/microbiología , Líquido del Lavado Bronquioalveolar/virología , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metagenómica/métodos , Femenino , Masculino , Persona de Mediana Edad , Adulto , Reacción en Cadena de la Polimerasa Multiplex/métodos , Anciano , Bacterias/aislamiento & purificación , Bacterias/genética , Bacterias/clasificación , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Infecciones del Sistema Respiratorio/microbiología , Virus/aislamiento & purificación , Virus/genética , Virus/clasificación , Técnicas de Diagnóstico Molecular/métodos , Adulto JovenRESUMEN
BACKGROUND: Metagenomics is a powerful approach for the detection of unknown and novel pathogens. Workflows based on Illumina short-read sequencing are becoming established in diagnostic laboratories. However, high sequencing depth requirements, long turnaround times, and limited sensitivity hinder broader adoption. We investigated whether we could overcome these limitations using protocols based on untargeted sequencing with Oxford Nanopore Technologies (ONT), which offers real-time data acquisition and analysis, or a targeted panel approach, which allows the selective sequencing of known pathogens and could improve sensitivity. METHODS: We evaluated detection of viruses with readily available untargeted metagenomic workflows using Illumina and ONT, and an Illumina-based enrichment approach using the Twist Bioscience Comprehensive Viral Research Panel (CVRP), which targets 3153 viruses. We tested samples consisting of a dilution series of a six-virus mock community in a human DNA/RNA background, designed to resemble clinical specimens with low microbial abundance and high host content. Protocols were designed to retain the host transcriptome, since this could help confirm the absence of infectious agents. We further compared the performance of commonly used taxonomic classifiers. RESULTS: Capture with the Twist CVRP increased sensitivity by at least 10-100-fold over untargeted sequencing, making it suitable for the detection of low viral loads (60 genome copies per ml (gc/ml)), but additional methods may be needed in a diagnostic setting to detect untargeted organisms. While untargeted ONT had good sensitivity at high viral loads (60,000 gc/ml), at lower viral loads (600-6000 gc/ml), longer and more costly sequencing runs would be required to achieve sensitivities comparable to the untargeted Illumina protocol. Untargeted ONT provided better specificity than untargeted Illumina sequencing. However, the application of robust thresholds standardized results between taxonomic classifiers. Host gene expression analysis is optimal with untargeted Illumina sequencing but possible with both the CVRP and ONT. CONCLUSIONS: Metagenomics has the potential to become standard-of-care in diagnostics and is a powerful tool for the discovery of emerging pathogens. Untargeted Illumina and ONT metagenomics and capture with the Twist CVRP have different advantages with respect to sensitivity, specificity, turnaround time and cost, and the optimal method will depend on the clinical context.
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Metagenómica , Virus , Metagenómica/métodos , Humanos , Virus/genética , Virus/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virosis/diagnóstico , Virosis/virología , Metagenoma , Sensibilidad y EspecificidadRESUMEN
Barramundi aquaculture is at risk of severe disease outbreaks and massive production losses. Here we used bioinformatics to screen 84 farmed barramundi transcriptomes to identify novel viruses that could threaten barramundi aquaculture and to establish a barramundi aquaculture virome. We discovered five novel viruses: latid herpesvirus 1 (LatHV-1) from the Alloherpesviridae family, barramundi parvovirus 1 (BParV1) from the Parvoviridae family, barramundi calicivirus 1 (BCaV1) from the Caliciviridae family, and barramundi associated picorna-like virus 1 and 2 (BPicV1 and BPicV2) from the Picornaviridae family. LatHV-1, BCaV1, and BParV1 are closely related to pathogenic viruses found in other fish species that can cause mass mortality in farms. To aid in future viral surveillance, we also designed and successfully tested an RT-PCR assay for the detection of BCaV1. Overall, we discovered a range of pathogenic viruses in barramundi aquaculture, paving the way for developing effective detection methods to assist early outbreak management.
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Acuicultura , Enfermedades de los Peces , Animales , Enfermedades de los Peces/virología , Enfermedades de los Peces/epidemiología , Australia/epidemiología , Asia/epidemiología , Filogenia , Perciformes/virología , Viroma/genética , Virus/genética , Virus/clasificación , Virus/aislamiento & purificación , Transcriptoma , Virosis/veterinaria , Virosis/virología , Virosis/epidemiología , Picornaviridae/genética , Picornaviridae/aislamiento & purificación , Picornaviridae/clasificaciónRESUMEN
The COVID-19 pandemic underscored the significance of omics technology and Wastewater-Based Epidemiology for epidemic preparedness. This study investigates the virosphere in wastewater samples from Natal (Brazil), aiming to understand its structure, relationships, and potential. Metaviromic analysis was used on DNA and RNA from weekly samples collected over a year (June/2021 to May/2022) from three wastewater treatment plants. The virosphere showed stability, particularly in viruses infecting microorganisms and plants. However, an alternation of representatives of viruses that infect animals has been observed. Among the most abundant viruses infecting microorganisms are genera associated with the bacterial genera Escherichia, Pseudomonas, and Caulobacte. Regarding the viruses infecting plants, Sobemovirus and Tobamovirus are the most abundant genera. Odontoglossum ringspot virus was identified as a possible RNA virus biomarker. Among DNA viruses infecting animals, genera Bocaparvovirus and Mastadenovirus are the most prevalent. Intriguingly, some Poxviridae family members were observed in the samples. Co-occurrence network analysis identified potential biomarkers like Volepox virus, Anatid herpesvirus 1, and Caviid herpesvirus 2. Among RNA viruses affecting animals, Mamastrovirus, Rotavirus, and Norovirus genera were the most abundant pathogens. Furthermore, members of the Coronaviridae family exhibited a high degree of centrality values in the co-occurrence network, even connecting with unclassified viruses. The study emphasizes the importance of research in understanding the roles of unclassified viruses. In addition, we observed an association between Coronaviridae reads, rainfall, and the number of reported COVID-19 cases. Our study highlights the diversity and complexity of the viral community in wastewater and the need for research to understand better the ecological roles unclassified viruses play. Such advances will significantly contribute to our preparedness and response to future viral threats. Furthermore, our study contributes to knowledge of virosphere dynamics, offering insights that can contribute to the direction of future public health policies and interventions.
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Aguas Residuales , Brasil , Aguas Residuales/virología , Virus/genética , Virus/aislamiento & purificación , Virus ARN/genética , Viroma , COVID-19/virologíaRESUMEN
BACKGROUND: Prokaryotic microbes have impacted marine biogeochemical cycles for billions of years. Viruses also impact these cycles, through lysis, horizontal gene transfer, and encoding and expressing genes that contribute to metabolic reprogramming of prokaryotic cells. While this impact is difficult to quantify in nature, we hypothesized that it can be examined by surveying virus-encoded auxiliary metabolic genes (AMGs) and assessing their ecological context. RESULTS: We systematically developed a global ocean AMG catalog by integrating previously described and newly identified AMGs and then placed this catalog into ecological and metabolic contexts relevant to ocean biogeochemistry. From 7.6 terabases of Tara Oceans paired prokaryote- and virus-enriched metagenomic sequence data, we increased known ocean virus populations to 579,904 (up 16%). From these virus populations, we then conservatively identified 86,913 AMGs that grouped into 22,779 sequence-based gene clusters, 7248 (~ 32%) of which were not previously reported. Using our catalog and modeled data from mock communities, we estimate that ~ 19% of ocean virus populations carry at least one AMG. To understand AMGs in their metabolic context, we identified 340 metabolic pathways encoded by ocean microbes and showed that AMGs map to 128 of them. Furthermore, we identified metabolic "hot spots" targeted by virus AMGs, including nine pathways where most steps (≥ 0.75) were AMG-targeted (involved in carbohydrate, amino acid, fatty acid, and nucleotide metabolism), as well as other pathways where virus-encoded AMGs outnumbered cellular homologs (involved in lipid A phosphates, phosphatidylethanolamine, creatine biosynthesis, phosphoribosylamine-glycine ligase, and carbamoyl-phosphate synthase pathways). CONCLUSIONS: Together, this systematically curated, global ocean AMG catalog and analyses provide a valuable resource and foundational observations to understand the role of viruses in modulating global ocean metabolisms and their biogeochemical implications. Video Abstract.
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Océanos y Mares , Agua de Mar , Agua de Mar/virología , Agua de Mar/microbiología , Metagenómica , Bacterias/genética , Bacterias/clasificación , Bacterias/metabolismo , Virus/genética , Virus/clasificación , Virus/aislamiento & purificación , Células Procariotas/metabolismo , Células Procariotas/virología , Metagenoma , Redes y Vías Metabólicas/genética , Transferencia de Gen Horizontal , Fosfatidiletanolaminas/metabolismoRESUMEN
A highly critical event in a virus's life cycle is successfully entering a given host. This process begins when a viral glycoprotein interacts with a target cell receptor, which provides the molecular basis for target virus-host cell interactions for novel drug discovery. Over the years, extensive research has been carried out in the field of virus-host cell interaction, generating a massive number of genetic and molecular data sources. These datasets are an asset for predicting virus-host interactions at the molecular level using machine learning (ML), a subset of artificial intelligence (AI). In this direction, ML tools are now being applied to recognize patterns in these massive datasets to predict critical interactions between virus and host cells at the protein-protein and protein-sugar levels, as well as to perform transcriptional and translational analysis. On the other end, deep learning (DL) algorithms-a subfield of ML-can extract high-level features from very large datasets to recognize the hidden patterns within genomic sequences and images to develop models for rapid drug discovery predictions that address pathogenic viruses displaying heightened affinity for receptor docking and enhanced cell entry. ML and DL are pivotal forces, driving innovation with their ability to perform analysis of enormous datasets in a highly efficient, cost-effective, accurate, and high-throughput manner. This review focuses on the complexity of virus-host cell interactions at the molecular level in light of the current advances of ML and AI in viral pathogenesis to improve new treatments and prevention strategies.
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Inteligencia Artificial , Humanos , Aprendizaje Automático , Interacciones Huésped-Patógeno , Virus/genética , Virus/metabolismo , Interacciones Microbiota-Huesped/genética , Aprendizaje Profundo , Algoritmos , AnimalesAsunto(s)
COVID-19 , Enfermedades Transmisibles Emergentes , Pandemias , Humanos , Pandemias/prevención & control , Enfermedades Transmisibles Emergentes/epidemiología , Enfermedades Transmisibles Emergentes/prevención & control , Enfermedades Transmisibles Emergentes/virología , COVID-19/epidemiología , COVID-19/prevención & control , COVID-19/virología , SARS-CoV-2/genética , Animales , Virus/genética , Virosis/epidemiología , Virosis/prevención & control , Virosis/virología , Preparación para una PandemiaRESUMEN
Waste pickers constitute a marginalized demographic engaged in the collection of refuse, facing considerable occupational hazards that heighten their susceptibility to contract infectious diseases. Moreover, waste pickers contend with societal stigmatization and encounter barriers to accessing healthcare services. To explore the viral profile of waste pickers potentially linked to their occupational environment, we conducted a metagenomic analysis on 120 plasma specimens sampled from individuals employed at the Cidade Estrutural dumpsite in Brasilia city, Brazil. In total, 60 blood donors served as a comparative control group. Specimens were pooled and subjected to Illumina NextSeq 2000 sequencing. Viral abundance among waste pickers revealed the presence of significant pathogens, including HIV, HCV, and Chikungunya, which were not detected in the control group. Additionally, elevated levels of anelloviruses and Human pegivirus-1 were noted, with a comparable incidence in the control group. These findings underscore the utility of metagenomics in identifying clinically relevant viral agents within underserved populations. The implications of this study extend to informing public health policies aimed at surveilling infectious diseases among individuals facing socioeconomic disparities and limited access to healthcare resources.
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Metagenómica , Humanos , Brasil , Masculino , Adulto , Femenino , Virus/genética , Virus/clasificación , Virus/aislamiento & purificación , Exposición Profesional , Estudios de Casos y Controles , Persona de Mediana Edad , Eliminación de ResiduosRESUMEN
The soil microbiome is recognized as an essential component of healthy soils. Viruses are also diverse and abundant in soils, but their roles in soil systems remain unclear. Here we argue for the consideration of viruses in soil microbial food webs and describe the impact of viruses on soil biogeochemistry. The soil food web is an intricate series of trophic levels that span from autotrophic microorganisms to plants and animals. Each soil system encompasses contrasting and dynamic physicochemical conditions, with labyrinthine habitats composed of particles. Conditions are prone to shifts in space and time, and this variability can obstruct or facilitate interactions of microorganisms and viruses. Because viruses can infect all domains of life, they must be considered as key regulators of soil food web dynamics and biogeochemical cycling. We highlight future research avenues that will enable a more robust understanding of the roles of viruses in soil function and health.
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Cadena Alimentaria , Microbiota , Microbiología del Suelo , Suelo , Virus , Virus/genética , Virus/clasificación , Virus/aislamiento & purificación , Suelo/química , Animales , Plantas/virología , Plantas/microbiología , Ecosistema , Bacterias/virología , Bacterias/metabolismo , Bacterias/genéticaRESUMEN
Zoonotic viruses are widely seen as the primary threat for future pandemics. Bats are the most diverse group of mammals, with more than 1400 species distributed across most habitats on Earth. So far, 31 known virus families were associated with bats, although the understanding of most viruses were insufficient. Continuous efforts to discover, understand and monitor these bats viruses, is thereby an area of public health interest. This systematic review was designed to catalogue publications reporting novel bat virus discoveries within PubMed, SCOPUS, and Web of Science databases, within a 5-year period from 2018 to 2022. Various experimental parameters, including sampling locations, methodology, bat species diversity, similarity to known viruses, species demarcation of new viruses, and genomic sequencing strategies, were extracted from 41 publications and analyzed. In total, 72 novel viruses from 19 virus families were identified between 2018 and 2022, particularly from Genomoviridae (DNA viruses) and Coronaviridae (RNA viruses). That said, only a limited number of bat families featured extensively despite noticeable shift towards next generation sequencing methods and metagenomics pipeline for virus identification across different sampling methods. This review aims to provide a comprehensive analysis of the global efforts made over the past five years to identify and characterize emerging viruses in bat species, and to provide a detailed overview of the current technologies and methodologies used in these studies.
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Quirópteros , Metagenómica , Quirópteros/virología , Animales , Metagenómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Virus/clasificación , Virus/genética , Virus/aislamiento & purificación , Genoma Viral , Virus ARN/genética , Virus ARN/clasificación , Virus ARN/aislamiento & purificación , Filogenia , Virus ADN/genética , Virus ADN/clasificación , Virus ADN/aislamiento & purificación , Virosis/virología , Virosis/veterinaria , Zoonosis Virales/virologíaRESUMEN
Viruses are the most abundant yet understudied members that may influence microbial metabolism in activated sludge treating antibiotic production wastewater. This study comprehensively investigated virome community characteristics under the selection pressure of nine types and different concentrations of antibiotics using a metagenomics approach. Of the 15,514 total viral operational taxonomic units (tOTUs) recovered, only 37.5 % were annotated. Antibiotics altered the original viral community structure in activated sludge. The proportion of some pathogenic viral families, including Herpesviridae_like, increased significantly in reactors treating erythromycin production wastewater. In total, 16.5 % of the tOTUs were associated with two or more hosts. tOTUs rarely carried antibiotic resistance genes (ARGs), and the ARG types in the tOTUs did not match the ARGs carried by the bacterial hosts. This suggests that transduction contributes little to the horizontal ARG transfer. Auxiliary metabolic genes (AMGs) were prevalent in tOTUs, and those involved in folate biosynthesis were particularly abundant, indicating their potential to mitigate antibiotic-induced host damage. This study provides comprehensive insights into the virome community in activated sludge treating antibiotic production wastewater and sheds light on the potential role of viral AMGs in mitigating antibiotic-induced stress.
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Antibacterianos , Reactores Biológicos , Aguas del Alcantarillado , Aguas Residuales , Aguas del Alcantarillado/virología , Eliminación de Residuos Líquidos , Farmacorresistencia Microbiana/genética , Virus/genéticaRESUMEN
Although long-read sequencing has enabled obtaining high-quality and complete genomes from metagenomes, many challenges still remain to completely decompose a metagenome into its constituent prokaryotic and viral genomes. This study focuses on decomposing an estuarine metagenome to obtain a more accurate estimate of microbial diversity. To achieve this, we developed a new bead-based DNA extraction method, a novel bin refinement method, and obtained 150 Gbp of Nanopore sequencing. We estimate that there are ~500 bacterial and archaeal species in our sample and obtained 68 high-quality bins (>90% complete, <5% contamination, ≤5 contigs, contig length of >100 kbp, and all ribosomal and tRNA genes). We also obtained many contigs of picoeukaryotes, environmental DNA of larger eukaryotes such as mammals, and complete mitochondrial and chloroplast genomes and detected ~40,000 viral populations. Our analysis indicates that there are only a few strains that comprise most of the species abundances. IMPORTANCE: Ocean and estuarine microbiomes play critical roles in global element cycling and ecosystem function. Despite the importance of these microbial communities, many species still have not been cultured in the lab. Environmental sequencing is the primary way the function and population dynamics of these communities can be studied. Long-read sequencing provides an avenue to overcome limitations of short-read technologies to obtain complete microbial genomes but comes with its own technical challenges, such as needed sequencing depth and obtaining high-quality DNA. We present here new sampling and bioinformatics methods to attempt decomposing an estuarine microbiome into its constituent genomes. Our results suggest there are only a few strains that comprise most of the species abundances from viruses to picoeukaryotes, and to fully decompose a metagenome of this diversity requires 1 Tbp of long-read sequencing. We anticipate that as long-read sequencing technologies continue to improve, less sequencing will be needed.
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Estuarios , Metagenómica , Microbiota , Virus , Microbiota/genética , Metagenómica/métodos , San Francisco , Virus/genética , Virus/clasificación , Virus/aislamiento & purificación , Metagenoma/genética , Bacterias/genética , Bacterias/clasificación , Archaea/genética , Archaea/virología , Eucariontes/genética , Genoma Viral/genéticaRESUMEN
Nucleic acid detection technology has become a crucial tool in cutting-edge research within the life sciences and clinical diagnosis domains. Its significance is particularly highlighted during the respiratory virus pandemic, where nucleic acid testing plays a pivotal role in accurately detecting the virus. Isothermal amplification technologies have been developed and offer advantages such as rapidity, mild reaction conditions and excellent stability. Among these methods, recombinase polymerase amplification (RPA) has gained significant attention due to its simple primer design and resistance to multiple reaction inhibitors. However, the detection of RPA amplicons hinders the widespread adoption of this technology, leading to a research focus on cost-effective and convenient detection methods for RPA nucleic acid testing. In this study, we propose a novel computational absorption spectrum approach that utilizes the polar GelRed dye to efficiently detect RPA amplicons. By exploiting the asymmetry of GelRed molecules upon binding with DNA, polar electric dipoles are formed, leading to precipitate formation through centrifugal vibration and electrostatic interaction. The quantification of amplicon content is achieved by measuring the residual GelRed concentration in the supernatant. Our proposed portable and integrated microfluidic device successfully detected five respiratory virus genes simultaneously. The optimized linear detection was achieved and the sensitivity for all the targets reached 100 copies/µL. The total experiment could be finished in 27 min. The clinical experiments demonstrated the practicality and accuracy. This cost-effective and convenient detection scheme presents a promising biosensor for rapid virus detection, contributing to the advancement of RPA technology.
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Técnicas de Amplificación de Ácido Nucleico , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , Dispositivos Laboratorio en un Chip , Centrifugación , Análisis Espectral/métodos , Virus/aislamiento & purificación , Virus/genética , Técnicas Analíticas Microfluídicas/métodos , Técnicas Analíticas Microfluídicas/instrumentaciónRESUMEN
OBJECTIVE: To explore the application value of the second-generation metagenomic next-generation sequencing (mNGS) in the detection of pathogens in patients with pulmonary infection. METHODS: We conducted a retrospective analysis of 65 pulmonary infection cases treated at our institution and the Fifth People's Hospital of Shanghai between January 2021 and May 2023. All subjects were subjected to mNGS, targeted next-generation sequencing (tNGS), and conventional microbiological culture. A comparative analysis was performed to evaluate the diversity and quantity of pathogens identified by these methodologies and to appraise their respective diagnostic capabilities in pulmonary infection diagnostics. RESULTS: The mNGS successfully identified etiological agents in 60 of the 65 cases, compared to tNGS, which yielded positive results in 42 cases, and conventional laboratory cultures, which detected pathogens in 24 cases. At the bacterial genus level, mNGS discerned 9 genera, 11 species, and 92 isolates of pathogenic bacteria, whereas tNGS identified 8 genera, 8 species, and 71 isolates. Conventional methods were less sensitive, detecting only 6 genera, 7 species, and 33 isolates. In terms of fungal detection, mNGS identified 4 fungal species, tNGS detected 4 isolates of the Candida genus, and conventional methods identified 2 isolates of the same genus. Viral detection at the species level revealed 10 species and 46 isolates by mNGS, whereas tNGS detected only 3 species and 7 isolates. The area under the receiver operating characteristic curve (AUC) with 95% confidence intervals for diagnosing pulmonary infections was 0.818 (0.671 to 0.966) for mNGS, 0.668 (0.475 to 0.860) for tNGS, and 0.721 (0.545 to 0.897) for conventional culture.The mNGS demonstrates superior diagnostic efficacy and pathogen detection breadth in critically ill patients with respiratory infections, offering a significant advantage by reducing the time to diagnosis. The enhanced sensitivity and comprehensive pathogen profiling of mNGS underscore its potential as a leading diagnostic tool in clinical microbiology.