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1.
Medicine (Baltimore) ; 99(2): e18504, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31914021

RESUMEN

We aimed to evaluate the clinical significance of bacterial coexistence and the coinfection dynamics between bacteria and respiratory viruses among young children. We retrospectively analyzed clinical data from children aged < 5 years hospitalized with a community-acquired single respiratory viral infection of influenza, adenovirus, or RSV during 2 recent consecutive influenza seasons. Remnant respiratory specimens were used for bacterial PCR targeting Moraxella catarrhalis, Haemophilus influenzae, Streptococcus pneumoniae, and Staphylococcus aureus.A total of 102 children were included; median age was 0.8 years and 44.1% had underlying comorbidities. Overall, 6.8% (7/102) of cases were classified as severe diseases requiring intensive care unit admission and/or mechanical ventilation and ranged from 8.8% for a patient with RSV and 7.6% for those with adenovirus to 0% for those with influenza viruses. The overall viral-bacterial codetection rate was 59.8% (61/102); M catarrhalis was the most frequent (33.3%), followed by H influenzae (31.4%). Influenza cases showed higher bacterial codetection rates (80.0%; 8/10) compared with those with adenoviruses (69.2%; 9/13) and RSV (55.7%; 44/79). S pneumoniae and H influenzae codetections were associated with reduced severity (aOR, 0.24; 95% CI, 0.07-0.89), and reduced risk of wheezing (aOR, 0.36; 95% CI, 0.13-0.98), respectively.We observed the interactions between respiratory viruses and bacteria and the clinical significance of viral-bacterial coexistence in upper airway on disease severity. Future study will be necessary to elucidate the active interactions between different viruses and bacteria and give clues to risk stratified strategy in the management of respiratory infections among young children.


Asunto(s)
Adenoviridae/aislamiento & purificación , Haemophilus influenzae/aislamiento & purificación , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Infecciones por Adenoviridae/diagnóstico , Infecciones por Adenoviridae/virología , Bacterias/genética , Preescolar , Coinfección/microbiología , Coinfección/virología , Infecciones Comunitarias Adquiridas/diagnóstico , Infecciones Comunitarias Adquiridas/microbiología , Infecciones Comunitarias Adquiridas/virología , Femenino , Hospitalización , Humanos , Lactante , Recién Nacido , Gripe Humana/diagnóstico , Gripe Humana/virología , Masculino , Prevalencia , Ruidos Respiratorios/diagnóstico , Ruidos Respiratorios/etiología , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/genética , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/epidemiología , Estudios Retrospectivos , Índice de Severidad de la Enfermedad , Staphylococcus aureus/aislamiento & purificación , Streptococcus pneumoniae/aislamiento & purificación , Virus/genética
2.
Phytopathology ; 110(1): 58-67, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31448996

RESUMEN

Potato mop top virus (PMTV) is a continuing threat to potato production throughout the world. It has the potential to persist in the soil for long periods in the sporosori of its vector Spongospora subterranea f. sp. subterranea, which is as an important source for PMTV infection and dissemination. In this study, we used real-time quantitative reverse-transcription PCR (qRT-PCR) and reverse-transcription droplet digital PCR (RT-ddPCR) assays of the total RNA extracted directly from the soil to develop a simple, fast, and sensitive method to detect PMTV in soil samples using a specific primer with high efficiency despite a minimal amount of viral RNA. The designed primers are resilient in the presence of various PCR inhibitors in the soil when RNA is extracted. Both assays detected PMTV in all soil types used and supported the detection of <10 PMTV copies µl-1 in the RNA sample. With qRT-PCR, detection was linear, with amplification efficiencies ranging from 93.3 to 105.3% for silt loam, loamy sand, sand, and sandy loam in various experiments with R2 > 0.99. Furthermore, the RT-ddPCR assay also demonstrated a high degree of linearity (R2 > 0.99 and P < 0.0001) with the RNA extracted from the soil samples representing different textures and physiochemical characteristics that were artificially spiked with infested S. subterranea f. sp. subterranea sporosori. Additionally, both assays successfully detected PMTV in different types of naturally infested soil with PMTV carrying S. subterranea f. sp. subterranea sporosori levels ranging from 6.2 × 102 g-1 to 1.2 × 106 g-1 in soils with pH ranging from 4.9 to 7.5 and organic matter ranging from 0.9 to 5.1%, demonstrating the potential to detect PMTV in a wide variety of soils. To our knowledge, this is the first report of the development of real-time PCR and ddPCR methods for the direct detection of a soilborne virus in soil.


Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa , Microbiología del Suelo , Virus , Enfermedades de las Plantas/virología , Plasmodiophorida , Reproducibilidad de los Resultados , Suelo/química , Virus/genética
3.
N Engl J Med ; 381(26): 2569-2580, 2019 12 26.
Artículo en Inglés | MEDLINE | ID: mdl-31881145

RESUMEN

Rapid advances in DNA sequencing technology ("next-generation sequencing") have inspired optimism about the potential of human genomics for "precision medicine." Meanwhile, pathogen genomics is already delivering "precision public health" through more effective investigations of outbreaks of foodborne illnesses, better-targeted tuberculosis control, and more timely and granular influenza surveillance to inform the selection of vaccine strains. In this article, we describe how public health agencies have been adopting pathogen genomics to improve their effectiveness in almost all domains of infectious disease. This momentum is likely to continue, given the ongoing development in sequencing and sequencing-related technologies.


Asunto(s)
Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos/epidemiología , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Gripe Humana/epidemiología , Salud Pública , Tuberculosis/epidemiología , Animales , Bacterias/genética , Enfermedades Transmitidas por los Alimentos/diagnóstico , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/parasitología , Humanos , Gripe Humana/diagnóstico , Gripe Humana/microbiología , Metagenómica , Parásitos/genética , Tuberculosis/diagnóstico , Virus/genética
4.
Emerg Microbes Infect ; 8(1): 1701-1710, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31749410

RESUMEN

We conducted this study to describe the dynamics of the acquisition of respiratory pathogens, their potential interactions and risk factors for possible lower respiratory tract infection symptoms (LRTI) among French pilgrims during the 2018 Hajj. Each participant underwent four successive systematic nasopharyngeal swabs before and during their stay in Saudi Arabia. Carriage of the main respiratory pathogens was assessed by PCR. 121 pilgrims were included and 93.4% reported respiratory symptoms during the study period. The acquisition of rhinovirus, coronaviruses and Staphylococcus aureus occurred soon after arrival in Saudi Arabia and rates decreased gradually after days 5 and 6. In contrast, Streptococcus pneumoniae and Klebsiella pneumoniae carriage increased progressively until the end of the stay in Saudi Arabia. Haemophilus influenzae and Moraxella catarrhalis carriage increased starting around days 12 and 13, following an initial clearance. Influenza viruses were rarely isolated. We observed an independent positive mutual association between S. aureus and rhinovirus carriage and between H. influenzae and M. catarrhalis carriage. Dual carriage of H. influenzae and M. catarrhalis was strongly associated with S. pneumoniae carriage (OR = 6.22). Finally, our model showed that M. catarrhalis carriage was negatively associated with K. pneumoniae carriage. Chronic respiratory disease was associated with symptoms of LRTI. K. pneumoniae, M. catarrhalis-S. aureus and H. influenzae-rhinovirus dual carriage was associated with LRTI symptoms. Our data suggest that RTIs at the Hajj are a result of complex interactions between a number of respiratory viruses and bacteria.


Asunto(s)
Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Virosis/virología , Virus/aislamiento & purificación , Adulto , Anciano , Anciano de 80 o más Años , Bacterias/clasificación , Bacterias/genética , Femenino , Francia , Humanos , Masculino , Persona de Mediana Edad , Arabia Saudita , Viaje , Virus/clasificación , Virus/genética
5.
Mikrobiyol Bul ; 53(4): 434-441, 2019 Oct.
Artículo en Turco | MEDLINE | ID: mdl-31709940

RESUMEN

Identification of viral agents causing central nervous system (CNS) infections increased by the application of nucleic acid tests. In this study, the results of polymerase chain reaction (PCR) for viral agents were evaluated in cerebrospinal fluid (CSF) samples taken from patients with CNS infection. CSF samples taken from 1185 patients between 2010 and 2017 were tested for the presence of Herpes simplex virus (HSV) type 1 and 2, Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), cytomegalovirus (CMV), adenovirus ve enterovirus by PCR in Dokuz Eylul University Hospital. Tests were performed according to the clinicians' orders and results were evaluated retrospectively. The number of tests performed were 1038 for HSV, 882 for adenovirus, 865 for enterovirus, 496 for VZV, 100 for EBV and 92 for CMV. Commercial tests were used for EBV, CMV (Artus QS-RGQ Kits, Qiagen, Germany) and enterovirus (GeneXpert, Cepheid, USA) while the other viruses (HSV, VZV, adenovirus) were tested by in-house real-time PCR assays. Ninety-one CSF (7.7%) samples were positive. The mean age was 13 (<1 to 76 years) while median was seven. The most frequently detected pathogens were enterovirus (63/91, 69%) and HSV-1 (14/91,15%). The number of patients positive for adenovirus, VZV, EBV and CMV were five, four, three and two, respectively. In one patient, both enterovirus (Ct: 29.5) and EBV (Ct: 38.53) were positive. The number of positive samples were increased in summer months. Enterovirus RNA positive patients (n= 60/63, 95.2%) were ≤ 18 years old while 29% were younger than one year of age. Enterovirus positive samples peaked in 2012 and 2014 and detected mainly in summer (60.3%) and autumn (20.6%) months. VZV was mostly detected in patients greater than 65 years of age. Mean Ct of the positive reactions was 31.87 ± 3.5 (22.88-40.32). The lowest and the highest Ct values were detected in HSV-1 assay. The mean Ct value of enterovirus assay (30.4; 25.7-35.9) was lower than the other pathogens' values. In the seven-year period, 7.7% of the1185 patients' CSF samples were positive for viral nucleic acids. As expected, enteroviruses were the most common pathogens in children and detected mainly in summer-autumn period. Syndromic approach in CNS infections could increase the viral pathogen detection.


Asunto(s)
Enfermedades Virales del Sistema Nervioso Central , Adolescente , Adulto , Anciano , Enfermedades Virales del Sistema Nervioso Central/líquido cefalorraquídeo , Enfermedades Virales del Sistema Nervioso Central/epidemiología , Enfermedades Virales del Sistema Nervioso Central/virología , Niño , Preescolar , ADN Viral/genética , Alemania/epidemiología , Humanos , Lactante , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Estudios Retrospectivos , Virus/genética , Adulto Joven
6.
Curr Top Microbiol Immunol ; 424: 75-83, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31650236

RESUMEN

Infectious disease emergence into humans from animals or the environment occurs primarily due to genetic changes in the microbe through mutation or re-assortment making it either more transmissible or virulent or through a change in the disease "ecosystem". Research into infectious disease emergence can be grouped into different strategic approaches. One strategic approach is to study a specific or model disease system to understand the ecology of an infectious disease and how is transmitted and propagated through the environment and different hosts and then extrapolate that disease system knowledge to related pathogens. The other strategic approach follows the genomics and phylogenetics-tracking how pathogens are evolving and changing at the amino acid level. Here we argue that for understanding complex zoonotic diseases and for the purposes of preventing emergence and re-emergence into humans, that the Return on Investment be considered for the best research strategy.


Asunto(s)
Enfermedades Transmisibles/economía , Enfermedades Transmisibles/epidemiología , Ecosistema , Monitoreo Epidemiológico , Filogenia , Virus/clasificación , Virus/patogenicidad , Animales , Enfermedades Transmisibles/clasificación , Enfermedades Transmisibles/virología , Humanos , Inversiones en Salud , Virus/genética , Zoonosis/virología
7.
Lett Appl Microbiol ; 69(5): 318-324, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31529706

RESUMEN

Peach is a major crop in China, and like any other stone fruit, virus and virus-like diseases can reduce the yield and quality of the fruit. Herein, we developed a multiplex RT-PCR (mRT-PCR) assay for simultaneously detecting three viruses known to infect peach: peach-associated luteovirus (PaLV), peach virus D (PeVD) and nectarine stem-pitting-associated virus (NSPaV). Plant nad5 mRNA was used as the internal control. Field samples that were co-infected with PaLV, PeVD and NSPaV were used; we identified three primer pairs to be the most specific for detecting these viruses, followed by determining the ideal concentration of each primer pair and optimizing the annealing temperature for mRT-PCR. We also assessed the detection limit using serial dilutions of RNA and cDNA. The newly developed mRT-PCR assay could simultaneously detect PaLV, PeVD and NSPaV. To validate the reliability of mRT-PCR for virus detection, mRT-PCR was used to detect viruses in the leaves of 21 peach plants collected in Liaoning Province, China. The obtained results revealed the presence of single and co-infections. To conclude, the mRT-PCR assay developed herein is sensitive, reliable and economical, and we believe that it can thus be used for large-scale surveys of PaLV, PeVD and NSPaV. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, we developed a multiplex reverse transcriptase PCR (mRT-PCR) assay for simultaneously detecting three viruses that infect peach: peach-associated luteovirus (PaLV), peach virus D (PeVD) and nectarine stem-pitting-associated virus (NSPaV). This assay is simple, easy to perform, reliable and cost-effective, and can thus be applied for large-scale surveys of PaLV, PeVD and NSPaV.


Asunto(s)
Reacción en Cadena de la Polimerasa Multiplex/métodos , Enfermedades de las Plantas/virología , Prunus persica/virología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Virus/aislamiento & purificación , China , Cartilla de ADN/genética , Hojas de la Planta/virología , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Virus/clasificación , Virus/genética
8.
Virol J ; 16(1): 111, 2019 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-31481063

RESUMEN

BACKGROUND: Viral respiratory tract infections are common during early childhood. How they impact cystic fibrosis lung disease history in young children is poorly known. The principal aim of our study was to determinate respiratory tract infections frequency in this cystic fibrosis young population. Secondary outcomes were nature of viral agents recovered and impact of such infections. METHODS: We conducted a prospective cohort study of 25 children affected by cystic fibrosis and aged less than 2 years. Nasal samplings were taken systematically monthly or bimonthly with additional samples taken during respiratory tract infections episodes. Ten pathogens were tested by a combination of five duplex RT-PCRs or PCRs: influenza A and B, respiratory syncytial virus (RSV), metapneumovirus (MPV), rhinovirus/enterovirus (RV/EV)), coronavirus (HKU1, NL63, 229E and OC43), parainfluenza virus (1-4), adenovirus and bocavirus (Respiratory Multi-Well System MWS r-gene®, BioMérieux, Marcy l'Étoile, France). Cycle thresholds (CTs) were reported for all positive samples and considered positive for values below 40. Quantitative variables were compared using a nonparametric statistical test (Wilcoxon signed rank for paired comparisons). Pearson's correlation coefficient (r) was used to assess relationships between two variables. Statistical analyses were performed using SAS v9.4 (SAS Institute, Cary, NC, USA) or GraphPad Prism V6.00 (GraphPad Software, La Jolla, CA, USA). The significance level was set at 0.05. RESULTS: The mean age at inclusion was 9.6 ± 6.7 months. The patients had 3.4 ± 1.7 respiratory tract infections episodes per child per year. Forty-four respiratory tract infections (69%) were associated with virus: rhinovirus and enterovirus (RV/EV) were implied in 61% of them and respiratory syncytial virus (RSV) in 14%. Only one patient required hospitalization for lower respiratory tract infections. 86% of the patients were treated by antibiotics for a mean of 13.8 ± 6.2 days. RSV infections (n = 6) were usually of mild severity. CONCLUSIONS: Respiratory tract infections in young children with cystic fibrosis were of mild severity, rarely requiring hospitalization. Unsurprisingly, RV/EV were the most frequent agents. RSV-related morbidity seems low in this population. This raises the question of the usefulness of RSV preventive medication in this young population.


Asunto(s)
Coinfección/virología , Fibrosis Quística/virología , Infecciones del Sistema Respiratorio/virología , Estaciones del Año , Virus/aislamiento & purificación , Infecciones por Coronavirus/complicaciones , Fibrosis Quística/complicaciones , Femenino , Francia , Humanos , Lactante , Masculino , Infecciones por Picornaviridae/complicaciones , Estudios Prospectivos , Infecciones por Virus Sincitial Respiratorio/complicaciones , Índice de Severidad de la Enfermedad , Virus/genética , Virus/patogenicidad
9.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 904-910, 2019 Aug 10.
Artículo en Chino | MEDLINE | ID: mdl-31484252

RESUMEN

Objective: To analyze the etiologic and epidemiological characteristics of adult acute respiratory infections in Shanghai during 2015-2017. Methods: Data was collected from outpatients with acute respiratory infections who visited the Fever Clinics in three hospitals of different levels in three administrative regions of Shanghai, from 2015 to 2017. Basic information and nasopharyngeal swabs were collected from cases in line with the inclusion criteria. Multiplex RT-PCR and bacterial cultures were performed to detect the respiratory pathogens. Results: A total of 806 individuals were enrolled from 2015 to 2017. Respiratory pathogens were identified in 73.45% (592/806) of the cases, with the virus detection rate as 66.75% (538/806). It was found that the major respiratory pathogens for virus detection were influenza A in 326 (40.45%), influenza B in 116 (14.39%), rhinovirus/enterovirus in 39 (4.84%) of the cases. The overall detection rate of bacteria was 16.13% (130/806), including Klebsiella pneumoniae in 90 (11.17%) cases, Staphylococcus Aureus in 46 (5.71%) cases. Other kind of bacteria were not detected in our study. The detection rates on Mycoplasma pneumoniae was 5.33% (43/806) and on Chlamydia pneumonia was 0.37% (3/806). Co-infection with multiple pathogens was detected in 18.61% (150/806) of the cases, including 135 with double infection (accounting for 90.00%), 14 with triple infection and 1 with quadruple infection (accounted for 9.33% and 0.67%, respectively). Among the 150 cases with co-infections, the main identified pathogens were influenza A, Klebsiella pneumoniae, Staphylococcus aureus, and Mycoplasma pneumoniae. Pathogens of acute respiratory infections that identified among the outpatients from the Fever Clinics at different time, region or population, the characteristics were different (P<0.001). Conclusions: In 2015-2017, outpatients with acute respiratory infections in Shanghai were mainly caused by influenza virus or other viruses, however dynamically with its composition, time, region and characteristics of the population. It is necessary to strengthen and combine related medical and preventive services and to develop the appropriate strategies regarding clinical diagnosis and treatment.


Asunto(s)
Bacterias/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Gripe Humana/diagnóstico , Reacción en Cadena de la Polimerasa Multiplex/métodos , Nasofaringe , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/etiología , Virosis/diagnóstico , Virus/aislamiento & purificación , Enfermedad Aguda , Adulto , Bacterias/genética , Infecciones Bacterianas/epidemiología , Infecciones Bacterianas/microbiología , China/epidemiología , Coinfección/diagnóstico , Enterovirus/genética , Enterovirus/aislamiento & purificación , Monitoreo Epidemiológico , Humanos , Incidencia , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Virus de la Influenza B/genética , Virus de la Influenza B/aislamiento & purificación , Gripe Humana/epidemiología , Gripe Humana/virología , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/aislamiento & purificación , Mycoplasma pneumoniae , Nasofaringe/microbiología , Nasofaringe/virología , Vigilancia de la Población , Infecciones del Sistema Respiratorio/diagnóstico , Rhinovirus/genética , Rhinovirus/aislamiento & purificación , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Virosis/epidemiología , Virosis/virología , Virus/genética
10.
Zhonghua Liu Xing Bing Xue Za Zhi ; 40(8): 911-916, 2019 Aug 10.
Artículo en Chino | MEDLINE | ID: mdl-31484253

RESUMEN

Objective: To understand the epidemiological and pathogenic characteristics of hospitalized severe acute respiratory infections (SARI) in Shanghai, China. Methods: From 2015 to 2017, one Tertiary hospital and one Secondary hospital were chosen as the surveillance sites. Two respiratory tract specimens per case were collected from SARI cases aged 15 years and older. One specimen was tested for 22 respiratory pathogens by RT-PCR, and the other specimen was cultured for 6 respiratory bacteria. Results: A total of 287 SARI cases were enrolled for sampling and lab testing. 70.73% of the cases were aged 60 years and older, with 41.46% (119/287) were positive for at least one pathogen. Influenza virus was the predominant pathogen, accounting for 17.77% (51/287) of all SARI cases. Human rhinovirus/Enterovirus and Coronavirus were both accounting for 7.32% (21/287), followed by Mycoplasma pneumoniae (5.57%, 16/287). The positive rates of parainfluenza virus, bocavirus, adenovirus, respiratory syncytial virus and human metapneumo virus were all less than 5%. Bacterial strains were identified in seven SARI cases, including Klebsiella pneumoniae (3 strains), Staphylococcus aureus (2 strains), Streptococcus pneumoniae (1 strain) and Pseudomonas aeruginosa (1 strain). Two or Three pathogens were co-detected from 40 cases, accounting for 33.61% of 119 positive cases. The most common co-detected pathogens were influenza virus and Mycoplasma pneumoniae (10 cases). Influenza cases peaked in winter-spring and summer. Mycoplasma pneumoniae peaked in winter-spring season and overlapped with influenza. The positive rates of pathogens were not significantly different between different age groups. Conclusions: Various respiratory pathogens can be detected from SARI cases aged 15 years and older. Influenza virus was the predominant pathogen and the co-detection of influenza virus with Mycoplasma pneumoniae the most common one.


Asunto(s)
Bacterias/aislamiento & purificación , Infecciones Bacterianas/diagnóstico , Gripe Humana/diagnóstico , Pacientes Internos/estadística & datos numéricos , Mycoplasma pneumoniae/aislamiento & purificación , Infecciones del Sistema Respiratorio , Virosis/diagnóstico , Virus/aislamiento & purificación , Enfermedad Aguda , Adolescente , Bacterias/genética , Infecciones Bacterianas/epidemiología , Infecciones Bacterianas/microbiología , China/epidemiología , Coinfección/epidemiología , Hospitalización , Humanos , Lactante , Gripe Humana/epidemiología , Gripe Humana/virología , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Virosis/epidemiología , Virosis/virología , Virus/genética
11.
Virol J ; 16(1): 109, 2019 09 02.
Artículo en Inglés | MEDLINE | ID: mdl-31477134

RESUMEN

The aim of this article is to summarise the virology content presented at the 9th Lorne Infection and Immunity Conference, Australia, in February 2019. The broad program included virology as a key theme, and the commentary herein highlights several key virology presentations at the meeting.


Asunto(s)
Virología , Virosis/inmunología , Animales , Australia , Quirópteros/virología , Congresos como Asunto , Reservorios de Enfermedades/virología , Humanos , Virosis/prevención & control , Virosis/transmisión , Virus/genética , Virus/patogenicidad
12.
Nucleic Acids Res ; 47(18): e104, 2019 10 10.
Artículo en Inglés | MEDLINE | ID: mdl-31418021

RESUMEN

Long-read next-generation amplicon sequencing shows promise for studying complete genes or genomes from complex and diverse populations. Current long-read sequencing technologies have challenging error profiles, hindering data processing and incorporation into downstream analyses. Here we consider the problem of how to reconstruct, free of sequencing error, the true sequence variants and their associated frequencies from PacBio reads. Called 'amplicon denoising', this problem has been extensively studied for short-read sequencing technologies, but current solutions do not always successfully generalize to long reads with high indel error rates. We introduce two methods: one that runs nearly instantly and is very accurate for medium length reads and high template coverage, and another, slower method that is more robust when reads are very long or coverage is lower. On two Mock Virus Community datasets with ground truth, each sequenced on a different PacBio instrument, and on a number of simulated datasets, we compare our two approaches to each other and to existing algorithms. We outperform all tested methods in accuracy, with competitive run times even for our slower method, successfully discriminating templates that differ by a just single nucleotide. Julia implementations of Fast Amplicon Denoising (FAD) and Robust Amplicon Denoising (RAD), and a webserver interface, are freely available.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Metagenómica , ARN Ribosómico 16S/genética , Virus/genética , Algoritmos , Técnicas de Visualización de Superficie Celular/métodos , VIH/genética , Filogenia , Alineación de Secuencia , Anticuerpos de Cadena Única/genética , Programas Informáticos
13.
Genome Biol ; 20(1): 153, 2019 08 02.
Artículo en Inglés | MEDLINE | ID: mdl-31375138

RESUMEN

We describe a method that adds long-read sequencing to a mix of technologies used to assemble a highly complex cattle rumen microbial community, and provide a comparison to short read-based methods. Long-read alignments and Hi-C linkage between contigs support the identification of 188 novel virus-host associations and the determination of phage life cycle states in the rumen microbial community. The long-read assembly also identifies 94 antimicrobial resistance genes, compared to only seven alleles in the short-read assembly. We demonstrate novel techniques that work synergistically to improve characterization of biological features in a highly complex rumen microbial community.


Asunto(s)
Farmacorresistencia Microbiana/genética , Metagenómica/métodos , Microbiota/genética , Análisis de Secuencia de ADN/métodos , Virus/genética , Animales , Bovinos , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Transferencia de Gen Horizontal , Genes Microbianos , Sistemas de Lectura Abierta , Profagos/genética , Rumen/microbiología , Rumen/virología , Virus/aislamiento & purificación
14.
Nucleic Acids Res ; 47(16): 8632-8648, 2019 09 19.
Artículo en Inglés | MEDLINE | ID: mdl-31392984

RESUMEN

CRISPR-Cas systems provide heritable immunity against viruses by capturing short invader DNA sequences, termed spacers, and incorporating them into the CRISPR loci of the prokaryotic host genome. Here, we investigate DNA elements that control accurate spacer uptake in the type II-A CRISPR locus of Streptococcus thermophilus. We determined that purified Cas1 and Cas2 proteins catalyze spacer integration with high specificity for CRISPR repeat junctions. We show that 10 bp of the CRISPR leader sequence is critical for stimulating polarized integration preferentially at the repeat proximal to the leader. Spacer integration proceeds through a two-step transesterification reaction where the 3' hydroxyl groups of the spacer target both repeat borders on opposite strands. The leader-proximal end of the repeat is preferentially targeted for the first site of integration through recognition of sequences spanning the leader-repeat junction. Subsequently, second-site integration at the leader-distal end of the repeat is specified by multiple determinants including a length-defining mechanism relying on a repeat element proximal to the second site of integration. Our results highlight the intrinsic ability of type II Cas1/Cas2 proteins to coordinate directional and site-specific spacer integration into the CRISPR locus to ensure precise duplication of the repeat required for CRISPR immunity.


Asunto(s)
Sistemas CRISPR-Cas , Endonucleasas/genética , Edición Génica , Genoma Bacteriano , Streptococcus thermophilus/genética , Secuencia de Bases , Endonucleasas/inmunología , Endonucleasas/metabolismo , Esterificación , Sitios Genéticos , Isoenzimas/genética , Isoenzimas/inmunología , Isoenzimas/metabolismo , Mutagénesis Insercional , Plásmidos/química , Plásmidos/metabolismo , Streptococcus thermophilus/inmunología , Streptococcus thermophilus/metabolismo , Streptococcus thermophilus/virología , Virus/genética , Virus/metabolismo
16.
Nat Commun ; 10(1): 3315, 2019 07 25.
Artículo en Inglés | MEDLINE | ID: mdl-31346176

RESUMEN

Channelrhodopsins (ChRs) are algal light-gated ion channels widely used as optogenetic tools for manipulating neuronal activity. ChRs desensitize under continuous bright-light illumination, resulting in a significant decline of photocurrents. Here we describe a metagenomically identified family of phylogenetically distinct anion-conducting ChRs (designated MerMAIDs). MerMAIDs almost completely desensitize during continuous illumination due to accumulation of a late non-conducting photointermediate that disrupts the ion permeation pathway. MerMAID desensitization can be fully explained by a single photocycle in which a long-lived desensitized state follows the short-lived conducting state. A conserved cysteine is the critical factor in desensitization, as its mutation results in recovery of large stationary photocurrents. The rapid desensitization of MerMAIDs enables their use as optogenetic silencers for transient suppression of individual action potentials without affecting subsequent spiking during continuous illumination. Our results could facilitate the development of optogenetic tools from metagenomic databases and enhance general understanding of ChR function.


Asunto(s)
Aniones/metabolismo , Bacterias/genética , Channelrhodopsins/genética , Familia de Multigenes , Virus/genética , Animales , Bacterias/clasificación , Bacterias/aislamiento & purificación , Bacterias/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Channelrhodopsins/química , Channelrhodopsins/metabolismo , Humanos , Cinética , Luz , Metagenoma , Neuronas/metabolismo , Optogenética , Filogenia , Agua de Mar/microbiología , Agua de Mar/virología , Proteínas Virales/genética , Proteínas Virales/metabolismo , Virus/clasificación , Virus/aislamiento & purificación , Virus/metabolismo
17.
Artículo en Inglés | MEDLINE | ID: mdl-31331104

RESUMEN

To assess the quality of shellfish harvest areas, bivalve mollusk samples from three coastal areas of the Campania region in Southwest Italy were evaluated for viruses over a three-year period (2015-2017). Screening of 289 samples from shellfish farms and other locations by qPCR and RT-qPCR identified hepatitis A virus (HAV; 8.9%), norovirus GI (NoVGI; 10.8%) and GII (NoVGII; 39.7%), rotavirus (RV; 9.0%), astrovirus (AsV; 20.8%), sapovirus (SaV; 18.8%), aichivirus-1 (AiV-1; 5.6%), and adenovirus (AdV, 5.6%). Hepatitis E virus (HEV) was never detected. Sequence analysis identified HAV as genotype IA and AdV as type 41. This study demonstrates the presence of different enteric viruses within bivalve mollusks, highlighting the limitations of the current EU classification system for shellfish growing waters.


Asunto(s)
Bivalvos/virología , Mariscos/virología , Virus/aislamiento & purificación , Animales , Monitoreo del Ambiente , Contaminación de Alimentos/análisis , Italia , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus/genética
18.
Emerg Microbes Infect ; 8(1): 1054-1065, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31335277

RESUMEN

Despite the advanced PCR-based assays available, a fraction of the pediatric respiratory infections remain unexplained every epidemic season, and there is a perception that novel viruses might be present in these specimens. We systematically collected samples from a prospective cohort of pediatric patients with respiratory infections, that returned negative results by validated molecular RT-PCR assays, and studied them with a target-independent, high-throughput sequencing-based approach. We also included a matched cohort of children with no symptoms of respiratory infection, as a contrast study population. More than fifty percent of the specimens from the group of patients with unexplained respiratory infections were resolved. However, the higher rate of detection was not due to the presence of novel viruses, but to the identification of well-known viral respiratory pathogens. Our results show that already known viral pathogens are responsible for the majority of cases that remain unexplained after the epidemic season. High-throughput sequencing approaches that use pathogen-specific probes are easier to standardize because they ensure reproducible library enrichment and sequencing. In consequence, these techniques might be desirable from a regulatory standpoint for diagnostic laboratories seeking to benefit from the many advantages of these sequencing technologies.


Asunto(s)
Infecciones del Sistema Respiratorio/virología , Virosis/virología , Virus/aislamiento & purificación , Adolescente , Niño , Preescolar , Femenino , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Estudios Prospectivos , Virus/clasificación , Virus/genética
19.
Adv Exp Med Biol ; 1215: 129-158, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31317499

RESUMEN

A virus particle consists of a genome contained within a protein shell. This shell (the capsid) plays multiple roles throughout the infectious cycle, from genome protection to host recognition to successful genome delivery. When capsids first assemble in the cell, most often an initial product is obtained that has not achieved its fully infectious form. To do so, it must undergo a final process called maturation. Virus maturation entails conformational and stability changes. These changes are often driven by proteolytic cleavages, and their main purpose is to ensure successful delivery of the virus genome to a new host cell. Recent advances in molecular, structural, and physical virology techniques are providing a wealth of detailed information and new points of view to understand the principles of virus maturation. Evidence showing that viral capsids are built with a limited set of structural solutions has prompted a new virus classification in structural lineages deriving from a few initial ancestors. This chapter summarizes the current knowledge on maturation for the main virus structural lineages, as well as for other relevant viruses not assigned to any particular lineage yet.


Asunto(s)
Cápside , Fenómenos Fisiológicos de los Virus , Cápside/química , Cápside/metabolismo , Proteínas de la Cápside/metabolismo , Genoma Viral , Conformación Molecular , Ensamble de Virus , Virus/clasificación , Virus/genética , Virus/metabolismo
20.
Methods Mol Biol ; 1910: 3-31, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31278660

RESUMEN

Organisms display astonishing levels of cell and molecular diversity, including genome size, shape, and architecture. In this chapter, we review how the genome can be viewed as both a structural and an informational unit of biological diversity and explicitly define our intended meaning of genetic information. A brief overview of the characteristic features of bacterial, archaeal, and eukaryotic cell types and viruses sets the stage for a review of the differences in organization, size, and packaging strategies of their genomes. We include a detailed review of genetic elements found outside the primary chromosomal structures, as these provide insights into how genomes are sometimes viewed as incomplete informational entities. Lastly, we reassess the definition of the genome in light of recent advancements in our understanding of the diversity of genomic structures and the mechanisms by which genetic information is expressed within the cell. Collectively, these topics comprise a good introduction to genome biology for the newcomer to the field and provide a valuable reference for those developing new statistical or computation methods in genomics. This review also prepares the reader for anticipated transformations in thinking as the field of genome biology progresses.


Asunto(s)
Biodiversidad , Eucariontes/genética , Genoma , Genómica , Archaea/genética , Bacterias/genética , Biología Computacional/métodos , Regulación de la Expresión Génica , Estructuras Genéticas , Genómica/métodos , Patrón de Herencia , Virus/genética
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