Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?  

作　　者：Selene Garcia-Garcia Maria Francesca Cortese David Tabernero Josep Gregori Marta Vila Beatriz Pacín Josep Quer Rosario Casillas Laura Castillo-Ribelles Roser Ferrer-Costa Ariadna Rando-Segura Jesús Trejo-Zahínos Tomas Pumarola Ernesto Casis Rafael Esteban Mar Riveiro-Barciela Maria Buti Francisco Rodríguez-Frías 

机构地区：[1]Clinical Biochemistry Research Group,Department of Biochemistry,Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron,Universitat Autònoma de Barcelona,Barcelona 08035,Spain [2]Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas,Instituto de Salud Carlos III,Madrid 28029,Spain [3]Liver Unit,Liver Disease Laboratory-Viral Hepatitis,Vall d'Hebron Institut Recerca-Hospital Universitari Vall d'Hebron,Vall d’Hebron Barcelona Hospital Campus,Barcelona 08035,Spain [4]Liver Pathology Unit,Departments of Biochemistry and Microbiology,Hospital Universitari Vall d’Hebron,Universitat Autònoma de Barcelona,Barcelona 08035,Spain [5]Department of Microbiology,Hospital Universitari Vall d'Hebron,Vall d'Hebron Barcelona Hospital Campus,Barcelona 08035,Spain [6]Liver Unit,Department of Internal Medicine,Hospital Universitari Vall d'Hebron,Universitat Autónoma de Barcelona,Barcelona 08035,Spain

出　　处：《World Journal of Gastroenterology》2021年第41期7144-7158,共15页世界胃肠病学杂志（英文版）

基　　金：by Instituto de Salud Carlos III,No.PI18/01436;and European Regional Development Fund(ERDF).

摘　　要：BACKGROUND Different forms of pregenomic and other hepatitis B virus(HBV)RNA have been detected in patients’sera.These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity,and predicting hepatitis B eantigen seroconversion or viral rebound after nucleos(t)ide analog cessation.Data on serum HBV-RNA quasispecies,however,is scarce.It is therefore important to develop methodologies to thoroughly analyze this quasispecies,ensuring the elimination of any residual HBV-DNA.Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection.AIM To establish a next-generation sequencing(NGS)methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies.METHODS Thirteen untreated chronic hepatitis B patients,showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA>5 Log10 IU/mL and HBV-RNA>4 Log10 copies/mL was taken from each patient.HBV-RNA was treated with DNAse I to remove any residual DNA,and the region between nucleotides(nt)1255-1611 was amplified using a 3-nested polymerase chain reaction protocol,and analyzed with NGS.Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies.RESULTS No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies.HBV quasispecies complexity showed heterogeneous behavior among patients.The Rare Haplotype Load at 1%was greater in DNA than in RNA quasispecies,with no statistically significant differences(P=0.1641).Regarding conservation,information content was equal in RNA and DNA quasispecies in most nt positions[218/357(61.06%)].In 102 of the remaining 139(73.38%),HBV-RNA showed slightly higher variability.Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies,3 of them coincided between the 2 quasispecies:nts 1258-1286,1545-1573 and 1575-1604.The 2 hyper-variable sequence fragments also coincided:nts 1311-1344 and 1461-1485.Sequences betwe

关 键 词：Hepatitis B virus RNA Hepatitis B X gene QUASISPECIES Next-generation sequencing Quasispecies conservation Quasispecies complexity 

分 类 号：R373.21[医药卫生—病原生物学]