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

Abstract

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 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. 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 Log 10 IU/mL and HBV-RNA > 4 Log 10 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 between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. CONCLUSION 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.

Diversity of the nucleic acid forms of circulating HBV in chronically infected patients and its impact on viral cycle

“…Strategies, which can be used to investigate this viral diversity include cloning/sequencing, 5'RACE-PCR or next-generation sequencing assays 41 .…”

Relationship Between Serum HBV-RNA Levels, Sero-biochemical Indices, and Clinicopathological Parameters in Patients with Hepatitis B Virus

“…In HBVinfected patients, failure to detect serological HBeAg levels and serum HBV DNA levels as indicators of virological response to treatment can increase the risk of virological rebound and disease recurrence, as recommended by AASLD 2015, China/APASL 2015, and EASL 2017 for NAs drug withdrawal (23). Additionally, for HBeAg-negative patients, long-term antiviral treatment results in only a few achieving negative HBsAg conversion, often leading to the need for long-term or lifelong medication (24). Studies have identified the persistence of HBV cccDNA in infected liver nuclei as the primary cause of chronic Hepatitis B (25), suggesting that HBV RNA, as the transcriptional form of cccDNA, could serve as a crucial indicator for the diagnosis, treatment, and monitoring of chronic HBV infectious diseases, as well as guiding safe drug withdrawal (25).…”