74 research outputs found
Full genome ultra-deep pyrosequencing associates G-to-A hypermutation of the hepatitis B virus genome with the natural progression of hepatitis
SUMMARY. Human APOBEC3 (A3) cytosine deaminases are antiviral restriction factors capable of editing the genome the hepatitis B virus (HBV). Despite the importance of the human A3 protein family for the innate immune response little is known about the clinical relevance for hepatitis B. The aim of this study was to utilize ultra-deep pyrosequencing (UDPS) data to analyse the phenomenon of G-to-A hypermutation of the complete HBV genome and to relate it to fundamental characteristics of patients with chronic hepatitis B. By analysing the viral population of 80 treatment na€ ıve patients (47 HBeAg-positive and 33 HBeAg-negative), we identified an unequal distribution of G-to-A hypermutations across the genome. Our data indicate that G-to-A hypermutation occurs predominantly in a region between nucleotide positions 600 and 1800 a region which is usually single stranded in matured HBV particles. This implies that A3 likely edits HBV in the virion. Hypermutation rates for HBeAg-negative patients were more than 10-fold higher than those of HBeAg-positive patients. For HBeAg-negative patients higher hypermutation rates were significantly associated with the degree of fibrosis. Additionally, we found that for HBeAg-positive chronic hepatitis G-to-A hypermutation rates were significantly associated with the relative prevalence of the G1764A mutation, which is related to HBeAg seroconversion. In total, our data imply an important association of hypermutation mediated by A3 deaminases with the natural progression of chronic hepatitis B infections both in terms of HBeAg seroconversion and disease progression towards cirrhosis
Applications of Next-Generation Sequencing Technologies to Diagnostic Virology
Novel DNA sequencing techniques, referred to as “next-generation” sequencing (NGS), provide high speed and throughput that can produce an enormous volume of sequences with many possible applications in research and diagnostic settings. In this article, we provide an overview of the many applications of NGS in diagnostic virology. NGS techniques have been used for high-throughput whole viral genome sequencing, such as sequencing of new influenza viruses, for detection of viral genome variability and evolution within the host, such as investigation of human immunodeficiency virus and human hepatitis C virus quasispecies, and monitoring of low-abundance antiviral drug-resistance mutations. NGS techniques have been applied to metagenomics-based strategies for the detection of unexpected disease-associated viruses and for the discovery of novel human viruses, including cancer-related viruses. Finally, the human virome in healthy and disease conditions has been described by NGS-based metagenomics
Characterization of hepatitis B virus X gene quasispecies complexity in mono-infection and hepatitis delta virus superinfection
Hepatitis B X gene; Hepatitis B virus; Hepatitis delta virusHepatitis B gen X; Virus d'hepatitis B; Virus d'hepatitis deltaHepatitis B gen X; Virus de hepatitis B; Virus de hepatitis deltaBACKGROUND:
Hepatitis delta virus (HDV) seems to strongly suppress hepatitis B virus (HBV) replication, although little is known about the mechanism of this interaction. Both these viruses show a dynamic distribution of mutants, resulting in viral quasispecies. Next-generation sequencing is a viable approach for analyzing the composition of these mutant spectra. As the regulatory hepatitis B X protein (HBx) is essential for HBV replication, determination of HBV X gene (HBX) quasispecies complexity in HBV/HDV infection compared to HBV mono-infection may provide information on the interactions between these two viruses.
AIM:
To compare HBV quasispecies complexity in the HBX 5' region between chronic hepatitis delta (CHD) and chronic HBV mono-infected patients.
METHODS:
Twenty-four untreated patients were included: 7/24 (29.2%) with HBeAg-negative chronic HBV infection (CI, previously termed inactive carriers), 8/24 (33.3%) with HBeAg-negative chronic hepatitis B (CHB) and 9/24 (37.5%) with CHD. A serum sample from each patient was first tested for HBV DNA levels. The HBX 5' region [nucleotides (nt) 1255-1611] was then PCR-amplified for subsequent next-generation sequencing (MiSeq, Illumina, United States). HBV quasispecies complexity in the region analyzed was evaluated using incidence-based indices (number of haplotypes and number of mutations), abundance-based indices (Hill numbers of order 1 and 2), and functional indices (mutation frequency and nucleotide diversity). We also evaluated the pattern of nucleotide changes to investigate which of them could be the cause of the quasispecies complexity.
RESULTS:
CHB patients showed higher median HBV-DNA levels [5.4 logIU/mL, interquartile range (IQR) 3.5-7.9] than CHD (3.4 logIU/mL, IQR 3-7.6) (P = n.s.) or CI (3.2 logIU/mL, IQR 2.3-3.5) (P < 0.01) patients. The incidence and abundance indices indicated that HBV quasispecies complexity was significantly greater in CI than CHB. A similar trend was observed in CHD patients, although only Hill numbers of order 2 showed statistically significant differences (CHB 2.81, IQR 1.11-4.57 vs CHD 8.87, 6.56-11.18, P = 0.038). There were no significant differences in the functional indices, but CI and CHD patients also showed a trend towards greater complexity than CHB. No differences were found for any HBV quasispecies complexity indices between CHD and CI patients. G-to-A and C-to-T nucleotide changes, characteristic of APOBEC3G, were higher in CHD and CI than in CHB in genotype A haplotypes, but not in genotype D. The proportion of nt G-to-A vs A-to-G changes and C-to-T vs T-to-C changes in genotype A and D haplotypes in CHD patients showed no significant differences. In CHB and CI the results of these comparisons were dependent on HBV genotype.
CONCLUSION:
The lower-replication CHD and CI groups show a trend to higher quasispecies complexity than the higher-replication CHB group. The mechanisms associated with this greater complexity require elucidation.Supported by the Instituto de Salud Carlos III, grants PI15/00856 and PI17/02233; co-financed by the European Regional Development Fund (ERDF
Determining and utilizing the quasispecies of the hepatitis B virus in clinical applications
Chronic hepatitis B caused by infection with the hepatitis B virus (HBV) affects about 240 million people worldwide and is one of the major causes of severe liver cirrhosis and liver cancer. Hepatitis B treatment options have improved dramatically in the last decade. Effective direct-acting antiviral drugs, so-called nucleos(t)ide analogs, and one effective immunomodulatory drug (pegylated interferon alpha-2a) are available presently. Current challenges for treating HBV involve the careful selection of patients who require therapy and the thoughtful choice of the treatment option tailored to each patient individually. Personalized medicine aims to optimize treatment decisions based on the analysis of host factors and virus characteristics.
The population of viruses within a host is called the viral quasispecies. This thesis provides statistical methods to infer relevant information about the viral quasispecies of HBV to support treatment decisions. We introduce a new genotyping methodology to identify dual infections, which can help to quantify the risk of interferon therapy failure. We present a method to infer short-range linkage information from Sanger sequencing chromatograms, a method to support treatment adjustment after the development of resistance to nucleos(t)ide analogs. Additionally, we provide the first full-genome analysis of the G-to-A hypermutation patterns of the HBV genome. Hypermutated viral genomes form a subpopulation of the quasispecies caused by proteins of the human innate immune system editing the genome of exogenous viral agents. We show that hypermutation is associated with the natural progression of hepatitis B, but does not correlate with treatment response to interferon.Die Hepatitis-B-Erkrankung wird durch eine Infektion mit dem Hepatitis-B-Virus (HBV) verursacht. Weltweit sind schätzungsweise 240 Millionen Menschen chronisch infiziert. Dabei stellt Hepatitis-B eine der häufigsten Ursachen für die Entwicklung von Leberzirrhose und Leberkrebs dar. Die Behandlungsmöglichkeiten wurden in den letzten zehn Jahren signifikant verbessert. Mittlerweile stehen effektive direkt antivirale Medikamente – sogenannte Nukleos(t)id-Analoga – und ein effektives immunmodulierendes Medikament (pegyliertes Interferon alpha-2a) für die Behandlung zur Verfügung. Zentrale Fragen bei der Behandlung von Hepatitis-B beinhalten die zielgerichtete Auswahl der Patienten, welche therapiert werden müssen, sowie die passgenaue Auswahl der Behandlungsoption. Die personalisierte Medizin verfolgt das Ziel, die Behandlung basierend auf der Analyse von Patientencharakteristika und Eigenschaften des Virus zu optimieren.
Die Gesamtheit der Viren innerhalb eines Wirtes wird als virale Quasispezies bezeichnet. Diese Arbeit stellt statistische Methoden zur Verfügung, um relevante Informationen über die Quasispezies von HBV zur Unterstützung von Therapieentscheidungen zu ermitteln. Wir entwickeln eine neue Methode zur Genotypisierung, welche Zweifachinfektionen mit HBV identifiziert und somit hilfreich sein kann, das Risiko eines Therapieversagens einer Interferonbehandlung korrekt einzuschätzen. Des Weiteren stellen wir eine Methode vor, welche Linkage-Informationen der viralen Quasispezies, basierend auf den Chromatogrammen der DNA-Sequenzierung nach Sanger, extrahieren kann. Diese Methode kann bei der Umstellung einer Therapie mit Nukleos(t)id-Analoga nach Resistenzentwicklung verwendet werden. Schließlich präsentieren wir die erste Vollgenomanalyse der G-zu-A Hypermutationsmuster von HBV. Hypermutierte virale Genome stellen eine Teilmenge der Quasispezies dar, welche durch von Proteinen der angeborenen Immunabwehr bewirkte Mutationen im viralen Genom entsteht. Wir zeigen, dass diese Subpopulation mit dem natürlichen Verlauf einer Hepatitis-B-Erkrankung, jedoch nicht mit dem Therapieansprechen auf Interferon, statistisch signifikant assoziiert werden kann
Comparison of the Mechanisms of Drug Resistance among HIV, Hepatitis B, and Hepatitis C
Human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) are the most prevalent deadly chronic viral diseases. HIV is treated by small molecule inhibitors. HBV is treated by immunomodulation and small molecule inhibitors. HCV is currently treated primarily by immunomodulation but many small molecules are in clinical development. Although HIV is a retrovirus, HBV is a double-stranded DNA virus, and HCV is a single-stranded RNA virus, antiviral drug resistance complicates the development of drugs and the successful treatment of each of these viruses. Although their replication cycles, therapeutic targets, and evolutionary mechanisms are different, the fundamental approaches to identifying and characterizing HIV, HBV, and HCV drug resistance are similar. This review describes the evolution of HIV, HBV, and HCV within individuals and populations and the genetic mechanisms associated with drug resistance to each of the antiviral drug classes used for their treatment
Hepatitis B Virus (HBV) - Induced Hepatocarcinogenesis, a Founding Framework of Cancer Evolution & Development (<em>Cancer Evo-Dev</em>)
In this chapter, we present the founding framework of a novel theory termed as Cancer Evolution-Development (Cancer Evo-Dev), based on the current understanding of hepatitis B virus (HBV) induced hepatocarcinogenesis. The interactions of genetic predispositions and HBV infection is responsible for the maintenance of chronic non-resolving inflammation. Under the inflammatory microenvironment, pro-inflammatory factors trans-activate the expression of cytidine deaminases and suppress the expression of uracil DNA glycosylase. The imbalance between the mutagenic forces and mutation-correcting forces facilitates the generations of somatic mutations, viral mutations, and viral integrations into the host genomes. The majority of cells with genomic mutations and mutated viruses are eliminated in survival competition. Only a small percentage of the mutated cells adapted to the hostile environment can survive, retro-differentiate, and function as cancer-initiating cells, representing a process of “mutation-selection-adaptation”. Cancer Evo-Dev lays the theoretical foundation for understanding the mechanisms by which chronic infection of HBV promotes hepatocarcinogenesis. This theory also plays an important role in specific prophylaxis, prediction, early diagnosis, and targeted treatment of cancers
Dynamique adaptative des virus hautement variables à un nouvel environnement réplicatif
La lutte pour les ressources est un phénomène qui a débuté dès l'apparition d'organismes reproductifs et dont la description a été initiée par Malthus puis remarquablement synthétisée et étendue à la biologie sous le terme d'évolution par Darwin en 1859 dans De l'origine des espèces . Si le concept est ancien à l'échelle des sciences biologiques, il continue à caractériser des domaines à l'époque insoupçonnés par son auteur tels que la virologie. En effet, les virus hautement variables tels que le virus de l'immunodéficience humaine (VIH), de l'hépatite B (VHB) et de l'hépatite C (VHC) sont présents sous forme de quasi espèce au sein de leur environnement réplicatif, c'est à dire qu'une multitude de virus génétiquement proche mais distincts coexistent au sein de cet espace qu'ils doivent partager selon les mêmes règles générales que les êtres vivants. Ainsi, lorsque des pressions de sélection s'exercent (immunitaires, antivirales ), une redistribution des variants majoritaires est observé au bénéfice de variants minoritaires mieux adaptés à cet environnement changeant. La modélisation mathématique et informatique de la capacité mutationnelle et la dynamique d'adaptation des variants minoritaires au travers de 6 études de cohortes de patients infectés, par la technique ultra-sensible de pyroséquençage haut débit associée à des logiciels originaux ont permis de mettre en évidence, caractériser et évaluer l'impact de marqueurs diagnostics permettant de prédire la résistance aux antiviraux mais aussi de caractériser de nouvelles cibles antivirales.Struggle for resources is a worldwide rule which has been first described by Malthus and extended to whole world of living organisms by Darwin in 1859 in Origin of species . Today, this concept has been enlarged to virological field, and is particularly adapted to describe highly variable viruses like Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) which have a quasispecies distribution in infected patients characterized by the co-existence of a number of distinct but related viral populations. Selection pressure on viral replicative environment (immune, antiviral drug treatment ), generally lead to a redistribution of the viral quasispecies with an increasing of the best adapted minor viral variants at the expense of major viral populations. Mathematical and bioinformatic modelization of this phenomenon through 6 infected patients cohorts by means of ultra-deep sequencing and an original bioinformatic package allowed discovery, characterization and evaluation of new diagnostic markers that could be used to prevent resistance emergence to antiviral drugs and to characterized new therapeutics antiviral targets.PARIS-EST-Université (770839901) / SudocPARIS12-Bib. électronique (940280011) / SudocSudocFranceF
Computational approaches for improving treatment and prevention of viral infections
The treatment of infections with HIV or HCV is challenging. Thus, novel drugs and new computational approaches that support the selection of therapies are required. This work presents methods that support therapy selection as well as methods that advance novel antiviral treatments. geno2pheno[ngs-freq] identifies drug resistance from HIV-1 or HCV samples that were subjected to next-generation sequencing by interpreting their sequences either via support vector machines or a rules-based approach. geno2pheno[coreceptor-hiv2] determines the coreceptor that is used for viral cell entry by analyzing a segment of the HIV-2 surface protein with a support vector machine. openPrimeR is capable of finding optimal combinations of primers for multiplex polymerase chain reaction by solving a set cover problem and accessing a new logistic regression model for determining amplification events arising from polymerase chain reaction. geno2pheno[ngs-freq] and geno2pheno[coreceptor-hiv2] enable the personalization of antiviral treatments and support clinical decision making. The application of openPrimeR on human immunoglobulin sequences has resulted in novel primer sets that improve the isolation of broadly neutralizing antibodies against HIV-1. The methods that were developed in this work thus constitute important contributions towards improving the prevention and treatment of viral infectious diseases.Die Behandlung von HIV- oder HCV-Infektionen ist herausfordernd. Daher werden neue Wirkstoffe, sowie neue computerbasierte Verfahren benötigt, welche die Therapie verbessern. In dieser Arbeit wurden Methoden zur Unterstützung der Therapieauswahl entwickelt, aber auch solche, welche neuartige Therapien vorantreiben. geno2pheno[ngs-freq] bestimmt, ob Resistenzen gegen Medikamente vorliegen, indem es Hochdurchsatzsequenzierungsdaten von HIV-1 oder HCV Proben mittels Support Vector Machines oder einem regelbasierten Ansatz interpretiert. geno2pheno[coreceptor-hiv2] bestimmt den HIV-2 Korezeptorgebrauch dadurch, dass es einen Abschnitt des viralen Oberflächenproteins mit einer Support Vector Machine analysiert. openPrimeR kann optimale Kombinationen von Primern für die Multiplex-Polymerasekettenreaktion finden, indem es ein Mengenüberdeckungsproblem löst und auf ein neues logistisches Regressionsmodell für die Vorhersage von Amplifizierungsereignissen zurückgreift. geno2pheno[ngs-freq] und geno2pheno[coreceptor-hiv2] ermöglichen die Personalisierung antiviraler Therapien und unterstützen die klinische Entscheidungsfindung. Durch den Einsatz von openPrimeR auf humanen Immunoglobulinsequenzen konnten Primersätze generiert werden, welche die Isolierung von breit neutralisierenden Antikörpern gegen HIV-1 verbessern. Die in dieser Arbeit entwickelten Methoden leisten somit einen wichtigen Beitrag zur Verbesserung der Prävention und Therapie viraler Infektionskrankheiten
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