Deep-sequencing reveals broad subtype-specific HCV resistance mutations associated with treatment failure

A percentage of hepatitis C virus (HCV)-infected patients fail direct acting antiviral (DAA)-based treatment regimens, often because of drug resistance-associated substitutions (RAS). The aim of this study was to characterize the resistance profile of a large cohort of patients failing DAA-based treatments, and investigate the relationship between HCV subtype and failure, as an aid to optimizing management of these patients. A new, standardized HCV-RAS testing protocol based on deep sequencing was designed and applied to 220 previously subtyped samples from patients failing DAA treatment, collected in 39 Spanish hospitals. The majority had received DAA-based interferon (IFN) a-free regimens; 79% had failed sofosbuvir-containing therapy. Genomic regions encoding the nonstructural protein (NS) 3, NS5A, and NS5B (DAA target regions) were analyzed using subtype-specific primers. Viral subtype distribution was as follows: genotype (G) 1, 62.7%; G3a, 21.4%; G4d, 12.3%; G2, 1.8%; and mixed infections 1.8%. Overall, 88.6% of patients carried at least 1 RAS, and 19% carried RAS at frequencies below 20% in the mutant spectrum. There were no differences in RAS selection between treatments with and without ribavirin. Regardless of the treatment received, each HCV subtype showed specific types of RAS. Of note, no RAS were detected in the target proteins of 18.6% of patients failing treatment, and 30.4% of patients had RAS in proteins that were not targets of the inhibitors they received. HCV patients failing DAA therapy showed a high diversity of RAS. Ribavirin use did not influence the type or number of RAS at failure. The subtype-specific pattern of RAS emergence underscores the importance of accurate HCV subtyping. The frequency of “extra-target” RAS suggests the need for RAS screening in all three DAA target regions

Whole-genome characterization and resistance-associated substitutions in a new HCV genotype 1 subtype

Georg von Massow,1 Damir Garcia-Cehic,1,2 Josep Gregori,1–3 Francisco Rodriguez-Frias,2,4 María Dolores Macià,5 Ana Escarda,6 Juan Ignacio Esteban,1–2,7 Josep Quer1–2,71Liver Unit, Liver Diseases – Viral Hepatitis, Vall d’Hebron Institut of Research (VHIR) – Hospital Universitari Vall d’Hebron (HUVH), Barcelona, Spain; 2Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid, Spain; 3Roche Diagnostics S.L., Sant Cugat del Vallès, Barcelona, Spain; 4Biochemistry and Microbiology Department, VHIR-HUVH, Barcelona, Spain; 5Unidad de Microbiología Molecular, Servicio de Microbiología, Instituto de Investigación Sanitaria de les Illes Balears (IdISBa), Hospital Universitario Son Espases, Mallorca, Spain; 6Digestive Department, Hospital Universitario Son Espases, Mallorca, Spain; 7Medicine Department, Universitat Autònoma de Barcelona, Barcelona, SpainAbstract: Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using phylogenetic analysis and analysis of genetic distances in sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing subtypes. The whole genome was analyzed to confirm that the new isolate could be classified as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, which could limit the use of some inhibitors for treating this subtype. RAS studies of new subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are reported. In our case, the patient has not yet been treated, and the RAS report will be used to design the most effective treatment.Keywords: subtype, direct-acting antivirals, HCV, genotype

Whole-genome characterization and resistance-associated substitutions in a new HCV genotype 1 subtype

Càrrega feta de Scopus d'articles UAB 2019 (Gold, hybrid o Bronze) procedents de l'Observatori d'Accés Obert (càrrega maig 2020). Compte! Cal comprovar la versió permesa per l'editor en els bronze.This study was supported by the Spanish Ministry of Health, Consumption and Social Welfare grant name "Plan Estratégico Nacional contra la Hepatitis C". This study was funded by Instituto de Salud Carlos III grants, PI15/00856, PI15/00829 and PI16/00337, and cofinanced by CIBERehd (Consorcio Centro de Investigación en Red de Enfermedades Hepaticas y Digestivas), which is funded by Instituto de Salud Carlos III and Centro para el Desarrollo Tecnológico Industrial-CDTI from the Spanish Ministry of Economy and Business, grant number, IDI-20151125.Hepatitis C virus (HCV) is a highly variable infectious agent, classified into 8 genotypes and 86 subtypes. Our laboratory has implemented an in-house developed high-resolution HCV subtyping method based on next-generation sequencing (NGS) for error-free classification of the virus using phylogenetic analysis and analysis of genetic distances in sequences from patient samples compared to reference sequences. During routine diagnostic, a sample from an Equatorial Guinea patient could not be classified into any of the existing subtypes. The whole genome was analyzed to confirm that the new isolate could be classified as a new HCV subtype. In addition, naturally occurring resistance-associated substitutions (RAS) were analyzed by NGS. Whole-genome analysis based on p-distances suggests that the sample belongs to a new HCV genotype 1 subtype. Several RAS in the NS3 (S122T, D168E and I170V) and NS5A protein (Q(1b)24K, R(1b)30Q and Y93L+Y93F) were found, which could limit the use of some inhibitors for treating this subtype. RAS studies of new subtypes are of great interest for tailoring treatment, as no data on treatment efficacy are reported. In our case, the patient has not yet been treated, and the RAS report will be used to design the most effective treatmen

Synaptic activation modifies microtubules underlying transport of postsynaptic cargo

Synaptic plasticity, the ability of synapses to change in strength, requires alterations in synaptic molecule compositions over time, and synapses undergo selective modifications on stimulation. Molecular motors operate in sorting/transport of neuronal proteins; however, the targeting mechanisms that guide and direct cargo delivery remain elusive. We addressed the impact of synaptic transmission on the regulation of intracellular microtubule (MT)-based transport. We show that increased neuronal activity, as induced through GlyR activity blockade, facilitates tubulin polyglutamylation, a posttranslational modification thought to represent a molecular traffic sign for transport. Also, GlyR activity blockade alters the binding of the MT-associated protein MAP2 to MTs. By using the kinesin (KIF5) and the postsynaptic protein gephyrin as models, we show that such changes of MT tracks are accompanied by reduced motor protein mobility and cargo delivery into neurites. Notably, the observed neurite targeting deficits are prevented on functional depletion or gene expression knockdown of neuronal polyglutamylase. Our data suggest a previously undescribed concept of synaptic transmission regulating MT-dependent cargo delivery

Microtubule Organization and Microtubule-Associated Proteins (MAPs)

Dendrites have a unique microtubule organization. In vertebrates, dendritic microtubules are organized in antiparallel bundles, oriented with their plus ends either pointing away or toward the soma. The mixed microtubule arrays control intracellular trafficking and local signaling pathways, and are essential for dendrite development and function. The organization of microtubule arrays largely depends on the combined function of different microtubule regulatory factors or generally named microtubule-associated proteins (MAPs). Classical MAPs, also called structural MAPs, were identified more than 20 years ago based on their ability to bind to and copurify with microtubules. Most classical MAPs bind along the microtubule lattice and regulate microtubule polymerization, bundling, and stabilization. Recent evidences suggest that classical MAPs also guide motor protein transport, interact with the actin cytoskeleton, and act in various neuronal signaling networks. Here, we give an overview of microtubule organization in dendrites and the role of classical MAPs in dendrite development, dendritic spine formation, and synaptic plasticity