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Dengue is a major health threat and the number of symptomatic infections caused by the four dengue serotypes is estimated to be 96 million1 with annually around 10,000 deaths2. However, no antiviral drugs are available for the treatment or prophylaxis of dengue. We recently described the interaction between non-structural proteins NS3 and NS4B as a promising target for the development of pan-serotype dengue virus (DENV) inhibitors3. Here we present JNJ-1802-a highly potent DENV inhibitor that blocks the NS3-NS4B interaction within the viral replication complex. JNJ-1802 exerts picomolar to low nanomolar in vitro antiviral activity, a high barrier to resistance and potent in vivo efficacy in mice against infection with any of the four DENV serotypes. Finally, we demonstrate that the small-molecule inhibitor JNJ-1802 is highly effective against viral infection with DENV-1 or DENV-2 in non-human primates. JNJ-1802 has successfully completed a phase I first-in-human clinical study in healthy volunteers and was found to be safe and well tolerated4. These findings support the further clinical development of JNJ-1802, a first-in-class antiviral agent against dengue, which is now progressing in clinical studies for the prevention and treatment of dengue.
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Antivirales , Virus del Dengue , Dengue , Primates , Proteínas no Estructurales Virales , Animales , Humanos , Ratones , Antivirales/efectos adversos , Antivirales/farmacología , Antivirales/uso terapéutico , Ensayos Clínicos Fase I como Asunto , Dengue/tratamiento farmacológico , Dengue/prevención & control , Dengue/virología , Virus del Dengue/clasificación , Virus del Dengue/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Farmacorresistencia Viral , Técnicas In Vitro , Terapia Molecular Dirigida , Primates/virología , Unión Proteica/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/metabolismo , Replicación ViralRESUMEN
Dengue virus causes approximately 96 million symptomatic infections annually, manifesting as dengue fever or occasionally as severe dengue1,2. There are no antiviral agents available to prevent or treat dengue. Here, we describe a highly potent dengue virus inhibitor (JNJ-A07) that exerts nanomolar to picomolar activity against a panel of 21 clinical isolates that represent the natural genetic diversity of known genotypes and serotypes. The molecule has a high barrier to resistance and prevents the formation of the viral replication complex by blocking the interaction between two viral proteins (NS3 and NS4B), thus revealing a previously undescribed mechanism of antiviral action. JNJ-A07 has a favourable pharmacokinetic profile that results in outstanding efficacy against dengue virus infection in mouse infection models. Delaying start of treatment until peak viraemia results in a rapid and significant reduction in viral load. An analogue is currently in further development.
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Antivirales/farmacología , Virus del Dengue/clasificación , Virus del Dengue/efectos de los fármacos , Dengue/virología , Proteínas de la Membrana/metabolismo , Proteínas no Estructurales Virales/metabolismo , Animales , Antivirales/farmacocinética , Antivirales/uso terapéutico , Dengue/tratamiento farmacológico , Virus del Dengue/genética , Virus del Dengue/metabolismo , Modelos Animales de Enfermedad , Femenino , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , Serina Endopeptidasas/metabolismo , Carga Viral/efectos de los fármacos , Proteínas no Estructurales Virales/antagonistas & inhibidores , Viremia/tratamiento farmacológico , Viremia/virología , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: In study TMC647055HPC2001, a 3-direct-acting-antiviral (DAA) regimen combining NS3/4A protease inhibitor simeprevir (SMV), non-nucleoside NS5B inhibitor TMC647055/ritonavir (RTV) and NS5A inhibitor JNJ-56914845 resulted in high sustained virologic response 12 weeks after actual end of treatment (SVR12) in chronic hepatitis C virus (HCV) genotype 1-infected patients. SVR12 rates were generally lower in the 2-DAA regimen SMV + TMC647055/RTV with or without ribavirin. The objective of this study was to identify and characterise pre-existing and emerging resistance-associated variants (RAVs) in patients enrolled in study TMC647055HPC2001. METHODS: HCV population sequencing analyses were performed on baseline isolates from all patients (n = 90) and post-baseline isolates from patients with virologic failure (n = 22). In addition, deep sequencing and phenotypic analyses were performed on selected baseline and post-baseline isolates. RESULTS: The majority of patients with virologic failure had emerging RAVs to all study drugs at the time of failure: in all 22 patients SMV RAVs emerged at NS3 positions 80, 155, 156 and/or 168, consistent with the known SMV resistance profile. Emerging TMC647055 RAVs at NS5B position 495 were detected in the majority of patients (16/22), and all 5 patients who failed the 3-DAA regimen had emerging JNJ-56914845 RAVs at NS5A positions 30 and/or 31. While at the end of study emerging SMV and TMC647055 RAVs were no longer observed by population sequencing in 40% (8/20) and 62.5% (10/16) of patients with follow-up data available, respectively, emerging JNJ-56914845 RAVs were still detected in all (5/5) patients. CONCLUSIONS: Virologic failure in the 2- and 3-DAA combinations was, in the majority of patients, associated with the emergence of RAVs to all study drugs. While emerging SMV and TMC647055 RAVs became undetectable during follow-up, JNJ-56914845 RAVs in NS5A were still observed at end of study. TRIAL REGISTRATION NUMBER: NCT01724086 (date of registration: September 26, 2012).
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Antivirales/uso terapéutico , Genotipo , Hepacivirus/aislamiento & purificación , Hepatitis C Crónica/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Ritonavir/uso terapéutico , Simeprevir/uso terapéutico , Sulfonamidas/uso terapéutico , Carbamatos/uso terapéutico , Farmacorresistencia Viral , Técnicas de Genotipaje , Hepacivirus/clasificación , Hepacivirus/genética , Hepatitis C Crónica/virología , Humanos , Ribavirina/uso terapéutico , Análisis de Secuencia de ADN , Insuficiencia del Tratamiento , Valina/análogos & derivados , Valina/uso terapéuticoRESUMEN
UNLABELLED: Opportunistic infection of oligodendrocytes by human JC polyomavirus may result in the development of progressive multifocal encephalopathy in immunocompromised individuals. Neurotropic JC virus generally harbors reorganized noncoding control region (NCCR) DNA interspersed on the viral genome between early and late coding genes. By applying 454 sequencing on NCCR DNA amplified from body fluid samples (urine, plasma, and cerebrospinal fluid [CSF]) from 19 progressive multifocal leukoencephalopathy (PML) patients, we attempted to reveal the composition of the JC polyomavirus population (the quasispecies, i.e., the whole of the consensus population and minor viral variants) contained in different body compartments and to better understand intrapatient viral dissemination. Our data demonstrate that in the CSF of PML patients, the JC viral population is often a complex mixture composed of multiple viral variants that contribute to the quasispecies. In contrast, urinary JC virus highly resembled the archetype virus, and urine most often did not contain minor viral variants. It also appeared that archetype JC virus could sporadically be identified in PML patient brain, although selection of rearranged JC virus DNA was favored. Comparison of the quasispecies from different body compartments within a given patient suggested a strong correlation between the viral population in plasma and CSF, whereas the viral population shed in urine appeared to be unrelated. In conclusion, it is shown that the representation of viral DNA in the CSF following the high-level DNA replication in the brain underlying PML has hitherto been much underestimated. Our data also underscore that the hematogenous route might play a pivotal role in viral dissemination from or toward the brain. IMPORTANCE: For the first time, the JC polyomavirus population contained in different body compartments of patients diagnosed with progressive multifocal encephalopathy has been studied by deep sequencing. Two main findings came out of this work. First, it became apparent that the complexity of the viral population associated with PML has been highly underestimated so far, suggestive of a highly dynamic process of reorganization of the noncoding control region of JC polyomavirus in vivo, mainly in CSF and blood. Second, evidence showing viral dissemination from and/or toward the brain via the hematogenous route was provided, confirming a hypothesis that was recently put forward in the field.
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Variación Genética , Virus JC/clasificación , Virus JC/genética , Leucoencefalopatía Multifocal Progresiva/virología , Líquido Cefalorraquídeo/virología , Análisis por Conglomerados , ADN Viral/química , ADN Viral/genética , Femenino , Humanos , Virus JC/aislamiento & purificación , Masculino , Datos de Secuencia Molecular , Filogenia , Plasma/virología , Análisis de Secuencia de ADN , Orina/virologíaRESUMEN
Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in the immunocompromised and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about strain diversity of the 235kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the available information for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hotspots of diversity in the genome. Importantly, 75% of strains are not genetically intact, but contain disruptive mutations in a diverse set of 26 genes, including immunomodulative genes UL40 and UL111A. These mutants are independent from culture passaging artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. Recombination density was significantly higher than in other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142 and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions. IMPORTANCE: Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased towards a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, they have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity.
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MOTIVATION: In virology, massively parallel sequencing (MPS) opens many opportunities for studying viral quasi-species, e.g. in HIV-1- and HCV-infected patients. This is essential for understanding pathways to resistance, which can substantially improve treatment. Although MPS platforms allow in-depth characterization of sequence variation, their measurements still involve substantial technical noise. For Illumina sequencing, single base substitutions are the main error source and impede powerful assessment of low-frequency mutations. Fortunately, base calls are complemented with quality scores (Qs) that are useful for differentiating errors from the real low-frequency mutations. RESULTS: A variant calling tool, Q-cpileup, is proposed, which exploits the Qs of nucleotides in a filtering strategy to increase specificity. The tool is imbedded in an open-source pipeline, VirVarSeq, which allows variant calling starting from fastq files. Using both plasmid mixtures and clinical samples, we show that Q-cpileup is able to reduce the number of false-positive findings. The filtering strategy is adaptive and provides an optimized threshold for individual samples in each sequencing run. Additionally, linkage information is kept between single-nucleotide polymorphisms as variants are called at the codon level. This enables virologists to have an immediate biological interpretation of the reported variants with respect to their antiviral drug responses. A comparison with existing SNP caller tools reveals that calling variants at the codon level with Q-cpileup results in an outstanding sensitivity while maintaining a good specificity for variants with frequencies down to 0.5%. AVAILABILITY: The VirVarSeq is available, together with a user's guide and test data, at sourceforge: http://sourceforge.net/projects/virtools/?source=directory.
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Algoritmos , Variación Genética/genética , Genómica/métodos , Hepacivirus/genética , Hepatitis C/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Genoma Viral , Hepatitis C/virología , HumanosRESUMEN
Minority variants (1.0-25.0%) were evaluated by deep sequencing (DS) at baseline and virological failure (VF) in a selection of antiretroviral treatment-naïve, HIV-1-infected patients from the rilpivirine ECHO/THRIVE phase III studies. Linkage between frequently emerging resistance-associated mutations (RAMs) was determined. DS (llIumina®) and population sequencing (PS) results were available at baseline for 47 VFs and time of failure for 48 VFs; and at baseline for 49 responders matched for baseline characteristics. Minority mutations were accurately detected at frequencies down to 1.2% of the HIV-1 quasispecies. No baseline minority rilpivirine RAMs were detected in VFs; one responder carried 1.9% F227C. Baseline minority mutations associated with resistance to other non-nucleoside reverse transcriptase inhibitors (NNRTIs) were detected in 8/47 VFs (17.0%) and 7/49 responders (14.3%). Baseline minority nucleoside/nucleotide reverse transcriptase inhibitor (NRTI) RAMs M184V and L210W were each detected in one VF (none in responders). At failure, two patients without NNRTI RAMs by PS carried minority rilpivirine RAMs K101E and/or E138K; and five additional patients carried other minority NNRTI RAMs V90I, V106I, V179I, V189I, and Y188H. Overall at failure, minority NNRTI RAMs and NRTI RAMs were found in 29/48 (60.4%) and 16/48 VFs (33.3%), respectively. Linkage analysis showed that E138K and K101E were usually not observed on the same viral genome. In conclusion, baseline minority rilpivirine RAMs and other NNRTI/NRTI RAMs were uncommon in the rilpivirine arm of the ECHO and THRIVE studies. DS at failure showed emerging NNRTI resistant minority variants in seven rilpivirine VFs who had no detectable NNRTI RAMs by PS.
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Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Rilpivirina/uso terapéutico , Adulto , Sustitución de Aminoácidos , Ensayos Clínicos Fase III como Asunto , Femenino , Humanos , Masculino , Mutación Missense , Selección Genética , Factores de Tiempo , Insuficiencia del TratamientoRESUMEN
BACKGROUND: Next generation sequencing enables studying heterogeneous populations of viral infections. When the sequencing is done at high coverage depth ("deep sequencing"), low frequency variants can be detected. Here we present QQ-SNV (http://sourceforge.net/projects/qqsnv), a logistic regression classifier model developed for the Illumina sequencing platforms that uses the quantiles of the quality scores, to distinguish true single nucleotide variants from sequencing errors based on the estimated SNV probability. To train the model, we created a dataset of an in silico mixture of five HIV-1 plasmids. Testing of our method in comparison to the existing methods LoFreq, ShoRAH, and V-Phaser 2 was performed on two HIV and four HCV plasmid mixture datasets and one influenza H1N1 clinical dataset. RESULTS: For default application of QQ-SNV, variants were called using a SNV probability cutoff of 0.5 (QQ-SNV(D)). To improve the sensitivity we used a SNV probability cutoff of 0.0001 (QQ-SNV(HS)). To also increase specificity, SNVs called were overruled when their frequency was below the 80(th) percentile calculated on the distribution of error frequencies (QQ-SNV(HS-P80)). When comparing QQ-SNV versus the other methods on the plasmid mixture test sets, QQ-SNV(D) performed similarly to the existing approaches. QQ-SNV(HS) was more sensitive on all test sets but with more false positives. QQ-SNV(HS-P80) was found to be the most accurate method over all test sets by balancing sensitivity and specificity. When applied to a paired-end HCV sequencing study, with lowest spiked-in true frequency of 0.5%, QQ-SNV(HS-P80) revealed a sensitivity of 100% (vs. 40-60% for the existing methods) and a specificity of 100% (vs. 98.0-99.7% for the existing methods). In addition, QQ-SNV required the least overall computation time to process the test sets. Finally, when testing on a clinical sample, four putative true variants with frequency below 0.5% were consistently detected by QQ-SNV(HS-P80) from different generations of Illumina sequencers. CONCLUSIONS: We developed and successfully evaluated a novel method, called QQ-SNV, for highly efficient single nucleotide variant calling on Illumina deep sequencing virology data.
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Infecciones por VIH/genética , VIH-1/genética , Hepacivirus/genética , Hepatitis C/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Polimorfismo de Nucleótido Simple/genética , Programas Informáticos , Algoritmos , Análisis por Conglomerados , Simulación por Computador , Genoma Viral , Infecciones por VIH/virología , Hepatitis C/virología , Humanos , Plásmidos/genética , Análisis de RegresiónRESUMEN
BACKGROUND: Deep-sequencing allows for an in-depth characterization of sequence variation in complex populations. However, technology associated errors may impede a powerful assessment of low-frequency mutations. Fortunately, base calls are complemented with quality scores which are derived from a quadruplet of intensities, one channel for each nucleotide type for Illumina sequencing. The highest intensity of the four channels determines the base that is called. Mismatch bases can often be corrected by the second best base, i.e. the base with the second highest intensity in the quadruplet. A virus variant model-based clustering method, ViVaMBC, is presented that explores quality scores and second best base calls for identifying and quantifying viral variants. ViVaMBC is optimized to call variants at the codon level (nucleotide triplets) which enables immediate biological interpretation of the variants with respect to their antiviral drug responses. RESULTS: Using mixtures of HCV plasmids we show that our method accurately estimates frequencies down to 0.5%. The estimates are unbiased when average coverages of 25,000 are reached. A comparison with the SNP-callers V-Phaser2, ShoRAH, and LoFreq shows that ViVaMBC has a superb sensitivity and specificity for variants with frequencies above 0.4%. Unlike the competitors, ViVaMBC reports a higher number of false-positive findings with frequencies below 0.4% which might partially originate from picking up artificial variants introduced by errors in the sample and library preparation step. CONCLUSIONS: ViVaMBC is the first method to call viral variants directly at the codon level. The strength of the approach lies in modeling the error probabilities based on the quality scores. Although the use of second best base calls appeared very promising in our data exploration phase, their utility was limited. They provided a slight increase in sensitivity, which however does not warrant the additional computational cost of running the offline base caller. Apparently a lot of information is already contained in the quality scores enabling the model based clustering procedure to adjust the majority of the sequencing errors. Overall the sensitivity of ViVaMBC is such that technical constraints like PCR errors start to form the bottleneck for low frequency variant detection.
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Algoritmos , Variación Genética/genética , Hepacivirus/genética , Hepatitis C/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación/genética , Programas Informáticos , Análisis por Conglomerados , Genoma Viral , Genómica/métodos , Hepatitis C/virología , Humanos , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodosRESUMEN
BACKGROUND: Population sequencing (PS) has shown that telaprevir-resistant variants are not typically detectable at baseline (prevalence, ≤5% of patients), and most variants present at the time of treatment failure are no longer detectable at the end of the study. METHODS: To gain insight into the evolution of telaprevir-resistant variants, their baseline prevalence and persistence after treatment was investigated using a more sensitive, deep-sequencing (DS) technique in a large number of treatment-experienced patients from the REALIZE study who were infected with hepatitis C virus genotype 1. RESULTS: Before treatment initiation, telaprevir-resistant variants (T54A, T54S, or R155K in 1%-2% of the viral population) were detected by DS in a fraction (2%) of patients for whom PS failed to detect resistance; these variants were not necessarily detected at the time of treatment failure. Of 49 patients in whom telaprevir-resistant variants were detected by PS at the time of treatment failure but not at the end of the study, DS revealed the presence of variants (V36A/L/M, T54S, or R155K in 1%-36% of the viral population) in 16 patients (33%) at the end of the study. CONCLUSIONS: Similar to PS findings, DS analysis revealed that the frequency of telaprevir-resistant variants before treatment was also low, and variants detected at the time of treatment failure were no longer detectable in the majority of patients during follow-up.
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Antivirales/uso terapéutico , Proteínas Portadoras/genética , Hepacivirus/enzimología , Hepacivirus/genética , Hepatitis C Crónica/tratamiento farmacológico , Oligopéptidos/uso terapéutico , Proteínas no Estructurales Virales/genética , Farmacorresistencia Viral , Hepacivirus/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Péptidos y Proteínas de Señalización Intracelular , Tasa de Mutación , PrevalenciaRESUMEN
OBJECTIVES: This study investigated strategies that may increase the yield of drug resistance testing prior to starting antiretroviral therapy (ART), and whether transmitted and polymorphic resistance-associated mutations (RAMs) correlated with virological outcomes. METHODS: We carried out retrospective testing of baseline samples from patients entering the SENSE trial of first-line ART in Europe, Russia and Israel. Prior to randomization to etravirine or efavirenz plus two nucleos(t)ide reverse transcriptase inhibitors (NRTIs), plasma samples underwent routine Sanger sequencing of HIV-1 RT and protease ((plasma)SS) in order to exclude patients with transmitted RAMs. Retrospectively, Sanger sequencing was repeated with HIV-1 DNA from baseline peripheral blood mononuclear cells (PBMCSS); baseline plasma samples were retested by allele-specific PCR targeting seven RT RAMs (AS-PCR) and ultra-deep RT sequencing (UDS). RESULTS: By (plasma)SS, 16/193 (8.3%) patients showed ≥ 1 transmitted RAM affecting the NRTIs (10/193, 5.2%), non-nucleoside reverse transcriptase inhibitors (4/193, 2.1%) or protease inhibitors (2/193, 1.0%). No additional RAMs were detected by AS-PCR (n = 152) and UDS (n = 24); PBMCSS (n =â 91) yielded two additional samples with one RAM each. Over 48 weeks, 4/79 (5.1%) patients on etravirine and 7/78 (9.0%) on efavirenz experienced virological failure; none had baseline RAMs. Conversely, 11/79 (13.9%) patients randomized to etravirine had one polymorphic RAM from the etravirine score in baseline plasma (V90I, V106I or E138A), without any impact on virological outcomes. CONCLUSIONS: The detection of resistance increased marginally with PBMC testing but did not increase with sensitive plasma testing. A careful consideration is required of the cost-effectiveness of different strategies for baseline HIV drug resistance testing.
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Fármacos Anti-VIH/uso terapéutico , ADN Viral/genética , Resistencia a Medicamentos , Técnicas de Genotipaje/métodos , Infecciones por VIH/virología , VIH-1/genética , ARN Viral/genética , Adolescente , Adulto , Anciano , Alquinos , Benzoxazinas/uso terapéutico , Ciclopropanos , ADN Viral/aislamiento & purificación , Europa (Continente) , Femenino , Infecciones por VIH/tratamiento farmacológico , VIH-1/aislamiento & purificación , Humanos , Israel , Masculino , Pruebas de Sensibilidad Microbiana/métodos , Persona de Mediana Edad , Nitrilos , Piridazinas/uso terapéutico , Pirimidinas , ARN Viral/aislamiento & purificación , Federación de Rusia , Resultado del Tratamiento , Adulto JovenRESUMEN
Respiratory syncytial virus (RSV) can cause pulmonary complications in infants, elderly and immunocompromised patients. While two vaccines and two prophylactic monoclonal antibodies are now available, treatment options are still needed. JNJ-7184 is a non-nucleoside inhibitor of the RSV-Large (L) polymerase, displaying potent inhibition of both RSV-A and -B strains. Resistance selection and hydrogen-deuterium exchange experiments suggest JNJ-7184 binds RSV-L in the connector domain. JNJ-7184 prevents RSV replication and transcription by inhibiting initiation or early elongation. JNJ-7184 is effective in air-liquid interface cultures and therapeutically in neonatal lambs, acting to drastically reverse the appearance of lung pathology.
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Antivirales , Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Replicación Viral , Antivirales/farmacología , Antivirales/química , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Infecciones por Virus Sincitial Respiratorio/virología , Animales , Humanos , Replicación Viral/efectos de los fármacos , Virus Sincitial Respiratorio Humano/efectos de los fármacos , Ovinos , Farmacorresistencia Viral , Proteínas Virales/antagonistas & inhibidores , Proteínas Virales/metabolismo , Proteínas Virales/genética , Pulmón/virologíaRESUMEN
Whereas most infants infected with respiratory syncytial virus (RSV) show no or only mild symptoms, an estimated 3 million children under five are hospitalized annually due to RSV disease. This study aimed to investigate biological mechanisms and associated biomarkers underlying RSV disease heterogeneity in young infants, enabling the potential to objectively categorize RSV-infected infants according to their medical needs. Immunophenotypic and functional profiling demonstrated the emergence of immature and progenitor-like neutrophils, proliferative monocytes (HLA-DRLow , Ki67+), impaired antigen-presenting function, downregulation of T cell response and low abundance of HLA-DRLow B cells in severe RSV disease. HLA-DRLow monocytes were found as a hallmark of RSV-infected infants requiring hospitalization. Complementary transcriptomics identified genes associated with disease severity and pointed to the emergency myelopoiesis response. These results shed new light on mechanisms underlying the pathogenesis and development of severe RSV disease and identified potential new candidate biomarkers for patient stratification.
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Mielopoyesis , Infecciones por Virus Sincitial Respiratorio , Lactante , Niño , Humanos , Mielopoyesis/genética , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Infecciones por Virus Sincitial Respiratorio/genética , Virus Sincitiales Respiratorios , Antígenos HLA-DR , BiomarcadoresRESUMEN
BACKGROUND: HIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment. To provide patients with maximal treatment options, massively parallel sequencing of the envelope V3 domain, in combination with tropism prediction tools, was evaluated as an alternative tropism determination strategy. Plasma samples from twelve HIV-1 infected individuals with failing phenotyping results were available. The samples were submitted to massive parallel sequencing and to confirmatory recombinant phenotyping using a fraction of the gp120 domain. RESULTS: A cut-off for sequence reads interpretation of 5 to10 times the sequencing error rate (0.2%) was implemented. On average, each sample contained 7 different V3 haplotypes. V3 haplotypes were submitted to tropism prediction algorithms, and 4/14 samples returned with presence of a dual/mixed (D/M) tropic virus, respectively at 3%, 10%, 11%, and 95% of the viral quasispecies. V3 tropism prediction was confirmed by gp120 phenotyping, except for two out of 4 D/M predicted viruses (with 3 and 95%) which were phenotypically R5-tropic. In the first case, the result was discordant due to the limit of detection for the phenotyping technology, while in the latter case the prediction algorithms were not computing the viral tropism correctly. CONCLUSIONS: Although only demonstrated on a limited set of samples, the potential of the combined use of "deep sequencing + prediction algorithms" in cases where routine gp160 phenotype testing cannot be employed was illustrated. While good concordance was observed between gp120 phenotyping and prediction of R5-tropic virus, the results suggest that accurate prediction of X4-tropic virus would require further algorithm development.
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Human cytomegalovirus (HCMV) is a betaherpesvirus which rarely presents problems in healthy individuals, yet may result in severe morbidity in immunocompromised patients and in immune-naïve neonates. HCMV has a large 235 kb genome with a coding capacity of at least 165 open reading frames (ORFs). This large genome allows complex gene regulation resulting in different sets of transcripts during lytic and latent infection. While latent virus mainly resides within monocytes and CD34+ progenitor cells, reactivation to lytic infection is driven by differentiation towards terminally differentiated myeloid dendritic cells and macrophages. Consequently, it has been suggested that macrophages and dendritic cells contribute to viral spread in vivo. Thus far only limited knowledge is available on the expression of HCMV genes in terminally differentiated myeloid primary cells and whether or not the virus exhibits a different set of lytic genes in primary cells compared with lytic infection in NHDF fibroblasts. To address these questions, we used Illumina next generation sequencing to determine the HCMV transcriptome in macrophages and dendritic cells during lytic infection and compared it to the transcriptome in NHDF fibroblasts. Here, we demonstrate unique expression profiles in macrophages and dendritic cells which significantly differ from the transcriptome in fibroblasts mainly by modulating the expression of viral transcripts involved in immune modulation, cell tropism and viral spread. In a head to head comparison between macrophages and dendritic cells, we observed that factors involved in viral spread and virion composition are differentially regulated suggesting that the plasticity of the virion facilitates the infection of surrounding cells. Taken together, this study provides the full transcript expression analysis of lytic HCMV genes in monocyte-derived type 1 and type 2 macrophages as well as in monocyte-derived dendritic cells. Thereby underlining the potential of HCMV to adapt to or influence different cellular environments to promote its own survival.
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Citomegalovirus/fisiología , Inmunomodulación/genética , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/citología , Transcriptoma/inmunología , Línea Celular , Humanos , Macrófagos/virología , Familia de MultigenesRESUMEN
BACKGROUND: We assessed etravirine resistance in treatment-experienced, HIV-1-infected children (n=41)/adolescents (n=60) who received twice-daily etravirine 5.2 mg/kg and a background regimen (boosted protease inhibitor plus nucleoside/nucleotide reverse transcriptase inhibitors, optional enfuvirtide/raltegravir) in a Phase II, open-label, multicentre trial (PIANO). METHODS: In addition to phenotypes, viral genotypes were assessed by population and deep sequencing (PS and DS) in virological failures (VFs; baseline and end point) and responders (baseline). Minority resistance-associated mutations (RAMs) were defined as those with frequencies above 1% and not detected with PS. RESULTS: By week 48, 41/101 (40.6%) patients experienced VF; 17/41 (41.5%) VFs and 22/54 (40.8%) responders had ≥1 baseline etravirine RAM by PS, mainly A98G, K101E, V106I and G190A. Baseline minority etravirine RAMs (n) were detected in 8/40 VFs (V90I [2], A98G [1], L100I [1], V106I [1], E138G [1] and Y181C [2]) and 5/38 responders (V90I [3], A98G [1], V106I [1] and E138G [1]). The most frequent emerging non-nucleoside reverse transcriptase inhibitor RAMs detected by PS (≥3 VFs; n) were the etravirine RAMs Y181C (8), V90I (3), L100I (3) and E138A (3). In 15 of 29 (51.7%) VFs with baseline DS/PS and end point PS data, ≥1 emerging etravirine RAM was detected by PS, which was not detected at baseline by DS in most cases (12/15 [80.0%]). In 10/26 (38.5%) VFs with baseline/end point DS data, ≥1 additional emerging minority etravirine RAM was detected. CONCLUSIONS: Patterns of etravirine resistance in adults, adolescents and children experiencing VF are similar. The presence of minority etravirine RAMs at baseline was not consistently associated with treatment failure. ClinicalTrials.gov: NCT00665847.
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Fármacos Anti-VIH/uso terapéutico , Farmacorresistencia Viral/genética , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Piridazinas/uso terapéutico , Adolescente , Fármacos Anti-VIH/farmacología , Niño , Evolución Molecular , Infecciones por VIH/tratamiento farmacológico , Humanos , Nitrilos , Piridazinas/farmacología , Pirimidinas , Insuficiencia del TratamientoRESUMEN
Background. The pre-existence of minority hepatitis C virus (HCV) variants and their impact on treatment outcome, as well as the persistence of emerging resistant variants posttreatment in patients failing treatment with simeprevir/peginterferon/ribavirin (SMV/PR), were assessed by deep sequencing (DS). Methods. Population sequencing (PS) and Illumina DS were performed on HCV genotype 1 isolates from patients treated with SMV/PR in Phase 2b (PILLAR [NCT00882908] and ASPIRE [NCT00980330]) and Phase 3 (QUEST-1 [NCT01289782], QUEST-2 [NCT01290679], and PROMISE [NCT01281839]) trials. Results. Minority polymorphisms (ie, detected pretreatment by DS only) reducing SMV activity in vitro were uncommon (3.6%, 19 of 534 patients). These SMV-resistant minority polymorphisms were detected in similar proportions of patients achieving (3.7%) and not achieving (3.3%) sustained virologic response with SMV/PR and generally did not emerge as major variants at time of failure. SMV-resistant variants emerging at time of failure were no longer detected at end of study in 69.3% and 52.0% of the patients by PS and DS, respectively. Conclusions. Minority polymorphisms did not impact outcome of SMV/PR treatment. The majority of emerging variants that became undetectable at end of study by PS were also undetectable by DS. These results suggest no added value of DS for clinical usage of SMV.
RESUMEN
Massively parallel sequencing (MPS) technology has opened new avenues to study viral dynamics and treatment-induced resistance mechanisms of infections such as human immunodeficiency virus (HIV) and hepatitis C virus (HCV). Whereas the Roche/454 platform has been used widely for the detection of low-frequent drug resistant variants, more recently developed short-read MPS technologies have the advantage of delivering a higher sequencing depth at a lower cost per sequenced base. This study assesses the performance characteristics of Illumina MPS technology for the characterization of genetic variability in viral populations by deep sequencing. The reported results from MPS experiments comprising HIV and HCV plasmids demonstrate that a 0.5-1% lower limit of detection can be achieved readily with Illumina MPS while retaining good accuracy also at low frequencies. Deep sequencing of a set of clinical samples (12 HIV and 9 HCV patients), designed at a similar budget for both MPS platforms, reveals a comparable lower limit of detection for Illumina and Roche/454. Finally, this study shows the possibility to apply Illumina's paired-end sequencing as a strategy to assess linkage between different mutations identified in individual viral subspecies. These results support the use of Illumina as another MPS platform of choice for deep sequencing of viral minority species.
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Variación Genética , VIH/clasificación , VIH/genética , Hepacivirus/clasificación , Hepacivirus/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virología/métodos , VIH/aislamiento & purificación , Infecciones por VIH/virología , Hepacivirus/aislamiento & purificación , Hepatitis C/virología , HumanosRESUMEN
Most antigen-specific mouse antibodies have been derived by hybridoma technology, predominantly through use of the Balb/c strain. Much of the Balb/c germline repertoire of variable genes (V regions) is known. However, there is little information about the background expressed repertoire of IgG antibodies in mice, which reflects the baseline against which antigen-specific antibodies are generated through immunization. To assess this baseline repertoire, RNA was isolated from splenic B-cells enriched for expression of IgG from three mice. The RNA was individually amplified with three distinct PCR primer sets for comprehensive recovery of the heavy and light chain variable regions. Each PCR product was independently subjected to deep sequencing using 454 pyro-sequencing technology and analysed for redundancy, open reading frame, germline representation, and CDR3 sequence of the heavy chain variable region (VH CDR3) within and across the primer sets and mice. A highly skewed abundance of heavy and light chain variable gene usage was observed for all three primers in all three mice. While showing considerable overlap, there were differences among these profiles indicative of primer bias and animal-to-animal variation. VH CDR3 sequences were likewise highly skewed indicating that the heavy chain genes profiles substantially reflected individual antibodies. This observation was confirmed through analysis of randomly selected complete heavy chain variable sequences. However, there was very little redundancy in VH CDR3 sequences across the different mice. We conclude that the background IgG repertoire in young, unimmunized mice is highly skewed within individual mice and is diverse among them, a pattern similar to that observed in highly immunized mice.