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 beta-thalassemial. 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 beta-thalassemia-patients and three involving both HCV-acute and chronic beta-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 beta-thalassemia-patients versus acute beta-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.

Bellocchi, M.C., Aragri, M., Carioti, L., Fabeni, L., Pipitone, R.M., Brancaccio, G., et al. (2019). NS5A Gene Analysis by Next Generation Sequencing in HCV Nosocomial Transmission Clusters of HCV Genotype 1b Infected Patients. CELLS, 8(7), 666 [10.3390/cells8070666].

NS5A Gene Analysis by Next Generation Sequencing in HCV Nosocomial Transmission Clusters of HCV Genotype 1b Infected Patients

Barbaliscia, Silvia;Di Maio, Velia Chiara;Perno, Carlo Federico;Ceccherini-Silberstein, Francesca
2019

Abstract

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 beta-thalassemial. 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 beta-thalassemia-patients and three involving both HCV-acute and chronic beta-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 beta-thalassemia-patients versus acute beta-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.
Pubblicato
Rilevanza internazionale
Articolo
Esperti anonimi
Settore MED/07
eng
HCV; NGS; acute infection; chronic infection; nosocomial transmission; sequencing; Acute Disease; Adult; Amino Acid Substitution; Antiviral Agents; Blood Transfusion; Chronic Disease; Cross Infection; Drug Resistance, Viral; Female; Genotype; Hepacivirus; Hepatitis C; High-Throughput Nucleotide Sequencing; Host-Pathogen Interactions; Humans; Interferons; Male; Middle Aged; Phylogeny; Polymorphism, Single Nucleotide; Viral Nonstructural Proteins; beta-Thalassemia
Bellocchi, M.C., Aragri, M., Carioti, L., Fabeni, L., Pipitone, R.M., Brancaccio, G., et al. (2019). NS5A Gene Analysis by Next Generation Sequencing in HCV Nosocomial Transmission Clusters of HCV Genotype 1b Infected Patients. CELLS, 8(7), 666 [10.3390/cells8070666].
Bellocchi, Mc; Aragri, M; Carioti, L; Fabeni, L; Pipitone, Rm; Brancaccio, G; Sorbo, Mc; Barbaliscia, S; Di Maio, Vc; Bronte, F; Grimaudo, S; Mazzucco, W; Frigeri, F; Cantone, M; Pinto, A; Perno, Cf; Craxì, A; Gaeta, Gb; Di Marco, V; Ceccherini-Silberstein, F
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2108/238603
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