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We present 109 near full-length HIV genomes amplified from blood serum samples obtained during early 1986 from across Uganda, which to our knowledge is the earliest and largest population sample from the initial phase of the HIV epidemic in Africa. Consensus sequences were made from paired-end Illumina reads with a target-capture approach to amplify HIV material following poor success with standard approaches. In comparisons with a smaller 'intermediate' genome dataset from 1998 to 1999 and a 'modern' genome dataset from 2007 to 2016, the proportion of subtype D was significantly higher initially, dropping from 67% (73/109), to 57% (26/46) to 17% (82/465) respectively (p < 0.0001). Subtype D has previously been shown to have a faster rate of disease progression than other subtypes in East African population studies, and to have a higher propensity to use the CXCR4 co-receptor ("X4 tropism"); associated with a decrease in time to AIDS. Here we find significant differences in predicted tropism between A1 and D subtypes in all three sample periods considered, which is particularly striking the 1986 sample: 66% (53/80) of subtype D env sequences were predicted to be X4 tropic compared with none of the 24 subtype A1. We also analysed the frequency of subtype in the envelope region of inter-subtype recombinants, and found that subtype A1 is over-represented in env, suggesting recombination and selection have acted to remove subtype D env from circulation. The reduction of subtype D frequency over three decades therefore appears to be a result of selective pressure against X4 tropism and its higher virulence. Lastly, we find a subtype D specific codon deletion at position 24 of the V3 loop, which may explain the higher propensity for subtype D to utilise X4 tropism.
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Infecciones por VIH , VIH-1 , Receptores CXCR4 , Tropismo Viral , Humanos , Pueblo Africano , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , Receptores CXCR4/genética , UgandaRESUMEN
A pathogen inactivation step during collection or processing of clinical samples has the potential to reduce infectious risks associated with diagnostic procedures. It is essential that these inactivation methods are demonstrated to be effective, particularly for non-traditional inactivation reagents or for commercial products where the chemical composition is undisclosed. This study assessed inactivation effectiveness of twenty-four next-generation (guanidine-free) nucleic acid extraction lysis buffers and twelve rapid antigen test buffers against SARS-CoV-2, the causative agent of COVID-19. These data have significant safety implications for SARS-CoV-2 diagnostic testing and support the design and evidence-based risk assessment of these procedures.
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Antivirales/farmacología , Prueba Serológica para COVID-19/métodos , SARS-CoV-2/efectos de los fármacos , Acetamidas , Tampones (Química) , COVID-19/diagnóstico , COVID-19/virología , Fluoroacetatos , Guanidina/efectos adversos , Humanos , Inactivación de Virus/efectos de los fármacosRESUMEN
Integrase strand transfer inhibitors (InSTIs) are recommended agents in first-line combination antiretroviral therapy (cART). We examined the evolution of drug resistance mutations throughout HIV-1 pol and the effects on InSTI susceptibility and viral fitness. We performed single-genome sequencing of full-length HIV-1 pol in a highly treatment-experienced patient, and determined drug susceptibility of patient-derived HIV-1 genomes using a phenotypic assay encompassing full-length pol gene. We show the genetic linkage of multiple InSTI-resistant haplotypes containing major resistance mutations at Y143, Q148 and N155 to protease inhibitor (PI) and reverse transcriptase inhibitor (RTI) resistance mutations. Phenotypic analysis of viruses expressing patient-derived IN genes with eight different InSTI-resistant haplotypes alone or in combination with coevolved protease (PR) and RT genes exhibited similar levels of InSTI susceptibility, except for three haplotypes that showed up to 3-fold increases in InSTI susceptibility (p ≤ 0.032). The replicative fitness of most viruses expressing patient-derived IN only significantly decreased, ranging from 8% to 56% (p ≤ 0.01). Interestingly, the addition of coevolved PR + RT significantly increased the replicative fitness of some haplotypes by up to 73% (p ≤ 0.024). Coevolved PR + RT contributes to the susceptibility and viral fitness of patient-derived IN viruses. Maintaining patients on failing cART promotes the selection of fitter resistant strains, and thereby limits future therapy options.
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Human respiratory syncytial virus (HRSV) is the leading viral cause of serious pediatric respiratory disease, and lifelong reinfections are common. Its 2 major subgroups, A and B, exhibit some antigenic variability, enabling HRSV to circulate annually. Globally, research has increased the number of HRSV genomic sequences available. To ensure accurate molecular epidemiology analyses, we propose a uniform nomenclature for HRSV-positive samples and isolates, and HRSV sequences, namely: HRSV/subgroup identifier/geographic identifier/unique sequence identifier/year of sampling. We also propose a template for submitting associated metadata. Universal nomenclature would help researchers retrieve and analyze sequence data to better understand the evolution of this virus.
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Infecciones por Virus Sincitial Respiratorio , Virus Sincitial Respiratorio Humano , Niño , Variación Genética , Genotipo , Humanos , Epidemiología Molecular , Filogenia , Virus Sincitial Respiratorio Humano/genéticaRESUMEN
The COVID-19 pandemic has necessitated a multifaceted rapid response by the scientific community, bringing researchers, health officials, and industry together to address the ongoing public health emergency. To meet this challenge, participants need an informed approach for working safely with the etiological agent, the novel human coronavirus SARS-CoV-2. Work with infectious SARS-CoV-2 is currently restricted to high-containment laboratories, but material can be handled at a lower containment level after inactivation. Given the wide array of inactivation reagents that are being used in laboratories during this pandemic, it is vital that their effectiveness is thoroughly investigated. Here, we evaluated a total of 23 commercial reagents designed for clinical sample transportation, nucleic acid extraction, and virus inactivation for their ability to inactivate SARS-CoV-2, as well as seven other common chemicals, including detergents and fixatives. As part of this study, we have also tested five filtration matrices for their effectiveness at removing the cytotoxic elements of each reagent, permitting accurate determination of levels of infectious virus remaining following treatment. In addition to providing critical data informing inactivation methods and risk assessments for diagnostic and research laboratories working with SARS-CoV-2, these data provide a framework for other laboratories to validate their inactivation processes and to guide similar studies for other pathogens.
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Betacoronavirus/efectos de los fármacos , Indicadores y Reactivos/farmacología , Inactivación de Virus/efectos de los fármacos , Animales , Betacoronavirus/aislamiento & purificación , COVID-19 , Prueba de COVID-19 , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , Filtración/instrumentación , Humanos , Pandemias , Neumonía Viral/diagnóstico , Neumonía Viral/virología , SARS-CoV-2 , Células VeroRESUMEN
Infants (under 1-year-old) are at most risk of life threatening respiratory syncytial virus (RSV) disease. RSV epidemiological data alone has been insufficient in defining who acquires infection from whom (WAIFW) within households. We investigated RSV genomic variation within and between infected individuals and assessed its potential utility in tracking transmission in households. Over an entire single RSV season in coastal Kenya, nasal swabs were collected from members of 20 households every 3-4 days regardless of symptom status and screened for RSV nucleic acid. Next generation sequencing was used to generate >90% RSV full-length genomes for 51.1% of positive samples (191/374). Single nucleotide polymorphisms (SNPs) observed during household infection outbreaks ranged from 0-21 (median: 3) while SNPs observed during single-host infection episodes ranged from 0-17 (median: 1). Using the viral genomic data alone there was insufficient resolution to fully reconstruct within-household transmission chains. For households with clear index cases, the most likely source of infant infection was via a toddler (aged 1 to <3 years-old) or school-aged (aged 6 to <12 years-old) co-occupant. However, for best resolution of WAIFW within households, we suggest an integrated analysis of RSV genomic and epidemiological data.
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ARN Viral/genética , Infecciones por Virus Sincitial Respiratorio/genética , Virus Sincitiales Respiratorios/fisiología , Niño , Preescolar , Trazado de Contacto , Brotes de Enfermedades , Composición Familiar , Femenino , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lactante , Recién Nacido , Kenia/epidemiología , Masculino , Polimorfismo de Nucleótido Simple , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/transmisiónRESUMEN
BACKGROUND: The epidemiology of HIV-1 drug resistance (HIVDR) determined by Sanger capillary sequencing, has been widely studied. However, much less is known about HIVDR detected using next generation sequencing (NGS) methods. We aimed to determine the presence, persistence and effect of pre-treatment HIVDR variants detected using NGS in HIV-1 infected antiretroviral treatment (ART) naïve participants from rural Coastal Kenya. METHODS: In a retrospective longitudinal study, samples from HIV-1 infected participants collected prior [n = 2 time-points] and after [n = 1 time-point] ART initiation were considered. An ultra-deep amplicon-based NGS assay, calling for nucleotide variants at >2.0% frequency of viral population, was used. Suspected virologic failure (sVF) was defined as a one-off HIV-1 viral load of >1000 copies/ml whilst on ART. RESULTS: Of the 50 eligible participants, 12 (24.0% [95% CI: 13.1-38.2]) had at least one detectable pre-treatment HIVDR variant against Protease Inhibitors (PIs, n = 6 [12%]), Nucleoside Reverse Transcriptase Inhibitors (NRTIs, n = 4 [8.0%]) and Non-NRTIs (n = 3 [6.0%]). Overall, 15 pre-treatment resistance variants were detected (frequency, range: 2.3-92.0%). A positive correlation was observed between mutation frequency and absolute load for NRTI and/or NNRTI variants (r = 0.761 [p = 0.028]), but not for PI variants (r = -0.117 [p = 0.803]). Participants with pre-treatment NRTI and/or NNRTI resistance had increased odds of sVF (OR = 6.0; 95% CI = 1.0-36.9; p = 0.054). CONCLUSIONS: Using NGS, pre-treatment resistance variants were common, though observed PI variants were unlikely transmitted, but rather probably generated de novo. Even when detected from a low frequency, pre-treatment NRTI and/or NNRTI resistance variants may adversely affect treatment outcomes.
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Antirretrovirales/uso terapéutico , Terapia Antirretroviral Altamente Activa , Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , Adulto , Antirretrovirales/farmacología , Terapia Antirretroviral Altamente Activa/métodos , Femenino , Variación Genética/efectos de los fármacos , Infecciones por VIH/epidemiología , Infecciones por VIH/virología , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Kenia/epidemiología , Estudios Longitudinales , Masculino , Filogenia , Estudios Retrospectivos , Población Rural , Adulto JovenRESUMEN
We report on infection patterns in 5 households (78 participants) delineating the natural history of human rhinovirus (HRV). Nasopharyngeal collections were obtained every 3-4 days irrespective of symptoms, over a 6-month period, with molecular screening for HRV and typing by sequencing VP4/VP2 junction. Overall, 311/3468 (8.9%) collections were HRV positive: 256 were classified into 3 species: 104 (40.6%) HRV-A; 14 (5.5%) HRV-B, and 138 (53.9%) HRV-C. Twenty-six known HRV types (13 HRV-A, 3 HRV-B, and 10 HRV-C) were identified (A75, C1, and C35 being most frequent). We observed continuous invasion and temporal clustering of HRV types in households (range 5-13 over 6 months). Intrahousehold transmission was independent of clinical status but influenced by age. Most (89.0%) of HRV infection episodes were limited to <14 days. Individual repeat infections were frequent (range 1-7 over 6 months), decreasing with age, and almost invariably heterotypic, indicative of lasting type-specific immunity and low cross-type protection.
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Resfriado Común/transmisión , Nasofaringe/virología , Infecciones por Picornaviridae/transmisión , Rhinovirus/clasificación , Rhinovirus/aislamiento & purificación , Adolescente , Adulto , Factores de Edad , Niño , Preescolar , Resfriado Común/epidemiología , Composición Familiar , Humanos , Lactante , Kenia/epidemiología , Infecciones por Picornaviridae/epidemiología , Estudios Prospectivos , Reacción en Cadena en Tiempo Real de la Polimerasa , Recurrencia , Factores de Tiempo , Adulto JovenRESUMEN
Background: Households are high-intensity close-contact environments favorable for transmission of respiratory viruses, yet little is known for low-income settings. Methods: Active surveillance was completed on 47 households in rural coastal Kenya over 6 months during a respiratory syncytial virus (RSV) season. Nasopharyngeal swabs (NPSs) were taken from 483 household members twice weekly irrespective of symptoms. Using molecular diagnostics, NPSs from 6 households were screened for 15 respiratory viruses and the remainder of households only for the most frequent viruses observed: rhinovirus (RV), human coronavirus (HCoV; comprising strains 229E, OC43, and NL63), adenovirus (AdV), and RSV (A and B). Results: Of 16928 NPSs tested for the common viruses, 4259 (25.2%) were positive for ≥1 target; 596 (13.8%) had coinfections. Detection frequencies were 10.5% RV (1780), 7.5% HCoV (1274), 7.3% AdV (1232), and 3.2% RSV (537). On average, each household and individual had 6 and 3 different viruses detected over the study period, respectively. Rhinovirus and HCoV were detected in all the 47 households while AdV and RSV were detected in 45 (95.7%) and 40 (85.1%) households, respectively. The individual risk of infection over the 6-month period was 93.4%, 80.1%, 71.6%, 61.5%, and 37.1% for any virus, RV, HCoV, AdV, and RSV, respectively. NPSs collected during symptomatic days and from younger age groups had higher prevalence of virus detection relative to respective counterparts. RSV was underrepresented in households relative to hospital admission data. Conclusions: In this household setting, respiratory virus infections and associated illness are ubiquitous. Future studies should address the health and economic implications of these observations.
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Coinfección/virología , Brotes de Enfermedades , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/virología , Población Rural , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Coinfección/epidemiología , Composición Familiar , Femenino , Humanos , Lactante , Kenia/epidemiología , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Nasofaringe/virología , Vigilancia en Salud Pública , Infecciones por Virus Sincitial Respiratorio/transmisión , Virus Sincitial Respiratorio Humano/aislamiento & purificación , Infecciones del Sistema Respiratorio/epidemiología , Infecciones del Sistema Respiratorio/transmisión , Estaciones del Año , Virus/genética , Virus/aislamiento & purificación , Adulto JovenRESUMEN
Detailed information on the source, spread and evolution of respiratory syncytial virus (RSV) during seasonal community outbreaks remains sparse. Molecular analyses of attachment (G) gene sequences from hospitalized cases suggest that multiple genotypes and variants co-circulate during epidemics and that RSV persistence over successive seasons is characterized by replacement and multiple new introductions of variants. No studies have defined the patterns of introduction, spread and evolution of RSV at the local community and household level. We present a whole genome sequence analysis of 131 RSV group A viruses collected during 6-month household-based RSV infection surveillance in Coastal Kenya, 2010 within an area of 12 km2. RSV infections were identified by regular symptom-independent screening of all household members twice weekly. Phylogenetic analysis revealed that the RSV A viruses in nine households were closely related to genotype GA2 and fell within a single branch of the global phylogeny. Genomic analysis allowed the detection of household-specific variation in seven households. For comparison, using only G gene analysis, household-specific variation was found only in one of the nine households. Nucleotide changes were observed both intra-host (viruses identified from same individual in follow-up sampling) and inter-host (viruses identified from different household members) and these coupled with sampling dates enabled a partial reconstruction of the within household transmission chains. The genomic evolutionary rate for the household dataset was estimated as 2.307 × 10 - 3 (95% highest posterior density: 0.935-4.165× 10 - 3) substitutions/site/year. We conclude that (i) at the household level, most RSV infections arise from the introduction of a single virus variant followed by accumulation of household specific variation and (ii) analysis of complete virus genomes is crucial to better understand viral transmission in the community. A key question arising is whether prevention of RSV introduction or spread within the household by vaccinating key transmitting household members would lead to a reduced onward community-wide transmission.
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In February 2012, the novel respiratory syncytial virus (RSV) group A, genotype ON1, was detected in Kilifi County, coastal Kenya. ON1 is characterized by a 72-nt duplication within the highly variable G gene (encoding the immunogenic attachment surface protein). Cases were diagnosed through surveillance of pneumonia in children at the county hospital. Analysis of epidemiologic, clinical, and sequence data of RSV-A viruses detected over 5 RSV seasons (2010/2011 to 2014/2015) indicated the following: 1) replacement of previously circulating genotype GA2 ON1, 2) an abrupt expansion in the number of ON1 variants detected in the 2014/2015 epidemic, 3) recently accumulation of amino acid substitutions within the ON1 duplicated sequence, and 4) no clear evidence of altered pathogenicity relative to GA2. The study demonstrates the public health importance of molecular surveillance in defining the spread, clinical effects, and evolution of novel respiratory virus variants.
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Evolución Molecular , Genotipo , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/genética , Sustitución de Aminoácidos , Preescolar , Femenino , Variación Genética , Historia del Siglo XXI , Humanos , Lactante , Recién Nacido , Kenia/epidemiología , Masculino , Oportunidad Relativa , Filogenia , Vigilancia en Salud Pública , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Infecciones por Virus Sincitial Respiratorio/historia , Virus Sincitial Respiratorio Humano/clasificación , Análisis de Secuencia de ADN , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND: Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. OBJECTIVES: Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. STUDY DESIGN: Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. RESULTS: N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. CONCLUSIONS: An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy.
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Variación Genética , Técnicas de Diagnóstico Molecular/métodos , Sondas de Oligonucleótidos/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Infecciones por Virus Sincitial Respiratorio/diagnóstico , Virus Sincitiales Respiratorios/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Sitios de Unión , Cartilla de ADN/genética , Reacciones Falso Negativas , Humanos , Kenia , Nucleoproteínas/genética , ARN Viral/genética , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios/genética , Sensibilidad y Especificidad , Análisis de Secuencia de ADN , Proteínas Virales de Fusión/genéticaRESUMEN
OBJECTIVES: To determine the prevalence of inferred low-frequency HIV-1 transmitted drug resistance (TDR) in MSM in the UK and its predicted effect on first-line therapy. METHODS: The HIV-1 pol gene was amplified from 442 newly diagnosed MSM identified as likely recently infected by serological avidity testing in 2011-13. The PCR products were sequenced by next-generation sequencing with a mutation frequency threshold of >2% and TDR mutations defined according to the 2009 WHO surveillance drug resistance mutations list. RESULTS: The majority (75.6%) were infected with subtype B and 6.6% with rare complex or unique recombinant forms. At a mutation frequency threshold of >20%, 7.2% (95% CI 5.0%-10.1%) of the sequences had TDR and this doubled to 15.8% (95% CI 12.6%-19.6%) at >2% mutation frequency (Pâ<â0.0001). The majority (26/42, 62%) of low-frequency variants were against PIs. The most common mutations detected at >20% and 2%-20% mutation frequency differed for each drug class, these respectively being: L90M (nâ=â7) and M46IL (nâ=â10) for PIs; T215rev (nâ=â9) and D67GN (nâ=â4) for NRTIs; and K103N (nâ=â5) and G190E (nâ=â2) for NNRTIs. Combined TDR was more frequent in subtype B than non-B (ORâ=â0.38; 95% CIâ=â0.17-0.88; Pâ=â0.024) and had minimal predicted effect on recommended first-line therapies. CONCLUSIONS: The data suggest differences in the types of low-frequency compared with majority TDR variants that require a better understanding of the origins and clinical significance of low-frequency variants. This will better inform diagnostic and treatment strategies.
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Farmacorresistencia Viral , Monitoreo Epidemiológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Adolescente , Adulto , Anciano , Genotipo , VIH-1/genética , VIH-1/aislamiento & purificación , Homosexualidad Masculina , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Reino Unido , Adulto Joven , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
UNLABELLED: The characteristic recurrent epidemics of human respiratory syncytial virus (RSV) within communities may result from the genetic variability of the virus and associated evolutionary adaptation, reducing the efficiency of preexisting immune responses. We analyzed the molecular evolutionary changes in the attachment (G) glycoprotein of RSV-A viruses collected over 13 epidemic seasons (2000 to 2012) in Kilifi (n = 649), Kenya, and contemporaneous sequences (n = 1,131) collected elsewhere within Kenya and 28 other countries. Genetic diversity in the G gene in Kilifi was dynamic both within and between epidemics, characterized by frequent new variant introductions and limited variant persistence between consecutive epidemics. Four RSV-A genotypes were detected in Kilifi: ON1 (11.9%), GA2 (75.5%), GA5 (12.3%), and GA3 (0.3%), with predominant genotype replacement of GA5 by GA2 and then GA2 by ON1. Within these genotypes, there was considerable variation in potential N-glycosylation sites, with GA2 and ON1 viruses showing up to 15 different patterns involving eight possible sites. Further, we identified 15 positively selected and 34 genotype-distinguishing codon sites, with six of these sites exhibiting both characteristics. The mean substitution rate of the G ectodomain for the Kilifi data set was estimated at 3.58 × 10(-3) (95% highest posterior density interval = 3.04 to 4.16) nucleotide substitutions/site/year. Kilifi viruses were interspersed in the global phylogenetic tree, clustering mostly with Kenyan and European sequences. Our findings highlight ongoing genetic evolution and high diversity of circulating RSV-A strains, locally and globally, with potential antigenic differences. Taken together, these provide a possible explanation on the nature of recurrent local RSV epidemics. IMPORTANCE: The mechanisms underlying recurrent epidemics of RSV are poorly understood. We observe high genetic diversity in circulating strains within and between epidemics in both local and global settings. On longer time scales (â¼7 years) there is sequential replacement of genotypes, whereas on shorter time scales (one epidemic to the next or within epidemics) there is a high turnover of variants within genotypes. Further, this genetic diversity is predicted to be associated with variation in antigenic profiles. These observations provide an explanation for recurrent RSV epidemics and have potential implications on the long-term effectiveness of vaccines.
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Epidemias , Evolución Molecular , Variación Genética , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/genética , Análisis por Conglomerados , Genes Virales , Genotipo , Humanos , Kenia/epidemiología , Simulación de Dinámica Molecular , FilogeniaRESUMEN
Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicativein vitroassay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of thegaggene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of this drug in pediatric patients.
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Farmacorresistencia Viral/genética , Productos del Gen gag/genética , Infecciones por VIH/tratamiento farmacológico , Inhibidores de la Proteasa del VIH/uso terapéutico , Proteasa del VIH/genética , VIH-1/efectos de los fármacos , Sustitución de Aminoácidos , Preescolar , Femenino , Expresión Génica , Productos del Gen gag/metabolismo , Infecciones por VIH/virología , Proteasa del VIH/metabolismo , VIH-1/genética , VIH-1/crecimiento & desarrollo , Humanos , Lactante , Lopinavir/uso terapéutico , Masculino , Mutagénesis Sitio-Dirigida , Mutación , Fenotipo , Ritonavir/uso terapéutico , Insuficiencia del TratamientoRESUMEN
UNLABELLED: Respiratory syncytial virus (RSV) is a global respiratory pathogen of humans, with infection occurring characteristically as recurrent seasonal epidemics. Unlike influenza viruses, little attention has been paid to the mechanism underlying worldwide spread and persistence of RSV and how this may be discerned through an improved understanding of the introduction and persistence of RSV in local communities. We analyzed 651 attachment (G) glycoprotein nucleotide sequences of RSV B collected over 11 epidemics (2002 to 2012) in Kilifi, Kenya, and contemporaneous data collected elsewhere in Kenya and 18 other countries worldwide (2002 to 2012). Based on phylogeny, genetic distance and clustering patterns, we set out pragmatic criteria to classify local viruses into distinct genotypes and variants, identifying those newly introduced and those locally persisting. Three genotypes were identified in the Kilifi data set: BA (n = 500), SAB1 (n = 148), and SAB4 (n = 3). Recurrent RSV epidemics in the local population were composed of numerous genetic variants, most of which have been newly introduced rather than persisting in the location from season to season. Global comparison revealed that (i) most Kilifi variants do not cluster closely with strains from outside Kenya, (ii) some Kilifi variants were closely related to those observed outside Kenya (mostly Western Europe), and (iii) many variants were circulating elsewhere but were never detected in Kilifi. These results are consistent with the hypothesis that year-to-year presence of RSV at the local level (i.e., Kilifi) is achieved primarily, but not exclusively, through introductions from a pool of variants that are geographically restricted (i.e., to Kenya or to the region) rather than global. IMPORTANCE: The mechanism by which RSV persists and reinvades local populations is poorly understood. We investigated this by studying the temporal patterns of RSV variants in a rural setting in tropical Africa and comparing these variants with contemporaneous variants circulating in other countries. We found that periodic seasonal RSV transmission at the local level appears to require regular new introductions of variants. However, importantly, the evidence suggests that the source of new variants is mostly geographically restricted, and we hypothesize that year-to-year RSV persistence is at the country level rather than the global level. This has implications for control.
Asunto(s)
Epidemias , Infecciones por Virus Sincitial Respiratorio/epidemiología , Virus Sincitial Respiratorio Humano/patogenicidad , Antígenos Virales/genética , Niño , Estudios de Cohortes , Variación Genética , Genotipo , Geografía , Humanos , Kenia/epidemiología , Datos de Secuencia Molecular , Filogenia , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitial Respiratorio Humano/clasificación , Virus Sincitial Respiratorio Humano/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
BACKGROUND: Respiratory syncytial virus (RSV) is globally ubiquitous, and infection during the first six months of life is a major risk for severe disease and hospital admission; consequently RSV is the most important viral cause of respiratory morbidity and mortality in young children. Development of vaccines for young infants is complicated by the presence of maternal antibodies and immunological immaturity, but vaccines targeted at older children avoid these problems. Vaccine development for young infants has been unsuccessful, but this is not the case for older children (> 6 m). Would vaccinating older children have a significant public health impact? We developed a mathematical model to explore the benefits of a vaccine against RSV. METHODS AND FINDINGS: We have used a deterministic age structured model capturing the key epidemiological characteristics of RSV and performed a statistical maximum-likelihood fit to age-specific hospitalization data from a developing country setting. To explore the effects of vaccination under different mixing assumptions, we included two versions of contact matrices: one from a social contact diary study, and the second a synthesised construction based on demographic data. Vaccination is assumed to elicit an immune response equivalent to primary infection. Our results show that immunisation of young children (5-10 m) is likely to be a highly effective method of protection of infants (<6 m) against hospitalisation. The majority benefit is derived from indirect protection (herd immunity). A full sensitivity and uncertainty analysis using Latin Hypercube Sampling of the parameter space shows that our results are robust to model structure and model parameters. CONCLUSIONS: This result suggests that vaccinating older infants and children against RSV can have a major public health benefit.
Asunto(s)
Inmunidad Colectiva , Infecciones por Virus Sincitial Respiratorio/prevención & control , Vacunas contra Virus Sincitial Respiratorio/uso terapéutico , Virus Sincitiales Respiratorios/inmunología , Adolescente , Adulto , Factores de Edad , Anciano , Niño , Preescolar , Simulación por Computador , Hospitalización , Humanos , Lactante , Kenia/epidemiología , Persona de Mediana Edad , Modelos Biológicos , Modelos Estadísticos , Pobreza , Infecciones por Virus Sincitial Respiratorio/epidemiología , Infecciones por Virus Sincitial Respiratorio/inmunología , Vacunas contra Virus Sincitial Respiratorio/inmunología , Vacunación , Adulto JovenRESUMEN
Structured treatment interruption (STI) has been trialed as an alternative to lifelong antiretroviral therapy (ART). We retrospectively performed single genome sequencing of the HIV-1 pol region from three patients representing different scenarios. They were either failing on continuous therapy (CT-F), failing STI (STI-F), or suppressing on STI (STI-S). Over 460 genomes were generated from three to five different time points over a 2-year period. We found multiple-linked-resistant mutations in both treatment failures. However, the CT-F patient showed a stepwise accumulation of diverse, linked mutations whereas the STI-F patient had lineage turnover between treatment periods with recirculation of wild-type and resistant variants from reservoirs. The STI-F patient showed a 7-fold increase in the third codon position substitution rate relative to the first and second positions compared to a 2-fold increase for CT-F and increased purifying selection in the pol gene (62 vs. 22 sites, respectively). An understanding of intrapatient viral dynamics could guide the future direction of treatment interruption strategies.