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1.
Antiviral Res ; 170: 104529, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31195019

RESUMO

Nucleoside analogues (NA) disrupt RNA viral RNA-dependent RNA polymerase (RdRP) function and fidelity for multiple viral families. The mechanism of action (MOA) of T-705 has been attributed alternatively or concurrently to chain termination and lethal mutagenesis depending on the viral species during in vitro studies. In this study, we evaluated the effect of T-705 on the viral population in non-human primates (NHPs) after challenge with Ebola virus (EBOV) or Marburg virus (MARV) to identify the predominant in vivo MOA. We used common virological assays in conjunction with deep sequencing to characterize T-705 effects. T-705 exhibited antiviral activity that was associated with a reduction in specific infectivity and an accumulation of low frequency nucleotide variants in plasma samples collected day 7 post infection. Stranded analysis of deep sequencing data to identify chain termination demonstrated no change in the transcriptional gradient in negative stranded viral reads and minimal changes in positive stranded viral reads in T-705 treated animals, questioning as a MOA in vivo. These findings indicate that lethal mutagenesis is a MOA of T-705 that may serve as an indication of therapeutic activity of NAs for evaluation in clinical settings. This study expands our understanding of MOAs of these compounds for the Filovirus family and provides further evidence that lethal mutagenesis could be a preponderant MOA for this class of therapeutic compounds.

2.
Viruses ; 11(4)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010201

RESUMO

Although Lloviu virus (LLOV) was discovered in the carcasses of insectivorous Schreiber's Bent-winged bats in the caves of Northern Spain in 2002, its infectivity and pathogenicity remain unclear. We examined the seroprevalence of LLOV in potentially exposed Schreiber's Bent-winged bats (n = 60), common serotine bats (n = 10) as controls, and humans (n = 22) using an immunoblot assay. We found antibodies against LLOV GP2 in all of Schreiber's Bent-winged bats serum pools, but not in any of the common serotine bats and human pools tested. To confirm this seroreactivity, 52 serums were individually tested using Domain Programmable Arrays (DPA), a phage display based-system serology technique for profiling filovirus epitopes. A serological signature against different LLOV proteins was obtained in 19/52 samples tested (36.5%). The immunodominant response was in the majority specific to LLOV-unique epitopes, confirming that the serological response detected was to LLOV. To our knowledge, this is the first serological evidence of LLOV exposure in live captured Schreiber's Bent-winged bats, dissociating LLOV circulation as the cause of the previously reported die-offs.

3.
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.

4.
Lancet Infect Dis ; 19(6): 641-647, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31000465

RESUMO

BACKGROUND: The 2018 Ebola virus disease (EVD) outbreak in Équateur Province, Democratic Republic of the Congo, began on May 8, and was declared over on July 24; it resulted in 54 documented cases and 33 deaths. We did a retrospective genomic characterisation of the outbreak and assessed potential therapeutic agents and vaccine (medical countermeasures). METHODS: We used target-enrichment sequencing to produce Ebola virus genomes from samples obtained in the 2018 Équateur Province outbreak. Combining these genomes with genomes associated with known outbreaks from GenBank, we constructed a maximum-likelihood phylogenetic tree. In-silico analyses were used to assess potential mismatches between the outbreak strain and the probes and primers of diagnostic assays and the antigenic sites of the experimental rVSVΔG-ZEBOV-GP vaccine and therapeutics. An in-vitro flow cytometry assay was used to assess the binding capability of the individual components of the monoclonal antibody cocktail ZMapp. FINDINGS: A targeted sequencing approach produced 16 near-complete genomes. Phylogenetic analysis of these genomes and 1011 genomes from GenBank revealed a distinct cluster, confirming a new Ebola virus variant, for which we propose the name "Tumba". This new variant appears to have evolved at a slower rate than other Ebola virus variants (0·69 × 10-3 substitutions per site per year with "Tumba" vs 1·06 × 10-3 substitutions per site per year without "Tumba"). We found few sequence mismatches in the assessed assay target regions and antigenic sites. We identified nine amino acid changes in the Ebola virus surface glycoprotein, of which one resulted in reduced binding of the 13C6 antibody within the ZMapp cocktail. INTERPRETATION: Retrospectively, we show the feasibility of using genomics to rapidly characterise a new Ebola virus variant within the timeframe of an outbreak. Phylogenetic analysis provides further indications that these variants are evolving at differing rates. Rapid in-silico analyses can direct in-vitro experiments to quickly assess medical countermeasures. FUNDING: Defense Biological Product Assurance Office.

5.
Viruses ; 11(1)2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30654482

RESUMO

We generated genome sequences from 218 cases of Ebola virus disease (EVD) in Sierra Leone (SLE) during 2014⁻2015 to complement available datasets, particularly by including cases from a period of low sequence coverage during peak transmission of Ebola virus (EBOV) in the highly-affected Western Area division of SLE. The combined dataset was utilized to produce phylogenetic and phylodynamic inferences, to study sink⁻source dynamics and virus dispersal from highly-populated transmission hotspots. We identified four districts in SLE where EBOV was introduced and transmission occurred without onward exportation to other districts. We also identified six districts that substantially contributed to the dispersal of the virus and prolonged the EVD outbreak: five of these served as major hubs, with lots of movement in and out, and one acted primarily as a source, exporting the virus to other areas of the country. Positive correlations between case numbers, inter-district transition events, and district population sizes reaffirm that population size was a driver of EBOV transmission dynamics in SLE. The data presented here confirm the role of urban hubs in virus dispersal and of a delayed laboratory response in the expansion and perpetuation of the EVD outbreak in SLE.


Assuntos
Ebolavirus/genética , Doença pelo Vírus Ebola/transmissão , Filogenia , Surtos de Doenças , Ebolavirus/classificação , Genoma Viral , Doença pelo Vírus Ebola/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Serra Leoa/epidemiologia
6.
Viruses ; 10(11)2018 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-30400182

RESUMO

The Egyptian rousette bat (ERB) is the only known Marburg virus (MARV) reservoir host. ERBs develop a productive MARV infection with low viremia and shedding but no overt disease, suggesting this virus is efficiently controlled by ERB antiviral responses. This dynamic would contrast with humans, where MARV-mediated interferon (IFN) antagonism early in infection is thought to contribute to the severe, often fatal disease. The newly-annotated ERB genome and transcriptome have now enabled us to use a custom-designed NanoString nCounter ERB CodeSet in conjunction with RNA-seq to investigate responses in a MARV-infected ERB cell line. Both transcriptomic platforms correlated well and showed that MARV inhibited the antiviral program in ERB cells, while an IFN antagonism-impaired MARV was less efficient at suppressing the response gene induction, phenotypes previously reported for primate cells. Interestingly, and despite the expansion of IFN loci in the ERB genome, neither MARV showed specific induction of almost any IFN gene. However, we detected an upregulation of putative, unannotated ERB antiviral paralogs, as well as an elevated basal expression in uninfected ERB cells of key antiviral genes.

7.
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.

8.
Cell ; 173(5): 1098-1110.e18, 2018 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-29706541

RESUMO

Bats harbor many viruses asymptomatically, including several notorious for causing extreme virulence in humans. To identify differences between antiviral mechanisms in humans and bats, we sequenced, assembled, and analyzed the genome of Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus. We found an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons, which dramatically differ from their functional counterparts in other mammals. Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense. An evaluation of the theoretical function of these genes suggests that an inhibitory immune state may exist in bats. Based on our findings, we hypothesize that tolerance of viral infection, rather than enhanced potency of antiviral defenses, may be a key mechanism by which bats asymptomatically host viruses that are pathogenic in humans.

9.
Hum Vaccin Immunother ; 13(12): 2883-2893, 2017 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-28699812

RESUMO

We performed epitope mapping studies on the major surface glycoprotein (GP) of Ebola virus (EBOV) using Chemically Linked Peptides on Scaffolds (CLIPS), which form linear and potential conformational epitopes. This method identified monoclonal antibody epitopes and predicted additional epitopes recognized by antibodies in polyclonal sera from animals experimentally vaccinated against or infected with EBOV. Using the information obtained along with structural modeling to predict epitope accessibility, we then constructed 2 DNA vaccines encoding immunodominant and subdominant epitopes predicted to be accessible on EBOV GP. Although a construct designed to produce a membrane-bound oligopeptide was poorly immunogenic, a construct generating a secreted oligopeptide elicited strong antibody responses in mice. When this construct was administered as a boost to a DNA vaccine expressing the complete EBOV GP gene, the resultant antibody response was focused largely toward the less immunodominant epitopes in the oligopeptide. Taken together, the results of this work suggest a utility for this method for immune focusing of antibody responses elicited by vaccination.


Assuntos
Anticorpos Antivirais/sangue , Antígenos Virais/imunologia , Vacinas contra Ebola/imunologia , Ebolavirus/imunologia , Mapeamento de Epitopos , Glicoproteínas/imunologia , Vacinas de DNA/imunologia , Animais , Formação de Anticorpos , Antígenos Virais/genética , DNA Viral , Vacinas contra Ebola/administração & dosagem , Vacinas contra Ebola/genética , Ebolavirus/genética , Epitopos/genética , Epitopos/imunologia , Glicoproteínas/genética , Esquemas de Imunização , Camundongos Endogâmicos BALB C , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
10.
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
11.
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
12.
Sci Adv ; 2(4): e1600378, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27386513

RESUMO

On 29 June 2015, Liberia's respite from Ebola virus disease (EVD) was interrupted for the second time by a renewed outbreak ("flare-up") of seven confirmed cases. We demonstrate that, similar to the March 2015 flare-up associated with sexual transmission, this new flare-up was a reemergence of a Liberian transmission chain originating from a persistently infected source rather than a reintroduction from a reservoir or a neighboring country with active transmission. Although distinct, Ebola virus (EBOV) genomes from both flare-ups exhibit significantly low genetic divergence, indicating a reduced rate of EBOV evolution during persistent infection. Using this rate of change as a signature, we identified two additional EVD clusters that possibly arose from persistently infected sources. These findings highlight the risk of EVD flare-ups even after an outbreak is declared over.


Assuntos
Ebolavirus/patogenicidade , Doença pelo Vírus Ebola/epidemiologia , Doença pelo Vírus Ebola/transmissão , Surtos de Doenças , Ebolavirus/genética , Genoma Viral/genética , Doença pelo Vírus Ebola/genética , Doença pelo Vírus Ebola/virologia , Humanos , Libéria
13.
Immunogenetics ; 68(6-7): 417-428, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27233955

RESUMO

Cynomolgus macaques (Macaca fascicularis) have become an important animal model for biomedical research. In particular, it is the animal model of choice for the development of vaccine candidates associated with emerging dangerous pathogens. Despite their increasing importance as animal models, the cynomolgus macaque genome is not fully characterized, hindering molecular studies for this model. More importantly, the lack of knowledge about the immunoglobulin (IG) locus organization directly impacts the analysis of the humoral response in cynomolgus macaques. Recent advances in next generation sequencing (NGS) technologies to analyze IG repertoires open the opportunity to deeply characterize the humoral immune response. However, the IG locus organization for the animal is required to completely dissect IG repertoires. Here, we describe the localization and organization of the rearranging IG heavy (IGH) genes on chromosome 7 of the cynomolgus macaque draft genome. Our annotation comprises 108 functional genes which include 63 variable (IGHV), 38 diversity (IGHD), and 7 joining (IGHJ) genes. For validation, we provide RNA transcript data for most of the IGHV genes and all of the annotated IGHJ genes, as well as proteomic data to validate IGH constant genes. The description and annotation of the rearranging IGH genes for the cynomolgus macaques will significantly facilitate scientific research. This is particularly relevant to dissect the immune response during vaccination or infection with dangerous pathogens such as Ebola, Marburg and other emerging pathogens where non-human primate models play a significant role for countermeasure development.


Assuntos
Genes de Cadeia Pesada de Imunoglobulina/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Região Variável de Imunoglobulina/genética , Macaca fascicularis/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia Líquida , Genes de Cadeia Pesada de Imunoglobulina/imunologia , Genoma , Humanos , Região Variável de Imunoglobulina/imunologia , Região Variável de Imunoglobulina/metabolismo , Macaca fascicularis/imunologia , Anotação de Sequência Molecular , Filogenia , Proteômica , Especificidade da Espécie , Espectrometria de Massas em Tandem
14.
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
15.
BMC Genomics ; 16: 1033, 2015 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-26643810

RESUMO

BACKGROUND: The Egyptian Rousette bat (Rousettus aegyptiacus), a common fruit bat species found throughout Africa and the Middle East, was recently identified as a natural reservoir host of Marburg virus. With Ebola virus, Marburg virus is a member of the family Filoviridae that causes severe hemorrhagic fever disease in humans and nonhuman primates, but results in little to no pathological consequences in bats. Understanding host-pathogen interactions within reservoir host species and how it differs from hosts that experience severe disease is an important aspect of evaluating viral pathogenesis and developing novel therapeutics and methods of prevention. RESULTS: Progress in studying bat reservoir host responses to virus infection is hampered by the lack of host-specific reagents required for immunological studies. In order to establish a basis for the design of reagents, we sequenced, assembled, and annotated the R. aegyptiacus transcriptome. We performed de novo transcriptome assembly using deep RNA sequencing data from 11 distinct tissues from one male and one female bat. We observed high similarity between this transcriptome and those available from other bat species. Gene expression analysis demonstrated clustering of expression profiles by tissue, where we also identified enrichment of tissue-specific gene ontology terms. In addition, we identified and experimentally validated the expression of novel coding transcripts that may be specific to this species. CONCLUSION: We comprehensively characterized the R. aegyptiacus transcriptome de novo. This transcriptome will be an important resource for understanding bat immunology, physiology, disease pathogenesis, and virus transmission.


Assuntos
Quirópteros/genética , Biologia Computacional , Anotação de Sequência Molecular , Transcriptoma , Animais , Análise por Conglomerados , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Reprodutibilidade dos Testes
16.
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
17.
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
18.
Hum Immunol ; 76(12): 891-6, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26028281

RESUMO

Single-molecule real-time (SMRT) sequencing technology with the Pacific Biosciences (PacBio) RS II platform offers the potential to obtain full-length coding regions (∼1100-bp) from MHC class I cDNAs. Despite the relatively high error rate associated with SMRT technology, high quality sequences can be obtained by circular consensus sequencing (CCS) due to the random nature of the error profile. In the present study we first validated the ability of SMRT-CCS to accurately identify class I transcripts in Mauritian-origin cynomolgus macaques (Macaca fascicularis) that have been characterized previously by cloning and Sanger-based sequencing as well as pyrosequencing approaches. We then applied this SMRT-CCS method to characterize 60 novel full-length class I transcript sequences expressed by a cohort of cynomolgus macaques from China. The SMRT-CCS method described here provides a straightforward protocol for characterization of unfragmented single-molecule cDNA transcripts that will potentially revolutionize MHC class I allele discovery in nonhuman primates and other species.


Assuntos
Alelos , Genes MHC Classe I , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Animais , Variação Genética , Haplótipos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Teste de Histocompatibilidade , Macaca fascicularis , Análise de Sequência de DNA
19.
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
20.
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
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