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1.
Nature ; 626(8001): 1094-1101, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38383783

RESUMO

Persistent SARS-CoV-2 infections may act as viral reservoirs that could seed future outbreaks1-5, give rise to highly divergent lineages6-8 and contribute to cases with post-acute COVID-19 sequelae (long COVID)9,10. However, the population prevalence of persistent infections, their viral load kinetics and evolutionary dynamics over the course of infections remain largely unknown. Here, using viral sequence data collected as part of a national infection survey, we identified 381 individuals with SARS-CoV-2 RNA at high titre persisting for at least 30 days, of which 54 had viral RNA persisting at least 60 days. We refer to these as 'persistent infections' as available evidence suggests that they represent ongoing viral replication, although the persistence of non-replicating RNA cannot be ruled out in all. Individuals with persistent infection had more than 50% higher odds of self-reporting long COVID than individuals with non-persistent infection. We estimate that 0.1-0.5% of infections may become persistent with typically rebounding high viral loads and last for at least 60 days. In some individuals, we identified many viral amino acid substitutions, indicating periods of strong positive selection, whereas others had no consensus change in the sequences for prolonged periods, consistent with weak selection. Substitutions included mutations that are lineage defining for SARS-CoV-2 variants, at target sites for monoclonal antibodies and/or are commonly found in immunocompromised people11-14. This work has profound implications for understanding and characterizing SARS-CoV-2 infection, epidemiology and evolution.


Assuntos
COVID-19 , Inquéritos Epidemiológicos , Infecção Persistente , SARS-CoV-2 , Humanos , Substituição de Aminoácidos , Anticorpos Monoclonais/imunologia , COVID-19/epidemiologia , COVID-19/virologia , Evolução Molecular , Hospedeiro Imunocomprometido/imunologia , Mutação , Infecção Persistente/epidemiologia , Infecção Persistente/virologia , Síndrome de COVID-19 Pós-Aguda/epidemiologia , Síndrome de COVID-19 Pós-Aguda/virologia , Prevalência , RNA Viral/análise , RNA Viral/genética , SARS-CoV-2/química , SARS-CoV-2/classificação , SARS-CoV-2/genética , SARS-CoV-2/imunologia , SARS-CoV-2/isolamento & purificação , Seleção Genética , Autorrelato , Fatores de Tempo , Carga Viral , Replicação Viral
2.
Bioinformatics ; 40(10)2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39360992

RESUMO

MOTIVATION: Target enrichment strategies generate genomic data from multiple pathogens in a single process, greatly improving sensitivity over metagenomic sequencing and enabling cost-effective, high-throughput surveillance and clinical applications. However, uptake by research and clinical laboratories is constrained by an absence of computational tools that are specifically designed for the analysis of multi-pathogen enrichment sequence data. Here we present an analysis pipeline, Castanet, for use with multi-pathogen enrichment sequencing data. Castanet is designed to work with short-read data produced by existing targeted enrichment strategies, but can be readily deployed on any BAM file generated by another methodology. Also included are an optional graphical interface and installer script. RESULTS: In addition to genome reconstruction, Castanet reports method-specific metrics that enable quantification of capture efficiency, estimation of pathogen load, differentiation of low-level positives from contamination, and assessment of sequencing quality. Castanet can be used as a traditional end-to-end pipeline for consensus generation, but its strength lies in the ability to process a flexible, pre-defined set of pathogens of interest directly from multi-pathogen enrichment experiments. In our tests, Castanet consensus sequences were accurate reconstructions of reference sequences, including in instances where multiple strains of the same pathogen were present. Castanet performs effectively on standard computers and can process the entire output of a 96-sample enrichment sequencing run (50M reads) using a single batch process command, in $<$2 h. AVAILABILITY AND IMPLEMENTATION: Source code freely available under GPL-3 license at https://github.com/MultipathogenGenomics/castanet, implemented in Python 3.10 and supported in Ubuntu Linux 22.04. The data underlying this article are available in Europe Nucleotide Archives, at https://www.ebi.ac.uk/ena/browser/view/PRJEB77004.


Assuntos
Software , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Análise de Sequência de DNA/métodos , Metagenômica/métodos
3.
PLoS Pathog ; 19(8): e1011461, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37578971

RESUMO

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burden, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. By analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior exposure, viral burden was 44% lower among Alpha variant infections, compared to those with the predecessor strain, B.1.177. Vaccination reduced viral burden by 67%, and among vaccinated individuals, viral burden was 286% higher among Delta variant, compared to Alpha variant, infections. In addition, viral burden increased by 17% for every 10-year age increment of the infected individual. In summary, within-host viral burden increases with age, is reduced by vaccination, and is influenced by the interplay of vaccination status and viral variant.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Viés de Seleção , SARS-CoV-2/genética , Carga Viral , COVID-19/epidemiologia , COVID-19/prevenção & controle , Vacinação
5.
Proc Natl Acad Sci U S A ; 118(17)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33883278

RESUMO

Cancer cells can survive chemotherapy-induced stress, but how they recover from it is not known. Using a temporal multiomics approach, we delineate the global mechanisms of proteotoxic stress resolution in multiple myeloma cells recovering from proteasome inhibition. Our observations define layered and protracted programs for stress resolution that encompass extensive changes across the transcriptome, proteome, and metabolome. Cellular recovery from proteasome inhibition involved protracted and dynamic changes of glucose and lipid metabolism and suppression of mitochondrial function. We demonstrate that recovering cells are more vulnerable to specific insults than acutely stressed cells and identify the general control nonderepressable 2 (GCN2)-driven cellular response to amino acid scarcity as a key recovery-associated vulnerability. Using a transcriptome analysis pipeline, we further show that GCN2 is also a stress-independent bona fide target in transcriptional signature-defined subsets of solid cancers that share molecular characteristics. Thus, identifying cellular trade-offs tied to the resolution of chemotherapy-induced stress in tumor cells may reveal new therapeutic targets and routes for cancer therapy optimization.


Assuntos
Neoplasias/tratamento farmacológico , Estresse Fisiológico/efeitos dos fármacos , Antineoplásicos/farmacologia , Autofagia/fisiologia , Linhagem Celular Tumoral , Humanos , Metaboloma/genética , Mitocôndrias/metabolismo , Mieloma Múltiplo/metabolismo , Neoplasias/metabolismo , Neoplasias/fisiopatologia , Inibidores de Proteassoma/farmacologia , Proteólise , Proteoma/genética , Análise de Sistemas , Transcriptoma/genética
6.
BMC Genomics ; 24(1): 381, 2023 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-37415108

RESUMO

BACKGROUND: Systematic description of library quality and sequencing performance of single-cell RNA sequencing (scRNA-seq) data is imperative for subsequent downstream modules, including re-pooling libraries. While several packages have been developed to visualise quality control (QC) metrics for scRNA-seq data, they do not include expression-based QC to discriminate between true variation and background noise. RESULTS: We present scQCEA (acronym of the single-cell RNA sequencing Quality Control and Enrichment Analysis), an R package to generate reports of process optimisation metrics for comparing sets of samples and visual evaluation of quality scores. scQCEA can import data from 10X or other single-cell platforms and includes functions for generating an interactive report of QC metrics for multi-omics data. In addition, scQCEA provides automated cell type annotation on scRNA-seq data using differential gene expression patterns for expression-based quality control. We provide a repository of reference gene sets, including 2348 marker genes, which are exclusively expressed in 95 human and mouse cell types. Using scRNA-seq data from 56 gene expressions and V(D)J T cell replicates, we show how scQCEA can be applied for the visual evaluation of quality scores for sets of samples. In addition, we use the summary of QC measures from 342 human and mouse shallow-sequenced gene expression profiles to specify optimal sequencing requirements to run a cell-type enrichment analysis function. CONCLUSIONS: The open-source R tool will allow examining biases and outliers over biological and technical measures, and objective selection of optimal cluster numbers before downstream analysis. scQCEA is available at https://isarnassiri.github.io/scQCEA/ as an R package. Full documentation, including an example, is provided on the package website.


Assuntos
Perfilação da Expressão Gênica , Software , Animais , Humanos , Camundongos , Análise de Sequência de RNA , Análise de Célula Única , Controle de Qualidade , RNA
7.
J Gen Virol ; 104(5)2023 05.
Artigo em Inglês | MEDLINE | ID: mdl-37196057

RESUMO

Hepatitis B virus (HBV) is one of the smallest human DNA viruses and its 3.2 Kb genome encodes multiple overlapping open reading frames, making its viral transcriptome challenging to dissect. Previous studies have combined quantitative PCR and Next Generation Sequencing to identify viral transcripts and splice junctions, however the fragmentation and selective amplification used in short read sequencing precludes the resolution of full length RNAs. Our study coupled an oligonucleotide enrichment protocol with state-of-the-art long read sequencing (PacBio) to identify the repertoire of HBV RNAs. This methodology provides sequencing libraries where up to 25 % of reads are of viral origin and enable the identification of canonical (unspliced), non-canonical (spliced) and chimeric viral-human transcripts. Sequencing RNA isolated from de novo HBV infected cells or those transfected with 1.3 × overlength HBV genomes allowed us to assess the viral transcriptome and to annotate 5' truncations and polyadenylation profiles. The two HBV model systems showed an excellent agreement in the pattern of major viral RNAs, however differences were noted in the abundance of spliced transcripts. Viral-host chimeric transcripts were identified and more commonly found in the transfected cells. Enrichment capture and PacBio sequencing allows the assignment of canonical and non-canonical HBV RNAs using an open-source analysis pipeline that enables the accurate mapping of the HBV transcriptome.


Assuntos
Vírus da Hepatite B , Transcriptoma , Humanos , Vírus da Hepatite B/genética , Sequenciamento de Nucleotídeos em Larga Escala , RNA Viral/genética
8.
Proc Biol Sci ; 290(2009): 20231284, 2023 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-37848057

RESUMO

The Office for National Statistics Coronavirus (COVID-19) Infection Survey (ONS-CIS) is the largest surveillance study of SARS-CoV-2 positivity in the community, and collected data on the United Kingdom (UK) epidemic from April 2020 until March 2023 before being paused. Here, we report on the epidemiological and evolutionary dynamics of SARS-CoV-2 determined by analysing the sequenced samples collected by the ONS-CIS during this period. We observed a series of sweeps or partial sweeps, with each sweeping lineage having a distinct growth advantage compared to their predecessors, although this was also accompanied by a gradual fall in average viral burdens from June 2021 to March 2023. The sweeps also generated an alternating pattern in which most samples had either S-gene target failure (SGTF) or non-SGTF over time. Evolution was characterized by steadily increasing divergence and diversity within lineages, but with step increases in divergence associated with each sweeping major lineage. This led to a faster overall rate of evolution when measured at the between-lineage level compared to within lineages, and fluctuating levels of diversity. These observations highlight the value of viral sequencing integrated into community surveillance studies to monitor the viral epidemiology and evolution of SARS-CoV-2, and potentially other pathogens.


Assuntos
COVID-19 , Epidemias , Humanos , COVID-19/epidemiologia , SARS-CoV-2 , Reino Unido/epidemiologia , Inquéritos e Questionários
9.
Int J Mol Sci ; 23(10)2022 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-35628247

RESUMO

The present investigation focuses on the analysis of the interactions among human lactoferrin (LF), SARS-CoV-2 receptor-binding domain (RBD) and human angiotensin-converting enzyme 2 (ACE2) receptor in order to assess possible mutual interactions that could provide a molecular basis of the reported preventative effect of lactoferrin against CoV-2 infection. In particular, kinetic and thermodynamic parameters for the pairwise interactions among the three proteins were measured via two independent techniques, biolayer interferometry and latex nanoparticle-enhanced turbidimetry. The results obtained clearly indicate that LF is able to bind the ACE2 receptor ectodomain with significantly high affinity, whereas no binding to the RBD was observed up to the maximum "physiological" lactoferrin concentration range. Lactoferrin, above 1 µM concentration, thus appears to directly interfere with RBD-ACE2 binding, bringing about a measurable, up to 300-fold increase of the KD value relative to RBD-ACE2 complex formation.


Assuntos
Enzima de Conversão de Angiotensina 2 , COVID-19 , Lactoferrina , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/metabolismo , COVID-19/virologia , Humanos , Lactoferrina/metabolismo , Peptidil Dipeptidase A/metabolismo , Domínios e Motivos de Interação entre Proteínas , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo
10.
New Phytol ; 231(2): 849-863, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33616943

RESUMO

Floral pigmentation patterning is important for pollinator attraction as well as aesthetic appeal. Patterning of anthocyanin accumulation is frequently associated with variation in activity of the Myb, bHLH and WDR transcription factor complex (MBW) that regulates anthocyanin biosynthesis. Investigation of two classic mutants in Antirrhinum majus, mutabilis and incolorata I, showed they affect a gene encoding a bHLH protein belonging to subclade bHLH-2. The previously characterised gene, Delila, which encodes a bHLH-1 protein, has a bicoloured mutant phenotype, with residual lobe-specific pigmentation conferred by Incolorata I. Both Incolorata I and Delila induce expression of the anthocyanin biosynthetic gene DFR. Rosea 1 (Myb) and WDR1 proteins compete for interaction with Delila, but interact positively to promote Incolorata I activity. Delila positively regulates Incolorata I and WDR1 expression. Hierarchical regulation can explain the bicoloured patterning of delila mutants, through effects on both regulatory gene expression and the activity of promoters of biosynthetic genes like DFR that mediate MBW regulation. bHLH-1 and bHLH-2 proteins contribute to establishing patterns of pigment distribution in A. majus flowers in two ways: through functional redundancy in regulating anthocyanin biosynthetic gene expression, and through differences between the proteins in their ability to regulate genes encoding transcription factors.


Assuntos
Antirrhinum , Antocianinas , Antirrhinum/genética , Antirrhinum/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Pigmentação/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
11.
Bioorg Med Chem Lett ; 30(24): 127559, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-32961320

RESUMO

The synthesis of a lead anti-viral cyclopropyl carboxy acyl hydrazone 4F17 (5) and three sequential arrays of structural analogues along with the initial assessment and optimization of the antiviral pharmacophore against the herpes simplex virus type 1 (HSV-1) are reported.


Assuntos
Antivirais/química , Antivirais/farmacologia , Herpesvirus Humano 1/efeitos dos fármacos , Hidrazonas/química , Hidrazonas/farmacologia , Antivirais/síntese química , Linhagem Celular , Técnicas de Química Sintética , Herpes Simples/tratamento farmacológico , Humanos , Hidrazonas/síntese química , Relação Estrutura-Atividade
12.
Mol Biol Evol ; 35(5): 1238-1252, 2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29688544

RESUMO

The platypus is an egg-laying mammal which, alongside the echidna, occupies a unique place in the mammalian phylogenetic tree. Despite widespread interest in its unusual biology, little is known about its population structure or recent evolutionary history. To provide new insights into the dispersal and demographic history of this iconic species, we sequenced the genomes of 57 platypuses from across the whole species range in eastern mainland Australia and Tasmania. Using a highly improved reference genome, we called over 6.7 M SNPs, providing an informative genetic data set for population analyses. Our results show very strong population structure in the platypus, with our sampling locations corresponding to discrete groupings between which there is no evidence for recent gene flow. Genome-wide data allowed us to establish that 28 of the 57 sampled individuals had at least a third-degree relative among other samples from the same river, often taken at different times. Taking advantage of a sampled family quartet, we estimated the de novo mutation rate in the platypus at 7.0 × 10-9/bp/generation (95% CI 4.1 × 10-9-1.2 × 10-8/bp/generation). We estimated effective population sizes of ancestral populations and haplotype sharing between current groupings, and found evidence for bottlenecks and long-term population decline in multiple regions, and early divergence between populations in different regions. This study demonstrates the power of whole-genome sequencing for studying natural populations of an evolutionarily important species.


Assuntos
Distribuição Animal , Ornitorrinco/genética , Animais , Austrália , Feminino , Variação Genética , Endogamia , Masculino , Taxa de Mutação , Dinâmica Populacional , Sequenciamento Completo do Genoma
13.
BMC Genomics ; 19(1): 115, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29390960

RESUMO

BACKGROUND: Transposable elements (TEs) are mobile genetic sequences that randomly propagate within their host's genome. This mobility has the potential to affect gene transcription and cause disease. However, TEs are technically challenging to identify, which complicates efforts to assess the impact of TE insertions on disease. Here we present a targeted sequencing protocol and computational pipeline to identify polymorphic and novel TE insertions using next-generation sequencing: TE-NGS. The method simultaneously targets the three subfamilies that are responsible for the majority of recent TE activity (L1HS, AluYa5/8, and AluYb8/9) thereby obviating the need for multiple experiments and reducing the amount of input material required. RESULTS: Here we describe the laboratory protocol and detection algorithm, and a benchmark experiment for the reference genome NA12878. We demonstrate a substantial enrichment for on-target fragments, and high sensitivity and precision to both reference and NA12878-specific insertions. We report 17 previously unreported loci for this individual which are supported by orthogonal long-read evidence, and we identify 1470 polymorphic and novel TEs in 12 additional samples that were previously undocumented in databases of insertion polymorphisms. CONCLUSIONS: We anticipate that future applications of TE-NGS alongside exome sequencing of patients with sporadic disease will reduce the number of unresolved cases, and improve estimates of the contribution of TEs to human genetic disease.


Assuntos
Algoritmos , Elementos de DNA Transponíveis , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Polimorfismo de Nucleotídeo Único , Biblioteca Gênica , Humanos
14.
J Virol ; 91(20)2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28768873

RESUMO

The predominant types of dendritic cells (DC) in the skin and mucosa are Langerhans cells (LC) and interstitial dermal DC (iDDC). LC and iDDC process cutaneous antigens and migrate out of the skin and mucosa to the draining lymph nodes to present antigens to T and B cells. Because of the strategic location of LC and iDDC and the ability of these cells to capture and process pathogens, we hypothesized that they could be infected with human herpesvirus 8 (HHV-8) (Kaposi's sarcoma [KS]-associated herpesvirus) and have an important role in the development of KS. We have previously shown that HHV-8 enters monocyte-derived dendritic cells (MDDC) through DC-SIGN, resulting in nonproductive infection. Here we show that LC and iDDC generated from pluripotent cord blood CD34+ cell precursors support productive infection with HHV-8. Anti-DC-SIGN monoclonal antibody (MAb) inhibited HHV-8 infection of iDDC, as shown by low expression levels of viral proteins and DNA. In contrast, blocking of both langerin and the receptor protein tyrosine kinase ephrin A2 was required to inhibit HHV-8 infection of LC. Infection with HHV-8 did not alter the cell surface expression of langerin on LC but downregulated the expression of DC-SIGN on iDDC, as we previously reported for MDDC. HHV-8-infected LC and iDDC had a reduced ability to stimulate allogeneic CD4+ T cells in the mixed-lymphocyte reaction. These results indicate that HHV-8 can target both LC and iDDC for productive infection via different receptors and alter their function, supporting their potential role in HHV-8 pathogenesis and KS.IMPORTANCE Here we show that HHV-8, a DNA tumor virus that causes Kaposi's sarcoma, infects three types of dendritic cells: monocyte-derived dendritic cells, Langerhans cells, and interstitial dermal dendritic cells. We show that different receptors are used by this virus to infect these cells. DC-SIGN is a major receptor for infection of both monocyte-derived dendritic cells and interstitial dermal dendritic cells, yet the virus fully replicates only in the latter. HHV-8 uses langerin and the ephrin A2 receptor to infect Langerhans cells, which support full HHV-8 lytic replication. This infection of Langerhans cells and interstitial dermal dendritic cells results in an impaired ability to stimulate CD4+ helper T cell responses. Taken together, our data show that HHV-8 utilizes alternate receptors to differentially infect and replicate in these tissue-resident DC and support the hypothesis that these cells play an important role in HHV-8 infection and pathogenesis.


Assuntos
Células Dendríticas/virologia , Herpesvirus Humano 8/fisiologia , Células de Langerhans/virologia , Antígenos CD/metabolismo , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Diferenciação Celular , Células Cultivadas , Células Dendríticas/imunologia , Efrina-A2/antagonistas & inibidores , Herpesvirus Humano 8/genética , Herpesvirus Humano 8/imunologia , Herpesvirus Humano 8/patogenicidade , Humanos , Células de Langerhans/imunologia , Células de Langerhans/patologia , Lectinas Tipo C/antagonistas & inibidores , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Teste de Cultura Mista de Linfócitos , Lectinas de Ligação a Manose/antagonistas & inibidores , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Sarcoma de Kaposi/virologia , Pele/citologia , Pele/imunologia , Pele/virologia , Linfócitos T Auxiliares-Indutores/imunologia , Replicação Viral
15.
Bioorg Med Chem Lett ; 27(20): 4601-4605, 2017 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-28943043

RESUMO

The discovery of antiviral activity of 2,3-disubstituted quinazolinones, prepared by a one-pot, three-component condensation of isatoic anhydride with amines and aldehydes, against Herpes Simplex Virus (HSV)-1 is reported. Sequential iterative synthesis/antiviral assessment allowed structure-activity relationship (SAR) generation revealing synergistic structural features required for potent anti-HSV-1 activity. The most potent derivatives show greater efficacy than acyclovir against acute HSV-1 infections in neurons and minimal toxicity to the host.


Assuntos
Herpesvirus Humano 1/efeitos dos fármacos , Quinazolinonas/química , Quinazolinonas/farmacologia , Aciclovir/farmacologia , Animais , Antivirais/química , Antivirais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Imunoprecipitação da Cromatina , Avaliação Pré-Clínica de Medicamentos , Humanos , Relação Estrutura-Atividade , Células Vero
16.
Emerg Infect Dis ; 22(4): 671-8, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26982117

RESUMO

Next-generation sequencing has critical applications in virus discovery, diagnostics, and environmental surveillance. We used metagenomic sequence libraries for retrospective screening of plasma samples for the recently discovered human hepegivirus 1 (HHpgV-1). From a cohort of 150 hepatitis C virus (HCV)-positive case-patients, we identified 2 persons with HHpgV-1 viremia and a high frequency of human pegivirus (HPgV) viremia (14%). Detection of HHpgV-1 and HPgV was concordant with parallel PCR-based screening using conserved primers matching groups 1 (HPgV) and 2 (HHPgV-1) nonstructural 3 region sequences. PCR identified 1 HHPgV-1-positive person with viremia from a group of 195 persons with hemophilia who had been exposed to nonvirally inactivated factor VII/IX; 18 (9%) were HPgV-positive. Relative to HCV and HPgV, active infections with HHpgV-1 were infrequently detected in blood, even in groups that had substantial parenteral exposure. Our findings are consistent with lower transmissibility or higher rates of virus clearance for HHpgV-1 than for other bloodborne human flaviviruses.


Assuntos
Infecções por Flaviviridae/virologia , Flaviviridae/classificação , Hemofilia A/virologia , Hepacivirus/classificação , Filogenia , Viremia/virologia , Coinfecção , Biologia Computacional , Fator VII/uso terapêutico , Flaviviridae/genética , Flaviviridae/isolamento & purificação , Infecções por Flaviviridae/complicações , Infecções por Flaviviridae/diagnóstico , Infecções por Flaviviridae/tratamento farmacológico , Hemofilia A/complicações , Hemofilia A/diagnóstico , Hemofilia A/tratamento farmacológico , Hepacivirus/genética , Hepacivirus/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Análise de Sequência de DNA , Viremia/complicações , Viremia/diagnóstico , Viremia/tratamento farmacológico
17.
N Engl J Med ; 369(13): 1195-205, 2013 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-24066741

RESUMO

BACKGROUND: It has been thought that Clostridium difficile infection is transmitted predominantly within health care settings. However, endemic spread has hampered identification of precise sources of infection and the assessment of the efficacy of interventions. METHODS: From September 2007 through March 2011, we performed whole-genome sequencing on isolates obtained from all symptomatic patients with C. difficile infection identified in health care settings or in the community in Oxfordshire, United Kingdom. We compared single-nucleotide variants (SNVs) between the isolates, using C. difficile evolution rates estimated on the basis of the first and last samples obtained from each of 145 patients, with 0 to 2 SNVs expected between transmitted isolates obtained less than 124 days apart, on the basis of a 95% prediction interval. We then identified plausible epidemiologic links among genetically related cases from data on hospital admissions and community location. RESULTS: Of 1250 C. difficile cases that were evaluated, 1223 (98%) were successfully sequenced. In a comparison of 957 samples obtained from April 2008 through March 2011 with those obtained from September 2007 onward, a total of 333 isolates (35%) had no more than 2 SNVs from at least 1 earlier case, and 428 isolates (45%) had more than 10 SNVs from all previous cases. Reductions in incidence over time were similar in the two groups, a finding that suggests an effect of interventions targeting the transition from exposure to disease. Of the 333 patients with no more than 2 SNVs (consistent with transmission), 126 patients (38%) had close hospital contact with another patient, and 120 patients (36%) had no hospital or community contact with another patient. Distinct subtypes of infection continued to be identified throughout the study, which suggests a considerable reservoir of C. difficile. CONCLUSIONS: Over a 3-year period, 45% of C. difficile cases in Oxfordshire were genetically distinct from all previous cases. Genetically diverse sources, in addition to symptomatic patients, play a major part in C. difficile transmission. (Funded by the U.K. Clinical Research Collaboration Translational Infection Research Initiative and others.).


Assuntos
Clostridioides difficile/genética , Infecções por Clostridium/transmissão , Infecção Hospitalar/transmissão , Idoso , Idoso de 80 Anos ou mais , Clostridioides difficile/isolamento & purificação , Infecções por Clostridium/epidemiologia , Infecções por Clostridium/microbiologia , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , DNA Bacteriano/análise , Transmissão de Doença Infecciosa , Feminino , Variação Genética , Estudo de Associação Genômica Ampla , Humanos , Incidência , Masculino , Análise de Sequência de DNA , Reino Unido
18.
J Clin Microbiol ; 54(10): 2470-84, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27385709

RESUMO

Affordable next-generation sequencing (NGS) technologies for hepatitis C virus (HCV) may potentially identify both viral genotype and resistance genetic motifs in the era of directly acting antiviral (DAA) therapies. This study compared the ability of high-throughput NGS methods to generate full-length, deep, HCV sequence data sets and evaluated their utility for diagnostics and clinical assessment. NGS methods using (i) unselected HCV RNA (metagenomics), (ii) preenrichment of HCV RNA by probe capture, and (iii) HCV preamplification by PCR implemented in four United Kingdom centers were compared. Metrics of sequence coverage and depth, quasispecies diversity, and detection of DAA resistance-associated variants (RAVs), mixed HCV genotypes, and other coinfections were compared using a panel of samples with different viral loads, genotypes, and mixed HCV genotypes/subtypes [geno(sub)types]. Each NGS method generated near-complete genome sequences from more than 90% of samples. Enrichment methods and PCR preamplification generated greater sequence depth and were more effective for samples with low viral loads. All NGS methodologies accurately identified mixed HCV genotype infections. Consensus sequences generated by different NGS methods were generally concordant, and majority RAVs were consistently detected. However, methods differed in their ability to detect minor populations of RAVs. Metagenomic methods identified human pegivirus coinfections. NGS provided a rapid, inexpensive method for generating whole HCV genomes to define infecting genotypes, RAVs, comprehensive viral strain analysis, and quasispecies diversity. Enrichment methods are particularly suited for high-throughput analysis while providing the genotype and information on potential DAA resistance.


Assuntos
Genoma Viral , Genótipo , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Farmacorresistência Viral , Hepacivirus/classificação , Humanos , Reino Unido
19.
Eur Arch Otorhinolaryngol ; 273(1): 27-35, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25537817

RESUMO

The aim of this study was to evaluate the cochlear implant (CI) performances in neurofibromatosis type 2 (NF2) patients with bilateral vestibular schwannoma (VS) and in patients with sporadic VS in the only or better hearing ear. All patients with bilateral VS or sporadic VS in the only or better hearing ear who underwent cochlear implantation, either simultaneous to VS surgery or staged after treatment for VS, in the tumor side were chosen for the study. Postimplantation audiometric scores (sound detection, closed-set and open-set discrimination scores) and device use patterns were the main outcome measures. 15 patients were implanted. Eight patients (53 %) were NF2 and seven patients had VS in the only or better hearing ear. One patient was explanted for cerebrospinal fluid leak. In the CI-only condition, the other 14 patients obtained sound detection, 64 % of them achieving open-set discrimination (mean 70 ± 38 %) and 85 % achieving closed-set discrimination (mean 41 ± 33 %). At the last follow-up 10 patients (67 %) were using the CI. Cochlear implantation provides hearing in particular cases of patients with bilateral VS or VS in the only or better hearing ear. As long as anatomic preservation of the cochlear nerve is achieved, cochlear implantation may offer improvement in communication skills for most patients.


Assuntos
Implante Coclear , Audição/fisiologia , Neurofibromatose 2/complicações , Neuroma Acústico/cirurgia , Adulto , Idoso , Audiometria , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Adulto Jovem
20.
J Clin Microbiol ; 53(10): 3155-64, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26202126

RESUMO

The importance of recombination in the evolution and genetic diversity of the hepatitis C virus (HCV) is currently uncertain. Only a small number of intergenotypic recombinants have been identified so far, and each has core and envelope genes classified as belonging to genotype 2. Here, we investigated two putative genotype 4/1 recombinants from southern Cameroon using a number of approaches, including standard Sanger sequencing, genotype-specific PCR amplification, and non-HCV-specific Illumina RNA sequencing (RNA-seq). Recombination between genotypes 1 and 4 was confirmed in both samples, and the parental lineages of each recombinant belong to HCV subtypes that are cocirculating at a high prevalence in Cameroon. Using the RNA-seq approach, we obtained a complete genome for one sample, which contained a recombination breakpoint at the E2/P7 gene junction. We developed and applied a new method, called Deep SimPlot, which can be used to visualize and identify viral recombination directly from the short sequence reads created by next-generation sequencing in conjunction with a consensus sequence.


Assuntos
Genótipo , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Recombinação Genética , Análise de Sequência de DNA/métodos , Idoso , Camarões , Análise por Conglomerados , Feminino , Hepacivirus/isolamento & purificação , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Filogenia
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