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1.
Lancet Infect Dis ; 19(6): 648-657, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31000464

RESUMO

BACKGROUND: The real-time generation of information about pathogen genomes has become a vital goal for transmission analysis and characterisation in rapid outbreak responses. In response to the recently established genomic capacity in the Democratic Republic of the Congo, we explored the real-time generation of genomic information at the start of the 2018 Ebola virus disease (EVD) outbreak in North Kivu Province. METHODS: We used targeted-enrichment sequencing to produce two coding-complete Ebola virus genomes 5 days after declaration of the EVD outbreak in North Kivu. Subsequent sequencing efforts yielded an additional 46 genomes. Genomic information was used to assess early transmission, medical countermeasures, and evolution of Ebola virus. FINDINGS: The genomic information demonstrated that the EVD outbreak in the North Kivu and Ituri Provinces was distinct from the 2018 EVD outbreak in Équateur Province of the Democratic Republic of the Congo. Primer and probe mismatches to Ebola virus were identified in silico for all deployed diagnostic PCR assays, with the exception of the Cepheid GeneXpert GP assay. INTERPRETATION: The first two coding-complete genomes provided actionable information in real-time for the deployment of the rVSVΔG-ZEBOV-GP Ebola virus envelope glycoprotein vaccine, available therapeutics, and sequence-based diagnostic assays. Based on the mutations identified in the Ebola virus surface glycoprotein (GP12) observed in all 48 genomes, deployed monoclonal antibody therapeutics (mAb114 and ZMapp) should be efficacious against the circulating Ebola virus variant. Rapid Ebola virus genomic characterisation should be included in routine EVD outbreak response procedures to ascertain efficacy of medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

2.
Cell Rep ; 24(4): 1050-1059.e5, 2018 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-30044972

RESUMO

Development of an effective vaccine became a worldwide priority after the devastating 2013-2016 Ebola disease outbreak. To qualitatively profile the humoral response against advanced filovirus vaccine candidates, we developed Domain Programmable Arrays (DPA), a systems serology platform to identify epitopes targeted after vaccination or filovirus infection. We optimized the assay using a panel of well-characterized monoclonal antibodies. After optimization, we utilized the system to longitudinally characterize the immunoglobulin (Ig) isotype-specific responses in non-human primates vaccinated with rVSV-ΔG-EBOV-glycoprotein (GP). Strikingly, we observed that, although the IgM response was directed against epitopes over the whole GP, the IgG and IgA responses were almost exclusively directed against the mucin-like domain (MLD) of the glycan cap. Further research will be needed to characterize this possible biased IgG and IgA response toward the MLD, but the results corroborate that DPA is a valuable tool to qualitatively measure the humoral response after vaccination.

3.
Nature ; 544(7650): 309-315, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28405027

RESUMO

The 2013-2016 West African epidemic caused by the Ebola virus was of unprecedented magnitude, duration and impact. Here we reconstruct the dispersal, proliferation and decline of Ebola virus throughout the region by analysing 1,610 Ebola virus genomes, which represent over 5% of the known cases. We test the association of geography, climate and demography with viral movement among administrative regions, inferring a classic 'gravity' model, with intense dispersal between larger and closer populations. Despite attenuation of international dispersal after border closures, cross-border transmission had already sown the seeds for an international epidemic, rendering these measures ineffective at curbing the epidemic. We address why the epidemic did not spread into neighbouring countries, showing that these countries were susceptible to substantial outbreaks but at lower risk of introductions. Finally, we reveal that this large epidemic was a heterogeneous and spatially dissociated collection of transmission clusters of varying size, duration and connectivity. These insights will help to inform interventions in future epidemics.


Assuntos
Ebolavirus/genética , Ebolavirus/fisiologia , Genoma Viral/genética , Doença pelo Vírus Ebola/transmissão , Doença pelo Vírus Ebola/virologia , Clima , Surtos de Doenças/estatística & dados numéricos , Ebolavirus/isolamento & purificação , Geografia , Doença pelo Vírus Ebola/epidemiologia , Humanos , Internacionalidade , Modelos Lineares , Epidemiologia Molecular , Filogenia , Viagem/legislação & jurisprudência , Viagem/estatística & dados numéricos
4.
PLoS One ; 12(2): e0171333, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28182717

RESUMO

Individual RNA viruses typically occur as populations of genomes that differ slightly from each other due to mutations introduced by the error-prone viral polymerase. Understanding the variability of RNA virus genome populations is critical for understanding virus evolution because individual mutant genomes may gain evolutionary selective advantages and give rise to dominant subpopulations, possibly even leading to the emergence of viruses resistant to medical countermeasures. Reverse transcription of virus genome populations followed by next-generation sequencing is the only available method to characterize variation for RNA viruses. However, both steps may lead to the introduction of artificial mutations, thereby skewing the data. To better understand how such errors are introduced during sample preparation, we determined and compared error baseline rates of five different sample preparation methods by analyzing in vitro transcribed Ebola virus RNA from an artificial plasmid-based system. These methods included: shotgun sequencing from plasmid DNA or in vitro transcribed RNA as a basic "no amplification" method, amplicon sequencing from the plasmid DNA or in vitro transcribed RNA as a "targeted" amplification method, sequence-independent single-primer amplification (SISPA) as a "random" amplification method, rolling circle reverse transcription sequencing (CirSeq) as an advanced "no amplification" method, and Illumina TruSeq RNA Access as a "targeted" enrichment method. The measured error frequencies indicate that RNA Access offers the best tradeoff between sensitivity and sample preparation error (1.4-5) of all compared methods.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Vírus de RNA/genética , RNA Viral/análise , Análise de Sequência de RNA , Manejo de Espécimes/métodos , Análise Mutacional de DNA/métodos , Análise Mutacional de DNA/normas , Erros de Diagnóstico , Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , Polimorfismo de Nucleotídeo Único , RNA Viral/genética , Projetos de Pesquisa , Análise de Sequência de RNA/métodos , Análise de Sequência de RNA/normas , Manejo de Espécimes/normas
5.
J Mol Biol ; 428(14): 2814-31, 2016 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-27216501

RESUMO

The lens epithelium-derived growth factor p75 (LEDGF/p75) is a chromatin-bound protein essential for efficient lentiviral integration. Genome-wide studies have located LEDGF/p75 inside actively transcribed genes where it mediates lentiviral integration. Although its role in HIV-1 integration is clearly established, the role of LEDGF/p75-associated proteins in HIV-1 infection remains unexplored. Using protein-protein interaction assays, we demonstrated that LEDGF/p75 complexes with a chromatin-remodeling complex facilitates chromatin transcription (FACT), a heterodimer of the structure-specific recognition protein 1 (SSRP1) and the human homolog of suppressor of Ty 16 (hSpt16). Detailed analysis of the interaction of LEDGF/p75 with the FACT complex indicates that LEDGF/p75 interacts with SSRP1 in an hSpt16-independent manner that requires the PWWP domain of LEDGF proteins and the HMG domain of SSRP1. Functional characterizations demonstrate a LEDGF/p75-independent role of SSRP1 in the regulation of HIV-1 replication. shRNA-mediated partial knockdown of SSRP1 reduces HIV-1 infection, but not Murine Leukemia Virus, in human CD4(+) T cells. Similarly, SSRP1 knockdown affects infection by HIV-1-derived viruses that express genes from the viral LTR but not from an internal immediate-early CMV promoter, suggesting a role of SSRP1 in LTR-driven gene expression but not in viral DNA integration. Together, our data demonstrate for the first time the association of LEDGF proteins with the FACT complex and give further support to a role of SSRP1 in HIV-1 infection.

6.
Nature ; 531(7594): 381-5, 2016 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-26934220

RESUMO

The most recent Ebola virus outbreak in West Africa, which was unprecedented in the number of cases and fatalities, geographic distribution, and number of nations affected, highlights the need for safe, effective, and readily available antiviral agents for treatment and prevention of acute Ebola virus (EBOV) disease (EVD) or sequelae. No antiviral therapeutics have yet received regulatory approval or demonstrated clinical efficacy. Here we report the discovery of a novel small molecule GS-5734, a monophosphoramidate prodrug of an adenosine analogue, with antiviral activity against EBOV. GS-5734 exhibits antiviral activity against multiple variants of EBOV and other filoviruses in cell-based assays. The pharmacologically active nucleoside triphosphate (NTP) is efficiently formed in multiple human cell types incubated with GS-5734 in vitro, and the NTP acts as an alternative substrate and RNA-chain terminator in primer-extension assays using a surrogate respiratory syncytial virus RNA polymerase. Intravenous administration of GS-5734 to nonhuman primates resulted in persistent NTP levels in peripheral blood mononuclear cells (half-life, 14 h) and distribution to sanctuary sites for viral replication including testes, eyes, and brain. In a rhesus monkey model of EVD, once-daily intravenous administration of 10 mg kg(-1) GS-5734 for 12 days resulted in profound suppression of EBOV replication and protected 100% of EBOV-infected animals against lethal disease, ameliorating clinical disease signs and pathophysiological markers, even when treatments were initiated three days after virus exposure when systemic viral RNA was detected in two out of six treated animals. These results show the first substantive post-exposure protection by a small-molecule antiviral compound against EBOV in nonhuman primates. The broad-spectrum antiviral activity of GS-5734 in vitro against other pathogenic RNA viruses, including filoviruses, arenaviruses, and coronaviruses, suggests the potential for wider medical use. GS-5734 is amenable to large-scale manufacturing, and clinical studies investigating the drug safety and pharmacokinetics are ongoing.


Assuntos
Alanina/análogos & derivados , Antivirais/uso terapêutico , Doença pelo Vírus Ebola/tratamento farmacológico , Macaca mulatta/virologia , Ribonucleotídeos/uso terapêutico , Alanina/farmacocinética , Alanina/farmacologia , Alanina/uso terapêutico , Sequência de Aminoácidos , Animais , Antivirais/farmacocinética , Antivirais/farmacologia , Linhagem Celular Tumoral , Ebolavirus/efeitos dos fármacos , Feminino , Células HeLa , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Pró-Fármacos/farmacocinética , Pró-Fármacos/farmacologia , Pró-Fármacos/uso terapêutico , Ribonucleotídeos/farmacocinética , Ribonucleotídeos/farmacologia
7.
PLoS One ; 11(3): e0150919, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27002733

RESUMO

The creation of licensed medical countermeasures against Select Agents such as Ebola virus (EBOV) is critically dependent on the use of standardized reagents, assays, and animal models. We performed full genome reconstruction, population genomics, contaminant analysis, and characterization of the glycoprotein gene editing site of historical United States Army Medical Research Institute of Infectious Diseases (USAMRIID) nonhuman-primate challenge stock Ebola virus Kikwit "R4368" and its 2014 replacement "R4415." We also provide characterization of the master stock used to create "R4415." The obtained data are essential to understanding the quality of the seed stock reagents used in pivotal animal studies that have been used to inform medical countermeasure development. Furthermore, these data might add to the understanding of the influence of EBOV variant populations on pathogenesis and disease outcome and inform attempts to avoid the evolution of EBOV escape mutants in response to current therapeutics. Finally, as the primary challenge stocks have changed over time, these data will provide a baseline for understanding and correlating past and future animal study results.


Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/virologia , Primatas/virologia , Academias e Institutos , Animais , Genômica/métodos , Glicoproteínas/genética , Estados Unidos
8.
Cell Host Microbe ; 18(6): 659-69, 2015 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-26651942

RESUMO

The 2013-present Western African Ebola virus disease (EVD) outbreak is the largest ever recorded with >28,000 reported cases. Ebola virus (EBOV) genome sequencing has played an important role throughout this outbreak; however, relatively few sequences have been determined from patients in Liberia, the second worst-affected country. Here, we report 140 EBOV genome sequences from the second wave of the Liberian outbreak and analyze them in combination with 782 previously published sequences from throughout the Western African outbreak. While multiple early introductions of EBOV to Liberia are evident, the majority of Liberian EVD cases are consistent with a single introduction, followed by spread and diversification within the country. Movement of the virus within Liberia was widespread, and reintroductions from Liberia served as an important source for the continuation of the already ongoing EVD outbreak in Guinea. Overall, little evidence was found for incremental adaptation of EBOV to the human host.


Assuntos
Ebolavirus/classificação , Ebolavirus/genética , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/transmissão , Análise por Conglomerados , Ebolavirus/isolamento & purificação , Variação Genética , Genoma Viral , Genótipo , Doença pelo Vírus Ebola/virologia , Humanos , Libéria/epidemiologia , Epidemiologia Molecular , Dados de Sequência Molecular , Filogeografia , Análise de Sequência de DNA , Homologia de Sequência
9.
N Engl J Med ; 373(25): 2448-54, 2015 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-26465384

RESUMO

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).


Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/transmissão , Sêmen/virologia , Adulto , Coito , Ebolavirus/isolamento & purificação , Feminino , Genoma Viral , Doença pelo Vírus Ebola/virologia , Humanos , Libéria , Masculino , RNA Viral/sangue , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sexo sem Proteção
10.
Cell Rep ; 12(12): 2111-20, 2015 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-26365189

RESUMO

MB-003, a plant-derived monoclonal antibody cocktail used effectively in treatment of Ebola virus infection in non-human primates, was unable to protect two of six animals when initiated 1 or 2 days post-infection. We characterized a mechanism of viral escape in one of the animals, after observation of two clusters of genomic mutations that resulted in five nonsynonymous mutations in the monoclonal antibody target sites. These mutations were linked to a reduction in antibody binding and later confirmed to be present in a viral isolate that was not neutralized in vitro. Retrospective evaluation of a second independent study allowed the identification of a similar case. Four SNPs in previously identified positions were found in this second fatality, suggesting that genetic drift could be a potential cause for treatment failure. These findings highlight the importance selecting different target domains for each component of the cocktail to minimize the potential for viral escape.


Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Antivirais/administração & dosagem , Ebolavirus/imunologia , Doença pelo Vírus Ebola/virologia , Evasão da Resposta Imune/genética , Imunização Passiva , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/biossíntese , Anticorpos Antivirais/biossíntese , Sequência de Bases , Ebolavirus/genética , Ebolavirus/patogenicidade , Epitopos/química , Epitopos/imunologia , Doença pelo Vírus Ebola/imunologia , Doença pelo Vírus Ebola/mortalidade , Doença pelo Vírus Ebola/prevenção & controle , Humanos , Macaca mulatta , Dados de Sequência Molecular , Mutação , Ligação Proteica , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/biossíntese , Estudos Retrospectivos , Análise de Sobrevida , Tabaco/genética , Replicação Viral
11.
Emerg Infect Dis ; 21(7): 1135-43, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26079255

RESUMO

To support Liberia's response to the ongoing Ebola virus (EBOV) disease epidemic in Western Africa, we established in-country advanced genomic capabilities to monitor EBOV evolution. Twenty-five EBOV genomes were sequenced at the Liberian Institute for Biomedical Research, which provided an in-depth view of EBOV diversity in Liberia during September 2014-February 2015. These sequences were consistent with a single virus introduction to Liberia; however, shared ancestry with isolates from Mali indicated at least 1 additional instance of movement into or out of Liberia. The pace of change is generally consistent with previous estimates of mutation rate. We observed 23 nonsynonymous mutations and 1 nonsense mutation. Six of these changes are within known binding sites for sequence-based EBOV medical countermeasures; however, the diagnostic and therapeutic impact of EBOV evolution within Liberia appears to be low.


Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/virologia , Antivirais/farmacologia , Antivirais/uso terapêutico , Análise Mutacional de DNA , Farmacorresistência Viral/genética , Evolução Molecular , Genes Virais , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/epidemiologia , Humanos , Libéria/epidemiologia
12.
Cell ; 161(7): 1516-26, 2015 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-26091036

RESUMO

The 2013-2015 Ebola virus disease (EVD) epidemic is caused by the Makona variant of Ebola virus (EBOV). Early in the epidemic, genome sequencing provided insights into virus evolution and transmission and offered important information for outbreak response. Here, we analyze sequences from 232 patients sampled over 7 months in Sierra Leone, along with 86 previously released genomes from earlier in the epidemic. We confirm sustained human-to-human transmission within Sierra Leone and find no evidence for import or export of EBOV across national borders after its initial introduction. Using high-depth replicate sequencing, we observe both host-to-host transmission and recurrent emergence of intrahost genetic variants. We trace the increasing impact of purifying selection in suppressing the accumulation of nonsynonymous mutations over time. Finally, we note changes in the mucin-like domain of EBOV glycoprotein that merit further investigation. These findings clarify the movement of EBOV within the region and describe viral evolution during prolonged human-to-human transmission.


Assuntos
Ebolavirus/genética , Ebolavirus/isolamento & purificação , Genoma Viral , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/virologia , Mutação , Evolução Biológica , Surtos de Doenças , Ebolavirus/classificação , Doença pelo Vírus Ebola/transmissão , Humanos , Serra Leoa/epidemiologia , Manejo de Espécimes
13.
MMWR Morb Mortal Wkly Rep ; 64(17): 479-81, 2015 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-25950255

RESUMO

On March 20, 2015, 30 days after the most recent confirmed Ebola Virus Disease (Ebola) patient in Liberia was isolated, Ebola was laboratory confirmed in a woman in Monrovia. The investigation identified only one epidemiologic link to Ebola: unprotected vaginal intercourse with a survivor. Published reports from previous outbreaks have demonstrated Ebola survivors can continue to harbor virus in immunologically privileged sites for a period of time after convalescence. Ebola virus has been isolated from semen as long as 82 days after symptom onset and viral RNA has been detected in semen up to 101 days after symptom onset. One instance of possible sexual transmission of Ebola has been reported, although the accompanying evidence was inconclusive. In addition, possible sexual transmission of Marburg virus, a filovirus related to Ebola, was documented in 1968. This report describes the investigation by the Government of Liberia and international response partners of the source of Liberia's latest Ebola case and discusses the public health implications of possible sexual transmission of Ebola virus. Based on information gathered in this investigation, CDC now recommends that contact with semen from male Ebola survivors be avoided until more information regarding the duration and infectiousness of viral shedding in body fluids is known. If male survivors have sex (oral, vaginal, or anal), a condom should be used correctly and consistently every time.


Assuntos
Ebolavirus/isolamento & purificação , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/transmissão , Doenças Virais Sexualmente Transmissíveis , Adulto , Surtos de Doenças , Feminino , Doença pelo Vírus Ebola/epidemiologia , Humanos , Libéria/epidemiologia , Masculino , Pessoa de Meia-Idade , RNA Viral , Sêmen/virologia , Sobreviventes , Sexo sem Proteção
14.
Genome Announc ; 3(2)2015 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-25908124

RESUMO

We obtained the complete coding genome of an eastern equine encephalitis virus (EEEV) strain, EEEV V105-00210, and the complete genome of a Venezuelan equine encephalitis virus (VEEV) strain, VEEV INH-9813. They were obtained from human cases and are proposed as reference challenge strains for vaccine and therapeutic development in animal models.

15.
MBio ; 6(1)2015 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-25604787

RESUMO

Until recently, Ebola virus (EBOV) was a rarely encountered human pathogen that caused disease among small populations with extraordinarily high lethality. At the end of 2013, EBOV initiated an unprecedented disease outbreak in West Africa that is still ongoing and has already caused thousands of deaths. Recent studies revealed the genomic changes this particular EBOV variant undergoes over time during human-to-human transmission. Here we highlight the genomic changes that might negatively impact the efficacy of currently available EBOV sequence-based candidate therapeutics, such as small interfering RNAs (siRNAs), phosphorodiamidate morpholino oligomers (PMOs), and antibodies. Ten of the observed mutations modify the sequence of the binding sites of monoclonal antibody (MAb) 13F6, MAb 1H3, MAb 6D8, MAb 13C6, and siRNA EK-1, VP24, and VP35 targets and might influence the binding efficacy of the sequence-based therapeutics, suggesting that their efficacy should be reevaluated against the currently circulating strain.


Assuntos
Ebolavirus/genética , Variação Genética , Doença pelo Vírus Ebola/virologia , Genômica , Doença pelo Vírus Ebola/terapia , Humanos , Mutação , RNA Interferente Pequeno , Proteínas Virais/genética , Proteínas Virais/metabolismo
16.
Genome Announc ; 2(6)2014 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-25414499

RESUMO

Ebola virus (EBOV) was discovered in 1976 around Yambuku, Zaire. A lack of nomenclature standards resulted in a variety of designations for each isolate, leading to confusion in the literature and databases. We sequenced the genome of isolate E718/ME/Ecran and unified the various designations under Ebola virus/H.sapiens-tc/COD/1976/Yambuku-Ecran.

17.
Viruses ; 6(11): 4760-99, 2014 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-25421896

RESUMO

In 2014, Ebola virus (EBOV) was identified as the etiological agent of a large and still expanding outbreak of Ebola virus disease (EVD) in West Africa and a much more confined EVD outbreak in Middle Africa. Epidemiological and evolutionary analyses confirmed that all cases of both outbreaks are connected to a single introduction each of EBOV into human populations and that both outbreaks are not directly connected. Coding-complete genomic sequence analyses of isolates revealed that the two outbreaks were caused by two novel EBOV variants, and initial clinical observations suggest that neither of them should be considered strains. Here we present consensus decisions on naming for both variants (West Africa: "Makona", Middle Africa: "Lomela") and provide database-compatible full, shortened, and abbreviated names that are in line with recently established filovirus sub-species nomenclatures.


Assuntos
Ebolavirus/classificação , Doença pelo Vírus Ebola/virologia , Terminologia como Assunto , República Democrática do Congo/epidemiologia , Surtos de Doenças , Ebolavirus/genética , Ebolavirus/isolamento & purificação , Guiné/epidemiologia , Doença pelo Vírus Ebola/epidemiologia , Humanos , Filogenia , RNA Viral/genética , Análise de Sequência de DNA
18.
MBio ; 5(3): e01360-14, 2014 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-24939889

RESUMO

Thanks to high-throughput sequencing technologies, genome sequencing has become a common component in nearly all aspects of viral research; thus, we are experiencing an explosion in both the number of available genome sequences and the number of institutions producing such data. However, there are currently no common standards used to convey the quality, and therefore utility, of these various genome sequences. Here, we propose five "standard" categories that encompass all stages of viral genome finishing, and we define them using simple criteria that are agnostic to the technology used for sequencing. We also provide genome finishing recommendations for various downstream applications, keeping in mind the cost-benefit trade-offs associated with different levels of finishing. Our goal is to define a common vocabulary that will allow comparison of genome quality across different research groups, sequencing platforms, and assembly techniques.


Assuntos
Genoma Viral , Sequenciamento de Nucleotídeos em Larga Escala/normas , Vírus/genética , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/métodos
19.
Emerg Infect Dis ; 20(2): 232-9, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24457084

RESUMO

Monkeypox virus is a zoonotic virus endemic to Central Africa. Although active disease surveillance has assessed monkeypox disease prevalence and geographic range, information about virus diversity is lacking. We therefore assessed genome diversity of viruses in 60 samples obtained from humans with primary and secondary cases of infection from 2005 through 2007. We detected 4 distinct lineages and a deletion that resulted in gene loss in 10 (16.7%) samples and that seemed to correlate with human-to-human transmission (p = 0.0544). The data suggest a high frequency of spillover events from the pool of viruses in nonhuman animals, active selection through genomic destabilization and gene loss, and increased disease transmissibility and severity. The potential for accelerated adaptation to humans should be monitored through improved surveillance.


Assuntos
Genoma Viral , Instabilidade Genômica , Vírus da Varíola dos Macacos/genética , Filogenia , Adaptação Biológica/genética , Sequência de Aminoácidos , Animais , República Democrática do Congo/epidemiologia , Monitoramento Epidemiológico , Deleção de Genes , Humanos , Dados de Sequência Molecular , Monkeypox/epidemiologia , Monkeypox/virologia , Vírus da Varíola dos Macacos/classificação , Análise de Sequência de DNA , Índice de Gravidade de Doença
20.
PLoS One ; 7(11): e50316, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23209706

RESUMO

To identify polymorphic sites that could be used as biomarkers of Ebola virus passage history, we repeatedly amplified Ebola virus (Kikwit variant) in vitro and in vivo and performed deep sequencing analysis of the complete genomes of the viral subpopulations. We then determined the sites undergoing selection during passage in Vero E6 cells. Four locations within the Ebola virus Kikwit genome were identified that together segregate cell culture-passaged virus and virus obtained from infected non-human primates. Three of the identified sites are located within the glycoprotein gene (GP) sequence: the poly-U (RNA editing) site at position 6925, as well as positions 6677, and 6179. One site was found in the VP24 gene at position 10833. In all cases, in vitro and in vivo, both populations (majority and minority variants) were maintained in the viral swarm, with rapid selections occurring after a few passages or infections. This analysis approach will be useful to differentiate whether filovirus stocks with unknown history have been passaged in cell culture and may support filovirus stock standardization for medical countermeasure development.


Assuntos
Ebolavirus/genética , Genoma Viral , Animais , Técnicas de Cultura de Células , Análise por Conglomerados , Marcadores Genéticos , Glicoproteínas/genética , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Mutação , Polimorfismo Genético , Polimorfismo de Nucleotídeo Único , Primatas/genética , RNA Viral/genética , Análise de Sequência de DNA , Análise de Sequência de RNA , Células Vero , Proteínas Virais/genética
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