Elsevier

Journal of Hepatology

Volume 68, Issue 1, January 2018, Pages 16-24
Journal of Hepatology

Research Article
Relationship between serum HBV-RNA levels and intrahepatic viral as well as histologic activity markers in entecavir-treated patients

https://doi.org/10.1016/j.jhep.2017.08.021Get rights and content

Highlights

  • Detectable HBV-RNA in serum of NUC successfully treated patients.

  • Serum HBV-RNA is indicative of the transcriptional activity of cccDNA.

  • Quasispecies of serum HBV-RNA and intrahepatic HBV-RNA are similar.

  • Levels of HBV-RNA correlate with histopathological scores.

Background & Aims

In diagnostics, serum hepatitis B virus (HBV)-RNA levels are valuable when the HBV-DNA load in circulation is effectively suppressed by nucleos(t)ide analogue (NUC) therapy. This study aimed to determine the intrahepatic viral replication activity reflected in serum HBV-RNA and whether HBV-RNA contributes to liver histological changes in patients treated with NUC.

Methods

A cross-sectional set of serum and liver biopsy samples was obtained from patients treated with entecavir, who had undetectable levels of serum HBV-DNA. The correlations between serum HBV-RNA concentration and levels of peripheral and intrahepatic viral replicative forms, as well as histological scores, were analyzed. Quasispecies of serum HBV-RNA and intrahepatic viral replicative forms were examined by deep sequencing. HBV-RNA-positive hepatocytes were visualized by in situ hybridization.

Results

Serum HBV-RNA was detected in 35 of 47 patients (74.47%, 2.33–4.80 log10 copies/ml). These levels correlated not only with the intrahepatic HBV-RNA level and the ratio of intrahepatic HBV-RNA to covalently closed circular DNA (cccDNA), but also with the histological scores for grading and staging. Regarding quasispecies, serum HBV-RNA was dynamic and more genetically homogenous to simultaneously sampled intrahepatic HBV-RNA than to the cccDNA pool. In situ histology revealed that HBV-RNA–positive hepatocytes were clustered in foci, sporadically distributed across the lobules, and co-localized with hepatitis B surface antigen.

Conclusion

Serum HBV-RNA levels reflect intrahepatic viral transcriptional activity and are associated with liver histopathology in patients receiving NUC therapy. Our study sheds light on the nature of HBV-RNA in the pathogenesis of chronic HBV infection and has implications for the management of chronic hepatitis B during NUC therapy.

Lay summary

Serum HBV-RNA levels are indicative of the intrahepatic transcriptional activity of covalently closed circular DNA and are associated with liver histological changes in patients with chronic B hepatitis who are receiving nucleos(t)ide analogue therapy.

Introduction

Hepatitis B virus (HBV) infection is associated with significant morbidity and mortality. At present, 240 million people are estimated to have chronic hepatitis B (CHB) infection worldwide.[1], [2] Considering its prevalence, monitoring viral replication activity is a vital part of the clinical management of CHB infection. Thus far, serum HBV-DNA load is the only licensed laboratory indicator that directly indicates the quantity of circulating viral particles, thereby reflecting the activity of viral production in the liver.[3], [4] Patients with higher levels of serum HBV-DNA are more susceptible to advanced liver disease than those with lower levels of HBV-DNA.[5], [6] Fortunately, with the availability of nucleos(t)ide analogues (NUCs), serum HBV-DNA levels can be potently suppressed to levels below the detection limits of sensitive PCR assays, within months of treatment initiation. Such virologic suppression leads to biochemical normalization, histological improvement, and prevention of complications, such as cirrhosis and hepatocellular carcinoma.[7], [8]

As polymerase inhibitors, NUCs can only curb HBV replication at reverse transcription; viral RNA transcription and protein translation machineries remain intact.9 Intrahepatic viral nucleic acids, such as total HBV-DNA and covalently closed circular DNA (cccDNA), persist even after prolonged NUC treatment.10 Therefore, NUCs are not capable of eradicating the virus from the liver reservoir. Owing to the low levels of intrahepatic viral products and residual ongoing viral replication, patients treated with NUCs continue to have higher mortality than the general population, with some of these patients progressing to end-stage liver disease.[7], [11], [12], [13], [14]

Apart from HBV-DNA and hepatitis B surface antigen (HBsAg), HBV-RNA containing virus-like particles represent an additional serum medium for the detection of viral replicative intermediates produced in the liver.[15], [16], [17] HBV-RNA is valuable in diagnostics because it carries genetic information and its quantity assessment is not influenced by immune complexes of antibodies and viral antigens. In the era of NUC therapy, the detection of serum HBV-RNA levels instead of HBV-DNA levels is more useful for predicting treatment response,[17], [18], [19] guiding discontinuation of therapy,16 and monitoring the emergence of viral mutations.[20], [21] However, whether the level of serum HBV-RNA correlates with the levels of intrahepatic HBV-RNA or other viral replicative intermediates remains unknown.

The cytopathic effects or immunopathology caused by HBV-RNA accumulation could be the driving force of liver disease progression in patients treated with NUC. The 5′-ε region of HBV pre-genomic RNA (pgRNA) induces the production of interferons and inflammatory cytokines in hepatocytes, which may lead to histological changes in patients receiving NUC therapy.22 In addition, the immune responses against viral proteins translated from HBV-RNA, albeit at a low level, are a coherent impetus for pathogenesis of CHB.

In this study, we aimed to determine whether serum HBV-RNA levels are correlated with the levels of intrahepatic viral replicative forms and the severity of histological necroinflammation and fibrosis in patients treated with NUC.

Section snippets

Patients and samples

A cross-sectional set of 47 patients with CHB infection who had received entecavir monotherapy for >1 year were included in this study (Table 1). A total of 25 patients were HB envelope antigen (HBeAg) positive and 22 patients were HBeAg negative. The study protocol was approved by the Ethical Committee of Huashan Hospital and was carried out in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. Serum samples were obtained from all patients

Levels of serum HBV-RNA and intrahepatic replicative forms during entecavir therapy

We examined the levels of serum and intrahepatic HBV-RNA in successfully treated patients. First, in seven patients with longitudinal consecutive samples taken from before treatment to 48 weeks after treatment, the HBV-RNA level dramatically declined within 4–12 weeks of entecavir treatment, because of suppression of viral replication by entecavir. Incomplete viral clearance by active immune responses against HBV products meant that the levels subsequently stabilized and slowly decreased (Fig. 1

Discussion

In this study, we have shown that HBV-RNA levels in circulation reflect their intrahepatic counterparts in terms of the amount and composition of quasispecies. For a proportion of patients treated with NUC in whom overwhelming active viral replication was effectively suppressed, HBV-RNA remained detectable in the serum and was correlated with intrahepatic HBV-RNA level, but not with cccDNA level. In addition, serum HBV-RNA levels were correlated with the ratio of the intrahepatic HBV-RNA to

Financial support

This work was supported by National Natural Science Foundation of China Grant (81373064, 81571981, 81670560), National Grand Program on Key Infectious Disease Control (2017ZX10302201-004-004), Specialized Project on Scientific Research within the health care industry of China (201302010), Chinese Foundation for Hepatitis Prevention and Control-TianQing Liver Disease Research Fund Subjects (TQGB20140064), Second Scientific Project of Yinzhou District of Ningbo (2014, No. 57).

Conflict of interest

All authors have no conflict of interest to disclose.

Please refer to the accompanying ICMJE disclosure forms for further details.

Authors’ contributions

Study concept and design: Wenhong Zhang, Jing Wang, Chao Qiu. Performing the experiments: Jing Wang, Chao Qiu, Guojun Li, Yanhong Jia, Xiao Zhang, Mengqi Zhu, Jiangjiang Zheng, Zhangzhang Song, Shaolong Chen, Jing Wu, Peiyu Qian, Xiaona Mao. Recruiting Patients and colleting specimens: Jing Wang, Guojun Li, Jianming Zheng, Chuan Shen, Lingyun Shao, Yuxian Huang, Caiyan Zhao, Jiming Zhang, Wenhong Zhang. Statistical analysis: Jing Wang, Chao Qiu, Yiqi Yu, Zhefeng Meng, Shaolong Chen, Chuan Shen,

Acknowledgments

We thank all of the study participants for their donation of blood samples and liver biopsy specimens. This work was supported by the National Natural Science Foundation of China Grant (81373064, 81571981, 81670560), National Grand Program on Key Infectious Disease Control (2017ZX10302201-004-004), Specialized Project on Scientific Research within the health care industry of China (201302010), Chinese Foundation for Hepatitis Prevention and Control-TianQing Liver Disease Research Fund Subjects

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