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 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 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.

Keywords: Hepatitis B X gene; Hepatitis B virus RNA; Next-generation sequencing; Quasispecies; Quasispecies complexity; Quasispecies conservation.

Figure 1

Quantitative polymerase chain reaction verification of residual hepatitis B virus-DNA elimination from hepatitis B virus-RNA isolations after DNAseI treatment. Fluorescence through quantitative polymerase chain reaction amplification samples is shown as green lines for DNAse I-treated RNA samples, and as red lines for their respective cDNA retrotranscribed samples.

Figure 2

Comparison of mean hepatitis B virus quasispecies complexity. Hepatitis B virus (HBV) quasispecies complexity analyzed by Rare Haplotype Load of all 13 patients in HBV-DNA quasispecies (blue-framed boxes) and HBV-RNA quasispecies (yellow-framed boxes). Differences were statistically non-significant (P = 0.1641, t test). RHL: Rare Haplotype Load; HBV: Hepatitis B virus.

Figure 3

Conservation and variability of 357 nucleotide positions analyzed. Information content of nucleotide positions from 1255 to 1611 for hepatitis B virus (HBV)-DNA (blue lines) and HBV-RNA (orange lines) quasispecies. IC: Information content; HBV: Hepatitis B virus.

Figure 4

Differences between information content of hepatitis B virus-DNA and RNA quasispecies. A: Nucleotide positions in which information content (IC) hepatitis B virus (HBV)-RNA > HBV-DNA are depicted in orange while positions where IC HBV-DNA > HBV-RNA in blue; B: Sliding window analysis of the subtraction of mean IC RNA-IC DNA values, in windows of 25 nucleotide positions, displaced in steps of 1 position between them. IC: Information content.

Figure 5

Representation by sequence logos of the information content of the most conserved regions in hepatitis B virus-DNA and hepatitis B virus-RNA quasispecies. The relative sizes of the letters in each stack, each of them representing a nucleotide (nt) position, indicate their relative frequencies at each position within the multiple alignments of nt haplotypes. The total height of each stack of letters depicts the IC of each nt position, measured in bits (Y-axis), therefore 0 bits is the minimum and 2 the maximum conservation. A: Hepatitis B virus-DNA; B: Hepatitis B virus-RNA.

Figure 6

Representation by sequence logos of the information content of the most variable regions in hepatitis B virus-DNA and hepatitis B virus-RNA quasispecies. The relative sizes of the letters in each stack, each of them representing a nucleotide (nt) position, indicate their relative frequencies at each position within the multiple alignments of nt haplotypes. The total height of each stack of letters depicts the IC of each nt position, measured in bits (Y-axis), therefore 0 bits is the minimum and 2 the maximum conservation. A: Hepatitis B virus-DNA; B: Hepatitis B virus-RNA.