Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA : Different quasispecies?
García-García, Selene (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Cortese, Maria Francesca (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Tabernero, David (Instituto de Salud Carlos II)
Gregori i Font, Josep (Instituto de Salud Carlos II)
Vila, Marta (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Pacín Ruiz, Beatriz (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Quer, Josep 1963- (Instituto de Salud Carlos II)
Casillas, Rosario (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Castillo-Ribelles, Laura (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Ferrer-Costa, Roser (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Rando-Segura, Ariadna (Hospital Universitari Vall d'Hebron)
Trejo-Zahínos, Jesús (Hospital Universitari Vall d'Hebron)
Pumarola Suñé, Tomàs (Hospital Universitari Vall d'Hebron)
Casis-Saez, Ernesto (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Esteban, Rafael (Instituto de Salud Carlos II)
Riveiro Barciela, Mar (Instituto de Salud Carlos II)
Buti, Maria (Instituto de Salud Carlos II)
Rodríguez Frías, Francisco (Hospital Universitari Vall d'Hebron. Institut de Recerca)
Universitat Autònoma de Barcelona

Date:	2021
Abstract:	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 e-antigen 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. To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies. 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 LogIU/mL and HBV-RNA > 4 Logcopies/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. 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 between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study.
Grants:	Instituto de Salud Carlos III PI18/01436
Rights:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original.
Language:	Anglès
Document:	Article ; recerca ; Versió publicada
Subject:	Hepatitis B virus RNA ; Hepatitis B X gene ; Quasispecies ; Next-generation sequencing ; Quasispecies conservation ; Quasispecies complexity
Published in:	World Journal of Gastroenterology, Vol. 27 (november 2021) , p. 7144-7158, ISSN 2219-2840

DOI: 10.3748/wjg.v27.i41.7144
PMID: 34887634

16 p, 1.9 MB

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