Hepatitis B virus serum RNA transcript isoform composition and proportion in chronic hepatitis B patients by nanopore long-read sequencing

Abstract

IntroductionSerum hepatitis B virus (HBV) RNA is a promising new biomarker to manage and predict clinical outcomes of chronic hepatitis B (CHB) infection. However, the HBV serum transcriptome within encapsidated particles, which is the biomarker analyte measured in serum, remains poorly characterized. This study aimed to evaluate serum HBV RNA transcript composition and proportionality by PCR-cDNA nanopore sequencing of samples from CHB patients having varied HBV genotype (gt, A to F) and HBeAg status.MethodsLongitudinal specimens from 3 individuals during and following pregnancy (approximately 7 months between time points) were also investigated. HBV RNA extracted from 16 serum samples obtained from 13 patients (73.3% female, 84.6% Asian) was sequenced and serum HBV RNA isoform detection and quantification were performed using three bioinformatic workflows; FLAIR, RATTLE, and a GraphMap-based workflow within the Galaxy application. A spike-in RNA variant (SIRV) control mix was used to assess run quality and coverage. The proportionality of transcript isoforms was based on total HBV reads determined by each workflow.ResultsAll chosen isoform detection workflows showed high agreement in transcript proportionality and composition for most samples. HBV pregenomic RNA (pgRNA) was the most frequently observed transcript isoform (93.8% of patient samples), while other detected transcripts included pgRNA spliced variants, 3′ truncated variants and HBx mRNA, depending on the isoform detection method. Spliced variants of pgRNA were primarily observed in HBV gtB, C, E, or F-infected patients, with the Sp1 spliced variant detected most frequently. Twelve other pgRNA spliced variant transcripts were identified, including 3 previously unidentified transcripts, although spliced isoform identification was very dependent on the workflow used to analyze sequence data. Longitudinal sampling among pregnant and post-partum antiviral-treated individuals showed increasing proportions of 3′ truncated pgRNA variants over time.ConclusionsThis study demonstrated long-read sequencing as a promising tool for the characterization of the serum HBV transcriptome. However, further studies are needed to better understand how serum HBV RNA isoform type and proportion are linked to CHB disease progression and antiviral treatment response.

Biogenesis of serum HBV RNA and clinical phenomena of serum HBV RNA in chronic hepatitis B patients before and after receiving nucleos(t)ide analogues therapy

“…Previously, serum HBV RNA has been characterized as pregenomic RNA (pgRNA), but more species have been demonstrated in recent years. Serum HBV RNA consists of full-length pgRNA, internally truncated pgRNA, [7][8][9][10][11] 3′ terminally truncated pgRNA, [12][13][14][15] various spliced pgRNA, 10,[13][14][15][16][17][18] subgenomic HBV RNA as well as HBV-human chimeric RNA (Figure 1). (1) Full-length and internally truncated pgRNA: pgRNA was the majority of serum HBV RNA, which was demonstrated through clonal sequencing and Northern blot.…”

“…These discrepancies likely derive from the variations of host factors or HBV genome, as studies reported that the generation of different spliced pgRNA variants during HBV infection was partly genotype-related. 14,[27][28][29] It would be interesting to determine whether serum HBV RNA was spliced differently from intrahepatic HBV RNA in the same individual; (4) subgenomic HBV RNA: circulating hepatitis B virus X protein (HBx) RNA was also observed in CHB patients, 14,18,30 but the subgenomic HBV RNA such as HBx RNA, if any, only represent the minority of total extracellular HBV RNA 10,14,18 ; (5) HBV-human chimeric RNA: recently, the presence of HBV-human chimeric RNA was reported in serum samples of CHB patients from integrated HBV DNA, including 5'-HBV-human-3' and 5'-human-HBV-3' transcripts accounting for a minimal proportion of serum HBV RNA. 10,31 Previous studies found that some HBV-human chimeric RNA could be detected in HBV-associated HCC tumour tissues which can promote hepatocarcinogenesis by functioning as a long noncoding RNA and depleting cellular miR-122.…”

Mechanisms of Hepatitis B Virus cccDNA and Minichromosome Formation and HBV Gene Transcription

“…This overlapping nature of HBV mRNAs, together with the high diversity of other non-canonical mRNAs produced during HBV infection, has been a bottleneck to studying the expression profiles of different HBV mRNA expressed during infection. In this context, long-read RNA sequencing has emerged as a promising tool for the characterization of the HBV transcriptome [ 46 , 47 ].…”