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1.
J Gen Virol ; 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-32134374

RESUMO

Hepatitis B virus (HBV) is a diverse, partially double-stranded DNA virus, with 9 genotypes (A-I), and a putative 10th genotype (J), characterized thus far. Given the broadening interest in HBV sequencing, there is an increasing requirement for a consistent, unified approach to HBV genotype and subgenotype classification. We set out to generate an updated resource of reference sequences using the diversity of all genomic-length HBV sequences available in public databases. We collated and aligned genomic-length HBV sequences from public databases and used maximum-likelihood phylogenetic analysis to identify genotype clusters. Within each genotype, we examined the phylogenetic support for currently defined subgenotypes, as well as identifying well-supported clades and deriving reference sequences for them. Based on the phylogenies generated, we present a comprehensive set of HBV reference sequences at the genotype and subgenotype level. All of the generated data, including the alignments, phylogenies and chosen reference sequences, are available online (https://doi.org/10.6084/m9.figshare.8851946) as a simple open-access resource.

2.
Elife ; 82019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31478835

RESUMO

Hepatitis C virus (HCV) is a highly variable pathogen that frequently establishes chronic infection. This genetic variability is affected by the adaptive immune response but the contribution of other host factors is unclear. Here, we examined the role played by interferon lambda-4 (IFN-λ4) on HCV diversity; IFN-λ4 plays a crucial role in spontaneous clearance or establishment of chronicity following acute infection. We performed viral genome-wide association studies using human and viral data from 485 patients of white ancestry infected with HCV genotype 3a. We demonstrate that combinations of host genetic variants, which determine IFN-λ4 protein production and activity, influence amino acid variation across the viral polyprotein - not restricted to specific viral proteins or HLA restricted epitopes - and modulate viral load. We also observed an association with viral di-nucleotide proportions. These results support a direct role for IFN-λ4 in exerting selective pressure across the viral genome, possibly by a novel mechanism.


Assuntos
Antivirais/metabolismo , Variação Genética , Hepacivirus/classificação , Hepacivirus/genética , Hepatite C/virologia , Fatores Imunológicos/metabolismo , Interleucinas/metabolismo , Grupo com Ancestrais do Continente Europeu , Estudo de Associação Genômica Ampla , Genótipo , Hepacivirus/isolamento & purificação , Hepatite C/imunologia , Interações Hospedeiro-Patógeno , Humanos , Interleucinas/genética , Seleção Genética , Carga Viral
3.
mBio ; 10(3)2019 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-31239374

RESUMO

HBsAg and HBeAg have gained traction as biomarkers of control and clearance during chronic hepatitis B virus infection (CHB). Improved understanding of the clearance correlates of these proteins could help inform improvements in patient-stratified care and advance insights into the underlying mechanisms of disease control, thus underpinning new cure strategies. We collected electronic clinical data via an electronic pipeline supported by the National Institute for Health Research Health Informatics Collaborative (NIHR HIC), adopting an unbiased approach to the generation of a robust longitudinal data set for adults testing HBsAg positive from a large UK teaching hospital over a 6-year period (2011 to 2016 inclusive). Of 553 individuals with CHB, longitudinal data were available for 319, representing >107,000 weeks of clinical follow-up. Among these 319 individuals, 13 (4%) cleared HBsAg completely. Among these 13, the HBsAg clearance rate in individuals on nucleos(t)ide analogue (NA) therapy (n = 4 [31%]; median clearance time,150 weeks) was similar to that in individuals not on NA therapy (n = 9 [69%]; median clearance time, 157 weeks). Those who cleared HBsAg were significantly older and less likely to be on NA therapy than nonclearers (P = 0.003 and P = 0.001, respectively). Chinese ethnicity was associated with HBeAg positivity (P = 0.025). HBeAg clearance occurred in individuals both on NA therapy (n = 24; median time, 49 weeks) and off NA therapy (n = 19; median time, 52 weeks). Improved insights into the dynamics of these biomarkers can underpin better prognostication and patient-stratified care. Our systematized approach to data collection paves the way for scaling up efforts to harness clinical data to address research questions and support improvements in clinical care.IMPORTANCE Advances in the diagnosis, monitoring, and treatment of hepatitis B virus (HBV) infection are urgently required if we are to meet international targets for elimination by the year 2030. Here we demonstrate how routine clinical data can be harnessed through an unbiased electronic pipeline, showcasing the significant potential for amassing large clinical data sets that can help to inform advances in patient care and provide insights that may help to inform new cure strategies. Our cohort from a large UK hospital includes adults from diverse ethnic groups that have previously been underrepresented in the literature. By tracking two protein biomarkers that are used to monitor chronic HBV infection, we provide new insights into the timelines of HBV clearance, both on and off treatment. These results contribute to improvements in individualized clinical care and may provide important clues into the immune events that underpin disease control.


Assuntos
Antígenos de Superfície da Hepatite B/sangue , Antígenos E da Hepatite B/sangue , Hepatite B Crônica/sangue , Adolescente , Adulto , Idoso , Antivirais/uso terapêutico , Biomarcadores/sangue , Criança , Estudos de Coortes , Feminino , Vírus da Hepatite B , Hepatite B Crônica/tratamento farmacológico , Hospitais/estatística & dados numéricos , Humanos , Estudos Longitudinais , Masculino , Informática Médica , Pessoa de Meia-Idade , Reino Unido , Adulto Jovem
4.
Sci Rep ; 9(1): 7081, 2019 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-31068626

RESUMO

Advancing interventions to tackle the huge global burden of hepatitis B virus (HBV) infection depends on improved insights into virus epidemiology, transmission, within-host diversity, drug resistance and pathogenesis, all of which can be advanced through the large-scale generation of full-length virus genome data. Here we describe advances to a protocol that exploits the circular HBV genome structure, using isothermal rolling-circle amplification to enrich HBV DNA, generating concatemeric amplicons containing multiple successive copies of the same genome. We show that this product is suitable for Nanopore sequencing as single reads, as well as for generating short-read Illumina sequences. Nanopore reads can be used to implement a straightforward method for error correction that reduces the per-read error rate, by comparing multiple genome copies combined into a single concatemer and by analysing reads generated from plus and minus strands. With this approach, we can achieve an improved consensus sequencing accuracy of 99.7% and resolve intra-sample sequence variants to form whole-genome haplotypes. Thus while Illumina sequencing may still be the most accurate way to capture within-sample diversity, Nanopore data can contribute to an understanding of linkage between polymorphisms within individual virions. The combination of isothermal amplification and Nanopore sequencing also offers appealing potential to develop point-of-care tests for HBV, and for other viruses.

5.
Viruses ; 11(4)2019 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-30987147

RESUMO

Using deep sequencing technologies such as Illumina's platform, it is possible to obtain reads from the viral RNA population revealing the viral genome diversity within a single host. A range of software tools and pipelines can transform raw deep sequencing reads into Sequence Alignment Mapping (SAM) files. We propose that interpretation tools should process these SAM files, directly translating individual reads to amino acids in order to extract statistics of interest such as the proportion of different amino acid residues at specific sites. This preserves per-read linkage between nucleotide variants at different positions within a codon location. The samReporter is a subsystem of the GLUE software toolkit which follows this direct read translation approach in its processing of SAM files. We test samReporter on a deep sequencing dataset obtained from a cohort of 241 UK HCV patients for whom prior treatment with direct-acting antivirals has failed; deep sequencing and resistance testing have been suggested to be of clinical use in this context. We compared the polymorphism interpretation results of the samReporter against an approach that does not preserve per-read linkage. We found that the samReporter was able to properly interpret the sequence data at resistance-associated locations in nine patients where the alternative approach was equivocal. In three cases, the samReporter confirmed that resistance or an atypical substitution was present at NS5A position 30. In three further cases, it confirmed that the sofosbuvir-resistant NS5B substitution S282T was absent. This suggests the direct read translation approach implemented is of value for interpreting viral deep sequencing data.

6.
BMC Med ; 17(1): 43, 2019 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-30786896

RESUMO

BACKGROUND: Sustainable Development Goals set a challenge for the elimination of hepatitis B virus (HBV) infection as a public health concern by the year 2030. Deployment of a robust prophylactic vaccine and enhanced interventions for prevention of mother to child transmission (PMTCT) are cornerstones of elimination strategy. However, in light of the estimated global burden of 290 million cases, enhanced efforts are required to underpin optimisation of public health strategy. Robust analysis of population epidemiology is particularly crucial for populations in Africa made vulnerable by HIV co-infection, poverty, stigma and poor access to prevention, diagnosis and treatment. METHODS: We here set out to evaluate the current and future role of HBV vaccination and PMTCT as tools for elimination. We first investigated the current impact of paediatric vaccination in a cohort of children with and without HIV infection in Kimberley, South Africa. Second, we used these data to inform a new parsimonious model to simulate the ongoing impact of preventive interventions. By applying these two approaches in parallel, we are able to determine both the current impact of interventions, and the future projected outcome of ongoing preventive strategies over time. RESULTS: Existing efforts have been successful in reducing paediatric prevalence of HBV infection in this setting to < 1%, demonstrating the success of the existing vaccine campaign. Our model predicts that, if consistently deployed, combination efforts of vaccination and PMTCT can significantly reduce population prevalence (HBsAg) by 2030, such that a major public health impact is possible even without achieving elimination. However, the prevalence of HBV e-antigen (HBeAg)-positive carriers will decline more slowly, representing a persistent population reservoir. We show that HIV co-infection significantly reduces titres of vaccine-mediated antibody, but has a relatively minor role in influencing the projected time to elimination. Our model can also be applied to other settings in order to predict impact and time to elimination based on specific interventions. CONCLUSIONS: Through extensive deployment of preventive strategies for HBV, significant positive public health impact is possible, although time to HBV elimination as a public health concern is likely to be substantially longer than that proposed by current goals.


Assuntos
Coinfecção/epidemiologia , Infecções por HIV/epidemiologia , Infecções por HIV/prevenção & controle , Vacinas contra Hepatite B/uso terapêutico , Transmissão Vertical de Doença Infecciosa/prevenção & controle , Adolescente , Adulto , Criança , Estudos de Coortes , Feminino , Vacinas contra Hepatite B/farmacologia , Humanos , Pessoa de Meia-Idade , Adulto Jovem
7.
AIDS ; 33(1): 67-75, Jan. 2019. ilus, tab
Artigo em Inglês | Sec. Est. Saúde SP, SESSP-IIERPROD, Sec. Est. Saúde SP | ID: biblio-1021225

RESUMO

BACKGROUND: Reports of posttreatment control following antiretroviral therapy (ART) have prompted the question of how common immune control of HIV infection is in the absence of ART. In contrast to adult infection, where elite controllers have been very well characterized and constitute approximately 0.5% of infections, very few data exist to address this question in paediatric infection. METHODS: We describe 11 ART-naive elite controllers from 10 cohorts of HIV-infected children being followed in South Africa, Brazil, Thailand, and Europe. RESULT: All but one of the elite controllers (91%) are females. The median age at which control of viraemia was achieved was 6.5 years. Five of these 11 (46%) children lost control of viraemia at a median age of 12.9 years. Children who maintained control of viraemia had significantly higher absolute CD4þ cell counts in the period of elite control than those who lost viraemic control. On the basis of data available from these cohorts, the prevalence of elite controllers in paediatric infection is estimated to be 5­10-fold lower than in adults. CONCLUSION: Although conclusions are limited by the study design, these data suggest that, whilst paediatric elite control can be achieved, compared with adult elite controllers, this occurs rarely, and takes some years after infection to achieve. Also, loss of immune control arises in a high proportion of children and often relatively rapidly. These findings are consistent with the more potent antiviral immune responses observed in adults and in females


Assuntos
Humanos , Masculino , Feminino , Criança , Infecções por HIV , Terapia Antirretroviral de Alta Atividade
8.
Hepatology ; 69(5): 1861-1872, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-29425396

RESUMO

Hepatitis C virus (HCV) genotype (gt) 3 is highly prevalent globally, with non-gt3a subtypes common in Southeast Asia. Resistance-associated substitutions (RASs) have been shown to play a role in treatment failure. However, the role of RASs in gt3 is not well understood. We report the prevalence of RASs in a cohort of direct-acting antiviral treatment-naive, gt3-infected patients, including those with rarer subtypes, and evaluate the effect of these RASs on direct-acting antivirals in vitro. Baseline samples from 496 gt3 patients enrolled in the BOSON clinical trial were analyzed by next-generation sequencing after probe-based enrichment for HCV. Whole viral genomes were analyzed for the presence of RASs to approved direct-acting antivirals. The resistance phenotype of RASs in combination with daclatasvir, velpatasvir, pibrentasvir, elbasvir, and sofosbuvir was measured using the S52 ΔN gt3a replicon model. The nonstructural protein 5A A30K and Y93H substitutions were the most common at 8.9% (n = 44) and 12.3% (n = 61), respectively, and showed a 10-fold and 11-fold increase in 50% effect concentration for daclatasvir compared to the unmodified replicon. Paired RASs (A30K + L31M and A30K + Y93H) were identified in 18 patients (9 of each pair); these combinations were shown to be highly resistant to daclatasvir, velpatasvir, elbasvir, and pibrentasvir. The A30K + L31M combination was found in all gt3b and gt3g samples. Conclusion: Our study reveals high frequencies of RASs to nonstructural protein 5A inhibitors in gt3 HCV; the paired A30K + L31M substitutions occur in all patients with gt3b and gt3g virus, and in vitro analysis suggests that these subtypes may be inherently resistant to all approved nonstructural protein 5A inhibitors for gt3 HCV. (Hepatology 2018).

9.
AIDS ; 33(1): 67-75, 2019 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-30325765

RESUMO

BACKGROUND: Reports of posttreatment control following antiretroviral therapy (ART) have prompted the question of how common immune control of HIV infection is in the absence of ART. In contrast to adult infection, where elite controllers have been very well characterized and constitute approximately 0.5% of infections, very few data exist to address this question in paediatric infection. METHODS: We describe 11 ART-naive elite controllers from 10 cohorts of HIV-infected children being followed in South Africa, Brazil, Thailand, and Europe. RESULTS: All but one of the elite controllers (91%) are females. The median age at which control of viraemia was achieved was 6.5 years. Five of these 11 (46%) children lost control of viraemia at a median age of 12.9 years. Children who maintained control of viraemia had significantly higher absolute CD4 cell counts in the period of elite control than those who lost viraemic control. On the basis of data available from these cohorts, the prevalence of elite controllers in paediatric infection is estimated to be 5-10-fold lower than in adults. CONCLUSION: Although conclusions are limited by the study design, these data suggest that, whilst paediatric elite control can be achieved, compared with adult elite controllers, this occurs rarely, and takes some years after infection to achieve. Also, loss of immune control arises in a high proportion of children and often relatively rapidly. These findings are consistent with the more potent antiviral immune responses observed in adults and in females.


Assuntos
Infecções por HIV/imunologia , Sobreviventes de Longo Prazo ao HIV , Fatores Sexuais , Brasil , Criança , Pré-Escolar , Europa (Continente) , Feminino , Humanos , Masculino , Prevalência , África do Sul , Tailândia
10.
Gastroenterology ; 156(2): 384-399, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30268787

RESUMO

Hepatitis B virus (HBV) is a unique, tiny, partially double-stranded, reverse-transcribing DNA virus with proteins encoded by multiple overlapping reading frames. The substitution rate is surprisingly high for a DNA virus, but lower than that of other reverse transcribing organisms. More than 260 million people worldwide have chronic HBV infection, which causes 0.8 million deaths a year. Because of the high burden of disease, international health agencies have set the goal of eliminating HBV infection by 2030. Nonetheless, the intriguing HBV genome has not been well characterized. We summarize data on the HBV genome structure and replication cycle, explain and quantify diversity within and among infected individuals, and discuss advances that can be offered by application of next-generation sequencing technology. In-depth HBV genome analyses could increase our understanding of disease pathogenesis and allow us to better predict patient outcomes, optimize treatment, and develop new therapeutics.


Assuntos
Genoma Viral , Vírus da Hepatite B/fisiologia , Hepatite B/terapia , Hepatite B/diagnóstico , Hepatite B/epidemiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Replicação Viral
11.
Sci Rep ; 8(1): 7224, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29740042

RESUMO

Hepatitis C virus (HCV)-specific T cell responses are critical for immune control of infection. Viral adaptation to these responses, via mutations within regions of the virus targeted by CD8+ T cells, is associated with viral persistence. However, identifying viral adaptation to HCV-specific CD4+ T cell responses has been difficult although key to understanding anti-HCV immunity. In this context, HCV sequence and host genotype from a single source HCV genotype 1B cohort (n = 63) were analyzed to identify viral changes associated with specific human leucocyte antigen (HLA) class II alleles, as these variable host molecules determine the set of viral peptides presented to CD4+ T cells. Eight sites across the HCV genome were associated with HLA class II alleles implicated in infection outcome in this cohort (p ≤ 0.01; Fisher's exact test). We extended this analysis to chronic HCV infection (n = 351) for the common genotypes 1A and 3A. Variation at 38 sites across the HCV genome were associated with specific HLA class II alleles with no overlap between genotypes, suggestive of genotype-specific T cell targets, which has important implications for vaccine design. Here we show evidence of HCV adaptation to HLA class II-restricted CD4+ T cell pressure across the HCV genome in chronic HCV infection without a priori knowledge of CD4+ T cell epitopes.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Genoma Viral , Hepacivirus/genética , Hepatite C Crônica/genética , Interações Hospedeiro-Patógeno/genética , Proteínas não Estruturais Virais/genética , Alelos , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/virologia , Estudos de Coortes , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/imunologia , Regulação da Expressão Gênica , Genótipo , Cadeias beta de HLA-DQ/genética , Cadeias beta de HLA-DQ/imunologia , Cadeias HLA-DRB1/genética , Cadeias HLA-DRB1/imunologia , Hepacivirus/imunologia , Hepatite C Crônica/imunologia , Hepatite C Crônica/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mutação , Proteínas não Estruturais Virais/imunologia
12.
Cell Host Microbe ; 23(6): 855-864.e7, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29805095

RESUMO

The Zika virus (ZIKV) epidemic in the Americas established ZIKV as a major public health threat and uncovered its association with severe diseases, including microcephaly. However, genetic epidemiology in some at-risk regions, particularly Central America and Mexico, remains limited. We report 61 ZIKV genomes from this region, generated using metagenomic sequencing with ZIKV-specific enrichment, and combine phylogenetic, epidemiological, and environmental data to reconstruct ZIKV transmission. These analyses revealed multiple independent ZIKV introductions to Central America and Mexico. One introduction, likely from Brazil via Honduras, led to most infections and the undetected spread of ZIKV through the region from late 2014. Multiple lines of evidence indicate biannual peaks of ZIKV transmission in the region, likely driven by varying local environmental conditions for mosquito vectors and herd immunity. The spatial and temporal heterogeneity of ZIKV transmission in Central America and Mexico challenges arbovirus surveillance and disease control measures.


Assuntos
Genoma Viral/genética , Mosquitos Vetores/virologia , Infecção por Zika virus/epidemiologia , Infecção por Zika virus/transmissão , Zika virus/genética , Adolescente , Adulto , Brasil/epidemiologia , América Central/epidemiologia , Criança , Pré-Escolar , Humanos , Imunidade Coletiva/imunologia , Metagenômica , México/epidemiologia , Filogenia , Análise de Sequência de RNA , Zika virus/imunologia , Infecção por Zika virus/sangue , Infecção por Zika virus/urina
13.
Hepatology ; 68(3): 859-871, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29534310

RESUMO

New directly acting antivirals (DAAs) provide very high cure rates in most patients infected by hepatitis C virus (HCV). However, some patient groups have been relatively harder to treat, including those with cirrhosis or infected with HCV genotype 3. In the recent BOSON trial, genotype 3, patients with cirrhosis receiving a 16-week course of sofosbuvir and ribavirin had a sustained virological response (SVR) rate of around 50%. In patients with cirrhosis, interferon lambda 4 (IFNL4) CC genotype was significantly associated with SVR. This genotype was also associated with a lower interferon-stimulated gene (ISG) signature in peripheral blood and in liver at baseline. Unexpectedly, patients with the CC genotype showed a dynamic increase in ISG expression between weeks 4 and 16 of DAA therapy, whereas the reverse was true for non-CC patients. Conclusion: These data provide an important dynamic link between host genotype and phenotype in HCV therapy also potentially relevant to naturally acquired infection. (Hepatology 2018; 00:000-000).


Assuntos
Antivirais/uso terapêutico , Hepatite C/tratamento farmacológico , Interleucinas/genética , Ribavirina/uso terapêutico , Sofosbuvir/uso terapêutico , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genótipo , Hepatite C/sangue , Hepatite C/genética , Humanos , Fígado/metabolismo , Cirrose Hepática/virologia , Resposta Viral Sustentada
14.
J Exp Med ; 214(11): 3239-3261, 2017 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-28983013

RESUMO

Recent studies have suggested greater HIV cure potential among infected children than adults. A major obstacle to HIV eradication in adults is that the viral reservoir is largely comprised of HIV-specific cytotoxic T lymphocyte (CTL) escape variants. We here evaluate the potential for CTL in HIV-infected slow-progressor children to play an effective role in "shock-and-kill" cure strategies. Two distinct subgroups of children were identified on the basis of viral load. Unexpectedly, in both groups, as in adults, HIV-specific CTL drove the selection of escape variants across a range of epitopes within the first weeks of infection. However, in HIV-infected children, but not adults, de novo autologous variant-specific CTL responses were generated, enabling the pediatric immune system to "corner" the virus. Thus, even when escape variants are selected in early infection, the capacity in children to generate variant-specific anti-HIV CTL responses maintains the potential for CTL to contribute to effective shock-and-kill cure strategies in pediatric HIV infection.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Evasão da Resposta Imune/imunologia , Adulto , Linfócitos T CD8-Positivos/metabolismo , Criança , Pré-Escolar , Epitopos de Linfócito T/imunologia , Epitopos de Linfócito T/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Antígenos HLA/imunologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interferon gama/imunologia , Interferon gama/metabolismo , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Carga Viral/imunologia , Produtos do Gene gag do Vírus da Imunodeficiência Humana
15.
Nat Genet ; 49(5): 666-673, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28394351

RESUMO

Outcomes of hepatitis C virus (HCV) infection and treatment depend on viral and host genetic factors. Here we use human genome-wide genotyping arrays and new whole-genome HCV viral sequencing technologies to perform a systematic genome-to-genome study of 542 individuals who were chronically infected with HCV, predominantly genotype 3. We show that both alleles of genes encoding human leukocyte antigen molecules and genes encoding components of the interferon lambda innate immune system drive viral polymorphism. Additionally, we show that IFNL4 genotypes determine HCV viral load through a mechanism dependent on a specific amino acid residue in the HCV NS5A protein. These findings highlight the interplay between the innate immune system and the viral genome in HCV control.


Assuntos
Imunidade Adaptativa/genética , Genoma Humano/genética , Genoma Viral/genética , Hepacivirus/genética , Hepatite C Crônica/genética , Imunidade Inata/genética , Alelos , Variação Genética , Genótipo , Antígenos HLA/genética , Hepacivirus/fisiologia , Hepatite C Crônica/virologia , Interações Hospedeiro-Patógeno/genética , Humanos , Interleucinas/genética , Modelos Logísticos , Análise de Componente Principal , Carga Viral/genética , Proteínas não Estruturais Virais/genética
16.
Microb Genom ; 2(1)2016 01 19.
Artigo em Inglês | MEDLINE | ID: mdl-27713836

RESUMO

Enterococcus faecium, a major cause of hospital-acquired infections, remains problematic because of its propensity to acquire resistance to vancomycin, which currently is considered first-line therapy. Here, we assess the evolution and resistance acquisition dynamics of E. faecium in a clinical context using a series of 132 bloodstream infection isolates from a single hospital. All isolates, of which 49 (37 %) were vancomycin-resistant, underwent whole-genome sequencing. E. faecium was found to be subject to high rates of recombination with little evidence of sequence importation from outside the local E. faecium population. Apart from disrupting phylogenetic reconstruction, recombination was frequent enough to invalidate MLST typing in the identification of clonal expansion and transmission events, suggesting that, where available, whole-genome sequencing should be used in tracing the epidemiology of E. faecium nosocomial infections and establishing routes of transmission. Several forms of the Tn1549-like element-vanB gene cluster, which was exclusively responsible for vancomycin resistance, appeared and spread within the hospital during the study period. Several transposon gains and losses and instances of in situ evolution were inferred and, although usually chromosomal, the resistance element was also observed on a plasmid background. There was qualitative evidence for clonal expansions of both vancomycin-resistant and vancomycin-susceptible E. faecium with evidence of hospital-specific subclonal expansion. Our data are consistent with continuing evolution of this established hospital pathogen and confirm hospital vancomycin-susceptible and vancomycin-resistant E. faecium patient transmission events, underlining the need for careful consideration before modifying current E. faecium infection control strategies.


Assuntos
Enterococcus faecium/genética , Genoma Bacteriano/genética , Resistência a Vancomicina/genética , Técnicas de Tipagem Bacteriana , Enterococcus faecium/isolamento & purificação , Evolução Molecular , Tipagem de Sequências Multilocus , Filogenia , Especificidade da Espécie
17.
Vaccines (Basel) ; 4(3)2016 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-27490575

RESUMO

An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV) infection, as an adjunct to newly developed directly-acting antivirals (DAA), or for the prevention of reinfection, would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3) vector and a modified vaccinia Ankara (MVA), encoding the non-structural proteins of HCV (NSmut), used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with interferon/ribavirin therapy), determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated with the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T-cell responses were rarely detected, and the overall magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV-specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFα production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression) compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced, showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T-cell response. Full-length, next-generation sequencing of the circulating virus demonstrated that T-cells were only induced by vaccination when there was a sequence mismatch between the autologous virus and the vaccine immunogen. However, these T-cells were not cross-reactive with the endogenous viral variant epitopes. Conversely, when there was complete homology between the immunogen and circulating virus at a given epitope T-cells were not induced. T-cell induction following vaccination had no significant impact on HCV viral load. In vitro T-cell culture experiments identified the presence of T-cells at baseline that could be expanded by vaccination; thus, HCV-specific T-cells may have been expanded from pre-existing low-level memory T-cell populations that had been exposed to HCV antigens during natural infection, explaining the partial T-cell dysfunction. In conclusion, vaccination with ChAd3-NSmut and MVA-NSmut prime/boost, a potent vaccine regimen previously optimized in healthy volunteers was unable to reconstitute HCV-specific T-cell immunity in HCV infected patients. This highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure.

18.
J Clin Microbiol ; 54(10): 2470-84, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27385709

RESUMO

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


Assuntos
Genoma Viral , Genótipo , Hepacivirus/genética , Hepatite C/diagnóstico , Hepatite C/virologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Análise de Sequência de DNA/métodos , Farmacorresistência Viral , Hepacivirus/classificação , Humanos , Reino Unido
19.
Genetics ; 204(1): 89-98, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27412711

RESUMO

The distribution of a phenotype on a phylogenetic tree is often a quantity of interest. Many phenotypes have imperfect heritability, so that a measurement of the phenotype for an individual can be thought of as a single realization from the phenotype distribution of that individual. If all individuals in a phylogeny had the same phenotype distribution, measured phenotypes would be randomly distributed on the tree leaves. This is, however, often not the case, implying that the phenotype distribution evolves over time. Here we propose a new model based on this principle of evolving phenotype distribution on the branches of a phylogeny, which is different from ancestral state reconstruction where the phenotype itself is assumed to evolve. We develop an efficient Bayesian inference method to estimate the parameters of our model and to test the evidence for changes in the phenotype distribution. We use multiple simulated data sets to show that our algorithm has good sensitivity and specificity properties. Since our method identifies branches on the tree on which the phenotype distribution has changed, it is able to break down a tree into components for which this distribution is unique and constant. We present two applications of our method, one investigating the association between HIV genetic variation and human leukocyte antigen and the other studying host range distribution in a lineage of Salmonella enterica, and we discuss many other potential applications.


Assuntos
Evolução Biológica , Estudos de Associação Genética/métodos , Modelos Genéticos , Fenótipo , Filogenia , Algoritmos , Animais , Teorema de Bayes , Simulação por Computador , Evolução Molecular , Humanos , Padrões de Herança
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