Superinfection and cure of infected cells as mechanisms for hepatitis C virus adaptation and persistence

Copyright © 2018 the Author(s). Published by PNAS. This open access article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).RNA viruses exist as a genetically diverse quasispecies with extraordinary ability to adapt to abrupt changes in the host environment. However, the molecular mechanisms that contribute to their rapid adaptation and persistence in vivo are not well studied. Here, we probe hepatitis C virus (HCV) persistence by analyzing clinical samples taken from subjects who were treated with a second-generation HCV protease inhibitor. Frequent longitudinal viral load determinations and large-scale single-genome sequence analyses revealed rapid antiviral resistance development, and surprisingly, dynamic turnover of dominant drug-resistant mutant populations long after treatment cessation. We fitted mathematical models to both the viral load and the viral sequencing data, and the results provided strong support for the critical roles that superinfection and cure of infected cells play in facilitating the rapid turnover and persistence of viral populations. More broadly, our results highlight the importance of considering viral dynamics and competition at the intracellular level in understanding rapid viral adaptation. Thus, we propose a theoretical framework integrating viral and molecular mechanisms to explain rapid viral evolution, resistance, and persistence despite antiviral treatment and host immune responses.info:eu-repo/semantics/publishedVersio

Unsuccessful therapy with adefovir and entecavir-tenofovir in a patient with chronic hepatitis B infection with previous resistance to lamivudine: a fourteen-year evolution of hepatitis B virus mutations

<p>Abstract</p> <p>Background</p> <p>Complex mutants can be selected under sequential selective pressure by HBV therapy. To determine hepatitis B virus genomic evolution during antiviral therapy we characterized the HBV quasi-species in a patient who did no respond to therapy following lamivudine breakthrough for a period of 14 years.</p> <p>Case Presentation</p> <p>The polymerase and precore/core genes were amplified and sequenced at determined intervals in a period of 14 years. HBV viral load and HBeAg/Anti-HBe serological profiles as well as amino transferase levels were also measured. A mixture of lamivudine-resistant genotype A2 HBV strains harboring the rtM204V mutation coexisted in the patient following viral breakthrough to lamivudine. The L180M+M204V dominant mutant displayed strong lamivudine-resistance. As therapy was changed to adefovir, then to entecavir, and finally to entecavir-tenofovir the viral load showed fluctuations but lamivudine-resistant strains continued to be selected, with minor contributions to the HBV quasi-species composition of additional resistance-associated mutations. At the end of the 14-year follow up period, high viral loads were predominant, with viral strains harboring the lamivudine-resistance signature rtL180M+M204V. The precore/core frame A1762T and G1764A double mutation was detected before treatment and remaining in this condition during the entire follow-up. Specific entecavir and tenofovir primary resistance-associated mutations were not detected at any time. Plasma concentrations of tenofovir indicated adequate metabolism of the drug.</p> <p>Conclusions</p> <p>We report the selection of HBV mutants carrying well-defined primary resistance mutations that escaped lamivudine in a fourteen-year follow-up period. With the exception of tenofovir resistance mutations, subsequent unselected primary resistance mutations were detected as minor populations into the HBV quasispecies composition during adefovir or entecavir monotherapies. Although tenofovir is considered an appropriate therapeutic alternative for the treatment of entecavir-unresponsive patients, its use was not effective in the case reported here.</p

Hepatitis C virus molecular evolution: Transmission, disease progression and antiviral therapy

Hepatitis C virus (HCV) infection represents an important public health problem worldwide. Reduction of HCV morbidity and mortality is a current challenge owned to several viral and host factors. Virus molecular evolution plays an important role in HCV transmission, disease progression and therapy outcome. The high degree of genetic heterogeneity characteristic of HCV is a key element for the rapid adaptation of the intrahost viral population to different selection pressures (e.g., host immune responses and antiviral therapy). HCV molecular evolution is shaped by different mechanisms including a high mutation rate, genetic bottlenecks, genetic drift, recombination, temporal variations and compartmentalization. These evolutionary processes constantly rearrange the composition of the HCV intrahost population in a staging manner. Remarkable advances in the understanding of the molecular mechanism controlling HCV replication have facilitated the development of a plethora of direct-acting antiviral agents against HCV. As a result, superior sustained viral responses have been attained. The rapidly evolving field of anti-HCV therapy is expected to broad its landscape even further with newer, more potent antivirals, bringing us one step closer to the interferon-free era.Fil: Preciado, María Victoria. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Valva, Pamela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Gobierno de la Ciudad de Buenos Aires. Hospital General de Niños ; ArgentinaFil: Escobar Gutierrez, Alejandro. Instituto de Diagnóstico y Referencia Epidemiológicos; MéxicoFil: Rahal, Paula. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Ruiz Tovar, Karina. Instituto de Diagnóstico y Referencia Epidemiológicos; MéxicoFil: Yamasaki, Lilian. Universidade Estadual Paulista Julio de Mesquita Filho; BrasilFil: Vazquez Chacon, Carlos. Instituto de Diagnóstico y Referencia Epidemiológicos; MéxicoFil: Martinez Guarneros, Armando. Instituto de Diagnóstico y Referencia Epidemiológicos; MéxicoFil: Carpio Pedroza, Juan Carlos. Instituto de Diagnóstico y Referencia Epidemiológicos; MéxicoFil: Fonseca Coronado, Salvador. Universidad Nacional Autónoma de México; MéxicoFil: Cruz Rivera, Mayra. Universidad Nacional Autónoma de México; Méxic

Replicative Homeostasis III: implications for antiviral therapy and mechanisms of response and non-response

While improved drug regimens have greatly enhanced outcomes for patients with chronic viral infection, antiviral therapy is still not ideal due to drug toxicities, treatment costs, primary drug failure and emergent resistance. New antiviral agents, alternative treatment strategies and a better understanding of viral pathobiology, host responses and drug action are desperately needed. Interferon (IFN) and ribavirin, are effective drugs used to treat hepatitis C (HCV), but the mechanism(s) of their action are uncertain. Error catastrophe (EC), or precipitous loss of replicative fitness caused by genomic mutation, is postulated to mediate ribavirin action, but is a deeply flawed hypothesis lacking empirical confirmation. Paradoxically ribavirin, a proven RNA mutagen, has no impact on HCV viraemia long term, suggesting real viruses, replicating in-vitro, as opposed to mathematical models, replicating in-silico, are likely to resist EC by highly selective replication of fit (~consensus sequence) genomes mediated, in part, by replicative homeostasis (RH), an epicyclic mechanism that dynamically links RNApol fidelity and processivity and other viral protein functions. Replicative homeostasis provides a rational explanation for the various responses seen during treatment of HCV, including genotype-specific and viral load-dependent differential response rates, as well as otherwise unexplained phenomena like the transient inhibition and rebound of HCV viraemia seen during ribavirin monotherapy. Replicative homeostasis also suggests a primarily non-immunological mechanism that mediates increased immune responsiveness during treatment with ribavirin (and other nucleos(t)ide analogues), explicating the enhanced second-phase clearance of HCV ribavirin promotes and, thus, the apparent immunomodulatory action of ribavirin. More importantly, RH suggests specific new antiviral therapeutic strategies

HepSEQ: International Public Health Repository for Hepatitis B

HepSEQ is a repository for an extensive library of public health and molecular data relating to hepatitis B virus (HBV) infection collected from international sources. It is hosted by the Centre for Infections, Health Protection Agency (HPA), England, United Kingdom. This repository has been developed as a web-enabled, quality-controlled database to act as a tool for surveillance, HBV case management and for research. The web front-end for the database system can be accessed from . The format of the database system allows for comprehensive molecular, clinical and epidemiological data to be deposited into a functional database, to search and manipulate the stored data and to extract and visualize the information on epidemiological, virological, clinical, nucleotide sequence and mutational aspects of HBV infection through web front-end. Specific tools, built into the database, can be utilized to analyse deposited data and provide information on HBV genotype, identify mutations with known clinical significance (e.g. vaccine escape, precore and antiviral-resistant mutations) and carry out sequence homology searches against other deposited strains. Further mechanisms are also in place to allow specific tailored searches of the database to be undertaken

Site-directed M2 proton channel inhibitors enable synergistic combination therapy for rimantadine-resistant pandemic influenza

Pandemic influenza A virus (IAV) remains a significant threat to global health. Preparedness relies primarily upon a single class of neuraminidase (NA) targeted antivirals, against which resistance is steadily growing. The M2 proton channel is an alternative clinically proven antiviral target, yet a near-ubiquitous S31N polymorphism in M2 evokes resistance to licensed adamantane drugs. Hence, inhibitors capable of targeting N31 containing M2 (M2-N31) are highly desirable. Rational in silico design and in vitro screens delineated compounds favouring either lumenal or peripheral M2 binding, yielding effective M2-N31 inhibitors in both cases. Hits included adamantanes as well as novel compounds, with some showing low micromolar potency versus pandemic “swine” H1N1 influenza (Eng195) in culture. Interestingly, a published adamantane-based M2-N31 inhibitor rapidly selected a resistant V27A polymorphism (M2-A27/N31), whereas this was not the case for non-adamantane compounds. Nevertheless, combinations of adamantanes and novel compounds achieved synergistic antiviral effects, and the latter synergised with the neuraminidase inhibitor (NAi), Zanamivir. Thus, site-directed drug combinations show potential to rejuvenate M2 as an antiviral target whilst reducing the risk of drug resistance

Modelling the genomic structure, and antiviral susceptibility of Human Cytomegalovirus

Human Cytomegalovirus (HCMV) is found ubiquitously in humans worldwide, and once acquired, the infection persists within the host throughout their life. Although Immunocompetent people rarely are affected by HCMV infections, their related diseases pose a major health problem worldwide for those with compromised or suppressed immune systems such as transplant recipients. Additionally, congenital transmission of HCMV is the most common infectious cause of birth defects globally and is associated with a substantial economic burden. This thesis explores the application of statistical modelling and genomics to unpick three key areas of interest in HCMV research. First, a comparative genomics analysis of global HCMV strains was undertaken to delineate the molecular population structure of this highly variable virus. By including in-house sequenced viruses of African origin and by developing a statistical framework to deconvolute highly variable regions of the genome, novel and important insights into the co-evolution of HCMV with its host were uncovered. Second, a rich database relating mutations to drug sensitivity was curated for all the antiviral treated herpesviruses. This structured information along with the development of a mutation annotation pipeline, allowed the further development of statistical models that predict the phenotype of a virus from its sequence. The predictive power of these models was validated for HSV1 by using external unseen mutation data provided in collaboration with the UK Health Security Agency. Finally, a nonlinear mixed effects model, expanded to account for Ganciclovir pharmacokinetics and pharmacodynamics, was developed by making use of rich temporal HCMV viral load data. This model allowed the estimation of the impact of immune-clearance versus antiviral inhibition in controlling HCMV lytic replication in already established infections post-haematopoietic stem cell transplant