NS5A Gene Analysis by Next Generation Sequencing in HCV Nosocomial Transmission Clusters of HCV Genotype 1b Infected Patients
Maria Concetta Bellocchi,
Marianna Aragri,
Luca Carioti,
Lavinia Fabeni,
Rosaria Maria Pipitone,
Giuseppina Brancaccio,
Maria Chiara Sorbo,
Silvia Barbaliscia,
Velia Chiara Di Maio,
Fabrizio Bronte,
Stefania Grimaudo,
Walter Mazzucco,
Ferdinando Frigeri,
Marco Cantone,
Antonio Pinto,
Carlo Federico Perno,
Antonio Craxì,
Giovanni Battista Gaeta,
Vito Di Marco,
Francesca Ceccherini-Silberstein
Affiliations
Maria Concetta Bellocchi
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Marianna Aragri
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Luca Carioti
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Lavinia Fabeni
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Rosaria Maria Pipitone
Health Promotion Sciences, Maternal and Infant Care, Internal Medicine and Medical Specialties (PROMISE) Department, University of Palermo, 90127 Palermo, Italy
Giuseppina Brancaccio
Infectious Diseases, Department of Mental and Physical Health and Preventive Medicine, Campania University “Luigi Vanvitelli”, 80138 Naples, Italy
Maria Chiara Sorbo
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Silvia Barbaliscia
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Velia Chiara Di Maio
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Fabrizio Bronte
Health Promotion Sciences, Maternal and Infant Care, Internal Medicine and Medical Specialties (PROMISE) Department, University of Palermo, 90127 Palermo, Italy
Stefania Grimaudo
Health Promotion Sciences, Maternal and Infant Care, Internal Medicine and Medical Specialties (PROMISE) Department, University of Palermo, 90127 Palermo, Italy
Walter Mazzucco
Health Promotion Sciences, Maternal and Infant Care, Internal Medicine and Medical Specialties (PROMISE) Department, University of Palermo, 90127 Palermo, Italy
Infectious Diseases, Department of Mental and Physical Health and Preventive Medicine, Campania University “Luigi Vanvitelli”, 80138 Naples, Italy
Antonio Pinto
Hematology Department, National Cancer Institute “Fondazione Pascale”, IRCCS, 80131 Naples, Italy
Carlo Federico Perno
Department of Microbiology and Clinic Microbiology, University of Milan, 20162 Milan, Italy
Antonio Craxì
Health Promotion Sciences, Maternal and Infant Care, Internal Medicine and Medical Specialties (PROMISE) Department, University of Palermo, 90127 Palermo, Italy
Giovanni Battista Gaeta
Infectious Diseases, Department of Mental and Physical Health and Preventive Medicine, Campania University “Luigi Vanvitelli”, 80138 Naples, Italy
Vito Di Marco
Health Promotion Sciences, Maternal and Infant Care, Internal Medicine and Medical Specialties (PROMISE) Department, University of Palermo, 90127 Palermo, Italy
Francesca Ceccherini-Silberstein
Department of Experimental Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Background: The aim of the study was to investigate the intra-host variability through next-generation-sequencing (NGS) of the NS5A-gene in nosocomial transmission-clusters observed in two Italian hospitals among hepatitis C virus (HCV)-genotype-1b infected patients. Methods: HCV-sequencing was performed by Sanger-sequencing (NS3 + NS5A + NS5B) and by NGS (NS5A, MiSeq-Illumina) in 15 HCV-1b infected patients [five acute with onco-hematologic-disease and 10 (4/6 acute/chronic) with β-thalassemia]. Resistance-associated-substitutions (RAS) were analysed by Geno2pheno-algorithm. Nucleotide-sequence-variability (NSV, at 1%, 2%, 5%, 10% and 15% NGS-cutoffs) and Shannon entropy were estimated. Phylogenetic analysis was performed by Mega6-software and Bayesian-analysis. Results: Phylogenetic analysis showed five transmission-clusters: one involving four HCV-acute onco-hematologic-patients; one involving three HCV-chronic β-thalassemia-patients and three involving both HCV-acute and chronic β-thalassemia-patients. The NS5A-RAS Y93H was found in seven patients, distributed differently among chronic/acute patients involved in the same transmission-clusters, independently from the host-genetic IL-28-polymorphism. The intra-host NSV was higher in chronic-patients versus acute-patients, at all cutoffs analyzed (p < 0.05). Even though Shannon-entropy was higher in chronic-patients, significantly higher values were observed only in chronic β-thalassemia-patients versus acute β-thalassemia-patients (p = 0.01). Conclusions: In nosocomial HCV transmission-clusters, the intra-host HCV quasispecies divergence in patients with acute-infection was very low in comparison to that in chronic-infection. The NS5A-RAS Y93H was often transmitted and distributed differently within the same transmission-clusters, independently from the IL-28-polymorphism.
