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1.
Proc Natl Acad Sci U S A ; 118(14)2021 04 06.
Artículo en Inglés | MEDLINE | ID: mdl-33811145

RESUMEN

Human respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory infection in children under 5 y of age. In the absence of a safe and effective vaccine and with limited options for therapeutic interventions, uncontrolled epidemics of RSV occur annually worldwide. Existing RSV reverse genetics systems have been predominantly based on older laboratory-adapted strains such as A2 or Long. These strains are not representative of currently circulating genotypes and have a convoluted passage history, complicating their use in studies on molecular determinants of viral pathogenesis and intervention strategies. In this study, we have generated reverse genetics systems for clinical isolates of RSV-A (ON1, 0594 strain) and RSV-B (BA9, 9671 strain) in which the full-length complementary DNA (cDNA) copy of the viral antigenome is cloned into a bacterial artificial chromosome (BAC). Additional recombinant (r) RSVs were rescued expressing enhanced green fluorescent protein (EGFP), mScarlet, or NanoLuc luciferase from an additional transcription unit inserted between the P and M genes. Mutations in antigenic site II of the F protein conferring escape from palivizumab neutralization (K272E, K272Q, S275L) were investigated using quantitative cell-fusion assays and rRSVs via the use of BAC recombineering protocols. These mutations enabled RSV-A and -B to escape palivizumab neutralization but had differential impacts on cell-to-cell fusion, as the S275L mutation resulted in an almost-complete ablation of syncytium formation. These reverse genetics systems will facilitate future cross-validation efficacy studies of novel RSV therapeutic intervention strategies and investigations into viral and host factors necessary for virus entry and cell-to-cell spread.


Asunto(s)
Farmacorresistencia Viral/genética , Mutación , Virus Sincitiales Respiratorios/genética , Animales , Antivirales/toxicidad , Chlorocebus aethiops , Farmacorresistencia Viral/inmunología , Células Hep G2 , Humanos , Palivizumab/toxicidad , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/inmunología , Virus Sincitiales Respiratorios/aislamiento & purificación , Virus Sincitiales Respiratorios/patogenicidad , Genética Inversa/métodos , Células Vero
2.
PLoS Pathog ; 15(5): e1007743, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-31059555

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) belongs to the subfamily of Gammaherpesvirinae and is the etiological agent of Kaposi's sarcoma as well as of two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman disease. The KSHV life cycle is divided into a latent and a lytic phase and is highly regulated by viral immunomodulatory proteins which control the host antiviral immune response. Among them is a group of proteins with homology to cellular interferon regulatory factors, the viral interferon regulatory factors 1-4. The KSHV vIRFs are known as inhibitors of cellular interferon signaling and are involved in different oncogenic pathways. Here we characterized the role of the second vIRF protein, vIRF2, during the KSHV life cycle. We found the vIRF2 protein to be expressed in different KSHV positive cells with early lytic kinetics. Importantly, we observed that vIRF2 suppresses the expression of viral early lytic genes in both newly infected and reactivated persistently infected endothelial cells. This vIRF2-dependent regulation of the KSHV life cycle might involve the increased expression of cellular interferon-induced genes such as the IFIT proteins 1, 2 and 3, which antagonize the expression of early KSHV lytic proteins. Our findings suggest a model in which the viral protein vIRF2 allows KSHV to harness an IFN-dependent pathway to regulate KSHV early gene expression.


Asunto(s)
Endotelio Vascular/virología , Regulación Viral de la Expresión Génica , Herpesvirus Humano 8/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Factores Reguladores del Interferón/metabolismo , Interferones/metabolismo , Sarcoma de Kaposi/virología , Proteínas Virales/metabolismo , Activación Viral , Células Cultivadas , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Humanos , Proteínas Inmediatas-Precoces/genética , Factores Reguladores del Interferón/genética , Interferones/genética , Sarcoma de Kaposi/genética , Sarcoma de Kaposi/metabolismo , Proteínas Virales/genética , Latencia del Virus
3.
Int J Mol Sci ; 21(17)2020 Sep 02.
Artículo en Inglés | MEDLINE | ID: mdl-32887347

RESUMEN

Recently an increasing number of new adenovirus types associated with type-dependent pathogenicity have been identified. However, identification of these clinical isolates represents the very first step to characterize novel pathogens. For deeper analyses, these adenoviruses need to be further characterized in basic virology experiments or they could be applied in translational research. To achieve this goal, it is essential to get genetic access and to enable genetic modification of these novel adenovirus genomes (deletion, insertion, and mutation). Here we demonstrate a high-throughput approach to get genetic access to new adenoviruses via homologous recombination. We first defined the cloning conditions regarding homology arm-length and input adenoviral genome amounts. Then we cloned four naturally occurring adenoviruses (Ad70, Ad73, Ad74, and Ad75) into easy-to-manipulate plasmids and genetically modified them by reporter gene insertion. Three recombinant adenoviruses (Ad70, Ad73, and Ad74) containing a reporter cassette were successfully reconstituted. These novel reporter-labeled adenoviruses were further characterized using the inserted luciferase reporter with respect to receptor usage, presence of anti-adenovirus antibodies, and tropism in vitro. The identified receptor usage, the relatively low prevalence of anti-adenovirus antibodies, and the various cancer cell line transduction pattern are important features of these new pathogens providing essential information for their therapeutic application.


Asunto(s)
Adenovirus Humanos/clasificación , Adenovirus Humanos/genética , Clonación Molecular/métodos , Genes Reporteros , Vectores Genéticos/genética , Genoma Viral , Ensayos Analíticos de Alto Rendimiento , Recombinación Homóloga , Humanos
4.
J Infect Dis ; 220(5): 781-791, 2019 07 31.
Artículo en Inglés | MEDLINE | ID: mdl-31050742

RESUMEN

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.


Asunto(s)
Secuencia de Bases , Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Genoma Viral , Recombinación Genética , ADN Viral/genética , Bases de Datos de Ácidos Nucleicos , Conjuntos de Datos como Asunto , Evolución Molecular , Genes Virales , Variación Genética , Genoma Viral/genética , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación , Análisis de Secuencia de ADN , Secuenciación Completa del Genoma
5.
Emerg Infect Dis ; 25(8): 1548-1551, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31310220

RESUMEN

We determined the complete genome sequence of a virus isolated from a mantled guereza that died of primary effusion lymphoma. The virus is closely related to Kaposi's sarcoma-associated herpesvirus (KSHV) but lacks some genes implicated in KSHV pathogenesis. This finding may help determine how KSHV causes primary effusion lymphoma in humans.


Asunto(s)
Herpesvirus Humano 8/clasificación , Herpesvirus Humano 8/genética , Linfoma/veterinaria , Enfermedades de los Monos/diagnóstico , Enfermedades de los Monos/virología , Animales , Biopsia , Colobus , Genoma Viral , Genómica , Herpesvirus Humano 8/aislamiento & purificación , Inmunohistoquímica , Masculino , Enfermedades de los Monos/epidemiología , Filogenia , Secuenciación Completa del Genoma
6.
PLoS Pathog ; 13(9): e1006639, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28938025

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) is the infectious cause of the highly vascularized tumor Kaposi's sarcoma (KS), which is characterized by proliferating spindle cells of endothelial origin, extensive neo-angiogenesis and inflammatory infiltrates. The KSHV K15 protein contributes to the angiogenic and invasive properties of KSHV-infected endothelial cells. Here, we asked whether K15 could also play a role in KSHV lytic replication. Deletion of the K15 gene from the viral genome or its depletion by siRNA lead to reduced virus reactivation, as evidenced by the decreased expression levels of KSHV lytic proteins RTA, K-bZIP, ORF 45 and K8.1 as well as reduced release of infectious virus. Similar results were found for a K1 deletion virus. Deleting either K15 or K1 from the viral genome also compromised the ability of KSHV to activate PLCγ1, Erk1/2 and Akt1. In infected primary lymphatic endothelial (LEC-rKSHV) cells, which have previously been shown to spontaneously display a viral lytic transcription pattern, transfection of siRNA against K15, but not K1, abolished viral lytic replication as well as KSHV-induced spindle cell formation. Using a newly generated monoclonal antibody to K15, we found an abundant K15 protein expression in KS tumor biopsies obtained from HIV positive patients, emphasizing the physiological relevance of our findings. Finally, we used a dominant negative inhibitor of the K15-PLCγ1 interaction to establish proof of principle that pharmacological intervention with K15-dependent pathways may represent a novel approach to block KSHV reactivation and thereby its pathogenesis.


Asunto(s)
Herpesvirus Humano 8/fisiología , Sarcoma de Kaposi/virología , Proteínas Virales/metabolismo , Replicación Viral/fisiología , Western Blotting , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Sarcoma de Kaposi/metabolismo , Activación Viral/fisiología , Latencia del Virus/fisiología
7.
J Infect Dis ; 215(11): 1673-1683, 2017 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-28368496

RESUMEN

Background: Advances in next-generation sequencing (NGS) technologies allow comprehensive studies of genetic diversity over the entire genome of human cytomegalovirus (HCMV), a significant pathogen for immunocompromised individuals. Methods: Next-generation sequencing was performed on target enriched sequence libraries prepared directly from a variety of clinical specimens (blood, urine, breast milk, respiratory samples, biopsies, and vitreous humor) obtained longitudinally or from different anatomical compartments from 20 HCMV-infected patients (renal transplant recipients, stem cell transplant recipients, and congenitally infected children). Results: De novo-assembled HCMV genome sequences were obtained for 57 of 68 sequenced samples. Analysis of longitudinal or compartmental HCMV diversity revealed various patterns: no major differences were detected among longitudinal, intraindividual blood samples from 9 of 15 patients and in most of the patients with compartmental samples, whereas a switch of the major HCMV population was observed in 6 individuals with sequential blood samples and upon compartmental analysis of 1 patient with HCMV retinitis. Variant analysis revealed additional aspects of minor virus population dynamics and antiviral-resistance mutations. Conclusions: In immunosuppressed patients, HCMV can remain relatively stable or undergo drastic genomic changes that are suggestive of the emergence of minor resident strains or de novo infection.


Asunto(s)
Infecciones por Citomegalovirus/virología , Citomegalovirus/genética , Genoma Viral/genética , Huésped Inmunocomprometido , Adulto , Anciano , Estudios de Cohortes , Citomegalovirus/clasificación , Infecciones por Citomegalovirus/inmunología , ADN Viral/análisis , ADN Viral/genética , Farmacorresistencia Viral/genética , Femenino , Variación Genética/genética , Genómica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Masculino , Persona de Mediana Edad , Receptores de Trasplantes
8.
Neurobiol Dis ; 99: 121-132, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-28017800

RESUMEN

Following intracerebral inoculation, the BeAn 8386 strain of Theiler's virus causes persistent infection and inflammatory demyelinating encephalomyelitis in the spinal cord of T-cell defective SJL/J mice, which is widely used as a model of multiple sclerosis. In contrast, C57BL/6 (B6) mice clear the virus and develop inflammation and lesions in the hippocampus, associated with acute and chronic seizures, representing a novel model of viral encephalitis-induced epilepsy. Here we characterize the geno- and phenotype of two naturally occurring variants of BeAn (BeAn-1 and BeAn-2) that can be used to further understand the viral and host factors involved in the neuropathogenesis in B6 and SJL/J mice. Next generation sequencing disclosed 15 single nucleotide differences between BeAn-1 and BeAn-2, of which 4 are coding changes and 3 are in the 5'-UTR (5'-untranslated region). The relatively minor variations in the nucleotide sequence of the two BeAn substrains led to marked differences in neurovirulence. In SJL/J mice, inflammatory demyelination in the spinal cord and its clinical consequences were significantly more marked following infection with BeAn-1 than with BeAn-2. Both BeAn substrains caused lymphocyte infiltration and increase of MAC3-positive cells in the hippocampus, but hippocampal damage and seizures were only observed in B6 mice. Seizures occurred in one third of BeAn-2 infected B6 mice, but not in BeAn-1 infected B6 mice. By comparing individual mice by receiver operating characteristic (ROC) curve analysis, the severity of hippocampal neurodegeneration and amount of MAC3-positive microglia/macrophages discriminated seizing from non-seizing B6 mice, whereas T-lymphocyte brain infiltration was not found to be a crucial factor. These data add novel evidence to the view that differential outcome of infection may be not invariably linked to a distinct viral burden but to a finely tuned balance between antiviral immune responses that although essential for host resistance can also contribute to immunopathology.


Asunto(s)
Encefalitis Viral/patología , Encefalomielitis Aguda Diseminada/patología , Epilepsia/patología , Esclerosis Múltiple/patología , Theilovirus , Animales , Encéfalo/inmunología , Encéfalo/patología , Encéfalo/virología , Modelos Animales de Enfermedad , Encefalitis Viral/inmunología , Encefalitis Viral/virología , Encefalomielitis Aguda Diseminada/inmunología , Encefalomielitis Aguda Diseminada/virología , Epilepsia/inmunología , Epilepsia/virología , Femenino , Interacciones Huésped-Patógeno , Ratones Endogámicos C57BL , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/virología , Fenotipo , Polimorfismo de Nucleótido Simple , ARN Viral/metabolismo , Especificidad de la Especie , Theilovirus/genética , Theilovirus/patogenicidad , Virulencia
9.
J Gen Virol ; 98(12): 3037-3045, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-29095687

RESUMEN

Species D is the largest of the seven species of human mastadenoviruses (HAdV), but few of its multiple types are associated with asevere disease, e.g. epidemic keratoconjunctivitis. Many other types are hardly ever associated with significant diseases in immunocompetent patients, but have been isolated from the diarrhoeal faeces of terminal AIDS patients suggesting their role as opportunistic pathogens. Three novel HAdV-D strains were isolated from the faeces of three immunocompromised adult patients (clinical diagnoses: lymphoma, myelodysplastic syndrome and AIDS CDC3B, respectively). These strains were not typeable by imputed serology of the hexon and fibre gene and therefore complete genomic sequences were generated by next-generation sequencing (NGS). All three strains were multiple recombinants and fulfilled the criteria for designation as types 73, 74 and 75 with the penton/hexon/fibre genotype codes P67H45F27, P70H74F51 and P75H26F29, respectively. A novel genomic backbone and also a novel hexon neutralization epitope sequence were discovered in type 74, and a novel penton sequence in type 75. At the complete genome level, types 73, 74 and 75 were closely related neither to each other nor to type 70, which was previously isolated in the same region. However, these four HAdV-D types were closely related to each other in single genes and gene regions, e.g. penton, E1 and E4 due to recombination events in their phylogeny. In conclusion, regional co-circulation of opportunistic HAdV-D types facilitated co- and super-infections, which are essential for homologous recombination, and thus resulted in the evolution of novel genotypes by lateral gene transfer.

10.
J Infect Dis ; 212(6): 871-80, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-25748322

RESUMEN

BACKGROUND: The circulation of human adenovirus type 21 (HAdV21) in the United States has been documented since the 1960s in association with outbreaks of febrile respiratory illness (FRI) in military boot camps and civilian cases of respiratory disease. METHODS: To describe the molecular epidemiology of HAdV21 respiratory infections across the country, 150 clinical respiratory isolates obtained from continuous surveillance of military recruit FRI, and 23 respiratory isolates recovered from pediatric and adult civilian cases of acute respiratory infection were characterized to compile molecular typing data spanning 37 years (1978-2014). RESULTS: Restriction enzyme analysis and genomic sequencing identified 2 clusters of closely related genomic variants readily distinguishable from the prototype and designated 21a-like and 21b-like. A-like variants predominated until 1999. A shift to b-like variants was noticeable by 2007 after a 7-year period (2000-2006) of cocirculation of the 2 genome types. US strains are phylogenetically more closely related to European and Asian strains isolated over the last 4 decades than to the Saudi Arabian prototype strain AV-1645 isolated in 1956. CONCLUSIONS: Knowledge of circulating HAdV21 variants and their epidemic behavior will be of significant value to local and global FRI surveillance efforts.


Asunto(s)
Infecciones por Adenoviridae/epidemiología , Infecciones por Adenoviridae/virología , Adenovirus Humanos/clasificación , Personal Militar , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Adenovirus Humanos/genética , Adenovirus Humanos/aislamiento & purificación , ADN Viral/genética , Brotes de Enfermedades , Variación Genética , Humanos , Epidemiología Molecular , Reacción en Cadena de la Polimerasa , Vigilancia de la Población , Factores de Tiempo , Estados Unidos/epidemiología
11.
J Gen Virol ; 96(9): 2734-2742, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-26002300

RESUMEN

A human mastadenovirus D (HAdV-D) isolated from diarrhoeal faeces of an allogeneic haematopoietic stem cell transplant (SCT) recipient was found to be non-typable by sequencing of loops 1 and 2 of the hexon main neutralization epitope ('imputed serology'). In contrast to HAdV-C, HAdV-D infections are rarely observed in SCT patients. Therefore, the whole genome of this isolate was sequenced and phylogenetically analysed. In addition, microneutralization testing with type-specific antisera was performed. A complete genomic sequence of 35.2 kb in length with a GC content of 57  % was obtained and found to be distantly related to HAdV-D27 (96.25 % identity). Imputed serology implicated a new type with a nucleotide sequence identity of only 96.11 % to HAdV-D37 (loop 1) and 95.76 % to HAdV-D30 and HAdV-D37 (loop 2). Microneutralization testing confirmed that this clinical isolate was not neutralized by HAdV-D37- or HAdV-D30-specific antisera. The penton base gene showed a novel sequence, which clustered with HAdV-D38, but bootscan analysis indicated an intra-penton recombination event with HAdV-D60. Another recombination event was detected within the early gene region E3 with the 12.2 kDa and CR1-α genes derived from HAdV-D58. Moreover, the E4 region was derived from HAdV-D13, but all these genes had evolved significantly from their ancestors. By contrast, the recombinant fibre gene was almost 100 % identical to HAdV-D29. In conclusion, the genomics of this novel HAdV, designated the HAdV-D70 [P70H70F29] prototype, supported the significance of multiple recombinations in the phylogeny of HAdV-D.


Asunto(s)
Infecciones por Adenoviridae/virología , Diarrea/virología , Heces/virología , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Mastadenovirus/genética , Mastadenovirus/aislamiento & purificación , Complicaciones Posoperatorias/virología , Recombinación Genética , Infecciones por Adenoviridae/etiología , Genoma Viral , Humanos , Mastadenovirus/clasificación , Persona de Mediana Edad , Datos de Secuencia Molecular , Filogenia , Proteínas Virales/genética
12.
Exp Dermatol ; 22(11): 725-9, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-24112647

RESUMEN

Patients treated with BRAF inhibitors (e.g. vemurafenib), a novel targeted therapy for advanced melanoma harbouring certain BRAF mutations, develop numerous adverse cutaneous side effects, including skin tumors such as squamous cell carcinoma or non-malignant verruciform keratinocyte proliferations, termed 'BRAF-inhibitor-associated verrucous keratosis (BAVK) lesions'. These keratinocyte proliferations are believed to be caused by paradoxical hyperactivation of the MAPK pathway in cells with wild-type BRAF, but mutated RAS. However, due to the clinical and histological verruca-like appearance of these lesions, additional aetiologic cofactors, such as infectious agents (i.e. oncogenic viruses), might be suspected. Therefore, we performed 454 high-throughput sequencing of BAVK lesions from vemurafenib-treated patients on the transcript level to identify actively transcribed viral sequences of known [e.g. human papilloma viruses (HPV)] or even yet-unknown viruses. Next-generation sequencing did not identify transcripts of any human viruses out of 1 595 161 reads obtained from BAVK lesions of four patients. Nevertheless, all controls were recognized correctly, and the detection of sequences derived from the cutaneous microbiome (e.g. skin commensals and bacterial phages) confirmed the validity and sensitivity of the sequencing data. Our results are consistent with preliminary histological and immunohistochemical findings recently reported by others, who also failed to detect the expression of HPV proteins in BAVK. Although the patient number is limited and we cannot exclude the possibility of having missed a viral transcript of very low abundance, our study argues against a viral aetiology of BRAF-inhibitor-associated verruciform keratoses occurring under vemurafenib.


Asunto(s)
Carcinoma Verrugoso/virología , Melanoma/tratamiento farmacológico , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/metabolismo , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/virología , Adulto , Anciano , Biopsia , Proliferación Celular , ADN Viral/análisis , Femenino , Biblioteca de Genes , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Indoles/uso terapéutico , Queratinocitos/citología , Masculino , Persona de Mediana Edad , Mutación , Papillomaviridae/genética , Neoplasias Cutáneas/complicaciones , Sulfonamidas/uso terapéutico , Vemurafenib
13.
iScience ; 25(5): 104276, 2022 May 20.
Artículo en Inglés | MEDLINE | ID: mdl-35573195

RESUMEN

To improve the identification and management of viral respiratory infections, we established a clinical and virologic surveillance program for pediatric patients fulfilling pre-defined case criteria of influenza-like illness and viral respiratory infections. The program resulted in a cohort comprising 6,073 patients (56% male, median age 1.6 years, range 0-18.8 years), where every patient was assessed with a validated disease severity score at the point-of-care using the ViVI ScoreApp. We used machine learning and agnostic feature selection to identify characteristic clinical patterns. We tested all patients for human adenoviruses, 571 (9%) were positive. Adenovirus infections were particularly common and mild in children ≥1 month of age but rare and potentially severe in neonates: with lower airway involvement, disseminated disease, and a 50% mortality rate (n = 2/4). In one fatal case, we discovered a novel virus: HAdV-80. Standardized surveillance leveraging digital technology helps to identify characteristic clinical patterns, risk factors, and emerging pathogens.

14.
JACC Cardiovasc Interv ; 15(12): 1191-1201, 2022 06 27.
Artículo en Inglés | MEDLINE | ID: mdl-35595673

RESUMEN

BACKGROUND: Currently, transradial access (TRA) is the recommended access for coronary procedures because of increased safety, with radial artery occlusion (RAO) being its most frequent complication, which will increasingly affect patients undergoing multiple procedures during their lifetimes. Recently, distal radial access (DRA) has emerged as a promising alternative access to minimize RAO risk. A large-scale, international, randomized trial comparing RAO with TRA and DRA is lacking. OBJECTIVES: The aim of this study was to assess the superiority of DRA compared with conventional TRA with respect to forearm RAO. METHODS: DISCO RADIAL (Distal vs Conventional Radial Access) was an international, multicenter, randomized controlled trial in which patients with indications for percutaneous coronary procedure using a 6-F Slender sheath were randomized to DRA or TRA with systematic implementation of best practices to reduce RAO. The primary endpoint was the incidence of forearm RAO assessed by vascular ultrasound at discharge. Secondary endpoints include crossover, hemostasis time, and access site-related complications. RESULTS: Overall, 657 patients underwent TRA, and 650 patients underwent DRA. Forearm RAO did not differ between groups (0.91% vs 0.31%; P = 0.29). Patent hemostasis was achieved in 94.4% of TRA patients. Crossover rates were higher with DRA (3.5% vs 7.4%; P = 0.002), and median hemostasis time was shorter (180 vs 153 minutes; P < 0.001). Radial artery spasm occurred more with DRA (2.7% vs 5.4%; P = 0.015). Overall bleeding events and vascular complications did not differ between groups. CONCLUSIONS: With the implementation of a rigorous hemostasis protocol, DRA and TRA have equally low RAO rates. DRA is associated with a higher crossover rate but a shorter hemostasis time.


Asunto(s)
Arteriopatías Oclusivas , Cateterismo Periférico , Intervención Coronaria Percutánea , Cateterismo Periférico/efectos adversos , Cateterismo Periférico/métodos , Angiografía Coronaria/efectos adversos , Angiografía Coronaria/métodos , Humanos , Intervención Coronaria Percutánea/efectos adversos , Intervención Coronaria Percutánea/métodos , Arteria Radial/diagnóstico por imagen , Resultado del Tratamiento
15.
J Crit Care ; 51: 99-104, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30798099

RESUMEN

Severe pneumonia and ARDS caused by human adenovirus B21 infections (HAdV-B21) is a rare, but a devastating disease with rapid progression to multiorgan failure and death. However, only a few cases were reported so far. Infections appear associated with increased disease severity and higher mortality in infected critically ill patients. Possible factors contributing to infection are underlying psychiatric disease resulting in institutionalization of respective patients, and polytoxicomania. Controlled data on the therapy of severe adenovirus infections are lacking and remains experimental. In conclusion, data on HAdV-B21 infections causing severe pneumonia or ARDS are scarce. Controlled clinical trials on the therapy of adenovirus pneumonia are non existent and thus there is no established therapy so far. ICU physicians should be aware of this potentially devastating disease and further studies are needed.


Asunto(s)
Infecciones por Adenovirus Humanos/diagnóstico , Adenovirus Humanos/aislamiento & purificación , Neumonía Viral/diagnóstico , Síndrome de Dificultad Respiratoria/diagnóstico , Infecciones por Adenovirus Humanos/complicaciones , Infecciones por Adenovirus Humanos/diagnóstico por imagen , Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/genética , Adulto , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neumonía Viral/complicaciones , Neumonía Viral/diagnóstico por imagen , Neumonía Viral/virología , Síndrome de Dificultad Respiratoria/complicaciones , Síndrome de Dificultad Respiratoria/diagnóstico por imagen , Síndrome de Dificultad Respiratoria/virología
16.
Sci Rep ; 9(1): 1039, 2019 01 31.
Artículo en Inglés | MEDLINE | ID: mdl-30705303

RESUMEN

Currently, 88 different Human Adenovirus (HAdV) types are grouped into seven HAdV species A to G. Most types (57) belong to species HAdV-D. Recombination between capsid genes (hexon, penton and fiber) is the main factor contributing to the diversity in species HAdV-D. Noteworthy, species HAdV-C contains so far only five types, although species HAdV-C is highly prevalent and clinically significant in immunosuppressed patients. Therefore, the evolution of species HAdV-C was studied by generating 51 complete genome sequences from circulating strains. Clustering of the whole genome HAdV-C sequences confirmed classical typing results (fifteen HAdV-C1, thirty HAdV-C2, four HAdV-C5, two HAdV-C6). However, two HAdV-C2 strains had a novel penton base sequence and thus were re-labeled as the novel type HAdV-C89. Fiber and early gene region 3 (E3) sequences clustered always with the corresponding prototype sequence but clustering of the E4 region indicated recombination events in 26 out of the 51 sequenced specimens. Recombination of the E1 gene region was detected in 16 circulating strains. As early gene region sequences are not considered in the type definition of HAdVs, evolution of HAdV-C remains on the subtype level. Nonetheless, recombination of the E1 and E4 gene regions may influence the virulence of HAdV-C strains.


Asunto(s)
Adenovirus Humanos/genética , Evolución Molecular , Infecciones por Adenovirus Humanos/virología , Biología Computacional , Genoma Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Filogenia , Recombinación Genética/genética , Análisis de Secuencia de ADN , Carga Viral
17.
Sci Rep ; 7: 40680, 2017 01 13.
Artículo en Inglés | MEDLINE | ID: mdl-28084428

RESUMEN

The German infectious disease surveillance system revealed an increase of epidemic keratoconjunctivitis (EKC) from an average of 320 cases/year (2001 to 2010) up to 2146 and 1986 cases in 2012 and 2013, respectively. From November 2011 until December 2013 (epidemic period) 85% of typed isolates were human adenovirus type 8 (HAdV-D8), whereas only low level circulation (19%) of HAdV-D8 was observed outside the epidemic period. In order to investigate whether a novel monophyletic HAdV-D8 strain prevailed during the epidemic period, complete genomic sequences of 23 HAdV-D8 isolates were generated by deep sequencing and analyzed phylogenetically. For comparison, eight HAdV-D8 isolates from outside the epidemic period were sequenced. HAdV-D8 isolates of the epidemic period had a very high sequence identity of at least 99.9% and formed a monophyletic cluster with two subclusters. A single outlier was closely related to HAdV-D8 strains isolated prior to the epidemic period. Circulation of the epidemic strain was detected as early as 2010 but not after the epidemic period in 2014. In conclusion, molecular phylogeny of complete genomic sequences proved a monophyletic HAdV-D8 epidemic. However, co-circulation of other HAdV types as well as better reporting may have contributed to the huge increase of reported cases.


Asunto(s)
Infecciones por Adenovirus Humanos/epidemiología , Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/clasificación , Adenovirus Humanos/genética , Genotipo , Queratoconjuntivitis/epidemiología , Queratoconjuntivitis/virología , Adulto , Anciano , ADN Viral , Brotes de Enfermedades , Femenino , Genoma Viral , Geografía , Alemania/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Oportunidad Relativa , Filogenia , Vigilancia de la Población , Análisis de Secuencia de ADN , Carga Viral , Secuenciación Completa del Genoma , Adulto Joven
19.
Nat Commun ; 7: 11995, 2016 06 22.
Artículo en Inglés | MEDLINE | ID: mdl-27329939

RESUMEN

Recombination plays a dominant role in the evolution of the bacterial pathogen Helicobacter pylori, but its dynamics remain incompletely understood. Here we use an in vitro transformation system combined with genome sequencing to study chromosomal integration patterns after natural transformation. A single transformation cycle results in up to 21 imports, and repeated transformations generate a maximum of 92 imports (8% sequence replacement). Import lengths show a bimodal distribution with averages of 28 and 1,645 bp. Reanalysis of paired H. pylori genomes from chronically infected people demonstrates the same bimodal import pattern in vivo. Restriction endonucleases (REases) of the recipient bacteria fail to inhibit integration of homeologous DNA, independently of methylation. In contrast, REases limit the import of heterologous DNA. We conclude that restriction-modification systems inhibit the genomic integration of novel sequences, while they pose no barrier to homeologous recombination, which reconciles the observed stability of the H. pylori gene content and its highly recombinational population structure.


Asunto(s)
Cromosomas Bacterianos , Infecciones por Helicobacter/microbiología , Helicobacter pylori/genética , Helicobacter pylori/metabolismo , ADN/genética , Enzimas de Restricción del ADN/metabolismo , Enzimas de Restricción-Modificación del ADN/genética , ADN Bacteriano/genética , Escherichia coli/genética , Variación Genética , Genoma Bacteriano , Genómica , Humanos , Mutagénesis , Polimorfismo de Nucleótido Simple , Recombinación Genética , Análisis de Secuencia de ADN
20.
J Virol Methods ; 217: 79-86, 2015 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-25736227

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) is a gamma-2-lymphotropic human oncogenic herpesvirus associated with Kaposi's sarcoma (KS) and two B-cell lymphoproliferative diseases, primary effusion lymphoma (PEL) and multicentric Castleman's disease (MCD). KSHV establishes latency soon after infection in vivo and in vitro. Consequently, it is technically difficult to generate high-titre virus stocks required for infection experiments in tissue culture. Currently used methods of KSHV stock production involve induction of the lytic/productive cycle in PEL cell lines or in adherent cell lines harbouring recombinant KSHV genomes. In this study, the BJAB-derived B-cell line BrK.219, which is infected latently with a recombinant KSHV (rKSHV.219), is used to produce high-titre virus stocks. BrK.219 cells enter the lytic KSHV replication cycle upon cross-linking of B-cell receptors (BCRs) with anti-IgM antibodies without the need for additional, potentially toxic chemical inducers. High cell concentrations can be cultured and induced easily in spinner flasks, saving time and resources. The established protocol allows the generation of KSHV virus stocks with titres of up to 10(6) IU/ml in unconcentrated culture supernatants, representing a 10(3)-10(4)-fold improvement compared to conventional methods.


Asunto(s)
Linfocitos B/virología , Herpesvirus Humano 8/crecimiento & desarrollo , Cultivo de Virus/métodos , Línea Celular , Herpesvirus Humano 8/genética , Humanos , Carga Viral
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