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

ARTICLE HIGHLIGHTS

Research background

Recent studies show that hepatitis B virus (HBV)-RNA detected in serum is mainly encapsidated pregenomic RNA (pgRNA), which could be a useful biomarker for monitoring covalently closed circular DNA activity. During nuclos(t)ide analogs (NA) therapy it is predictive of hepatitis B e-antigen seroconversion and for following viral rebound after treatment cessation. However, few studies have analyzed the serum HBV-RNA quasispecies, a complex mutant spectrum constituted by closely related, but not identical, viral populations. Analysis of serum circulating HBV-RNA quasispecies in those previous studies showed no significant difference between HBV-RNA and HBV-DNA quasispecies complexity before antiviral treatment and indicated that serum HBV-RNA is genetically homogenous with intrahepatic HBV-RNA, which is the main target for targeted gene therapy. Such therapies should be directed to hyper-conserved sequence targets, ideally located at hepatitis B X gene (HBX), located next to the 3’ end of all the HBV mRNAs.

Research motivation

Composition and evolution over time of HBV quasispecies is closely linked to liver disease progression, and serum circulating HBV-RNA is potentially useful for its analysis, even in situations with low or undetectable serum level of HBV-DNA. However, HBV-RNA has not been subjected to reverse transcription, which is the main source of variability in the HBV genome and it may therefore present significant differences with respect to the serum HBV-DNA quasiespecies. Nonetheless, little data is available on the comparison of genetic variability/conservation and complexity of both quasispecies. Moreover, analysis of serum HBV-RNA quasispecies may be a very useful tool when looking for hyper-conserved targets for targeted gene therapy, due to its similarities with intrahepatic HBV-RNA, the main target of this kind of antiviral therapy. Thus, in previous studies, we identified hyper-conserved sequence stretches in the circulating HBV-DNA quasispecies, which we aimed to analyze at the circulating HBV-RNA level. Studying circulating HBV-RNA quasispecies may thus favor achieving functional cure of HBV infection. However, it is necessary to establish a reliable methodology to thoroughly analyze this quasispecies, without interference from serum HBV-DNA.

Research objectives

This study aimed to establish a methodology to achieve an in-depth analysis serum HBV-RNA quasispecies without interference from circulating HBV-DNA, using next-generation sequencing (NGS). With this methodology, we aimed to compare both serum HBV-RNA and DNA quasispecies in a group of untreated chronic hepatitis B (CHB) patients. Considering the potential of HBV-RNA for analyzing HBV quasispecies, even in situations of low or undetectable serum HBV-DNA, this thorough analysis may serve as prognostic factor for clinical follow-up of CHB patients. Furthermore, it may be a useful tool for looking for hyper-conserved targets for targeted gene therapy.

Research methods

Serum samples were taken from 13 untreated CHB patients attending the outpatient clinic of Vall d’Hebron University Hospital (Barcelona, Spain). HBV-DNA levels > 5 log₁₀ IU/mL, HBV-RNA levels > 4 Log₁₀ copies/mL and heterogeneity in terms of HBV genotypes and degrees of severity of liver disease were considered as inclusion criteria. HBV-RNA and DNA were extracted differently using specific manual isolation protocols. In addition, HBV-RNA, which was quantified by an in-house method, was treated with DNAse I (Life Technologies, Austin, United Ststes) to remove any residual DNA present. The elimination of residual DNA after DNAse I digestion was verified by means of quantitative PCR (qPCR). In parallel, these samples were retrotranscribed into cDNA, and along with DNA isolates the 5’ end region of HBX, between nucleotides 1255-1611 (the amplicon analysed), was amplified by a 3-nested PCR protocol and later sequenced using NGS (MiSeq, Illumina, United States). HBV-RNA and DNA quasispecies complexity was evaluated using Rare Haplotype Load (RHL) index, which measures enrichment of quasispecies in minority genomes. Sequence conservation and variability was determined by calculating the information content (IC) of each position by aligning all unique sequences covering the full amplicon (i.e., haplotypes) in a multiple alignment. After this analysis, the most conserved and variable sequence stretches were represented as sequence logos, which were compared between both quasispecies.

Research results

After treatment of RNA isolates with DNase I, we confirmed that no residual HBV-DNA was present in the HBV-RNA isolates and that contamination by HBV-DNA in sequences obtained from HBV-RNA was therefore unlikely. HBV quasispecies complexity showed heterogeneous behavior among patients. While RHL was greater in DNA than in RNA quasispecies, differences were not statistically significant. This tendency of HBV DNA quasispecies toward higher quasispecies complexity than HBV-RNA needs to be studied further in larger groups of patients. In general, conservation was highly coincident between HBV-RNA and DNA quasispecies; the majority of nt positions showed the same IC value in both of them. Interestingly, HBV-RNA quasispecies was slightly less conserved than DNA and displayed more positions with some variability (IC < 2 bits). Sliding window analysis in HBV-DNA and RNA quasispecies showed 4 sequence fragments as the most conserved in each of them. Of those fragments 3 coincided between both quasispecies, but 1 was found to be among the most conserved only in HBV-DNA and 1 only in HBV-RNA. The most variable sequence fragments coincided in both quasispecies.

Research conclusions

In this study, we describe a methodology for analyzing serum HBV-RNA quasispecies without HBV-DNA interference. This methodology allowed us to compare HBV-DNA and RNA quasispecies. For quasispecies complexity, we analyzed the RHL index, a new reliable diversity index to diagnose mutant spectrum expansions, such as may have occurred with the error-prone reverse transcription of HBV-RNA into DNA. However, although we detected a tendency to greater quasispecies complexity in HBV-DNA than in RNA, the differences were statistically non-significant, which may indicate an increased presence of low-frequency HBV genomes in DNA quasispecies. For this reason, we suggest that further studies in larger groups of patients be performed to confirm this observation. We also found a highly coincident conservation between HBV-RNA and DNA quasispecies in the HBX 5’ region. Interestingly, HBV-RNA quasispecies showed slightly higher variability than HBV-DNA quasispecies, which may indicate that only a fraction of packaged pgRNA is reverse-transcribed and released. The hyper-conserved sequences identified were highly coincident to what was observed in previous studies by our group in HBV-DNA quasispecies. Nevertheless, we observed that some sequence fragments were differently conserved between HBV-DNA and RNA quasispecies, which suggest that serum HBV-RNA quasispecies should also be considered when looking for targets for directed gene therapy, specially taking into account the similarities between serum and intrahepatic HBV-RNA.

Research perspectives

In this study, data were obtained from patients with a broad range of HBV genotypes and degrees of severity of liver disease. This allowed us to make a preliminary comparison of complexity and conservation of HBV-DNA and RNA quasispecies. However, further studies with a larger sample size are warranted to confirm our results. The main goal of the methodology for HBV-RNA quasispecies described here is to use it in groups of patients where HBV-DNA levels are usually too low for PCR amplification (e.g., patients under NA treatment, since the generation of HBV-RNA is not inhibited by NA directly), in order to be able to monitor HBV quasispecies in those patients.