A standardized assay for the quantitative detection of serum HBV RNA in chronic hepatitis B patients

Abstract

Serum hepatitis B virus (HBV) pregenomic RNA (pgRNA) is a surrogate marker for reflecting the transcriptional activity of covalently closed circular DNA. However, there is still no standardized assay for the quantitative detection of serum HBV RNA in chronic hepatitis B patients. In this study, quantitative polymerase chain reactions for detecting the preC/C-RNA (preC/C region HBV pgRNA), SF-RNA (splicing variants-free pgRNA) and XR-RNA (X region remained pgRNA) regions were set up. The dynamic changes of serum pgRNA splicing variants and 3' terminal truncations were analysed in three retrospective cohorts: 35 treatment-naive chronic HBV-infected patients (cohort A), 52 chronic hepatitis B (CHB) patients who received nucleos(t)ide analogs (NAs) therapy for 48 weeks (cohort B) and eight CHB patients who are under long-term NAs treatment (cohort C). The accuracy and sensitivity of HBV RNA detection were assessed by the National Standard of HBV RNA. We confirmed that high proportions of pgRNA splicing variants and 3' terminal truncations were present and significantly affect the quantitative detection of serum HBV RNA in both treatment-naive and NAs-treated CHB patients. To achieve the higher accuracy and sensitivity on the detection of HBV RNA level, the primers and probes should be designed at the 5' terminal region of HBV genome and outside the mainly spliced sequence of pgRNA, especially for CHB patients under long-term NAs treatment. This study would help to better understand the significance of the pgRNA splicing variants and 3' terminal truncations, and further guide the clinical detection of serum HBV RNA.

Keywords: 3′ terminal truncations; HBV RNA; nucleos(t)ide analogs; quantitative detection; splicing variants.

Figure 1.

The accuracy and sensitivity of HBV RNA detection at preC/C-RNA, SF-RNA and XR-RNA regions were assessed by the National Standard of HBV RNA. (A) The diagram of HBV pgRNA and the relative locations of preC/C-RNA, SF-RNA and XR-RNA regions targeted by qPCR primers. SP: pgRNA splicing variant; *SP1-3 were the pgRNA splicing variants identified in our previous study.²⁶ (B) The accuracy on the detection of HBV RNA at preC/C-RNA, SF-RNA and XR-RNA regions was assessed by HBV RNA standards. (C) The sensitivity on the detection of HBV RNA at preC/C-RNA, SF-RNA and XR-RNA regions was precisely analysed by HBV RNA standards. The lowest concentration of 95% positive was considered as the LLOD.

Figure 2.

Comparative analysis of HBV RNA levels detected at preC/C-RNA, SF-RNA and XR-RNA regions in the sera of treatment-naïve chronic HBV-infected individuals. (A) The levels of serum HBV RNA in the sera of 35 treatment-naïve chronic HBV-infected individuals were detected at preC/C-RNA, SF-RNA and XR-RNA regions. (B) The levels of HBV RNA between HBeAg-positive (n = 20) and HBeAg-negative groups (n = 15) were detected at preC/C-RNA, SF-RNA and XR-RNA regions. (C) The bias of serum HBV RNA levels detected at preC/C-RNA and SF-RNA, preC/C-RNA and XR-RNA regions were analysed by Bland Altman plot method. (D) Serum HBV RNA was reverse-transcribed and amplified by nested-PCR. The PCR products were analysed by 1.0% agarose gel electrophoresis. (E) Sequencing analysis of SP1. Statistical significance shown as the p value was evaluated by a One-way ANOVA with Bonferroni’s multiple comparisons and Paired Mann–Whitney U test. eff%: amplification efficiency.

Figure 3.

Comparative analysis of HBV RNA levels detected at preC/C-RNA, SF-RNA and XR-RNA regions in the sera of CHB patients under NAs treatment. (A) The levels of HBV RNA were quantitatively detected at preC/C-RNA, SF-RNA and XR-RNA regions in the sera of CHB patients (n = 52) under NAs treatment for 48 weeks. (B) The bias of serum HBV RNA levels detected at preC/C-RNA and SF-RNA regions were analysed by Bland Altman plot method. (C) The bias of serum HBV RNA levels detected at preC/C-RNA and XR-RNA regions were analysed by Bland Altman plot method. The mean difference was given (y axis) for the mean results (x axis). (D) The levels of HBV RNA were quantitatively detected at preC/C-RNA, SF-RNA and XR-RNA regions in rapid decline group and (E) slow decline group. (F) The levels of HBV RNA were detected at preC/C-RNA, SF-RNA and XR-RNA regions in the sera of CHB patients (n = 8) under NAs treatment for 5 years. (G) The levels of serum HBV RNA in each CHB patient under NAs treatment for 5 years were quantitatively detected at preC/C-RNA, SF-RNA and XR-RNA regions. The white block represented the level was lower than LLOD. Statistical significance shown as the p value was evaluated by a Paired Mann–Whitney U test.

Figure 4.

Analysis on the reverse transcription efficiency between pgRNA splicing variants and unspliced pgRNA. (A) HBV RNA in the sera of two CHB patients was reverse-transcribed and amplified by nested-PCR at baseline and 12 weeks post NAs treatment. The PCR products were analysed by 1.0% agarose gel electrophoresis. (B) The gray value analysis on the levels of unspliced pgRNA and SP1 with ImageJ Software. To avoid the difference in band strength due to the difference of amplification efficiency, the comparisons of fold changes between 0 and 12 weeks were conducted in the same sets. (C) The proportions of intracellular 3.5 kb HBV RNA splicing variants, extracellular pgRNA splicing variants and HBV DNA splicing variants were analysed in Huh-7 cells transfected with prcccDNA plasmids and (D) HepAD38 cells. (E) The ratio of splicing variants in serum HBV DNA and RNA in 7 CHB patients of cohort A. SP1: pgRNA splicing variant 1. Statistical significance shown as the p value was evaluated by a Paired Mann–Whitney U test.