Potential use of serum HBV RNA in antiviral therapy for chronic hepatitis B in the era of nucleos(t)ide analogs

Abstract

Although the efficacy of nucleos(t)ide analogue (NA) has been confirmed for treatment of chronic hepatitis B, long-term therapy has been recommended due to the high frequency of off-therapy viral DNA rebound and disease relapse. In this review, the RNA virion-like particles of hepatitis B virus (HBV) are integrated into the life cycle of HBV replication, and the potential significance of serum HBV RNA is systematically described. The production of HBV RNA virion-like particles should not be blocked by NA; in this regard, serum HBV RNA is found to be a suitable surrogate marker for the activity of intrahepatic covalently closed circular DNA (cccDNA), particularly among patients receiving NA therapy. Therefore, the concept of virological response is redefined as persistent loss of serum HBV DNA and HBV RNA. In contrast to hepatitis B surface antigen (HBsAg) that can originate from either the cccDNA or the integrated HBV DNA fragment, serum HBV RNA, with pregenomic RNA origination, can only be transcribed from cccDNA. Therefore, the loss of serum HBV RNA would likely be a promising predicator for safe drug discontinuation. The clinical status of consistent loss of serum HBV RNA accompanied with low serum HBsAg levels might be implicated as a “para-functional cure,” a status nearly close to the functional cure of chronic hepatitis B, to distinguish the “functional cure” characterized as serum HBsAg loss with or without anti-HBs seroconversion.

Keywords

chronic hepatitis B / serum HBV RNA / nucleos(t)ide analogs / virological response / para-functional cure

Introduction

Hepatitis B virus (HBV), the causative factor of chronic hepatitis B (CHB), belongs to the Hepadnaviridae family. Given that an estimated number of 650 000 people die annually from CHB and relevant diseases, including cirrhosis and hepatocellular carcinoma, chronic HBV infection remains the 10th leading cause of human death [1–3]. The eradication of covalently closed circular DNA (cccDNA) has been suggested to be the therapeutic goal in chronic HBV infection because the persistence of cccDNA in infected hepatocytes causes off-therapy DNA rebound and makes CHB difficult to cure [4]. The two kinds of antiviral agents used for treatment of CHB are pegylated interferon (Peg-IFN-α) and nucleos(t)ide analogue (NA); however, they do not directly target cccDNA [5,6]. Although NA have been confirmed to be highly effective for treatment of CHB, long-term or even life-long NA treatment has been recommended in almost all guidelines for the management of CHB to prevent viral rebound and disease relapse [7–10]. However, poor compliance and increased risk of side effects are the challenges of life-long NA therapy.

The recent discovery of HBV RNA virion-like particles completes the life cycle of HBV infection [11,12], offering an opportunity to rethink the nature of the real viral response. Why do off-therapy HBV DNA rebound and disease relapse frequently occur after consolidation therapy? Does undetectable serum HBV DNA reflect the true virological response (VR) to antiviral therapy, particularly in patients receiving NA therapy? What is the real sustained virological response (SVR)?

In this review, suggestions on redefining VR and SVR were put forward, and the concept and potential significance of “para-functional cure” were elaborated.

Discovery and potential clinical significance of HBV RNA virion-like particle

More than 20 years ago, in addition to HBV DNA, HBV RNA was also found in the serum of chronic HBV-infected individuals [13]. Several groups found that serum HBV RNA could be used as a new marker for monitoring the efficacies of NA and interferon therapy [11,14–18], a predictor of early emergence of the YMDD mutant in patients treated with lamivudine [19], and a useful marker for the safe discontinuation of NA treatments [12,20]. However, the nature of serum HBV RNA has not been sufficiently investigated until recently. First, serum HBV RNA was confirmed to be encapsidated and enveloped because it could be pulled down by hepatitis B core protein (HBcAg)-specific antibodies, and its level increased after removing the HBV envelope [11]. Moreover, using Northern blot and 5′ rapid amplification of cDNA ends, we confirmed that serum HBV RNA was the HBV pregenomic RNA (pgRNA) and was present in the virion-like particles, which was validated by sucrose density gradient centrifugation and electron microscopy assays [12]. Accordingly, serum HBV RNA was present in HBV RNA virion-like particles.

HBV RNA virion-like particle supplemented the HBV life cycle

With an enveloped 3.2-kb-long relaxed circular DNA (rcDNA) genome, HBV is traditionally classified as a DNA virus [21]. As shown in Fig. 1, HBV virions enter hepatocytes through a high-affinity interaction between the myristoylated preS1 region of HBV and a functional receptor, sodium taurocholate co-transporting polypeptide [22]. During entry, a low-affinity interaction between the major hydrophilic region of viral small S protein and heparan sulfate proteoglycan is also required for infectivity [23,24]. After entering into hepatocytes, rcDNA translocates into the nucleus where it is converted to cccDNA. The cccDNA persists as a minichromosome and serves as the template for the transcription of five viral mRNAs. Among them, the 3.5-kb-long pgRNA also serves as the template for the reverse transcription of HBV DNA minus strand. The binding of HBV DNA polymerase to pgRNA provides the packaging signal and subsequently initiates the assembly of viral capsid using core proteins. Next, the encapsidated pgRNA undergoes reverse transcription to produce the minus strand of viral DNA, and the plus strand is incompletely synthesized from the minus strand [25]. The newly synthesized rcDNAs are enveloped by the viral surface proteins and released as Dane particles or re-enter the nucleus to replenish the cccDNA pool [26,27]. Except for rcDNA, the double-stranded linear DNA (DSL-DNA), a replicative intermediate, can also be generated when failing to translocate the RNA primer needed to prime the plus-stranded DNA synthesis [28]. The DSL-DNA is often integrated into the host genome through recombination using host enzymes [29]. Consequently, the integrated viral DNA fragments frequently end in the DR-1/DR-2 regions of the viral genome [30,31]. Theoretically, the 3.5-kb-long pgRNA can only be produced from the circular cccDNA. By contrast, because the open reading frame of the S gene with its regulatory elements remains intact in the integrated sequences, hepatitis B surface antigen (HBsAg) can also be produced from the integrated HBV DNA fragments [24,32,33]. Therefore, HBsAg has two sources: cccDNA and integrated HBV DNA (Fig. 1).

As previous studies have already suggested that nucleocapsid maturation may not be dependent on DNA synthesis [34–37], the pgRNA containing nucleocapsids may also be enveloped and released via multivesicular bodies. The discovery of HBV RNA virion-like particle provides a supplementary to the traditional HBV replication cycle (Fig. 1). The infection of hepatitis D virus (HDV) is mediated by HBV envelope proteins [38,39]. Theoretically, after entry into hepatocytes, the encapsidated pgRNA might restart its reverse transcription to form rcDNA and cccDNA and subsequently establish HBV infection. The infection potential of HBV RNA virion-like particles needs experimental evidences.

Para-functional cure of CHB

In recent years, studies have revealed that the half-life of cccDNA is less than two months [40,41]. Obviously, the replenishment of the cccDNA pool through either an extracellular or intracellular (recycling) way, or both, is essential for persistent viral infection. Thus, it is reasonable to postulate that though they do not directly act on cccDNA, via blocking rcDNA formation, NA can efficiently inhibit the replenishment of cccDNA pool [42,43]. Theoretically, the eventual eradication of cccDNA would be expectable after prolonged consolidation NA therapy. Indeed, a number of clinical studies have verified the significant decrease of cccDNA level in CHB patients after receiving long-term NA therapies [44–46].

Given that complete decay of cccDNA means the elimination of viral infection, a status nearly close to the complete cure of CHB [47], to avoid viral DNA rebound, guaranteeing the silence or elimination of intrahepatic cccDNA before suspending NA treatment is imperative. However, restricted by invasive liver biopsy, regular detection of intrahepatic cccDNA is not feasible in clinical practice [48,49]. Different from the frequent occurrence of off-therapy HBV DNA rebound and disease relapse when the consistent loss of serum HBV DNA is used as a pre-requirement for NA withdrawal, relapse is quite uncommon for patients who had already reached HBsAg loss with or without anti-HBs seroconversion. Therefore, “loss of serum HBsAg” has been recommended as an ideal endpoint of therapy. Unfortunately, with the currently available anti-HBV agents, the loss of serum HBsAg is difficult to achieve. A recent study reported that the rate and durability of HBsAg seroclearance induced by NA are comparable to those developed spontaneously [50]. As a consequence, most patients would have to receive life-long NA therapy, leading to problems such as drug resistance, adverse events, adherence, and expenses.

“Loss of serum HBsAg” has been recommended to be an effective endpoint of antiviral treatment for CHB patients, in whom antiviral treatment could be safely discontinued. On the other hand, a part of NA-treated patients could safely discontinue NA therapy after consolidation therapy, even though their serum HBsAg remains positive [51,52]. A recent study has demonstrated that the correlation of serum HBsAg with cccDNA is not found in CHB patients who have received NA therapy [53]. In this study, though intrahepatic cccDNAs were undetectable in 49% (21/43) of CHB patients, only one patient achieved serum HBsAg loss. Moreover, a majority of the biopsies were negative for viral core protein and cccDNA, which strongly indicated that intrahepatic cccDNA might be silenced or exhausted after receiving long-term NA therapy.

The following question comes into mind: where does HBsAg come from in those patients whose cccDNA reservoirs were either exhausted or transcriptionally silenced? Given the fact that the integrated HBV DNA fragments were frequently detected in HBV-infected hepatocytes [30,32,54], the integrated HBV DNA could act as a cellular gene to produce viral protein HBsAg [24,33,55,56]. All these observations indicated that, after a prolonged consolidation NA therapy, even after cccDNA had been eliminated or epigenetically silenced, serum HBsAg could remain at low levels in a small but certain fraction of patients. This is because serum HBsAg might originate from integrated HBV DNA fragments. The term “para-functional cure” is suggested to describe such a clinical status of potential elimination or transcriptional silencing of cccDNA defined by persistent loss of serum HBV RNA [57]. Different from the already used terms such as “functional cure” or “clinical cure” in which HBsAg disappeared, a patient achieving “para-functional cure” could be positive for low-level serum HBsAg. However, though the status of “para-functional cure” is theoretically close to “functional cure” and might greatly lower the risk of NA off-therapy disease relapse, it must be emphasized that this is not an ideal endpoint of treatment because patients who achieve “functional cure” characterized by serum HBsAg loss would have a favorable clinical course and very low risk for hepatocellular carcinoma [58,59].

Redefining the virological response of CHB patients receiving NA therapy

To assess the VR for NA therapy, monitoring the dynamic change of a patient’s serum HBV DNA levels during therapy is important. Nowadays, VR has been defined as undetectable serum HBV DNA during therapy, and achieving VR (serum HBV DNA below the lower limit of detection by a sensitive PCR-based assay) is one of the prerequisites for discontinuation of NA therapy in clinical practice guidelines [7–10]. However, off-therapy virological rebound and hepatitis relapse still occur at high frequency, even after prolonged therapy to consolidate the constant suppression of viral DNA replication. Even worse, disease progression occasionally occurs to patients whose serum HBV DNA remains undetectable. As mentioned above, the presence and transcription of cccDNA were not directly affected by NA. Thus, the detection of serum HBV DNA would not comprehensively reflect the activity of cccDNA in patients under NA therapy. Therefore, understanding why frequent off-therapy viral rebound and CHB disease relapse occur is not difficult when only serum HBV DNA loss is adopted as a prerequisite for discontinuation of NA therapy.

As shown in Fig. 1, HBV RNA virion-like particle is confirmed to be an additional by-product of HBV viral replication. Theoretically, the formation of rcDNA, but not the production of HBV RNA virion-like particle, is directly blocked by NA [12,20]. Therefore, under NA treatment, serum HBV RNA may better reflect the activity of intrahepatic cccDNA than serum HBV DNA [20,57,60]. Based on the discoveries mentioned above, instead of solely measuring serum HBV DNA, simultaneously monitoring serum HBV DNA and HBV RNA for CHB patients under NA therapy may better reflect whether true VR is being achieved or not. Accordingly, VR should be redefined as persistent loss (below the lower limit of detection by a sensitive PCR-based assay) of HBV DNA and HBV RNA.

Suggestion on the safe discontinuation of NA treatment

According to current clinical practice with loss of serum HBsAg as treatment goal for CHB management, only 2%–13% of CHB patients can attain functional cure with the current antiviral therapy roadmap [7–10]. The finding means that most patients will have to receive life-long NA therapy, and the cost will be a considerable burden to patients and society. As serum HBV RNA is a potential indicator of cccDNA activity, the loss of serum HBV RNA might indicate exhausting or transcription silencing of cccDNA reservoirs [57,60]. Moreover, serum HBV RNA can be used as a potential marker for the safe discontinuation of NA treatments [12,20]. Therefore, as shown in Table 1, in addition to serum HBsAg level, serum HBV RNA should be monitored simultaneously during consolidation NA therapy. In HBeAg-positive CHB patients without liver cirrhosis, safe discontinuation of NA therapy might be recommended to those who have reached HBeAg seroconversion and have persistently undetectable serum HBV DNA and HBV RNA during consolidation therapy and persistently normal ALT levels. In HBeAg-negative CHB patients without liver cirrhosis, discontinuation of NA therapy might be done to those with persistently undetectable serum HBV DNA and HBV RNA and persistently normal ALT levels. However, serum HBsAg could be positive in HBeAg-positive and HBeAg-negative patients at low levels. As low HBsAg is correlated with low risk of off-therapy viral DNA rebound and disease relapse, and the chance of HBsAg loss would be increased in CHB patients with HBsAg<1500 IU/mL at the time of switching from NA to Peg-IFN-α therapy [61,62], HBsAg level less than 1500 IU/mL is suggested in Table 1.

Recently, HBcrAg and empty virions have also been reported to be potential surrogate markers used to reflect intrahepatic cccDNA activity [63,64]. How to integrate these new markers and HBsAg to monitor VR, predict disease progression, and guide safe discontinuation of NA treatment need to be studied in the future.

Conclusions

In this review, the life cycle of HBV replication has been supplemented with HBV RNA virion-like particles, and the definition of HBV DNA-based VR has been modified by combined monitoring of serum HBV DNA and HBV RNA. Based on the suggested novel concept of “para-functional cure,” “persistent loss of serum HBV RNA” has been put forward as an indicator for safe discontinuation of NA therapy to patients who finished consolidation NA therapy. However, a multi-centered and large-scale cohort study should be conducted to testify the feasibility of safe withdrawal among CHB patients receiving consolidation NA therapy in the future.

References

[1] Trépo C, Chan HL, Lok A. Hepatitis B virus infection. Lancet 2014; 384(9959): 2053–2063

[2] WHO guidelines for the prevention, care and treatment of persons with chronic hepatitis B infection. Geneva: World Health Organization, 2015. Available from: URL: 

[3] Lu FM, Zhuang H. Management of hepatitis B in China. Chin Med J (Engl) 2009; 122(1): 3–4

[4] Petersen J, Thompson AJ, Levrero M. Aiming for cure in HBV and HDV infection. J Hepatol 2016; 65(4): 835–848

[5] Lau GK, Piratvisuth T, Luo KX, Marcellin P, Thongsawat S, Cooksley G, Gane E, Fried MW, Chow WC, Paik SW, Chang WY, Berg T, Flisiak R, McCloud P, Pluck N; Peginterferon Alfa-2a HBeAg-Positive Chronic Hepatitis B Study Group. Peginterferon Alfa-2a, lamivudine, and the combination for HBeAg-positive chronic hepatitis B. N Engl J Med 2005; 352(26): 2682–2695

[6] Wei L, Kao JH. Benefits of long-term therapy with nucleos(t)ide analogues in treatment-naïve patients with chronic hepatitis B. Curr Med Res Opin 2017; 33(3): 495–504

[7] Sarin SK, Kumar M, Lau GK, Abbas Z, Chan HL, Chen CJ, Chen DS, Chen HL, Chen PJ, Chien RN, Dokmeci AK, Gane E, Hou JL, Jafri W, Jia J, Kim JH, Lai CL, Lee HC, Lim SG, Liu CJ, Locarnini S, Al Mahtab M, Mohamed R, Omata M, Park J, Piratvisuth T, Sharma BC, Sollano J, Wang FS, Wei L, Yuen MF, Zheng SS, Kao JH. Asian-Pacific clinical practice guidelines on the management of hepatitis B: a 2015 update. Hepatol Int 2016; 10(1): 1–98

[8] Lampertico P, Agarwal K, Berg T, Buti M, Janssen HLA, Papatheodoridis G, Zoulim F, Tacke F; European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J Hepatol 2017; 67(2): 370–398

[9] Terrault NA, Bzowej NH, Chang KM, Hwang JP, Jonas MM, Murad MH; American Association for the Study of Liver Diseases.AASLD guidelines for treatment of chronic hepatitis B. Hepatology 2016; 63(1): 261–283

[10] Chinese Society of Infectious Diseases, Chinese Medical Association; Hou JL, wei L. The guideline of prevention and treatment for chronic hepatitis B: a 2015 update. Chin J Hepatol (Zhonghua Gan Zang Bing Za Zhi) 2015; 23(12): 888–905 (in Chinese)

[11] Jansen L, Kootstra NA, van Dort KA, Takkenberg RB, Reesink HW, Zaaijer HL. Hepatitis B virus pregenomic RNA is present in virions in plasma and is associated with a response to pegylated interferon alfa-2a and nucleos(t)ide analogues. J Infect Dis 2016; 213(2): 224–232

[12] Wang J, Shen T, Huang X, Kumar GR, Chen X, Zeng Z, Zhang R, Chen R, Li T, Zhang T, Yuan Q, Li PC, Huang Q, Colonno R, Jia J, Hou J, McCrae MA, Gao Z, Ren H, Xia N, Zhuang H, Lu F. Serum hepatitis B virus RNA is encapsidated pregenome RNA that may be associated with persistence of viral infection and rebound. J Hepatol 2016; 65(4): 700–710

[13] Köck J, Theilmann L, Galle P, Schlicht HJ. Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus. Hepatology 1996; 23(3): 405–413 (PubMed: 8617418)

[14] Rokuhara A, Matsumoto A, Tanaka E, Umemura T, Yoshizawa K, Kimura T, Maki N, Kiyosawa K. Hepatitis B virus RNA is measurable in serum and can be a new marker for monitoring lamivudine therapy. J Gastroenterol 2006; 41(8): 785–790

[15] Huang YW, Chayama K, Tsuge M, Takahashi S, Hatakeyama T, Abe H, Hu JT, Liu CJ, Lai MY, Chen DS, Yang SS, Kao JH. Differential effects of interferon and lamivudine on serum HBV RNA inhibition in patients with chronic hepatitis B. Antivir Ther 2010; 15(2): 177–184

[16] Huang YW, Takahashi S, Tsuge M, Chen CL, Wang TC, Abe H, Hu JT, Chen DS, Yang SS, Chayama K, Kao JH. On-treatment low serum HBV RNA level predicts initial virological response in chronic hepatitis B patients receiving nucleoside analogue therapy. Antivir Ther 2015; 20(4): 369–375

[17] van Bömmel F, Bartens A, Mysickova A, Hofmann J, Krüger DH, Berg T, Edelmann A. Serum hepatitis B virus RNA levels as an early predictor of hepatitis B envelope antigen seroconversion during treatment with polymerase inhibitors. Hepatology 2015; 61(1): 66–76

[18] Tsuge M, Chayama K. Availability of monitoring serum HBV DNA plus RNA during nucleot(s)ide analogue therapy. J Gastroenterol 2013; 48(6): 779–780

[19] Hatakeyama T, Noguchi C, Hiraga N, Mori N, Tsuge M, Imamura M, Takahashi S, Kawakami Y, Fujimoto Y, Ochi H, Abe H, Maekawa T, Kawakami H, Yatsuji H, Aisaka Y, Kohno H, Aimitsu S, Chayama K. Serum HBV RNA is a predictor of early emergence of the YMDD mutant in patients treated with lamivudine. Hepatology 2007; 45(5): 1179–1186

[20] Tsuge M, Murakami E, Imamura M, Abe H, Miki D, Hiraga N, Takahashi S, Ochi H, Nelson Hayes C, Ginba H, Matsuyama K, Kawakami H, Chayama K. Serum HBV RNA and HBeAg are useful markers for the safe discontinuation of nucleotide analogue treatments in chronic hepatitis B patients. J Gastroenterol 2013; 48(10): 1188–1204

[21] Summers J, O’Connell A, Millman I. Genome of hepatitis B virus: restriction enzyme cleavage and structure of DNA extracted from Dane particles. Proc Natl Acad Sci USA 1975; 72(11): 4597–4601

[22] Yan H, Zhong G, Xu G, He W, Jing Z, Gao Z, Huang Y, Qi Y, Peng B, Wang H, Fu L, Song M, Chen P, Gao W, Ren B, Sun Y, Cai T, Feng X, Sui J, Li W. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. eLife 2012; 1: e00049

[23] Sureau C, Salisse J. A conformational heparan sulfate binding site essential to infectivity overlaps with the conserved hepatitis B virus a-determinant. Hepatology 2013; 57(3): 985–994

[24] Cornberg M, Wong VW, Locarnini S, Brunetto M, Janssen HL, Chan HL. The role of quantitative hepatitis B surface antigen revisited. J Hepatol 2017; 66(2): 398–411

[25] Yang HC, Kao JH. Persistence of hepatitis B virus covalently closed circular DNA in hepatocytes: molecular mechanisms and clinical significance. Emerg Microbes Infect 2014; 3(9): e64

[26] Tuttleman JS, Pourcel C, Summers J. Formation of the pool of covalently closed circular viral DNA in hepadnavirus-infected cells. Cell 1986; 47(3): 451–460

[27] Zhang YY, Zhang BH, Theele D, Litwin S, Toll E, Summers J. Single-cell analysis of covalently closed circular DNA copy numbers in a hepadnavirus-infected liver. Proc Natl Acad Sci USA 2003; 100(21): 12372–12377

[28] Yang W, Summers J. Illegitimate replication of linear hepadnavirus DNA through nonhomologous recombination. J Virol 1995; 69(7): 4029–4036

[29] Bill CA, Summers J. Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration. Proc Natl Acad Sci USA 2004; 101(30): 11135–11140

[30] Li X, Zhang J, Yang Z, Kang J, Jiang S, Zhang T, Chen T, Li M, Lv Q, Chen X, McCrae MA, Zhuang H, Lu F. The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma. J Hepatol 2014; 60(5): 975–984

[31] Wang HP, Rogler CE. Topoisomerase I-mediated integration of hepadnavirus DNA in vitro. J Virol 1991; 65(5): 2381–2392

[32] Mason WS, Gill US, Litwin S, Zhou Y, Peri S, Pop O, Hong ML, Naik S, Quaglia A, Bertoletti A, Kennedy PT. HBV DNA integration and clonal hepatocyte expansion in chronic hepatitis B patients considered immune tolerant. Gastroenterology 2016; 151(5): 986–998.e4

[33] Rivkina MB, Lunin VG, Mahov AM, Tikchonenko TI, Kukain RA. Nucleotide sequence of integrated hepatitis B virus DNA and human flanking regions in the genome of the PLC/PRF/5 cell line. Gene 1988; 64(2): 285–296

[34] Kaplan PM, Ford EC, Purcell RH, Gerin JL. Demonstration of subpopulations of Dane particles. J Virol 1976; 17(3): 885–893

[35] Sakamoto Y, Yamada G, Mizuno M, Nishihara T, Kinoyama S, Kobayashi T, Takahashi T, Nagashima H. Full and empty particles of hepatitis B virus in hepatocytes from patients with HBsAg-positive chronic active hepatitis. Lab Invest 1983; 48(6): 678–682

[36] Ning X, Nguyen D, Mentzer L, Adams C, Lee H, Ashley R, Hafenstein S, Hu J. Secretion of genome-free hepatitis B virus—single strand blocking model for virion morphogenesis of para-retrovirus. PLoS Pathog 2011; 7(9): e1002255

[37] Luckenbaugh L, Kitrinos KM, Delaney WE 4th, Hu J. Genome-free hepatitis B virion levels in patient sera as a potential marker to monitor response to antiviral therapy. J Viral Hepat 2015; 22(6): 561–570

[38] Littlejohn M, Locarnini S, Yuen L. Origins and evolution of hepatitis B virus and hepatitis D virus. Cold Spring Harb Perspect Med 2016; 6(1): a021360

[39] Huang CR, Lo SJ. Hepatitis D virus infection, replication and cross-talk with the hepatitis B virus. World J Gastroenterol 2014; 20(40): 14589–14597

[40] Li M, Hongyan C, Huaxing Z, Wei L, Daru L. Locked nucleic acid couples with Fok I nucleases to target and cleave hepatitis B virus’s gene in vitro. Yi Chuan 2016; 38(4): 350–359

[41] Zhu Y, Yamamoto T, Cullen J, Saputelli J, Aldrich CE, Miller DS, Litwin S, Furman PA, Jilbert AR, Mason WS. Kinetics of hepadnavirus loss from the liver during inhibition of viral DNA synthesis. J Virol 2001; 75(1): 311–322

[42] Dandri M, Locarnini S. New insight in the pathobiology of hepatitis B virus infection. Gut 2012; 61(Suppl 1): i6–i17

[43] Nguyen T, Locarnini S. Hepatitis: monitoring drug therapy for hepatitis B—a global challenge? Nat Rev Gastroenterol Hepatol 2009; 6(10): 565–567

[44] Werle-Lapostolle B, Bowden S, Locarnini S, Wursthorn K, Petersen J, Lau G, Trepo C, Marcellin P, Goodman Z, Delaney WE 4th, Xiong S, Brosgart CL, Chen SS, Gibbs CS, Zoulim F. Persistence of cccDNA during the natural history of chronic hepatitis B and decline during adefovir dipivoxil therapy. Gastroenterology 2004; 126(7): 1750–1758

[45] Wong DK, Yuen MF, Ngai VW, Fung J, Lai CL. One-year entecavir or lamivudine therapy results in reduction of hepatitis B virus intrahepatic covalently closed circular DNA levels. Antivir Ther 2006; 11(7): 909–916

[46] Wursthorn K, Lutgehetmann M, Dandri M, Volz T, Buggisch P, Zollner B, Longerich T, Schirmacher P, Metzler F, Zankel M, Fischer C, Currie G, Brosgart C, Petersen J. Peginterferon alpha-2b plus adefovir induce strong cccDNA decline and HBsAg reduction in patients with chronic hepatitis B. Hepatology 2006; 44(3): 675–684

[47] Durantel D, Zoulim F. New antiviral targets for innovative treatment concepts for hepatitis B virus and hepatitis delta virus. J Hepatol 2016; 64(1 Suppl): S117–S131

[48] Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut 2015; 64(12): 1972–1984

[49] Petersen J, Thompson AJ, Levrero M. Aiming for cure in HBV and HDV infection. J Hepatol 2016; 65(4): 835–848

[50] Yip TC, Wong VW, Tse YK, Chan HL, Wong GL. Hepatitis B surface antigen seroclearance in a cohort of 154,740 patients with chronic hepatitis B: a 15-year follow-up study. Hepatol Int 2017; 11: S75

[51] Liu ZR, Liu F, Wang L, Liu YD, Zhang M, Li T. Clinical characteristics and outcomes of patients with recurrent chronic hepatitis B after nucleos(t)ide analog withdrawal with stringent cessation criteria: a prospectivecohort study. Hepatol Res 2017; 47(10): 1000–1007

[52] Petersen J, Buggisch P, Hinrichsen H, Berg T, Wedemeyer H, Cornberg M, Stoehr A. Stopping long-term NA-therapy before HBsAg loss in HBeAg negative patients: follow-up of long-term responders. J Hepatol 2013; 58: S313–S314

[53] Lai CL, Wong D, Ip P, Kopaniszen M, Seto WK, Fung J, Huang FY, Lee B, Cullaro G, Chong CK, Wu R, Cheng C, Yuen J, Ngai V, Yuen MF. Reduction of covalently closed circular DNA with long-term nucleos(t)ide analogue treatment in chronic hepatitis B. J Hepatol 2017; 66(2): 275–281

[54] Jiang S, Yang Z, Li W, Li X, Wang Y, Zhang J, Xu C, Chen PJ, Hou J, McCrae MA, Chen X, Zhuang H, Lu F. Re-evaluation of the carcinogenic significance of hepatitis B virus integration in hepatocarcinogenesis. PLoS One 2012; 7(9): e40363

[55] Freitas N, Cunha C, Menne S, Gudima SO. Envelope proteins derived from naturally integrated hepatitis B virus DNA support assembly and release of infectious hepatitis delta virus particles. J Virol 2014; 88(10): 5742–5754

[56] Wooddell CI, Yuen MF, Chan HL, Gish RG, Locarnini SA, Chavez D, Ferrari C, Given BD, Hamilton J, Kanner SB, Lai CL, Lau JYN, Schluep T, Xu Z, Lanford RE, Lewis DL. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci Trans Med 2017; 9(409): eaan0241 160;

[57] Wang J, Du M, Huang H, Chen R, Niu J, Jiang J, Zhuang H, Lu F. Reply to: “Serum HBV pgRNA as a clinical marker for cccDNA activity”: Consistent loss of serum HBV RNA might predict the “para-functional cure” of chronic hepatitis B. J Hepatol 2017; 66(2): 462–463

[58] Yip TC, Chan HL, Wong VW, Tse YK, Lam KL, Wong GL. Impact of age and gender on risk of hepatocellular carcinoma after hepatitis B surface antigen seroclearance. J Hepatol 2017; 67(5): 902–908

[59] Liu J, Yang HI, Lee MH, Lu SN, Jen CL, Batrla-Utermann R, Wang LY, You SL, Hsiao CK, Chen PJ, Chen CJ; R.E.V.E.A.L.-HBV Study Group. Spontaneous seroclearance of hepatitis B seromarkers and subsequent risk of hepatocellular carcinoma. Gut 2014; 63(10): 1648–1657

[60] Giersch K, Allweiss L, Volz T, Dandri M, Lütgehetmann M. Serum HBV pgRNA as a clinical marker for cccDNA activity. J Hepatol 2017; 66(2): 460–462

[61] Hu P, Jia S, Zhang W, Gong G, Li Y, Chen X, Jiang J, Xie Q, Dou X, Sun Y, Li Y, Liu Y, Liu G, Mao D, Chi X, Tang H, Li X, Xie Y, Chen X, Jiang J, Zhao P, Hou J, Gao Z, Fan H, Ding J, Ren H. A multi-center randomized study on the efficacy and safety of switching to peginterferon alpha-2a (40KD) for 48 or 96 weeks in HBeAg positive CHB patients with a prior NUC history for 1 to 3 years: an interim analysis of NEW SWITCH study. Hepatology 2014; 60: 1273A–1274A

[62] Ning Q, Han M, Sun Y, Jiang J, Tan D, Hou J, Tang H, Sheng J, Zhao M. Switching from entecavir to PegIFN alfa-2a in patients with HBeAg-positive chronic hepatitis B: a randomised open-label trial (OSST trial). J Hepatol 2014; 61(4): 777–784

[63] Chen EQ, Feng S, Wang ML, Liang LB, Zhou LY, Du LY, Yan LB, Tao CM, Tang H. Serum hepatitis B core-related antigen is a satisfactory surrogate marker of intrahepatic covalently closed circular DNA in chronic hepatitis B. Sci Rep 2017; 7(1): 173

[64] Hu J, Liu K. Complete and incomplete hepatitis B virus particles: formation, function, and application. Viruses 2017; 9(3): E56

RIGHTS & PERMISSIONS

Higher Education Press and Springer-Verlag GmbH Germany