hepatitis b virus literature review

Chapter 1: Literature Review

• In book: Molecular Characterization of Full Genome HBV sequences from an urban hospital cohort in Pretoria, South Africa [thesis] (pp.1-48)
• Edition: 1st
• Publisher: University of Pretoria

• French National Centre for Scientific Research

Abstract and Figures

Discover the world's research

• 25+ million members
• 160+ million publication pages
• 2.3+ billion citations
• Recruit researchers
• Join for free
• Login Email Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google Welcome back! Please log in. Email · Hint Tip: Most researchers use their institutional email address as their ResearchGate login Password Forgot password? Keep me logged in Log in or Continue with Google No account? Sign up

Loading metrics

Open Access

Peer-reviewed

Research Article

A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: A call for urgent action

Roles Conceptualization, Data curation, Formal analysis, Funding acquisition, Methodology, Project administration, Visualization, Writing – original draft, Writing – review & editing

Affiliation Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom

Roles Data curation, Methodology, Writing – review & editing

Roles Visualization

Affiliation Oxford University Academic IT Department, Oxford, United Kingdom

Roles Writing – review & editing

Affiliation Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom

Affiliation Division of Virology, University of the Free State/National Health Laboratory Service, Bloemfontein, Republic of South Africa

Roles Conceptualization, Formal analysis, Funding acquisition, Methodology, Project administration, Resources, Supervision, Visualization, Writing – review & editing

* E-mail: [email protected]

Affiliations Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom, Department of Microbiology and Infectious Diseases, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Headington, Oxford, United Kingdom

• Jolynne Mokaya, 
• Anna L. McNaughton, 
• Martin J. Hadley, 
• Apostolos Beloukas, 
• Anna-Maria Geretti, 
• Dominique Goedhals, 
• Philippa C. Matthews

• Published: August 6, 2018
• https://doi.org/10.1371/journal.pntd.0006629
• See the preprint
• Reader Comments

International sustainable development goals for the elimination of viral hepatitis as a public health problem by 2030 highlight the pressing need to optimize strategies for prevention, diagnosis and treatment. Selected or transmitted resistance associated mutations (RAMs) and vaccine escape mutations (VEMs) in hepatitis B virus (HBV) may reduce the success of existing treatment and prevention strategies. These issues are particularly pertinent for many settings in Africa where there is high HBV prevalence and co-endemic HIV infection, but lack of robust epidemiological data and limited education, diagnostics and clinical care. The prevalence, distribution and impact of RAMs and VEMs in these populations are neglected in the current literature. We therefore set out to assimilate data for sub-Saharan Africa through a systematic literature review and analysis of published sequence data, and present these in an on-line database ( https://livedataoxford.shinyapps.io/1510659619-3Xkoe2NKkKJ7Drg/ ). The majority of the data were from HIV/HBV coinfected cohorts. The commonest RAM was rtM204I/V, either alone or in combination with associated mutations, and identified in both reportedly treatment-naïve and treatment-experienced adults. We also identified the suite of mutations rtM204V/I + rtL180M + rtV173L, that has been associated with vaccine escape, in over 1/3 of cohorts. Although tenofovir has a high genetic barrier to resistance, it is of concern that emerging data suggest polymorphisms that may be associated with resistance, although the precise clinical impact of these is unknown. Overall, there is an urgent need for improved diagnostic screening, enhanced laboratory assessment of HBV before and during therapy, and sustained roll out of tenofovir in preference to lamivudine alone. Further data are needed in order to inform population and individual approaches to HBV diagnosis, monitoring and therapy in these highly vulnerable settings.

Author summary

The Global Hepatitis Health Sector Strategy is aiming for the elimination of viral hepatitis as a public health threat by 2030. However, mutations associated with drug resistance and vaccine escape may reduce the success of existing treatment and prevention strategies. In the current literature, the prevalence, distribution and impact of hepatitis B virus (HBV) mutations in many settings in Africa are neglected, despite the high prevalence of HBV and co-endemic HIV infection. This systematic review describes the frequency, prevalence and co-occurrence of mutations associated with HBV drug resistance and vaccine escape mutations in Africa. The findings suggest a high prevalence of these mutations in some populations in sub-Saharan Africa. Scarce resources have contributed to the lack of HBV diagnostic screening, inconsistent supply of drugs, and poor access to clinical monitoring, all of which contribute to drug and vaccine resistance. Sustainable long-term investment is required to expand consistent drug and vaccine supply, to provide screening to diagnose infection and to detect drug resistance, and to provide appropriate targeted clinical monitoring for treated patients.

Citation: Mokaya J, McNaughton AL, Hadley MJ, Beloukas A, Geretti A-M, Goedhals D, et al. (2018) A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: A call for urgent action. PLoS Negl Trop Dis 12(8): e0006629. https://doi.org/10.1371/journal.pntd.0006629

Editor: Manuel Schibler, University of Geneva Hospitals, SWITZERLAND

Received: February 8, 2018; Accepted: June 22, 2018; Published: August 6, 2018

Copyright: © 2018 Mokaya et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: JM is funded by a Leverhulme Mandela Rhodes Doctoral Scholarship. PCM is funded by a Wellcome Trust Intermediate Fellowship (grant number 110110), https://wellcome.ac.uk/ . The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

In 2015, the World Health Organisation (WHO) estimated that 3.5% of the world’s population (257 million people) were living with Hepatitis B virus (HBV) infection, resulting in 887,000 deaths each year, mostly from complications including cirrhosis and hepatocellular carcinoma (HCC) [ 1 ]. United Nations Sustainable Development Goals set out the challenge of elimination of viral hepatitis as a public health threat by the year 2030 [ 2 ]. One of the existing strategies in the elimination toolbox is use of antiviral drugs in the form of nucleos(t)ide analogues (NAs). Suppression of viraemia not only reduces inflammatory and fibrotic liver disease in the individual receiving treatment but also reduces the risk of transmission. However, the emergence of HBV resistance-associated mutations (RAMs) is a potentially significant concern for the success of this strategy.

Africa is the continent with the second largest number of individuals with chronic HBV (CHB) infection, with an estimated 6.1% of the adult population infected [ 1 ]. However, there is little commitment and resource invested into the burden of this disease, and many barriers are contributing to the epidemic [ 3 , 4 ]. Globally, less than 10% of the population with CHB are diagnosed, with an even smaller proportion on treatment [ 1 , 4 ]. This proportion is likely to be even lower in Africa. The situation in Africa is further complicated by the substantial public health challenge of coendemic human immunodeficiency virus (HIV) and HBV; coinfection worsens the prognosis in dually infected individuals [ 5 ]. There is also a lack of robust epidemiological data on HBV from Africa [ 3 , 4 ].

Widespread use of antiretroviral therapy (ART) for HIV, incorporating NAs that also have activity against HBV, may have an impact on HBV through improved rates of viraemic suppression, but also potentially by driving the selection of RAMs. The WHO recommends screening for Hepatitis B virus surface antigen (HBsAg) in all HIV-1 infected individuals prior to ART initiation, and for all pregnant women during antenatal visits, to improve the clinical outcomes of people living with CHB and to enhance interventions that reduce the incidence of new cases [ 6 ]. However, screening of HBsAg is not routinely performed in many settings in Africa, with lack of implementation at least partially driven by cost and lack of programmes for HBV treatment outside the setting of HIV coinfection. HBV infected patients either remain untreated (most typical in the setting of monoinfection), or are exposed to antiviral drugs without proper monitoring and often intermittently, putting them at risk of developing RAMs (more likely in the setting of HIV coinfection) [ 4 , 7 – 10 ].

HBV is a DNA virus that replicates via an RNA intermediate, with reverse transcriptase (RT) catalysing the transcription of RNA into DNA [ 7 ]. NAs that inhibit RT are therefore used to prevent HBV replication, including lamivudine (3TC), entecavir (ETV) and tenofovir (conventionally in the form of tenofovir disoproxil fumarate (TDF), but more recently available as the prodrug, tenofovir alafenamide fumarate (TAF)), with mostly historical use of other agents including telbivudine (LdT) and adefovir (ADV) [ 6 , 11 ]. Choice of TDF/TAF or ETV is determined by availability, cost, safety profile and barrier to resistance [ 4 ]. In Africa, the choice of agent is usually limited to 3TC or TDF. Emergence of mutations happens because the RT enzyme is error-prone and lacks the proofreading function required to repair errors during transcription [ 7 ]; when these mutations confer a selective advantage by allowing the virus to escape the effect of drug therapy, they will become amplified in the viral population. Some RAMs confer resistance to one agent only, while others are associated with resistance to several agents ( Fig 1 ).

• PPT PowerPoint slide
• PNG larger image
• TIFF original image

HBV genes are shown in the coloured ovals. TDF = tenofovir, ETV = entecavir, 3TC = lamivudine. This figure incorporates data from eight studies; three were identified by the systematic review presented in this manuscript [ 12 – 14 ] and five from the wider literature [ 7 , 15 – 18 ].

https://doi.org/10.1371/journal.pntd.0006629.g001

3TC was originally seen as a major breakthrough in treating HBV [ 19 ]. However, it is now known to have a low genetic barrier to resistance and its long-term effectiveness is limited as a result of resistance mutations in the ‘YMDD’ motif (tyrosine, methionine, aspartate, aspartate; amino acids 203–206) in domain C of the viral polymerase (Pol). These occur with associated upstream mutations in Pol domains A, B and in the B-C interdomain [ 7 , 15 , 16 ]. Among chronic HBV monoinfected patients, incidence of HBV resistance to 3TC is as high as 20% per year. In HIV/HBV coinfected patients, this can reach 90% over 5 years of treatment, as development of resistance is accelerated in HIV coinfection [ 5 , 20 ]. 3TC has also been associated with the induction of cross-resistance to emtricitabine (FTC), LdT, and at least partially ETV, thus reducing the options for subsequent treatment [ 10 ].

TDF is widely used in treatment of both HIV and HBV and is generally well tolerated. TDF has a high genetic barrier to resistance and maintains effective suppression of HBV in both monoinfected and HIV/HBV coinfected individuals [ 5 , 7 , 10 , 21 , 22 ]. Although it has a recognised association with nephrotoxicity in HIV treatment, current literature suggests it may be better tolerated in HBV infection [ 11 ]. Conversely, African populations have a higher background of renal disease [ 23 ] and could be potentially more vulnerable to nephrotoxitiy from TDF [ 24 ]. TAF delivers equally potent viraemic suppression at lower plasma levels, and is therefore associated with reduced nephrotoxicity [ 25 ], but is not available in Africa at present. HBV resistance to TDF is not well characterised, but there are emerging data from in vitro studies associating Pol mutations rtA194T and rtN236T with decreased susceptibility [ 11 , 21 ]. Virological breakthrough on TDF therapy has been reported in two patients harbouring rtS78T/sC69 mutations [ 17 ], and in another patient with multi-site polymerase mutations; rtL80M, rtL180M, rtM204V/I, rtA200V, rtF221Y, rtS223A, rtT184A/L, rtR153Q, and rtV191I [ 26 ]. The significance of these mutations needs to be further explored in clinical studies.

First line ART treatment regimens for HIV in sSA now almost universally include TDF, and current guidelines also recommend TDF-based regimens in individuals with HBV/HIV coinfection [ 27 ]. Accordingly, in both HIV monoinfection and HBV/HIV coinfection, use of TDF has increased across much of Africa. Nevertheless, it remains the case that 3TC is used as the only HBV-active agent in some settings [ 7 , 8 ], as well as in second line regimens, exemplified by South Africa where second line ART substitutes Zidovudine (AZT) for TDF leaving only 3TC coverage for HBV [ 28 ]. Among HBV/HIV coinfected children in South Africa treated with regimens including 3TC and/or TDF, HBV viraemia has been demonstrated, highlighting potential underlying HBV drug resistance [ 29 ].

ETV is another active agent, and is safe and well tolerated. However it is not active against HIV and therefore has to be added to ART regimens rather than being part of the primary backbone, is not recommended in pregnancy, and is not routinely available in most African settings [ 30 ]. Resistance arises more commonly in the context of prior 3TC exposure [ 11 , 31 ], which may limit its future potential in Africa, particularly in HIV endemic populations.

As a component of the Expanded Programme on Immunization (EPI), HBV preventive vaccines have been rolled out in Africa since 1995 [ 4 ]. HBV vaccine is highly effective in prevention of mother to child transmission (PMTCT); when administered to infants within 24 hours of birth followed by a dose given at 6 and another at 14 weeks to complete the primary series, it reduces the rate of mother to child transmission by 85%–95% [ 32 , 33 ]. However, by 2016 only 11 countries in Africa had adopted birth dose HBV vaccination as part of the routine infant immunisation schedule [ 34 ]. Changes in the S protein can result in vaccine escape mutants (VEMs) [ 16 , 18 ], and also diagnostic escape mutations which result in false negative HBsAg testing [ 16 ]. Mutations in HBV Pol can also lead to amino acid changes in the Surface (S) protein due to overlapping reading frames (ORFs) in the genome [ 16 ]. Whilst the S protein mutation sG145R has been identified as the major VEM, recently other mutations in S protein have been associated with immune escape [ 16 ] Fig 1 . There are very few data for VEMs in Africa, but in other settings of high endemicity, VEMs can be common, as evidenced by a reported prevalence of 28% in vaccinated HBV-infected children in Taiwan [ 35 ].

To date, no systematic review has assessed the geography and prevalence of HBV RAMs and VEMs in Africa. An understanding of the extent to which these mutations circulate in Africa is essential to improving HBV therapy in patients with and without HIV coinfection. We therefore set out to describe the frequency, co-occurrence and distribution of RAMs and VEMs in Africa, and to suggest whether changes are needed in recommendations for laboratory diagnostics and/or approaches to drug therapy or vaccine deployment. This will underpin further research to identify and track relevant mutations in these populations.

Search strategy

Between October 2017 and January 2018, we searched the published literature, in MEDLINE (PubMed; https://www.ncbi.nlm.nih.gov/pubmed ), SCOPUS ( https://www.elsevier.com/solutions/scopus ) and EMBASE ( https://www.elsevier.com/en-gb/solutions/embase-biomedical-research ). Our search strategy is detailed in S1 Table (documenting use of PRISMA criteria and selection of studies) and S2 Table (listing our search criteria). The earliest paper we identified on HBV drug resistance in Africa was published in 2007. We reviewed the titles and abstracts matching the search terms and only included those relating to drug or vaccine resistance in HBV infection, including only those that presented original data and had undergone peer review. All retrieved articles were in English, therefore no exclusion in relation to language was required.

For each publication we recorded reference, publication year, study design, sample size, study population, proportion of participants who tested HBsAg+ or HBV DNA+, country, year(s) of specimen collection, genotype identified, antiviral treatment, sequencing method, gene sequenced, number of sequenced samples, participant recruitment site and sequence accession number. Data were curated using MS Excel software (Microsoft, Redmond, WA).

RAMs reported in published sequences not represented in primary studies

We expanded our search for evidence of RAMs by identifying publicly available HBV sequences from Africa, that had not been included in the results of our primary literature search. We used both the Hepatitis B Virus database ( https://hbvdb.ibcp.fr/ [ 36 ] and Hepatitis Virus Diversity Research Alignments database ( http://hvdr.bioinf.wits.ac.za/alignments/ ) [ 37 ].

In order to determine the prevalence of RAMs and VEMs, we first reported these using the denominator (total number of HBV positive patients) and numerator (total number of HBV positive patients with the specified mutation) as reported in published studies. We also pooled data by country in order to provide regional estimates. Downloaded sequences were managed using Sequence editor, database and analysis platform, SSE version 1.3, for analysis [ 38 ].

Data visualisation

We developed an R package, gene.alignment.tables, for the visualisation of the sequence data in this study; this is available on Github [ 39 ] and can be used for visualising generic gene sequence datasets. The package was developed by University of Oxford’s Interactive Data Network and a specific instance of the visualisation is hosted as a Shiny app which can be viewed here: https://livedataoxford.shinyapps.io/1510659619-3Xkoe2NKkKJ7Drg/ [ 40 ].

The initial search yielded 56 articles in MEDLINE, 150 in SCOPUS and 150 in EMBASE. Of these, 32, 136 and 119 were excluded from search results of MEDLINE, SCOPUS and EMBASE respectively, as they did not did not meet the inclusion criteria. After de-duplication, 37 articles were included. 27 articles identified from MEDLINE, SCOPUS and EMBASE were identical; five unique articles were included from EMBASE, four from SCOPUS and one from MEDLINE. A total of 37 articles were downloaded in full ( S1 Table (part II); S3 Table ).

Study characteristics

Epidemiological data for HBV represented by the 37 studies we identified are summarised in Table 1 . Studies included were from Southern Africa (Botswana, Mozambique, South Africa, Zambia and Zimbabwe), East Africa (Ethiopia, Kenya, Malawi, Sudan and Uganda), West Africa (Cote d’Ivoire, Gambia, Ghana, Guinea-Bissau and Nigeria) and Central Africa (Cameroon, Gabon). There was considerable heterogeneity in recruitment protocols and exposure to anti-viral treatment. Twenty-six studies recruited from hospitals, three studies recruited from the community [ 8 , 41 , 42 ] and eight studies did not specify where recruitment was undertaken [ 10 , 43 – 49 ]. All studies were observational.

https://doi.org/10.1371/journal.pntd.0006629.t001

Study populations were categorised as follows:

• HBV/HIV coinfected patients: (n = 28 studies), [ 8 – 10 , 12 – 14 , 20 , 43 – 48 , 50 – 56 , 58 – 62 , 64 , 67 , 70 ];
• HBV infected with and without HIV coinfection: (n = 8 studies), [ 41 – 43 , 57 , 63 , 65 , 66 , 68 ];
• Chronic HBV monoinfection: (n = 1 study), [ 69 ].

Antiviral treatment exposure varied as follows:

• Treatment-naïve: (n = 8 studies), [ 14 , 46 – 48 , 63 , 64 , 68 , 70 ];
• 3TC-based regimen only: (n = 10 studies), [ 8 , 9 , 20 , 44 , 45 , 49 , 53 , 59 , 61 , 69 ];
• Regimens including 3TC or TDF: (n = 6 studies), [ 10 , 13 , 51 , 52 , 55 , 65 ];
• Mixed regimen where some received 3TC, others TDF, while others left untreated; (n = 7 studies), [ 12 , 50 , 54 , 56 , 60 , 62 , 66 ];
• Treatment regimen not specified: (n = 6 studies), [ 41 – 43 , 57 , 58 , 67 ].

HBV amino acid polymorphisms were studied from within the following proteins;

• Pol only (n = 13 studies), [ 8 , 9 , 12 – 14 , 20 , 44 , 53 , 55 , 56 , 59 , 63 , 68 ]; only one of these used a deep sequencing method [ 20 ];
• S only (n = 3 studies), [ 43 , 57 , 65 ];
• Pol and S (n = 12 studies),[ 10 , 41 , 45 , 48 , 51 , 52 , 54 , 58 , 60 , 62 , 64 , 67 ];
• Pol, S and PC/BCP (n = 4 studies), [ 47 , 50 , 61 , 69 ];
• S and PC/BCP region (n = 3 studies), [ 42 , 46 , 70 ];
• Whole genome (n = 2 studies), [ 49 , 66 ].

All studies, except for two [ 53 , 68 ], specified the HBV genotype ( S3 Table & S2 Fig ).

Prevalence of HBsAg, HBeAg and HDV coinfection

The prevalence rates of HBsAg in these study cohorts ranged from 3%-26%; however, the populations included were highly selected and therefore not necessarily representative of the general population, particularly as a result of a strong bias towards HIV-infection ( Table 1 ). Only three studies included in this review reported on HDV prevalence: two studies did not detect any HDV antibodies [ 63 , 65 ], whereas the other study reported a HDV prevalence of 25% in Guinea-Bissau [ 56 ].

RAMs identified in African cohorts

The co-occurrence and distribution of HBV RAMs and VEMs are summarised according to the region where they were identified ( Fig 2 ). This illustrates the patchy and limited data that are available, with South Africa, Ghana and Cameroon best represented, but with large areas (especially in northern and central Africa) not represented at all in the literature.

Mutations identified from 33 studies of African cohorts published between 2007 and 2017 (inclusive). Four studies identified by our systematic literature review were not represented here as they did not report any RAMs. Full details of each citation can be found in Table 1 .

https://doi.org/10.1371/journal.pntd.0006629.g002

Although 35 studies specified the HBV genotype, it was only possible to group RAMs according to genotype in fourteen studies [ 8 , 9 , 13 , 14 , 44 , 46 , 47 , 50 , 51 , 56 , 60 – 62 , 69 ] ( S1 Fig ; S2 Fig ). The remaining 21 studies generally reported the genotypes identified, but did not specifically state the genotype of HBV within which RAMs were identified.

We have developed an interactive tool to display the genomic positions of RAMs identified through our literature review alongside relevant metadata. This can be accessed on-line here: https://livedataoxford.shinyapps.io/1510659619-3Xkoe2NKkKJ7Drg/ [ 40 ].

Overall, the most prevalent RAM was rtM204V/I in both treatment experienced and treatment naïve individuals, and occurring either alone or in combination with other polymorphisms rtL80I/V, rtV173L, rtL180M, rtA181S, rtT184S, rtA200V and/or rtS202S ( Fig 3 ); mutations among individuals with and without exposure to HBV therapy are listed in S4 Table and S5 Table , respectively). This mutation was present in 29 studies at a highly variable prevalence of between 0.4% [ 12 ] and 76% [ 69 ]. Across all cohorts, the mutation was present in 208/2569 (8%) of all individuals represented. The mutation, by itself, was most prevalent in South Africa; on pooling data for three studies from this setting, it was present in both treatment experienced and treatment naïve patients (n = 13/17, 76% [ 69 ] and n = 16/72, 22% [ 67 , 68 ] respectively). In addition to South Africa, rtM204I/V was also frequent in Malawi among treatment experienced patients (n = 24/154, 16% [ 20 , 45 ]) ( Fig 3 ), and in genotype non-A infection: in this setting, the mutation was detected in genotype C infection (n = 2/17, 12% [ 69 ]) ( S2 Fig ).

These data are derived from 27 studies of HBV drug resistance in Africa published between 2007 and 2017 (inclusive). The countries represented are listed in alphabetical order. A detailed summary of RAMs identified from each study is presented ( Fig 2 , S4 Table , S5 Table ). Prevalence of RAMs for a specific country was determined by grouping all studies from that country that reported a specific mutation. We used all individuals who tested HBsAg positive to generate a denominator in order to provide a conservative estimate of RAM prevalence, and the numerator was the total number of individuals with that specific mutation from these studies. A: treatment naïve; B: treatment experienced.

https://doi.org/10.1371/journal.pntd.0006629.g003

The rtM204I/V mutation by itself confers resistance to 3TC; in combination with A194T it may also be associated with reduced efficacy to TDF, and in combination with L180M and V173L with vaccine escape, through corresponding substitutions in the surface antigen sites targeted by neutralising antibodies. Although TDF has a high genetic barrier to resistance, and is associated with reliable suppression of HBV viraemia [ 7 , 10 , 21 , 22 ], mutations rtN236T and rtA194T, which have been linked with resistance to both TDF and ADV [ 7 ], have been identified in Southern Africa in both treatment naïve [ 14 ] and treatment experienced [ 10 ] patients.

WHO guidelines recommend a first-line regimen including TDF in HIV/HBV coinfected patients [ 6 ], and the South African Department of Health HIV/AIDS treatment guideline included TDF as first-line regimen from 2010 [ 71 ], however we found a minority of studies (9/37, 24%) reporting TDF-containing regimens for HIV/HBV coinfected individuals. As anticipated, most of the studies that did use TDF were carried out after 2010, whereas those that used 3TC were generally earlier ( S3 Table ).

From this dataset, it is difficult to ascertain whether RAMs are genuinely more prevalent in genotype A infection, or this simply reflects enrichment of genotype A in sub-Saharan African populations ( S2 Fig ). Interpreting RAMs according to sub-genotypes was difficult since most studies did not specify sub-genotype and others did not indicate which RAMs were identified in which genotype. Of concern is the detection of RAMs even in reportedly treatment naïve individuals ( Fig 3 & S4 Table ), suggesting that RAMs are being transmitted. A study in South Africa that recruited 3TC-naïve HBV infected adults with or without HIV, reported rtM204I in 13/35 (37%) individuals [ 68 ].

HBV RAMs in published sequences from Africa

We searched the Hepatitis B Virus database and GenBank to identify HBV sequences derived from Africa, from studies not already included in our review. We identified an additional 69 isolates: 23 had undergone full length genome sequencing whereas 46 isolates represented either the polymerase (n = 3) or S region (n = 43) of the HBV genome Table 2 . To avoid duplication of results, we excluded fourteen studies already identified by our literature review that had submitted their sequences to GenBank ( S3 Table ). RAMs in the additional 69 isolates were as follows:

• rtM204V in genotype A (2/69, 2.9% of sequences), this occurred in combination with rtL180M;
• rtM204V + rtL180M in genotype E (1/69, 1.5%);
• rt180M + rtA181V in genotype E (1/69 (1.5%);
• rtQ215S identified in genotype D (4/69, 5.8%).

All these mutations are associated with 3TC resistance; rtA181V has also been associated with reduced susceptibility to TDF [ 7 , 15 ].

In the S gene, the most prevalent mutations were:

• sD144A/E/G occurring in genotype A (6/69, 8.7%), D (10/69, 14.5%) and E (7/69, 10.1%) associated with VEM;
• sI110L occurring in genotype A (3/69, 4.3%), D (4/69, 5.8%) and E (11/69, 15.9%) associated with immunoglobulin resistance.

https://doi.org/10.1371/journal.pntd.0006629.t002

VEMs were identified in Central, East, West and Southern Africa ( Fig 2 ). However, it was not possible to ascertain whether individuals harbouring these mutations had been vaccinated against HBV infection. The most common VEM was the triple mutation rtV173L + rtL180M + rtM204I/V, found in the Pol gene. This suite of mutations was identified in 14 studies [ 12 , 13 , 20 , 44 , 50 – 55 , 58 – 60 , 62 ], at a pooled prevalence of 4% (57/1462). Another significant VEM, sG145K/R [ 16 ], was identified in six studies [ 12 , 42 , 47 , 57 , 60 , 62 ] and sM133L/T, associated with VEM, immunoglobulin and diagnostic escape mutation [ 12 , 48 ], was identified in seven studies [ 12 , 41 , 47 , 48 , 57 , 62 , 70 ] ( Fig 2 ).

To our knowledge, this is the first systematic review that assesses RAMs and VEMs for HBV in Africa. The high rates of HBV infection among HIV infected individuals in some locations including Cameroon [ 60 ] and South Africa [ 10 ] could be an indication that HBV infection has been previously under-reported, possibly due to lack of routine screening, poor awareness, stigma, high costs and limited clinical and laboratory infrastructure [ 4 , 8 – 10 , 45 , 53 ]. The literature suggests a widespread exposure of the HIV-infected population to 3TC-based treatment. This may be changing over time in line with current ART treatment recommendations (regimens for Africa summarised in S6 Table ), but the introduction of TDF-based regimens for HIV treatment has been inconsistent, and TDF monotherapy is not consistently available for HBV infection in the absence of HIV.

In keeping with other settings, the most common RAM identified here was rtM204I/V, either alone or in combination with compensatory mutations rtL180M ± rtV173L. Of concern, rtM204V/I was seen in 76% of treatment experienced patients [ 69 ] and 22% of treatment naïve patients [ 67 , 68 ] in South Africa. A review of worldwide incidence of RAMs among treatment naïve patients also described rtM204V/I as the most frequent, but with a much lower prevalence of 5% [ 72 ]. The contribution of unreported or undocumented 3TC exposure in the reportedly treatment naïve populations remains to be determined. A European study demonstrated that the most frequent primary mutation was rtM204V/I, found in 49% of treatment experienced patients [ 73 ], while in China rtM204I, rtN236T and rtL180M+rtM204V+rtV173L/rtS202G were also the most prevalent RAMs [ 74 ].

The triple mutation rtM204V + rtL180M + rtV173L has been identified in East, West and Central Africa [ 20 , 44 , 51 – 54 , 59 ]. This combination of polymorphisms is associated with both vaccine escape and resistance to 3TC and other L-nucleoside analogues [ 20 , 44 , 51 , 54 , 59 , 60 ]. Interestingly, this triple mutation has not been reported in the Southern African region to date, which is likely to reflect the composition of the study populations.

Clinical and public health significance of RAMs

Apart from the nature of drugs being used for HBV treatment, other reported predictors of HBV drug resistance include HBV viral load, HBV intra host heterogeneity, HBeAg status, host body mass index and serum alanine aminotransferase (ALT) activity [ 20 , 75 , 76 ]. Individuals with rtM204V/I plus compensatory mutations typically exhibit high HBV DNA levels [ 20 ] and are therefore highly infectious to others. The spread of RAMs may lead to a rise in drug resistance in treatment naïve chronic HBV infection, representing a substantial challenge for Africa and highlighting an imperative to ensure routine use of TDF in preference to, or in combination with, 3TC-based therapy.

Although these data provide a preliminary picture of the prevalence of RAMs in some settings, there are no recommendations to stipulate any specific prevalence threshold above which HBV drug resistance mutations represent a significant barrier to successful treatment at a population level, and/or RAM prevalence thresholds that should trigger a switch to alternative first-line therapy. For HIV, surveillance for transmission of RAMs is based on screening recently infected, treatment naive individuals, and classifies drug resistance using thresholds of <5%, 5–15%, and >15% to stratify the risk to public health [ 77 ]. Similar thresholds and recommendations for HBV could help to underpin the assimilation of epidemiological data and to unify treatment approaches.

TDF resistance

The identification of mutations associated with reduced TDF susceptibility are of concern, as they suggest the potential for increasing prevalence of polymorphisms that confer partial or complete viral escape from a drug that to date has not been widely associated with resistance. There is now potential for increasing selection of TDF resistance as this drug becomes more widely used. However, as a new first line single tablet option incorporating 3TC, TDF and Dolutegravir (DTG) (triple therapy abbreviated to ‘LTD’) emerges as a recommended option for HIV treatment in Africa, surveillance is needed to determine the clinical outcomes for HBV [ 78 ].

If clinically significant, TDF resistance mutations may still represent a particular problem for many African settings, as resource constraints make it unrealistic to provide baseline screening for RAMs, or to monitor patients on treatment with serial viral load measurements. Despite these potential concerns, it has been shown that TDF is effective even in the presence of RAMs and that there is comparable efficacy among 3TC-experienced and NA-naïve patients [ 79 ].

VEM were identified in 16 different countries in East, West, Central and Southern Africa. Information on vaccine exposure was not available, but there are two strands of evidence to support significant population exposure to HBV vaccination. First, vaccination has been progressively rolled out in most countries in sSA since the mid-1990’s; second, most HBsAg mutations reported by these studies are located within the common immunodominant B cell epitope (aa 124–147) in which selection of polymorphisms is associated with HBV vaccination [ 80 , 81 ].

VEM have been more robustly reported from Asia, in settings where the HBV infant vaccination programme is well established; for example, in Taiwan, VEM prevalence among vaccinated children increased from 7.8% to 23.1% within 15 years of the launch of the universal vaccine program, although the decline in VEM prevalence thereafter may be partly related to a smaller HBV carrier pool [ 80 ]. HBV infection despite immunoprophylaxis can occur either as a consequence of MTCT of pre-existing VEM, or as a result of de novo selection of escape mutations from vaccine- induced immune responses, particularly in the setting of delayed vaccination [ 80 , 81 ].The HBV genotype sequence used for vaccines may potentially have an influence on immunogenicity against non-vaccine genotypes, but there are limited data to support this [ 82 ]. Only 11 African countries recommend the first HBV vaccine dose at birth, in contrast to the majority of African countries in which HBV vaccination is delayed until 6 weeks of age [ 33 ]. It is likely that this delay not only provides a window of infection but also increases the possibility of transmitted VEM and/or emergence of new escape mutations.

High maternal HBV viral load and immunosuppression are other risk factors associated with VEM among infants [ 80 ]; both of these are pertinent for emergence of VEMs in Africa given that HBV viral load testing is not routinely available, and HIV is highly prevalent in some populations. Effective PMTCT strategies in Africa, including screening and treating antenatal women, increasing access to viral load monitoring, and introducing HBV birth dose vaccine will help to decrease the prevalence of VEM [ 4 , 33 , 83 ].

HDV/HBV coinfection

One study from our literature review reported a high HDV prevalence of 25%; however, in this cohort, RAMs occurred in individuals with HBV monoinfection [ 56 ]. Given that HDV is characteristically associated with decreased HBV replication [ 84 ], it is possible that emergence of HBV RAMs is altered in this setting. However, as the true prevalence and impact of HDV in sSA is not known [ 85 ], further studies are needed to determine the impact of HDV coinfection on HBV RAMs.

Limitations of current data

Screening for HBV infection is not routinely performed in many African settings and therefore the true prevalence and characteristics of HBV infection are not known [ 4 , 7 – 10 ]. We identified very few published studies; only a minority of patients had HBV sequencing undertaken, and there were no data from certain regions of Africa. This highlights the substantial problem of HBV neglect in Africa, and a specific blind-spot relating to sequence data [ 4 ]. Identifying the true prevalence of resistance mutations, and characterising the populations in which these are selected and enriched, is currently not possible due to sparse data and lack of clear descriptions of the denominator population. Most such studies do not perform a truly systematic assessment, but focus on high risk groups–particularly including those with HIV/HBV coinfection: of the 37 studies included here, only one exclusively reported on participants who were HBV mono-infected [ 69 ]. Although we have made every effort to assimilate the relevant data to build up a regional picture for Africa, the heterogeneity between studies makes it difficult to draw robust conclusions from pooled data. These findings are a reflection of the little attention paid towards the burden of this disease in Africa and the neglect in robust epidemiological data.

Only nine studies undertook a longitudinal approach to detection of drug resistance [ 8 – 10 , 20 , 44 , 45 , 49 , 52 , 53 ]. The results of the other 28 studies that undertook a cross-sectional approach could be skewed by the timing of recruitment of study participants, with a risk of under-representation of drug resistance if screening is undertaken only at baseline, and potentially an over-representation if screening is undertaken in patients with HIV coinfection, who are more at risk of advanced disease and prolonged drug exposure. As most of these studies recruited individuals from hospital settings, this raises the latter possibility.

Mutations across the whole genome might be relevant in determining resistance [ 86 ]. However, most of the included studies analysed only defined genes from within the HBV genome; only two sequenced the whole genome, and these determined consensus sequence. This potentially results in an under-representation of RAMs and VEMs that may be present as low numbers of quasispecies, but could become significant if selected out by exposure to drug or vaccine.

In studies that reported RAMs among treatment naïve individuals, the literature suggests that sequence analysis was performed prior to ART initiation. However, we cannot exclude the possibility that that some of these participants had prior ART exposure. Due to the nature of the cohorts that have been studied, most of the RAMs identified were from HIV/HBV coinfected individuals. It is possible that HIV increases the risk of HBV RAMs both in terms of drug exposure, and also as a function of increased HBV viral loads. A study from Malawi demonstrated the rapid emergence of 3TC resistance in HIV coinfection, with virtually all treatment naïve HBeAg positive individuals starting antiviral treatment showing emergence of rtM204I by six months. Likewise, a study carried out in Italy revealed that patients with HIV coinfection were more likely to harbour the rtM204V mutation and to show multiple mutations compared to HBV monoinfected patients [ 87 ]. It would be worth further exploration of this observation in Africa, as there are currently very limited data.

Challenges and opportunities for Africa

A major challenge for Africa is to improve coverage rates of infant vaccination, deploy catch-up vaccination programmes for older children and adults, adopt widespread screening and develop treatment programmes for HBV. While HBV vaccine is effective, gaps in vaccine coverage in Africa can be demonstrated by the high perinatal transmission rate of HBV in sSA (estimated at 38% among women with a high HBV viral load) and the observation that up to 1% of newborns in sSA are still infected with HBV [ 88 ]. Sustained efforts are required to build robust PMTCT programmes that deliver screening and treatment for antenatal women, and timely administration of HBV birth vaccine for their babies [ 33 , 83 ].

Although the WHO recommends monitoring for the development of drug resistance once on therapy [ 6 ], implementation remains challenging as viral load monitoring and sequencing are both rarely available [ 7 ]; despite the advancement and availability of HIV testing and monitoring, in many settings it remains uncommon to monitor HIV viral load after ART initiation [ 89 , 90 ]. Affordable, accessible and sustainable platforms for quantifying both HIV and HBV viral loads remain an important priority for many settings in Africa, given the lack of on-treatment monitoring in many settings. Given the simplicity and relative ease of collection, preparation and transport of dried-blood-spot (DBS) samples [ 91 ], adopting DBS testing could improve access to HBV diagnosis, viral load monitoring and linkage to care, especially in areas with limited access to laboratory facilities.

Development of a cheap, rapid test for the detection of the most frequently observed RAMs and VEMs should be considered as a potentially cost-effective strategy for Africa. Proof of principle for a rapid test for diagnosis and detection of resistance has been demonstrated by the GeneXpert MTB/RIF assay for Mycobacterium tuberculosis (MTB) [ 92 ]. A similar approach has been applied for HBV through use of a multiplex ligation-dependent probe real time PCR (MLP-RT-PCR) [ 93 ]. Although this assay is able to detect RAMs quickly and cheaply, there are still limitations as the test requires high viral load samples, is based on detection of known RAMs from within discrete regions of the genome, and may not identify RAMs that are present as minor quasispecies.

New metagenomic sequencing platforms, such as Illumina and Nanopore, provide the opportunity for whole deep genome sequencing, which can reveal the full landscape of HBV mutations in individual patients, quantify the prevalence of drug resistance mutations among HBV quasi-species, and determine the relationship between these polymorphisms and treatment outcomes [ 87 ]. Nanopore technology also has the potential to develop into an efficient point of care test that could detect viral infection and coinfection, as well as determining the presence of VEMs and RAMs [ 94 ], but is currently limited by cost and concerns about high error rates.

There have been few studies looking at the correlation between genotype, clinical outcomes of disease, response to antiviral therapy and RAMs/VEMs, but none from Africa. Studies outside Africa have shown that genotype A is more prone to immune/vaccine escape mutants, pre-S mutants associated with immune suppression, drug associated mutations and HCC in HIV/HBV coinfected participants [ 46 , 87 , 95 ]. Studies investigating the role of genotypes in predicting response to antiviral therapy and their association with various types of mutations are urgently needed in Africa, particularly in light of the high frequency of genotype A infection and high population exposure to antiviral agents that have been rolled out over the past two decades as a component of first-line ART.

Existing infrastructure for diagnosis, clinical monitoring and drug therapy for HIV represents an opportunity for linkage with HBV care. Particularly in settings of limited resource, joining up services for screening and management of blood-borne virus infection could be a cost-effective pathway to service improvements.

Conclusions

This review highlights the very limited data for HBV RAMs and VEMs that are available from Africa. Scarce resources resulting in lack of diagnostic screening, inconsistent supply of HBV drugs and vaccines, and poor access to clinical monitoring contribute to drug and vaccine resistance, potentially amplifying the risk of ongoing transmission and adding to the long-term burden of HBV morbidity and mortality in Africa. We call for urgent action to gather and analyse better data, particularly representing the HBV monoinfected population, and for improved access to TDF.

HBV RAMs and VEMs have been identified in several African countries among HIV/HBV coinfected and HBV monoinfected patients, before and during treatment with NAs but the data are currently insufficient to allow us to form a clear picture of the prevalence, distribution or clinical significance of these mutations. Overall, the data we describe suggest a significantly higher prevalence of drug resistance in some African populations than has been described elsewhere, and that is not confined only to drug-exposed populations, highlighting an urgent need for better population screening, assessment of HBV infection before and during therapy, and increasing roll out of TDF in preference to 3TC. At present, TDF accessibility is largely confined to HIV/HBV coinfected individuals; we now need to advocate to make monotherapy available for HBV monoinfected individuals. However, there are uncertainties as to whether its long-term use might result in nephrotoxicity, and potentially in an increase in selection of TDF RAMs.

We should ideally aim for the goals of a combined HBV test that includes diagnosis of infection, genotype and presence of RAMs/VEMs; new sequencing platforms such as Nanopore make this technically possible, although cost remains a significant barrier at present. Sustainable long-term investment is required to expand consistent drug and vaccine supply, to provide screening infection and for drug resistance, and to provide appropriate targeted clinical monitoring for treated patients.

Supporting information

S1 fig. hbv drug resistance associated mutations (rams) grouped according to genotype..

Data summarised from fourteen studies published between 2009–2017 (inclusive). 21 studies were not represented here as they did not specifically indicate which genotype individuals with RAMs belonged to. Available at https://doi.org/10.6084/m9.figshare.5774091 [ 96 ].

https://doi.org/10.1371/journal.pntd.0006629.s001

S2 Fig. Distribution of HBV genotypes and prevalence of HBV resistance associated mutations (RAMs) in Pol/RT proteins in geno-A and geno-non-A samples.

A: Distribution of HBV genotypes derived from 35 studies reporting resistance associated mutations (RAMs) in Africa published between 2009 to 2017 (inclusive); B: Prevalence of HBV resistance associated mutations (RAMs) in Pol/RT proteins in geno-A and geno-non-A samples. These data are derived from 14 studies of HBV drug resistance in Africa published between 2007 and 2017 (inclusive). 21 studies were not represented here as they did not specifically indicate which genotype individuals with RAMs belonged to. We had more geno-A samples represented than other samples, we therefore combined samples from other genotypes that had RAMs (B, C, D, E, D/E) to form geno-non-A samples. We then compared prevalence of Pol/RT mutation between geno-A samples to geno-non-A samples. Prevalence of RT/Pol mutations for a specific genotype(geno-A/geno-non-A) was determined by grouping all studies with geno-A/geno-non-A infection that reported a specific mutation; the denominator was the total number of individuals infected with geno-A/geno-non-A from these studies and the numerator was the total number of individuals infected with geno-A/geno-non-A with that specific mutation. Available at https://doi.org/10.6084/m9.figshare.5774091 [ 96 ].

https://doi.org/10.1371/journal.pntd.0006629.s002

S1 Table. PRISMA (preferred reporting items for systematic reviews and meta-analyses) criteria for a systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa.

Available at https://doi.org/10.6084/m9.figshare.5774091 [ 96 ].

I. Checklist to demonstrate how PRISMA criteria (2009) have been met in this review;

II. Flow diagram illustrating identification and inclusion of studies for a systematic review of drug and vaccine resistance mutations in Africa.

https://doi.org/10.1371/journal.pntd.0006629.s003

S2 Table. Details of search strategy used to identify studies on HBV resistance associated mutations (RAMs) and vaccine escape mutations (VEMs) conducted in Africa.

A: PubMed database; B: SCOPUS and EMBASE database. Available at https://doi.org/10.6084/m9.figshare.5774091 [ 96 ].

https://doi.org/10.1371/journal.pntd.0006629.s004

S3 Table. Full details of 37 studies identified by a systematic literature search of HBV resistance associated mutations (RAMs) and vaccine escape mutations (VEMs) from African cohorts published between 2007 and 2017 (inclusive).

https://doi.org/10.1371/journal.pntd.0006629.s005

S4 Table. HBV Pol/RT mutations among treatment-naïve HBV infected patients in Africa from 12 studies published between 2007 and 2017 (inclusive).

https://doi.org/10.1371/journal.pntd.0006629.s006

S5 Table. HBV Pol/RT mutations among treatment-experienced HBV infected patients in Africa, from 25 studies published between 2009 and 2017 (inclusive).

https://doi.org/10.1371/journal.pntd.0006629.s007

S6 Table. First line ART regimen for adults in Africa, and overlap with HBV therapy.

Information derived from published ART guidelines in all cases where these are available in the public domain. This information was collated in May 2018. Available at https://doi.org/10.6084/m9.figshare.5774091 [ 96 ].

https://doi.org/10.1371/journal.pntd.0006629.s008

• 1. World Health Organization. Global Hepatitis Programme. Global hepatitis report, 2017. http://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/
• 2. WHO Combating Hepatitis B and C to reach elimination by 2030. Advocacy brief. 2016. http://apps.who.int/iris/bitstream/10665/206453/1/WHO_HIV_2016.04_eng.pdf
• View Article
• PubMed/NCBI
• Google Scholar
• 6. WHO Hepatitis B treatment guidelines. 2015 http://www.who.int/mediacentre/news/releases/2015/hepatitis-b-guideline/en/
• 7. Beloukas A, Geretti AM. Hepatitis B Virus Drug Resistance. In: Antimicrobial Drug Resistance; 2017.p.1227–42.
• 27. Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection: Recommendations for a public health approach—Second edition http://www.who.int/hiv/pub/arv/chapter4.pdf?ua=1
• 28. World Health Organisation–South Africa HIV Country Profile: 2016 http://www.who.int/hiv/data/Country_profile_South_Africa.pdf
• 31. Locarnini SA. The Hepatitis B Virus and Antiviral Drug Resistance: Causes, Patterns and Mechanisms. In: Antimicrobial Drug Resistance; 2017.p. 565–77.
• 34. WHO UNICEF coverage estimates WHO World Health Organization: Immunization, Vaccines And Biologicals. Vaccine preventable diseases Vaccines monitoring system 2017 Global Summary Reference Time Series: HEPB3. http://apps.who.int/immunization_monitoring/globalsummary/timeseries/tswucoveragehepb3.html
• 39. Mokaya J, Hadley M, Matthews P. gene.alignment.tables. Figshare. 2017. https://doi.org/10.6084/m9.figshare.5729229
• 40. Mokaya J, Hadley M, Matthews P. On-line tool to visualise sites of drug and vaccine escape mutations within the HBV genome. 2018. https://livedataoxford.shinyapps.io/1510659619-3Xkoe2NKkKJ7Drg/
• 71. WHO Clinical guidelines for the management of HIV & AIDS in adults and adolescents. 2010. http://www.who.int/hiv/pub/guidelines/south_africa_art.pdf
• 77. WHO Surveillance of transmitted HIV drug resistance. 2013. http://www.who.int/hiv/topics/drugresistance/surveillance/en/
• 83. McNaughton A, Lourenco J, Hattingh L, Adland E, Daniels S, Zyl A van, et al. Can we meet global challenges for elimination of Hepatitis B Virus infection by 2030? Vaccine-mediated immunity in a South African cohort and a model of transmission and prevention. bioRxiv. 2017;162594.
• 94. Oxford Nanopore Technologies: https://nanoporetech.com/

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

• View all journals
• Explore content
• About the journal
• Publish with us
• Sign up for alerts
• Review Article
• Published: 28 May 2020

The evolution and clinical impact of hepatitis B virus genome diversity

• Peter A. Revill 1 , 2 ,
• Thomas Tu   ORCID: orcid.org/0000-0002-0482-4387 3 , 4 , 5 ,
• Hans J. Netter 1 ,
• Lilly K. W. Yuen 1 ,
• Stephen A. Locarnini 1 &
• Margaret Littlejohn   ORCID: orcid.org/0000-0002-8206-2664 1 , 2  

Nature Reviews Gastroenterology & Hepatology volume  17 ,  pages 618–634 ( 2020 ) Cite this article

5158 Accesses

106 Citations

28 Altmetric

Metrics details

• Hepatitis B
• Molecular medicine

The global burden of hepatitis B virus (HBV) is enormous, with 257 million persons chronically infected, resulting in more than 880,000 deaths per year worldwide. HBV exists as nine different genotypes, which differ in disease progression, natural history and response to therapy. HBV is an ancient virus, with the latest reports greatly expanding the host range of the Hepadnaviridae (to include fish and reptiles) and casting new light on the origins and evolution of this viral family. Although there is an effective preventive vaccine, there is no cure for chronic hepatitis B, largely owing to the persistence of a viral minichromosome that is not targeted by current therapies. HBV persistence is also facilitated through aberrant host immune responses, possibly due to the diverse intra-host viral populations that can respond to host-mounted and therapeutic selection pressures. This Review summarizes current knowledge on the influence of HBV diversity on disease progression and treatment response and the potential effect on new HBV therapies in the pipeline. The mechanisms by which HBV diversity can occur both within the individual host and at a population level are also discussed.

Hepatitis B virus (HBV) is an ancient virus with deep ancestry in the animal kingdom.

HBV seems to undergo very little long-term mutational variation despite multiple host–virus factors driving short-term viral variations.

Viral diversity is generated by features of the unique replication cycle of HBV as well as by cellular host factors.

A possible bottleneck in the establishment of new viral variants could be the limited number of HBV-susceptible hepatocytes in the chronically infected liver.

HBV viral diversity contributes to variations in natural history, disease progression and treatment response in those with chronic infection.

Viral diversity must be considered in the development of new therapeutic regimens.

This is a preview of subscription content, access via your institution

Access options

Access Nature and 54 other Nature Portfolio journals

Get Nature+, our best-value online-access subscription

24,99 € / 30 days

cancel any time

Subscribe to this journal

Receive 12 print issues and online access

195,33 € per year

only 16,28 € per issue

Buy this article

• Purchase on SpringerLink
• Instant access to full article PDF

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

Genetic variability of hepatitis B virus in acute and in different phases of chronic infection in Brazil

Immunobiology and pathogenesis of hepatitis B virus infection

Breakthroughs in hepatitis C research: from discovery to cure

Polaris Observatory Collaborators. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. Lancet Gastroenterol. Hepatol. 3 , 383–403 (2018).

Google Scholar  

World Health Organization. Global Hepatitis Report, 2019 https://www.who.int/hepatitis/publications/global-hepatitis-report2017/en/ (WHO, 2017).

Parkin, D. M. Global cancer statistics in the year 2000. Lancet Oncol. 2 , 533–543 (2001).

CAS   PubMed   Google Scholar  

Revill, P. A. et al. A global scientific strategy to cure hepatitis B. Lancet Gastroenterol. Hepatol. 4 , 545–558 (2019).

PubMed   PubMed Central   Google Scholar  

Alter, H. et al. A research agenda for curing chronic hepatitis B virus infection. Hepatology 67 , 1127–1131 (2018).

PubMed   Google Scholar  

Revill, P., Testoni, B., Locarnini, S. & Zoulim, F. Global strategies are required to cure and eliminate HBV infection. Nat. Rev. Gastroenterol. Hepatol. 13 , 239–248 (2016). First ‘call to arms’ for hepatitis B cure, that led to the formation of the International Coalition to Eliminate Hepatitis B (ICE-HBV) .

Wei, L. & Lok, A. S. Impact of new hepatitis C treatments in different regions of the world. Gastroenterology 146 , 1145–1150 (2014).

Salpini, R. et al. A snapshot of virological presentation and outcome of immunosuppression-driven HBV reactivation from real clinical practice: evidence of a relevant risk of death and evolution from silent to chronic infection. J. Viral Hepat. 26 , 846–855 (2019).

Hoofnagle, J. H. Reactivation of hepatitis B. Hepatology 49 , S156–S165 (2009).

Comas, I. et al. Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nat. Genet. 45 , 1176–1182 (2013).

CAS   PubMed   PubMed Central   Google Scholar  

Linz, B. et al. An African origin for the intimate association between humans and Helicobacter pylori. Nature 445 , 915–918 (2007).

Wolfe, N. D., Dunavan, C. P. & Diamond, J. Origins of major human infectious diseases. Nature 447 , 279–283 (2007).

Blaser, M. J. & Kirschner, D. The equilibria that allow bacterial persistence in human hosts. Nature 449 , 843–84 (2007).

Kahila Bar-Gal, G. et al. Tracing hepatitis B virus to the 16th century in a Korean mummy. Hepatology 56 , 1671–1680 (2012). First report of ancient HBV genome isolated from ancient human remains, a 400-year-old Korean mummy .

Patterson Ross, Z. et al. The paradox of HBV evolution as revealed from a 16th century mummy. PLoS Pathog. 14 , e1006750 (2018).

Muhlemann, B. et al. Ancient hepatitis B viruses from the Bronze age to the Medieval period. Nature 557 , 418–423 (2018). First report of Bronze age HBV genomes extracted from a number of archaeological sites across Europe, including a now extinct human genotype .

Krause-Kyora, B. et al. Neolithic and medieval virus genomes reveal complex evolution of hepatitis B. Elife 7 , e36666 (2018).

Yuen, L. K. W. et al. Tracing ancient human migrations into Sahul using hepatitis B virus genomes. Mol. Biol. Evol. 36 , 942–954 (2019). First example of HBV evolution and diversity being used to trace human migration .

MacLachlan, J. H. & Cowie, B. C. Hepatitis B virus epidemiology. Cold Spring Harb. Perspect. Med. 5 , a021410 (2015).

Graham, S. et al. Chronic hepatitis B prevalence among Aboriginal and Torres Strait Islander Australians since universal vaccination: a systematic review and meta-analysis. BMC Infect. Dis. 13 , 403 (2013).

Parker, C. et al. Hepatocellular carcinoma in Australia’s Northern Territory: high incidence and poor outcome. Med. J. Aust. 201 , 470–474 (2014).

Ormaeche, M., Whittembury, A., Pun, M. & Suarez-Ognio, L. Hepatitis B virus, syphilis, and HIV seroprevalence in pregnant women and their male partners from six indigenous populations of the Peruvian Amazon Basin, 2007-2008. Int. J. Infect. Dis. 16 , e724–e730 (2012).

Viana, S., Parana, R., Moreira, R. C., Compri, A. P. & Macedo, V. High prevalence of hepatitis B virus and hepatitis D virus in the western Brazilian Amazon. Am. J. Trop. Med. Hyg. 73 , 808–814 (2005).

Schweitzer, A., Mmath, J. H., Mikolaiczyk, R. T., Krause, G. & Ott, J. J. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet 386 , 1546–1555 (2015).

Bouckaert, R., Simons, B. C., Krarup, H., Friesen, T. M. & Osiowy, C. Tracing hepatitis B virus (HBV) genotype B5 (formerly B6) evolutionary history in the circumpolar Arctic through phylogeographic modelling. PeerJ. 5 , e3757 (2017).

McMahon, B. J. et al. Elimination of hepatocellular carcinoma and acute hepatitis B in children 25 years after a hepatitis B newborn and catch-up immunization program. Hepatology 54 , 801–807 (2011).

Robinson, T., Bullen, C., Humphries, W., Hornell, J. & Moyes, C. The New Zealand hepatitis B screening programme: screening coverage and prevalence of chronic hepatitis B infection. NZ Med. J. 118 , U1345 (2005).

Huang, C. F. et al. HBV infection in indigenous children, 20 years after immunization in Taiwan: a community-based study. Prev. Med. 48 , 397–400 (2009).

Wilson, N., Ruff, T. A., Rana, B. J., Leydon, J. & Locarnini, S. The effectiveness of the infant hepatitis B immunisation program in Fiji, Kiribati, Tonga and Vanuatu. Vaccine 18 , 3059–3066 (2000).

McMahon, B. J. Viral hepatitis in the Arctic. Int. J. Circumpolar Health 63 (Suppl. 2), 41–48 (2004).

Kramvis, A. Molecular characteristics and clinical relevance of African genotypes and subgenotypes of hepatitis B virus. S. Afr. Med. J. 108 , S17–S21 (2018).

CAS   Google Scholar  

European Association for the Study of the Liver. EASL 2017 Clinical Practice Guidelines on the management of hepatitis B virus infection. J. Hepatol. 67 , 370–398 (2017). The current European Clinical Practice Guidelines for the management of hepatitis B virus infection .

Bayliss, J. et al. Deep sequencing shows that HBV basal core promoter and precore variants reduce the likelihood of HBsAg loss following tenofovir disoproxil fumarate therapy in HBeAg-positive chronic hepatitis B. Gut 66 , 2013–2023 (2017).

Sonneveld, M. J. et al. Presence of precore and core promoter mutants limits the probability of response to peginterferon in hepatitis B e antigen-positive chronic hepatitis B. Hepatology 56 , 67–75 (2012).

Seeger, C., Zoulim, F. & Mason, W. S. in Fields Virology (eds Knipe, D. M. & Howley, P. M.) 2185–2221 (Lippincott Williams & Wilkins, 2013).

International Committee on Taxonomy of Viruses. ICTV https://talk.ictvonline.org (2019).

Hahn, C. M. et al. Characterization of a novel hepadnavirus in the white sucker (Catostomus commersonii) from the Great Lakes region of the United States. J. Virol. 89 , 11801–11811 (2015).

Dill, J. A. et al. Distinct viral lineages from fish and amphibians reveal the complex evolutionary history of hepadnaviruses. J. Virol. 90 , 7920–7933 (2016).

Littlejohn, M., Locarnini, S. & Yuen, L. Origins and evolution of hepatitis B virus and hepatitis D virus. Cold Spring Harb. Perspect. Med. 6 , a021360 (2016). Detailed review summarizing and discussing current theories on the origin and evolution of HBV and HDV .

Lauber, C. et al. Deciphering the origin and evolution of hepatitis B viruses by means of a family of non-enveloped fish viruses. Cell Host Microbe 22 , 387–399 (2017).

de Carvalho Dominguez Souza, B. F. et al. A novel hepatitis B virus species discovered in capuchin monkeys sheds new light on the evolution of primate hepadnaviruses. J. Hepatology 68 , 1114–1122 (2018).

Aghazadeh, M. et al. A novel hepadnavirus identified in an immunocompromised domestic cat in Australia. Viruses 10 , E269 (2018).

Geoghegan, J. L. et al. Hidden diversity and evolution of viruses in market fish. Virus Evol. 4 , vey031 (2018).

Gong, Z. & Han, G. Z. Insect retroelements provide novel insights into the origin of hepatitis B viruses. Mol. Biol. Evol. 35 , 2254–2259 (2018).

Gilbert, C. & Feschotte, C. Genomic fossils calibrate the long-term evolution of hepadnaviruses. PLoS Biol. 8 , e1000495 (2010). First report of endogenous hepadnaviruses, discovered in the genome of the zebra finch .

Gilbert, C. et al. Endogenous hepadnaviruses, bornaviruses and circoviruses in snakes. Proc. Biol. Sci. 281 , 20141122 (2014).

Suh, A. et al. Early mesozoic coexistence of amniotes and hepadnaviridae. PLoS Genet. 10 , e1004559 (2014).

Geoghegan, J. L., Duchene, S. & Holmes, E. C. Comparative analysis estimates the relative frequencies of co-divergence and cross-species transmission within viral families. PLoS Pathog. 13 , e1006215 (2017).

Drexler, J. F. et al. Bats carry pathogenic hepadnaviruses antigenically related to hepatitis B virus and capable of infecting human hepatocytes. Proc. Natl Acad. Sci. USA 110 , 16151–16156 (2013). Report of the discovery of a number of HBV-like viruses in bats, including experiments showing that bat HBV can infect human hepatocytes .

Wieland, S. F. The chimpanzee model for hepatitis B virus infection. Cold Spring Harb. Perspect. Med. 5 , a021469 (2015).

Dupinay, T. et al. Discovery of naturally occurring transmissible chronic hepatitis B virus infection among Macaca fascicularis from Mauritius Island. Hepatology 58 , 1610–1620 (2013).

Dickens, C., Kew, M. C., Purcell, R. H. & Kramvis, A. Occult hepatitis B virus infection in chacma baboons, South Africa. Emerg. Infect. Dis. 19 , 598–605 (2013).

Tatematsu, K. et al. A genetic variant of hepatitis B virus divergent from known human and ape genotypes isolated from a Japanese patient and provisionally assigned to new genotype J. J. Virol. 83 , 10538–10547 (2009). Description of the single isolate of HBV ‘putative genotype’ J .

Begun, D. R. Planet of the apes. Sci. Am. 289 , 74–83 (2003).

Begun, D. R., Nargolwalla, M. C. & Kordos, L. European Miocene hominids and the origin of the African ape and human clade. Evol. Anthropol. 21 , 10–23 (2012).

Zhou, Y. & Holmes, E. C. Bayesian estimates of the evolutionary rate and age of hepatitis B virus. J. Mol. Evol. 65 , 197–205 (2007).

Orito, E. et al. Host-independent evolution and a genetic classification of the hepadnavirus family based on nucleotide sequences. Proc. Natl Acad. Sci. USA 86 , 7059–7062 (1989).

Paraskevis, D. et al. Dating the origin of hepatitis B virus reveals higher substitution rate and adaptation on the branch leading to F/H genotypes. Mol. Phylogenet. Evol. 93 , 44–54 (2015).

Paraskevis, D. et al. Dating the origin and dispersal of hepatitis B virus infection in humans and primates. Hepatology 57 , 908–916 (2013).

Tedder, R. S., Bissett, S. L., Myers, R. & Ijaz, S. The ‘Red Queen’ dilemma – running to stay in the same place: reflections on the evolutionary vector of HBV in humans. Antivir. Ther. 18 , 489–496 (2013). An interesting paper outlining experiments confirming a theory explaining the slow evolutionary rate of human HBV .

Simmonds, P. & Smith, D. B. in Viral Hepatitis 4th edn (eds Thomas, H. C., Lok, A. S. F., Locarnini, S. A. & Zuckerman, A. J.) 575–586 (Wiley, 2014).

Simmonds, P., Aiewsakun, P. & Katzourakis, A. Prisoners of war – host adaptation and its constraints on virus evolution. Nat. Rev. Microbiol. 17 , 321–328 (2018).

PubMed Central   Google Scholar  

Lim, S. G. et al. Viral quasi-species evolution during hepatitis Be antigen seroconversion. Gastroenterology 133 , 951–958 (2007).

Homs, M. et al. Clinical application of estimating hepatitis B virus quasispecies complexity by massive sequencing: correlation between natural evolution and on-treatment evolution. PLoS One 9 , e112306 (2014).

Charuworn, P. et al. Baseline interpatient hepatitis B viral diversity differentiates HBsAg outcomes in patients treated with tenofovir disoproxil fumarate. J. Hepatol. 62 , 1033–1039 (2015).

Kramvis, A. Genotypes and genetic variability of hepatitis B virus. Intervirology 57 , 141–150 (2014). Excellent summary of human HBV genotype and sub-genotype classification, including a set of minimum criteria for future proposals of novel genotypes and sub-genotypes .

Kim, B. K., Revill, P. A. & Ahn, S. H. HBV genotypes: relevance to natural history, pathogenesis and treatment of chronic hepatitis B. Antivir. Ther. 16 , 1169–1186 (2011).

Chan, H. L. et al. Genotype C hepatitis B virus infection is associated with an increased risk of hepatocellular carcinoma. Gut 53 , 1494–1498 (2004).

Yang, H. I. et al. Associations between hepatitis B virus genotype and mutants and the risk of hepatocellular carcinoma. J. Natl Cancer Inst. 100 , 1134–1143 (2008).

Wong, G. L. et al. Meta-analysis: the association of hepatitis B virus genotypes and hepatocellular carcinoma. Aliment. Pharmacol. Ther. 37 , 517–526 (2013).

Kramvis, A. & Kew, M. C. Molecular characterization of subgenotype A1 (subgroup Aa) of hepatitis B virus. Hepatol. Res. 37 , S27–S32 (2007). First characterization of the African A1 HBV subgenotype describing specific sequence elements that may contribute to the increased risk of HCC in patients infected with HBV-A1 .

Gounder, P. P. et al. Hepatocellular carcinoma risk in Alaska native children and young adults with hepatitis B virus: retrospective cohort analysis. J. Pediatr. 178 , 206–213 (2016).

Orito, E. & Mizokami, M. Differences of HBV genotypes and hepatocellular carcinoma in Asian countries. Hepatol. Res. 37 , S33–S35 (2007).

Krarup, H. B. et al. Benign course of long-standing hepatitis B virus infection among Greenland Inuit? Scand. J. Gastroenterol. 43 , 334–343 (2008).

Minuk, G. Y. et al. A paucity of liver disease in Canadian Inuit with chronic hepatitis B virus, subgenotype B6 infection. J. Viral Hepat. 20 , 890–896 (2013).

Roman, S., Fierro, N., Moreno-Luna, L. & Panduro, A. Hepatitis B virus genotype H and environmental factors associated to the low prevalence of hepatocellular carcinoma in Mexico. J. Cancer Ther. 4 , 367–376 (2013).

Sozzi, V. et al. In vitro studies identify a low replication phenotype for hepatitis B virus genotype H generally associated with occult HBV and less severe liver disease. Virology 519 , 190–196 (2018).

Sozzi, V. et al. In vitro studies show that sequence variability contributes to marked variation in hepatitis B virus replication, protein expression, and function observed across genotypes. J. Virol. 90 , 10054–10064 (2016).

Sugiyama, M. et al. Influence of hepatitis B virus genotypes on the intra- and extracellular expression of viral DNA and antigens. Hepatology 44 , 915–924 (2006).

Bhoola, N. H., Reumann, K., Kew, M. C., Will, H. & Kramvis, A. Construction of replication competent plasmids of hepatitis B virus subgenotypes A1, A2 and D3 with authentic endogenous promoters. J. Virol. Methods 203 , 54–64 (2014).

Flink, H. J. et al. Treatment with Peg-interferon α-2b for HBeAg-positive chronic hepatitis B: HBsAg loss is associated with HBV genotype. Am. J. Gastroenterol. 101 , 297–303 (2006).

Buster, E. H. et al. Factors that predict response of patients with hepatitis B e antigen-positive chronic hepatitis B to peginterferon-alfa. Gastroenterology 137 , 2002–2009 (2009).

Shen, F. et al. Hepatitis B virus sensitivity to interferon-α in hepatocytes is more associated with cellular interferon response than with viral genotype. Hepatology 67 , 1237–1252 (2018).

Marcellin, P. et al. Tenofovir disoproxil fumarate versus adefovir dipivoxil for chronic hepatitis B. N. Engl. J. Med. 359 , 2442–2455 (2008).

Bihl, F. et al. HBV genotypes and response to tenofovir disoproxil fumarate in HIV/HBV-coinfected persons. BMC Gastroenterol. 15 , 79 (2015).

Marcellin, P. et al. Kinetics of hepatitis B surface antigen loss in patients with HBeAg-positive chronic hepatitis B treated with tenofovir disoproxil fumarate. J. Hepatol. 61 , 1228–1237 (2014).

Yao, C. C. et al. Incidence and predictors of HBV relapse after cessation of nucleoside analogues in HBeAg-negative patients with HBsAg ≤ 200 IU/mL. Sci. Rep. 7 , 1839 (201 7 ).

Chen, C. H. et al. The role of hepatitis B surface antigen quantification in predicting HBsAg loss and HBV relapse after discontinuation of lamivudine treatment. J. Hepatol. 61 , 515–522 (2014).

Kramvis, A., Kostaki, E. G., Hatzakis, A. & Paraskevis, D. Immunomodulatory function of HBeAg related to short-sighted evolution, transmissibility, and clinical manifestation of hepatitis B virus. Front. Microbiol. 9 , 2521 (2018).

Buckwold, V. E., Xu, Z., Chen, M., Yen, T. S. & Ou, J. H. Effects of a naturally occurring mutation in the hepatitis B virus basal core promoter on precore gene expression and viral replication. J. Virol. 70 , 5845–5851 (1996).

Warner, N. & Locarnini, S. The antiviral drug selected hepatitis B virus rtA181T/sW172* mutant has a dominant negative secretion defect and alters the typical profile of viral rebound. Hepatology 48 , 88–98 (2008).

Kay, A. & Zoulim, F. Hepatitis B virus genetic variability and evolution. Virus Res. 127 , 164–176 (2007).

Zoulim, F. & Locarnini, S. Optimal management of chronic hepatitis B patients with treatment failure and antiviral drug resistance. Liver Int. 33 (Suppl. 1), 116–124 (2013).

Terrault, N. A. et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology 67 , 1560–1599 (2018). Most recent American HBV treatment guidelines from the AASLD .

Littlejohn, M. Antiviral drug resistance and hepatitis B: a continuing public health problem. Antivir. Ther. 22 , 643–644 (2017).

Liu, Y. et al. No detectable resistance to tenofovir disoproxil fumarate in HBeAg+ and HBeAg− patients with chronic hepatitis B after 8 years of treatment. J. Viral Hepat. 24 , 68–74 (2017).

Park, E. S. et al. Identification of a quadruple mutation that confers tenofovir resistance in chronic hepatitis B patients. J. Hepatol. 70 , 1093–1102 (2019).

Cathcart, A. L. et al. No resistance to tenofovir alafenamide detected through 96 weeks of treatment in patients with chronic hepatitis B infection. Antimicrob. Agents Chemother . 62 , e01064-18 (2018).

Lok, A. S., Zoulim, F., Dusheiko, G. & Ghany, M. G. Hepatitis B cure: from discovery to regulatory approval. Hepatology 66 , 1296–1313 (2017).

Zeisel, M. B. et al. Towards an HBV cure: state-of-the-art and unresolved questions—report of the ANRS workshop on HBV cure. Gut 64 , 1314–1326 (2015).

Revill, P. A., Penicaud, C., Brechot, C. & Zoulim, F. Meeting the challenge of eliminating chronic hepatitis B infection. Genes 10 , 260 (2019).

CAS   PubMed Central   Google Scholar  

Petersen, J. et al. Prevention of hepatitis B virus infection in vivo by entry inhibitors derived from the large envelope protein. Nat. Biotechnol. 26 , 335–341 (2008).

Thi, E. P. et al. ARB-1740, a RNA interference therapeutic for chronic hepatitis B infection. ACS Infect. Dis. 5 , 725–737 (2018).

Wooddell, C. I. et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci. Transl. Med. 9 , eaan0241 (2017). Groundbreaking study demonstrating that in HBeAg-negative individuals, most of the secreted HBsAg is produced by RNA transcribed from integrated HBV DNA .

Bazinet, M. et al. Safety and efficacy of REP 2139 and pegylated interferon alfa-2a for treatment-naive patients with chronic hepatitis B virus and hepatitis D virus co-infection (REP 301 and REP 301-LTF): a non-randomised, open-label, phase 2 trial. Lancet Gastroenterol. Hepatol. 2 , 877–889 (2017).

Roehl, I. et al. Nucleic acid polymers with accelerated plasma and tissue clearance for chronic hepatitis B therapy. Mol. Ther. Nucleic Acids 8 , 1–12 (2017).

Long, K. R. et al. Efficacy of hepatitis B virus ribonuclease H inhibitors, a new class of replication antagonists, in FRG human liver chimeric mice. Antivir. Res. 149 , 41–47 (2018).

Klumpp, K. et al. Efficacy of NVR 3-778, alone and in combination with pegylated interferon, vs entecavir In uPA/SCID mice with humanized livers and HBV infection. Gastroenterology 154 , 652–662 (2018).

Lam, A. M. et al. Preclinical characterization of NVR 3-778, a first-in-class capsid assembly modulator against hepatitis B virus. Antimicrob. Agents Chemother. 63 , e01734-18 (2019).

Berke, J. M. et al. Antiviral profiling of the capsid assembly modulator BAY41-4109 on full-length HBV genotype A-H clinical isolates and core site-directed mutants in vitro. Antivir. Res. 144 , 205–215 (2017).

Ruan, L., Hadden, J. A. & Zlotnick, A. Assembly properties of hepatitis B virus core protein mutants correlate with their resistance to assembly-directed antivirals. J. Virol. 92 , e01082-18 (2018).

Wang, J. et al. Influences on viral replication and sensitivity to GLS4, a HAP compound, of naturally occurring T109/V124 mutations in hepatitis B virus core protein. J. Med. Virol. 89 , 1804–1810 (2017).

Zhou, Z. et al. Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) inhibit hepatitis B virus replication by different molecular mechanisms. Sci. Rep. 7 , 42374 (2017).

Seeger, C. & Sohn, J. A. Complete spectrum of CRISPR/Cas9-induced mutations on HBV cccDNA. Mol. Ther. 24 , 1258–1266 (2016).

Carman, W. F. et al. Vaccine-induced escape mutant of hepatitis B virus. Lancet 336 , 325–329 (1990).

Carman, W. F. The clinical significance of surface antigen variants of hepatitis B virus. J. Viral Hepat. 4 (Suppl. 1), 11–20 (1997).

Hsu, H. Y., Chang, M. H., Ni, Y. H. & Chen, H. L. Survey of hepatitis B surface variant infection in children 15 years after a nationwide vaccination programme in Taiwan. Gut 53 , 1499–1503 (2004).

Wang, J. et al. Molecular evolution of hepatitis B vaccine escape variants in China, during 2000–2016. Vaccine 35 , 5808–5813 (2017).

Mokaya, J. et al. A systematic review of hepatitis B virus (HBV) drug and vaccine escape mutations in Africa: a call for urgent action. PLoS Negl. Trop. Dis. 12 , e0006629 (2018).

Locarnini, S. & Shouval, D. Commonly found variations/mutations in the HBsAg of hepatitis B virus in the context of effective immunization programs: questionable clinical and public health significance. J. Virol. 88 , 6532 (2014).

Jilg, W. et al. Reduced prevalence of HBsAg variants following a successful immunization program in China. J. Virol. 88 , 4605–4606 (2014).

Tu, T., Buhler, S. & Bartenschlager, R. Chronic viral hepatitis and its association with liver cancer. Biol. Chem. 398 , 817–837 (2017).

Orito, E. et al. Geographic distribution of hepatitis B virus (HBV) genotype in patients with chronic HBV infection in Japan. Hepatology 34 , 590–594 (2001).

Orito, E. & Mizokami, M. Hepatitis B virus genotypes and hepatocellular carcinoma in Japan. Intervirology 46 , 408–412 (2003).

Ding, X. et al. Hepatitis B virus genotype distribution among chronic hepatitis B virus carriers in Shanghai, China. Intervirology 44 , 43–47 (2001).

Pan, M. H. et al. Hepatitis B splice variants are strongly associated with and are indeed predictive of hepatocellular carcinoma. J. Hepatol. 68 , s474–s475 (2018).

Bayliss, J. et al. Hepatitis B virus splicing is enhanced prior to development of hepatocellular carcinoma. J. Hepatol. 59 , 1022–1028 (2013). First study to identify an association between the presence of HBV splice variants in patient serum and liver cancer .

Baptista, M., Kramvis, A. & Kew, M. C. High prevalence of 1762(T) 1764(A) mutations in the basic core promoter of hepatitis B virus isolated from black Africans with hepatocellular carcinoma compared with asymptomatic carriers. Hepatology 29 , 946–953 (1999).

Kao, J. H. Hepatitis B virus genotypes and hepatocellular carcinoma in Taiwan. Intervirology 46 , 400–407 (2003).

Fang, Z. L. et al. HBV core promoter mutations prevail in patients with hepatocellular carcinoma from Guangxi, China. J. Med. Virol. 56 , 18–24 (1998).

Kim, D. W., Lee, S. A., Hwang, E. S., Kook, Y. H. & Kim, B. J. Naturally occurring precore/core region mutations of hepatitis B virus genotype C related to hepatocellular carcinoma. PLoS One 7 , e47372 (2012).

Ni, Y. H., Chang, M. H., Hsu, H. Y. & Tsuei, D. J. Different hepatitis B virus core gene mutations in children with chronic infection and hepatocellular carcinoma. Gut 52 , 122–125 (2003).

Sung, F. Y. et al. Hepatitis B virus core variants modify natural course of viral infection and hepatocellular carcinoma progression. Gastroenterology 137 , 1687–1697 (2009).

Liu, W. C. et al. Hepatocellular carcinoma-associated single-nucleotide variants and deletions identified by the use of genome-wide high-throughput analysis of hepatitis B virus. J. Pathol. 243 , 176–192 (2017).

Yen, C. J. et al. Hepatitis B virus surface gene pre-S2 mutant as a high-risk serum marker for hepatoma recurrence after curative hepatic resection. Hepatology 68 , 815–826 (2018).

Tsai, H. W. et al. Resistance of ground glass hepatocytes to oral antivirals in chronic hepatitis B patients and implication for the development of hepatocellular carcinoma. Oncotarget 7 , 27724–27734 (2016).

Yen, T. T. et al. Hepatitis B virus PreS2-mutant large surface antigen activates store-operated calcium entry and promotes chromosome instability. Oncotarget 7 , 23346–23360 (2016).

Hsieh, Y. H. et al. Hepatitis B virus pre-S2 mutant large surface protein inhibits DNA double-strand break repair and leads to genome instability in hepatocarcinogenesis. J. Pathol. 236 , 337–347 (2015).

Lai, M. W. et al. Hepatocarcinogenesis in transgenic mice carrying hepatitis B virus pre-S/S gene with the sW172* mutation. Oncogenesis 5 , e273 (2016).

Su, I. J., Wang, H. C., Wu, H. C. & Huang, W. Y. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection. J. Gastroenterol. Hepatol. 23 , 1169–1174 (2008).

Fan, Y. F. et al. Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection. Hepatology 33 , 277–286 (2001).

Lamontagne, R. J., Bagga, S. & Bouchard, M. J. Hepatitis B virus molecular biology and pathogenesis. Hepatoma Res. 2, 163–186 (2016).

Salpini, R. et al. The novel HBx mutation F30V correlates with hepatocellular carcinoma in vivo, reduces hepatitis B virus replicative efficiency and enhances anti-apoptotic activity of HBx N terminus in vitro. Clin. Microbiol. Infect. 25 , 906.e1–906.e7 (2019).

Raimondo, G. et al. Occult hepatitis B virus infection. Dig. Liver Dis. 32 , 822–826 (2000).

Raimondo, G. et al. Update of the statements on biology and clinical impact of occult hepatitis B virus infection. J. Hepatol. 71 , 397–408 (2019). Comprehensive summary on the importance of occult HBV infection .

Ramachandran, S. et al. Recent and occult hepatitis B virus infections among blood donors in the United States. Transfusion 59 , 601–611 (2019).

Deguchi, M. et al. Evaluation of the highly sensitive chemiluminescent enzyme immunoassay ‘‘Lumipulse HBsAg-HQ’’ for hepatitis B virus screening. J. Clin. Lab. Anal. 32 , e22334 (2018).

Zhang, K. et al. Antigenicity reduction contributes mostly to poor detectability of HBsAg by hepatitis B virus (HBV) S-gene mutants isolated from individuals with occult HBV infection. J. Med. Virol. 90 , 263–270 (2018).

Hass, M. et al. Functional analysis of hepatitis B virus reactivating in hepatitis B surface antigen-negative individuals. Hepatology 42 , 93–103 (2005).

Huang, F. Y. et al. Sequence variations of full-length hepatitis B virus genomes in Chinese patients with HBsAg-negative hepatitis B infection. PLoS One 9 , e99028 (2014).

Ponde, R. A. Molecular mechanisms underlying HBsAg negativity in occult HBV infection. Eur. J. Clin. Microbiol. Infect. Dis. 34 , 1709–1731 (2015).

McNaughton, A. L. et al. Insights from deep sequencing of the HBV genome — unique, tiny, and misunderstood. Gastroenterology 156 , 384–399 (2019).

Bill, C. A. & Summers, J. Genomic DNA double-strand breaks are targets for hepadnaviral DNA integration. Proc. Natl Acad. Sci. USA 101 , 11135–11140 (2004).

Lucifora, J. et al. Specific and nonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science 343 , 1221–1228 (2014).

Nair, S. & Zlotnick, A. Asymmetric modification of hepatitis B virus (HBV) genomes by an endogenous cytidine deaminase inside HBV cores informs a model of reverse transcription. J. Virol. 92 , e02190–17 (2018).

Suspene, R. et al. Extensive editing of both hepatitis B virus DNA strands by APOBEC3 cytidine deaminases in vitro and in vivo. Proc. Natl Acad. Sci. USA 102 , 8321–8326 (2005).

Vartanian, J. P. et al. Massive APOBEC3 editing of hepatitis B viral DNA in cirrhosis. PLoS Pathog. 6 , e1000928 (2010).

Gout, J. F., Thomas, W. K., Smith, Z., Okamoto, K. & Lynch, M. Large-scale detection of in vivo transcription errors. Proc. Natl Acad. Sci. USA 110 , 18584–18589 (2013).

Imashimizu, M., Oshima, T., Lubkowska, L. & Kashlev, M. Direct assessment of transcription fidelity by high-resolution RNA sequencing. Nucleic Acids Res. 41 , 9090–9104 (2013).

Carey, L. B. RNA polymerase errors cause splicing defects and can be regulated by differential expression of RNA polymerase subunits. Elife 4 , e09945 (2015).

Park, S. G., Kim, Y., Park, E., Ryu, H. M. & Jung, G. Fidelity of hepatitis B virus polymerase. Eur. J. Biochem. 270 , 2929–2936 (2003).

Preikschat, P. et al. Complex HBV populations with mutations in core promoter, C gene, and pre-S region are associated with development of cirrhosis in long-term renal transplant recipients. Hepatology 35 , 466–477 (2002).

Yeung, P. et al. Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in chronic hepatitis B. J. Infect. Dis. 203 , 646–654 (2011).

Zhao, X. L. et al. Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV. Gut 65 , 502–511 (2016).

Lewellyn, E. B. & Loeb, D. D. Base pairing between cis-acting sequences contributes to template switching during plus-strand DNA synthesis in human hepatitis B virus. J. Virol. 81 , 6207–6215 (2007).

Liu, N., Ji, L., Maguire, M. L. & Loeb, D. D. cis-Acting sequences that contribute to the synthesis of relaxed-circular DNA of human hepatitis B virus. J. Virol. 78 , 642–649 (2004).

Brechot, C., Gozuacik, D., Murakami, Y. & Paterlini-Brechot, P. Molecular bases for the development of hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). Semin. Cancer Biol. 10 , 211–231 (2000).

Tu, T., Budzinska, M. A., Vondran, F. W. R., Shackel, N. A. & Urban, S. Hepatitis B virus DNA integration occurs early in the viral life cycle in an in vitro infection model via NTCP-dependent uptake of enveloped virus particles. J. Virol. 92 , e02007–17 (2018). This study changed the dogma that HBV integration only occurs late in chronic disease by demonstrating that HBV integration occurs very early after infection .

Lan, P., Zhang, C., Han, Q., Zhang, J. & Tian, Z. Therapeutic recovery of hepatitis B virus (HBV)-induced hepatocyte-intrinsic immune defect reverses systemic adaptive immune tolerance. Hepatology 58 , 73–85 (2013).

Michler, T. et al. Blocking sense-strand activity improves potency, safety and specificity of anti-hepatitis B virus short hairpin RNA. EMBO Mol. Med. 8 , 1082–1098 (2016).

Li, B. et al. Suppression of hepatitis B virus antigen production and replication by wild-type HBV dependently replicating HBV shRNA vectors in vitro and in vivo. Antivir. Res. 134 , 117–129 (2016).

Tu, T., Budzinska, M. A., Shackel, N. A. & Urban, S. HBV DNA integration: molecular mechanisms and clinical implications. Viruses 9 , 75 (2017).

Sommer, G. & Heise, T. Posttranscriptional control of HBV gene expression. Front. Biosci. 13 , 5533–5547 (2008).

Su, T. S. et al. Hepatitis B virus transcript produced by RNA splicing. J. Virol. 63 , 4011–4018 (1989).

Gunther, S., Sommer, G., Iwanska, A. & Will, H. Heterogeneity and common features of defective hepatitis B virus genomes derived from spliced pregenomic RNA. Virology 238 , 363–371 (1997).

Chen, J. et al. Hepatitis B virus spliced variants are associated with an impaired response to interferon therapy. Sci. Rep. 5 , 16459 (2015).

Lam, A. M. et al. Hepatitis B virus capsid assembly modulators, but not nucleoside analogs, inhibit the production of extracellular pregenomic RNA and spliced RNA variants. Antimicrob. Agents Chemother . 61 , e00680-17 (2017).

Ziemer, M., Garcia, P., Shaul, Y. & Rutter, W. J. Sequence of hepatitis B virus DNA incorporated into the genome of a human hepatoma cell line. J. Virol. 53 , 885–892 (1985). One of the first studies showing HBV integration into the human genome .

Soussan, P. et al. In vivo expression of a new hepatitis B virus protein encoded by a spliced RNA. J. Clin. Invest. 105 , 55–60 (2000). First identification and characterization of a novel protein produced by HBV splice variants .

Soussan, P. et al. Expression of defective hepatitis B virus particles derived from singly spliced RNA is related to liver disease. J. Infect. Dis. 198 , 218–225 (2008).

Soussan, P. et al. The expression of hepatitis B spliced protein (HBSP) encoded by a spliced hepatitis B virus RNA is associated with viral replication and liver fibrosis. J. Hepatol. 38 , 343–348 (2003).

Chen, W. N. et al. Interaction of the hepatitis B spliced protein with cathepsin B promotes hepatoma cell migration and invasion. J. Virol. 86 , 13533–13541 (2012).

Shi, W. et al. Identification of novel inter-genotypic recombinants of human hepatitis B viruses by large-scale phylogenetic analysis. Virology 427 , 51–59 (2012).

Simmonds, P. & Midgley, S. Recombination in the genesis and evolution of hepatitis B virus genotypes. J. Virol. 79 , 15467–15476 (2005). This study was the first to demonstrate that significant recombination events occurred in the evolution of HBV .

Yang, J., Xing, K., Deng, R., Wang, J. & Wang, X. Identification of hepatitis B virus putative intergenotype recombinants by using fragment typing. J. Gen. Virol. 87 , 2203–2215 (2006).

Bowyer, S. M. & Sim, J. G. Relationships within and between genotypes of hepatitis B virus at points across the genome: footprints of recombination in certain isolates. J. Gen. Virol. 81 , 379–392 (2000).

Araujo, N. M. Hepatitis B virus intergenotypic recombinants worldwide: an overview. Infect. Genet. Evol. 36 , 500–510 (2015).

Sugauchi, F. et al. Hepatitis B virus of genotype B with or without recombination with genotype C over the precore region plus the core gene. J. Virol. 76 , 5985–5992 (2002).

Littlejohn, M. et al. Molecular virology of hepatitis B virus, sub-genotype C4 in northern Australian indigenous populations. J. Med. Virol. 86 , 695–706 (2014). This is the first description of the sequence and characterization of the novel C4 subgenotype in indigenous Australian peoples .

Cui, C. et al. The dominant hepatitis B virus genotype identified in Tibet is a C/D hybrid. J. Gen. Virol. 83 , 2773–2777 (2002).

Cheng, Y., Guindon, S., Rodrigo, A. & Lim, S. G. Increased viral quasispecies evolution in HBeAg seroconverter patients treated with oral nucleoside therapy. J. Hepatol. 58 , 217–224 (2013).

Wu, S. et al. Evolution of hepatitis B genotype C viral quasi-species during hepatitis B e antigen seroconversion. J. Hepatol. 54 , 19–25 (2011).

Nie, H., Evans, A. A., London, W. T., Block, T. M. & Ren, X. D. Quantitative dynamics of hepatitis B basal core promoter and precore mutants before and after HBeAg seroconversion. J. Hepatol. 56 , 795–802 (2012).

Stevens, C. E., Neurath, R. A., Beasley, R. P. & Szmuness, W. HBeAg and anti-HBe detection by radioimmunoassay: correlation with vertical transmission of hepatitis B virus in Taiwan. J. Med. Virol. 3 , 237–241 (1979).

Yan, H. et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus. Elife 1 , e00049 (2012). This is the first report of the identification and description of the HBV entry receptor — a major discovery for the HBV field .

Meier, A., Mehrle, S., Weiss, T. S., Mier, W. & Urban, S. Myristoylated PreS1-domain of the hepatitis B virus L-protein mediates specific binding to differentiated hepatocytes. Hepatology 58 , 31–42 (2013).

Hasegawa, K., Huang, J., Rogers, S. A., Blum, H. E. & Liang, T. J. Enhanced replication of a hepatitis B virus mutant associated with an epidemic of fulminant hepatitis. J. Virol. 68 , 1651–1659 (1994).

Baumert, T. F., Rogers, S. A., Hasegawa, K. & Liang, T. J. Two core promotor mutations identified in a hepatitis B virus strain associated with fulminant hepatitis result in enhanced viral replication. J. Clin. Invest. 98 , 2268–2276 (1996).

Nishizawa, T. et al. Enhanced pregenomic RNA levels and lowered precore mRNA transcription efficiency in a genotype A hepatitis B virus genome with C1766T and T1768A mutations obtained from a fulminant hepatitis patient. J. Gen. Virol. 97 , 2643–2656 (2016).

Seeger, C. & Mason, W. S. Molecular biology of hepatitis B virus infection. Virology 479–480 , 672–686 (2015).

Tu, T. & Urban, S. Virus entry and its inhibition to prevent and treat hepatitis B and hepatitis D virus infections. Curr. Opin. Virol. 30 , 68–79 (2018).

Allweiss, L. et al. Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo. Gut 67 , 542–552 (2018).

Farci, P. & Niro, G. A. Current and future management of chronic hepatitis D. Gastroenterol. Hepatol. 14 , 342–351 (2018).

Wranke, A. et al. Clinical and virological heterogeneity of hepatitis delta in different regions world-wide: the Hepatitis Delta International Network (HDIN). Liver Internatl. 38 , 842–850 (2018).

Magnius, L. et al. ICTV virus taxonomy profile: deltavirus. J. Gen. Virol. 99 , 1565–1566 (2018).

Le Gal, F. et al. Genetic diversity and worldwide distribution of the deltavirus genus: a study of 2,152 clinical strains. Hepatology 66 , 1826–1841 (2017).

Ryu, W. S., Bayer, M. & Taylor, M. Assembly of hepatitis delta virus. J. Virol. 66 , 2310–2315 (1992).

Wang, C. J., Chen, P. J., Wu, J. C., Patel, D. & Chen, D. S. Small-form hepatitis B surface antigen is sufficient to help in the assembly of hepatitis delta virus-like particles. J. Virol. 65 , 6630–6636 (1991).

Polo, J. M. et al. Transgenic mice support replication of hepatitis delta virus RNA in multiple tissues, particularly in skeletal muscle. J. Virol. 69 , 4880–4887 (1995).

Kristensen, L. S. et al. The biogenesis, biology and characterization of circular RNAs. Nat. Rev. Genet. 20 , 675–691 (2019).

Barrett, S. P. & Salzman, J. Circular RNAs: analysis, expression and potential functions. Development 143 , 1838–1847 (2016).

Perrotta, A. T. & Been, M. D. A pseudoknot-like structure required for efficient self-cleavage of hepatitis delta virus RNA. Nature 350 , 434–436 (1991).

Ferre-D’Amare, A. R., Zhou, K. & Doudna, J. A. Crystal structure of a hepatitis delta virus ribozyme. Nature 395 , 567–574 (1998).

Webb, C. H., Nguyen, D., Myszka, M. & Luptak, A. Topological constraints of structural elements in regulation of catalytic activity in HDV-like self-cleaving ribozymes. Sci. Rep. 6 , 28179 (2016).

Webb, C. H. & Luptak, A. HDV-like self-cleaving ribozymes. RNA Biol. 8 , 719–727 (2011).

Salehi-Ashtiani, K., Luptak, A., Litovchick, A. & Szostak, J. W. A genomewide search for ribozymes reveals an HDV-like sequence in the human CPEB3 gene. Science 313 , 1788–1792 (2006).

Wille, M. et al. A divergent hepatitis D-like agent in birds. Viruses 10 , E720 (2018).

Hetzel, U. et al. Identification of a novel deltavirus in boa constrictors. MBio 10 , e00014–19 (2019).

Chang, W. S. et al. Novel hepatitis D-like agents in vertebrates and invertebrates. Virus Evol. 5 , vez021 (2019).

Perez-Vargas, J. et al. Enveloped viruses distinct from HBV induce dissemination of hepatitis D virus in vivo. Nat. Commun. 10 , 2098 (2019).

Weller, M. L. et al. Hepatitis delta virus detected in salivary glands of Sjögren’s syndrome patients and recapitulates a Sjögren’s syndrome-like phenotype in vivo. Pathog. Immun. 1 , 12–40 (2016).

Szirovicza, L. et al. Snake deltavirus utilizes envelope proteins of different viruses to generate infectious particles. M Bio 11 , e03250-19 (2019).

Rizzetto, M. Hepatitis D virus: introduction and epidemiology. Cold Spring Harb. Perspect. Med. 5 , a021576 (2015).

Wang, T. C. & Chao, M. RNA recombination of hepatitis delta virus in natural mixed-genotype infection and transfected cultured cells. J. Virol. 79 , 2221–2229 (2005).

Lin, C. C. et al. RNA recombination in hepatitis delta virus: identification of a novel naturally occurring recombinant. J. Microbiol. Immunol. Infect. 50 , 771–780 (2017).

Hsu, S. C. et al. Varied assembly and RNA editing efficiencies between genotypes I and II hepatitis D virus and their implications. Hepatology 35 , 665–672 (2002).

Wu, J. C. et al. Genotyping of hepatitis D virus by restriction-fragment length polymorphism and relation to outcome of hepatitis D. Lancet 346 , 939–941 (1995).

Ivaniushina, V. et al. Hepatitis delta virus genotypes I and II cocirculate in an endemic area of Yakutia, Russia. J. Gen. Virol. 82 , 2709–2718 (2001).

Casey, J. L. et al. Hepatitis B virus (HBV)/hepatitis D virus (HDV) coinfection in outbreaks of acute hepatitis in the Peruvian Amazon basin: the roles of HDV genotype III and HBV genotype F. J. Infect. Dis. 174 , 920–926 (1996).

Watanabe, H. et al. Chronic hepatitis delta virus infection with genotype IIb variant is correlated with progressive liver disease. J. Gen. Virol. 84 , 3275–3289 (2003).

Madejon, A. et al. Hepatitis B and D viruses replication interference: influence of hepatitis B genotype. World J. Gastroenterol. 22 , 3165–3174 (2016).

Kiesslich, D. et al. Influence of hepatitis B virus (HBV) genotype on the clinical course of disease in patients coinfected with HBV and hepatitis delta virus. J. Infect. Dis. 199 , 1608–1611 (2009).

Schaper, M. et al. Quantitative longitudinal evaluations of hepatitis delta virus RNA and hepatitis B virus DNA shows a dynamic, complex replicative profile in chronic hepatitis B and D. J. Hepatol. 52 , 658–664 (2010).

Su, C. W. et al. Genotypes and viremia of hepatitis B and D viruses are associated with outcomes of chronic hepatitis D patients. Gastroenterology 130 , 1625–1635 (2006).

Smedile, A. et al. Hepatitis B virus replication modulates pathogenesis of hepatitis D virus in chronic hepatitis D. Hepatology 13 , 413–416 (1991).

Quintero, A. et al. Hepatitis delta virus genotypes I and III circulate associated with hepatitis B virus genotype F in Venezuela. J. Med. Virol. 64 , 356–359 (2001).

Shih, H. H. et al. Hepatitis B surface antigen levels and sequences of natural hepatitis B virus variants influence the assembly and secretion of hepatitis d virus. J. Virol. 82 , 2250–2264 (2008).

Vietheer, P. T., Netter, H. J., Sozzi, T. & Bartholomeusz, A. Failure of the lamivudine-resistant rtM204I hepatitis B virus mutants to efficiently support hepatitis delta virus secretion. J. Virol. 79 , 6570–6573 (2005).

Colombo, P. et al. Smouldering hepatitis B virus replication in patients with chronic liver disease and hepatitis delta virus superinfection. J. Hepatol. 12 , 64–69 (1991).

Colagrossi, L. et al. HDV can constrain HBV genetic evolution in HBsAg: implications for the identification of innovative pharmacological targets. Viruses 10 , E363 (2018).

Jilbert, A. J. & Locarnini, S. A. in Viral Hepatitis 3rd edn (eds Thomas, H. C. Lemon, S. & Zuckerman, A. J.) 193–209 (Wiley, 2005).

Erhardt, A. et al. Response to interferon alfa is hepatitis B virus genotype dependent: genotype A is more sensitive to interferon than genotype D. Gut 54 , 1009–1013 (2005).

Kao, J. H., Wu, N. H., Chen, P. J., Lai, M. Y. & Chen, D. S. Hepatitis B genotype and the response to interferon therapy. J. Hepatol. 33 , 998–1002 (2000).

Chu, C. J., Hussain, M. & Lok, A. S. Hepatitis B virus genotype B is associated with earlier HBeAg seroconversion compared with hepatitis B virus genotype C. Gastroenterology 122 , 1756–1762 (2002).

Osiowy, C., Gordon, D., Borlang, J., Giles, E. & Villeneuve, J. P. Hepatitis B virus genotype G epidemiology and co-infection with genotype A in Canada. J. Gen. Virol. 89 , 3009–3015 (2008).

Huy, T. T. T., Ngoc, T. T. & Abe, K. New complex recombinant genotype of hepatitis B virus identified in Vietnam. J. Virol. 82 , 5657–5663 (2008).

Nguyen, L. T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32 , 268–274 (2015).

Kalyaanamoorthy, S., Minh, B. Q., Wong, T. K. F., von Haeseler, A. & Jermiin, L. S. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat. Methods 14 , 587–589 (2017).

Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35 , 518–522 (2018).

Li, J., Buckwold, V. E., Hon, M. W. & Ou, J. H. Mechanism of suppression of hepatitis B virus precore RNA transcription by a frequent double mutation. J. Virol. 73 , 1239–1244 (1999).

Carman, W. F. et al. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet 2 , 588–591 (1989).

Okamoto, H. et al. Hepatitis B viruses with precore region defects prevail in persistently infected hosts along with seroconversion to the antibody against e antigen. J. Virol. 64 , 1298–1303 (1990).

Chotiyaputta, W. & Lok, A. S. Hepatitis B virus variants. Nat. Rev. Gastroenterol. Hepatol. 6 , 453–462 (2009).

Zoulim, F. & Locarnini, S. Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology 137 , 1593–1608 (2009).

Li, X. et al. PreS deletion profiles of hepatitis B virus (HBV) are associated with clinical presentations of chronic HBV infection. J. Clin. Virol. 82 , 27–32 (2016).

Lin, C. L. et al. Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma. J. Gastroenterol. Hepatol. 22 , 1098–1103 (2007).

Blondot, M. L., Bruss, V. & Kann, M. Intracellular transport and egress of hepatitis B virus. J. Hepatol. 64 , S49–S59 (2016).

Qi, Y. et al. DNA polymerase kappa is a key cellular factor for the formation of covalently closed circular DNA of hepatitis B virus. PLoS Pathog. 12 , e1005893 (2016).

Walters, K. A., Joyce, M. A., Addison, W. R., Fischer, K. P. & Tyrrell, D. L. Superinfection exclusion in duck hepatitis B virus infection is mediated by the large surface antigen. J. Virol. 78 , 7925–7937 (2004).

Ni, Y. et al. The effect of integration on cell susceptibility to HBV and HDV. Presented at the 2019 International Meeting for the Molecular Biology of Hepatitis Viruses (IHBV, 2019).

Bogomolov, P. et al. Treatment of chronic hepatitis D with the entry inhibitor myrcludex B: first results of a phase Ib/IIa study. J. Hepatol. 65 , 490–498 (2016).

Allweiss, L. et al. Strong intrahepatic decline of hepatitis D virus RNA and antigen after 24 weeks of treatment with Myrcludex B in combination with tenofovir in chronic HBV/HDV infected patients: interim results from a multicenter, open-label phase 2b clinical trial [abstract PS-162]. J. Hepatol. 68 , S90 (2018).

Wedemeyer, H. et al. Final results of a multicenter, open-label phase 2b clinical trial to assess safety and efficacy of Myrcludex B in combination with tenofovir in patients with chronic HBV/HDV co-infection [abstract GS-005]. J. Hepatol. 68 , S3 (2018). The final results of the clinical trial that demonstrated treatment with the entry inhibitor Myrcludex B showed efficacy against HDV infection .

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

Mason, W. S., Liu, C., Aldrich, C. E., Litwin, S. & Yeh, M. M. Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection. J. Virol. 84 , 8308–8315 (2010).

Tu, T. et al. Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection. J. Viral Hepat. 22 , 737–753 (2015).

Download references

Acknowledgements

T.T. is financially supported by the German Centre for Infection Research (DZIF), TTU Hepatitis Projects 5.807 and 5.704, the Deutsche Forschungsgemeinschaft (DFG) TRR179 (TP 15 and the Australian Centre for HIV and Hepatitis Virology Research). The authors thank R. Schinazi for providing helpful information on capsid inhibitors.

Author information

Authors and affiliations.

Victorian Infectious Diseases Reference Laboratory, Royal Melbourne Hospital, at the Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia

Peter A. Revill, Hans J. Netter, Lilly K. W. Yuen, Stephen A. Locarnini & Margaret Littlejohn

Department of Microbiology and Immunology, University of Melbourne, Melbourne, Victoria, Australia

Peter A. Revill & Margaret Littlejohn

Department of Infectious Diseases, Molecular Virology, Heidelberg Hospital University, Heidelberg, Germany

German Center for Infection Research (DZIF), Heidelberg Partner Site, Heidelberg, Germany

Storr Liver Centre, Westmead Institute for Medical Research, Westmead Hospital and University of Sydney, Sydney, New South Wales, Australia

You can also search for this author in PubMed   Google Scholar

Contributions

The authors contributed equally to all aspects of the article.

Corresponding author

Correspondence to Margaret Littlejohn .

Ethics declarations

Competing interests.

The authors declare no competing interests.

Additional information

Peer review information.

Nature Reviews Gastroenterology & Hepatology thanks T. Block, P. Matthews and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

(HBsAg). HBV encodes three surface (envelope) proteins: large (L), middle (M) and small (S) HBsAg, which share the C-terminal domain but differ in their N-terminal extensions.

Based on >7.5% nucleotide divergence, HBV has been phylogenetically classified into nine genotypes (A–I) and one putative genotype (J).

HBV genotypes have been further classified into >35 sub-genotypes based on approximately 4–8% nucleotide divergence.

(HBeAg). A secreted protein of the nucleocapsid gene of HBV. Seroconversion to HBeAg-negative is an important clinical marker in the natural history of HBV infection.

Occurring or sampled at different times.

A variant with nucleotide changes in the basal core promoter of HBV that result in reduced expression of HBeAg.

A variant with nucleotide changes in the pre-core region of the HBeAg that result in loss of expression of HBeAg.

Changes in the virus sequence that may or may not have consequences.

Variants produced as a result of splicing of HBV pregenomic RNA. These are incapable of autonomous replication but replicate in the presence of wild-type HBV.

Insertions or deletions of bases in the virus genome.

(HBcAg). The nucleocapsid (core) protein of HBV. A phosphoprotein that associates with the viral polymerase, DNA and RNA.

The population of related viral species occurring within a patient.

Generation of viruses (or viral vectors) in combination with foreign viral envelope proteins.

Rights and permissions

Reprints and permissions

About this article

Cite this article.

Revill, P.A., Tu, T., Netter, H.J. et al. The evolution and clinical impact of hepatitis B virus genome diversity. Nat Rev Gastroenterol Hepatol 17 , 618–634 (2020). https://doi.org/10.1038/s41575-020-0296-6

Download citation

Accepted : 20 March 2020

Published : 28 May 2020

Issue Date : October 2020

DOI : https://doi.org/10.1038/s41575-020-0296-6

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

This article is cited by

Pten deficiency potentiates hbv-associated liver cancer development through augmented gp73/golm1.

• Fuqiang Huang
• Hongbing Zhang

Journal of Translational Medicine (2024)

Hepatitis B virus reactivation associated with CAR T-cell therapy

• Haolong Lin
• Xiaoxi Zhou

Holistic Integrative Oncology (2024)

Origin and dispersal history of Hepatitis B virus in Eastern Eurasia

• Aida Andrades Valtueña

Nature Communications (2024)

Dose–response relationship between serum N-glycan markers and liver fibrosis in chronic hepatitis B

• Wei-Feng Liang

Hepatology International (2024)

Emerging Therapies for Chronic Hepatitis B and the Potential for a Functional Cure

• Ming-Ling Chang
• Yun-Fan Liaw

Drugs (2023)

Quick links

• Explore articles by subject
• Guide to authors
• Editorial policies

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

• Open access
• Published: 15 August 2024

The impact of integrated hepatitis B virus DNA on oncogenesis and antiviral therapy

• Mingming Zhang 1 , 2   na1 ,
• Han Chen 1 , 2   na1 ,
• Huan Liu 1 , 2 &
• Hong Tang 1 , 2  

Biomarker Research volume  12 , Article number:  84 ( 2024 ) Cite this article

256 Accesses

7 Altmetric

Metrics details

The global burden of hepatitis B virus (HBV) infection remains high, with chronic hepatitis B (CHB) patients facing a significantly increased risk of developing cirrhosis and hepatocellular carcinoma (HCC). The ultimate objective of antiviral therapy is to achieve a sterilizing cure for HBV. This necessitates the elimination of intrahepatic covalently closed circular DNA (cccDNA) and the complete eradication of integrated HBV DNA. This review aims to summarize the oncogenetic role of HBV integration and the significance of clearing HBV integration in sterilizing cure. It specifically focuses on the molecular mechanisms through which HBV integration leads to HCC, including modulation of the expression of proto-oncogenes and tumor suppressor genes, induction of chromosomal instability, and expression of truncated mutant HBV proteins. The review also highlights the impact of antiviral therapy in reducing HBV integration and preventing HBV-related HCC. Additionally, the review offers insights into future objectives for the treatment of CHB. Current strategies for HBV DNA integration inhibition and elimination include mainly antiviral therapies, RNA interference and gene editing technologies. Overall, HBV integration deserves further investigation and can potentially serve as a biomarker for CHB and HBV-related HCC.

Chronic Hepatitis B (CHB) refers to a persistent liver infection caused by the hepatitis B virus (HBV) that lasts for more than 6 months [ 1 ]. Among these individuals with CHB, a staggering 820,000 cases experienced adverse outcomes such as liver failure, liver cirrhosis, HBV-related hepatocellular carcinoma (HCC), or other HBV-associated diseases. The risk of developing HCC in patients with CHB is significantly elevated, ranging from 10 to 100 times higher compared to non-infected individuals. Integrated HBV DNA refers to the incorporation of HBV DNA into the host cell’s genome, which is one of the important factors contributing to HBV-related carcinogenesis. HBV integration can induce genetic damage and chromosomal instability, leading to tumor progression via the activation of oncogenes or inactivation of tumor suppressor genes [ 2 , 3 , 4 ]. It may persist even after successful hepatitis B surface antigen (HBsAg) seroconversion, which can contribute to HBsAg re-positivity and increase the risk of developing HCC [ 5 ]. Remarkably, integrated HBV DNA is identified in around 85-90% of HCC cases associated with hepatitis B virus infection [ 6 ]. Targeting the HBV DNA integration process and eliminating integrated HBV DNA from the host genome becomes crucial in preventing the progression of chronic hepatitis B. The treatment of CHB now includes options such as nucleos(t)ide analogues (NAs) and interferon-alpha (IFN-α), which could effectively inhibit HBV replication and new integrations. While IFN is administered for a finite duration, NAs are typically prescribed for extended periods, often lifelong [ 1 , 7 , 8 ]. The primary objective of CHB treatment is to suppress HBV replication to the maximum extent possible. Hepatocyte inflammation and necrosis, liver fibrosis and hyperplasia can be attenuated by inhibiting HBV replication, thereby delaying and reducing the occurrence of severe complications such as liver failure, liver cirrhosis, and HCC [ 9 , 10 ]. The types of CHB cure are categorized as functional cure (also known as clinical cure or immunological cure) and sterilizing cure (also known as virological cure). A “functional” cure was defined as sustained HBsAg loss and HBV DNA less than the lower limit of quantitation (LLOQ) 24 weeks off-treatment [ 11 ]. On the other hand, a “sterilizing” cure was defined as all traces of HBV infection would be eliminated, including cccDNA and integrated HBV DNA [ 11 , 12 ]. For eligible patients, the pursuit of functional cure should be considered [ 7 , 13 , 14 ]. The sterilizing cure of HBV is to achieve sterilizing cure, which necessitates the elimination of intrahepatic cccDNA and the complete eradication of integrated HBV DNA. However, this remains a challenging feat due to the persistence of viral reservoirs, weak immune response, long-term medication requirements, variable treatment responses, and the presence of advanced liver disease [ 1 , 15 ]. In animal models, the frequency of integration events is estimated to be approximately one in 10 3 -10 4 hepatocytes [ 16 , 17 ]. However, the precise mechanisms of integration remain unclear, necessitating further attention and investigation. This review aims to provide a comprehensive overview of HBV DNA integration, including its molecular mechanisms, detection methods, research models, oncogenetic roles in HCC, and potential treatment strategies for eliminating HBV DNA integration.

HBV integration occurs during HBV replication

The structure and life cycle of hbv dna.

The HBV is a DNA virus that belongs to the Hepadnaviridae family [ 1 ]. Dane particles are infectious virions characterized by a lipid membrane that encapsulates a nucleocapsid. The lipid membrane contains three HBsAg, which include large (L-HBs), middle (M-HBs), and small (S-HBs) forms. The nucleocapsid within the Dane particle is composed of hepatitis B core protein (HBc), viral polymerase (Pol), and the viral genome DNA. The HBc protein provides structural integrity to the nucleocapsid, while the viral polymerase is responsible for replicating the viral genome during the viral life cycle (Fig.  1 A).

The viral genome of the HBV is a partially double-stranded DNA (dsDNA) structure. It consists of one complete coding minus(-) strand and one incomplete non-coding plus(+) strand. The viral genome contains four overlapping open reading frames (ORFs) that are responsible for encoding various viral proteins including: (1) HBV DNA polymerase (pol), which is involved in viral replication and synthesis of the viral genome. (2) HBsAg, which exists in three forms: Large, Medium, and Small. These antigens are crucial for viral attachment and entry into host cells. (3) HBV core antigen (HBcAg), providing structural integrity to the nucleocapsid of the virus. (4) HBV e antigen (HBeAg), whose exact function is not fully understood but is associated with immune tolerance and viral replication. (5) HBV X protein (HBx), a multifunctional protein involved in regulating viral replication, cell proliferation, and immune response modulation. Each of these viral proteins contributes to different aspects of the HBV life cycle, including viral replication, virion assembly, and immune evasion (Fig.  1 B) [ 18 ].

After the HBV virion entering hepatocytes, the nucleocapsid is released into the cytoplasm and the relaxed circular DNA (rcDNA) enters the nucleus where it will convert into cccDNA [ 19 , 20 , 21 , 22 ]. The cccDNA then serves as a template for the synthesis of five transcripts (3.5Kb pregenomic RNA, 3.5Kb precore mRNA, 2.4Kb preS1 mRNA, 2.1Kb preS2/S mRNA, and 0.7Kb HBx mRNA), which are transcribed by host RNA polymerase II. Among these transcripts, the 3.5Kb pregenomic RNA (pgRNA) plays a crucial role in viral reverse transcription and replication processes. Following the utilization of pgRNA as a template, the minus(-) strand is synthesized, and subsequently, the synthesis of the plus(+) strand proceeds using the minus(-) strand as a template. This process gives rise to two products during plus(+) strand synthesis: partially circular rcDNA and double-stranded linear DNA (dslDNA). Rather than being encapsulated and secreted as virions, dslDNA has the potential to re-enter the nucleus and integrate into the human genome [ 5 ] (Fig.  1 C).

The structure of HBV DNA genome and HBV life cycle. (A) The schematic diagram of Dane particle. (B) The circular schematic diagram of genotype C HBV genome. (C) After HBV Dane particles entering hepatocytes, uncoating takes place and genome is released. RcDNA is repaired to form cccDNA which is transcribed to pgRNA and 3.5Kb/3.5Kb/2.4Kb/2.1Kb/0.7Kb transcripts, HBV RNA transcripts are translated into proteins such as HBeAg, Core protein and HBx protein. Polymerase binds to the pgRNA with the recruitment of Core protein to assemble nucleocapsid and package pgRNA, pgRNA serves as the template to reverse transcription synthesize the HBV minus(-)-strand DNA. Polymerase translocates accurately to synthesize the HBV plus(+) strand DNA. Polymerase translocates mistakenly results the synthesis of dslDNA and the integration of dslDNA into the host genome

The procedure of HBV integration

During reverse transcription, the HBV DNA polymerase utilizes pgRNA as a template to transcribe the minus(-) strand DNA. This process involves the generation of DNA oligonucleotides (TGAA or GAA), which serve as primers for synthesizing the minus(-) strand. These primers are produced within a ε stem-loop structure located at the 3’ terminal of the pgRNA. During the process of extending the minus(-) strand DNA, most of the pgRNA undergoes degradation mediated by RNase H, except for the capped 5’ end of pgRNA. Remarkably, these undegraded RNA oligonucleotides play a vital role as primers for the synthesis of the plus(+) strand DNA. Among these fragments, one containing the direct repeat 1 (DR1) sequence acts as a primer for the synthesis of the plus(+) strand DNA [ 23 , 24 , 25 ]. Typically, this primer binds to the direct repeat 2 (DR2) region of the newly synthesized minus(-) strand DNA, which is complementary to the DR1 segment of the residual pgRNA. This binding guides the synthesis of plus(+) strand DNA, resulting in the formation of partially circular rcDNA (90–95%). However, if the initiation of the plus(+) strand synthesis occurs in situ without binding to the DR2 region (in-situ priming), the synthesis of double-stranded dslDNA takes place (5–10%) [ 26 ]. This small probability of primer translocation failure might be due to the mutation of DR2 region which causing reduced complementarity between DR2 region and RNA primer [ 27 ]. Another study suggests that cis-acting elements mutants in HBV genome are related to the proportion of dslDNA generation [ 28 , 29 ].

Since the initial discovery of HBV integration in the early 1980s [ 30 , 31 ], several hypotheses have emerged to shed light on the mechanisms underlying this integration process [ 32 , 33 ]. The dslDNA provides HBV DNA fragments that can integrate into the host genome. Upon entering the nucleus, dslDNA is inserted randomly into hepatocyte chromosomes through DNA repair pathways [ 23 ]. The oxidative damage caused by hepatitis can induce DNA breakages in the host genome, creating breakpoints for the integration process [ 34 , 35 , 36 , 37 , 38 ]. Considering the limited homologous sequences between viral DNA and the human genome, the most likely mechanisms for HBV integration are non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ) DNA repair pathways [ 38 , 39 ]. These pathways facilitate the joining of DNA ends during the repair process, allowing the integration of HBV DNA fragments into the host genome. In the NHEJ pathway, DNA breaks lacking significant homology undergo modification and subsequent ligation, leading to the generation of deletions or insertions [ 40 ]. On the other hand, MMEJ pathway is a distinct mechanism for end joining that operates separately from NHEJ [ 41 , 42 ]. MMEJ relies on the presence of microhomology and utilizes longer stretches of microhomology (5–25 bp) compared to NHEJ [ 41 ]. Furthermore, the dslDNA can undergo circularization through the NHEJ DNA repair pathway. However, this process can lead to the formation of non-functional molecules due to the error-prone nature of NHEJ (Fig.  2 ) [ 43 ].

The breakpoints of HBV integration in the viral genome display several distinctive characteristics: (1) Integration often takes place near the DR1 or DR2 sites. (2) Integrated HBV fragments show a range of sizes, varying from 28 bp to 3215 bp. Long integration fragments(> 2000 bp) are observed more frequently than short ones. (3) It is common to observe small deletions within viral sequences at the joining site [ 16 , 38 , 44 , 45 , 46 , 47 ]. These characteristics highlight the specific patterns and variations in HBV integration events within the viral genome. Previous studies have shown that HBV has a preference for integrating into genic regions such as exons, introns, and promoters, as well as gene-rich areas [ 48 ]. Notably, certain genes such as hTERT , MLL4 , and CCNE1 have been frequently identified as targets of HBV integration [ 44 , 48 , 49 , 50 , 51 , 52 ]. This biased selection of integration sites has been observed in both tumor and adjacent tissues with a higher frequency of integration occurring in tumor tissues compared to non-tumor tissues [ 44 , 53 , 54 ]. These findings highlight the specific genomic locations where HBV integration tends to occur and suggest its potential impact on specific genes in both tumor and non-tumor tissues.

Schematic diagram about synthesis of dslDNA and HBV DNA integration. (A) ε stem-loop structure forms and P protein primes at the ε stem-loop structure to form P-ε ribonucleoprotein (RNP) complex. The terminal protein (TP) domain of the P protein binds with the first deoxyribonucleotide in ε stem-loop structure near the 5’cap of pgRNA. After the first four(TGAA) or three (GAA) nucleotides of the new minus(-) strand DNA generated in ε stem-loop structure, the DNA oligo then transferred to DR1 at the 3’end of pgRNA with TP and the synthesis of minus(-) strand starts. (B) pgRNA is degraded by RNase H domain of P protein while the minus(-) strand is synthesizing. (C) The DNA oligomer binds to the direct repeat 2 (DR2) region of the newly synthesized minus(-) strand DNA to guide the synthesis of plus(+) strand DNA, forming a partially circular rcDNA (90–95%). (D) The RNA primer directly initiated in situ without binding to the DR2 region (5–10%), dslDNA will be generated. (E) Inflammation and oxidative stress induce host genomic DNA double-stranded breaking, which provides breakpoints for integration through NHEJ or MMEJ

HBV integration occurs early in infection

The occurrence of integration during the early stages of HBV infection has been supported by multiple studies, which aligns with experimental evidence from cell infection models and animal liver infection models. For instance, in ducklings experimentally infected with the avian hepadnavirus duck hepatitis B virus (DHBV), integrated HBV DNA was detected as early as 6 days post-infection [ 55 ]. Similarly, in the woodchuck infection model, integration of woodchuck hepatitis virus (WHV) was observed within 1–3 h post-infection, indicating immediate genomic integration of WHV DNA into hepatocytes upon natural viral invasion [ 45 , 56 ]. These findings highlight the early occurrence of HBV integration and provide valuable insights into the dynamics of viral integration in different infection models. Moreover, numerous investigations utilizing primary human hepatocytes (PHH), HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells have consistently demonstrated rapid viral integration after infection [ 35 ]. Furuta et al. conducted a study using a chimeric mouse model consisting of human hepatocytes infected with HBV, where they found that HBV integration could occur between 23 and 49 days post-infection through MMEJ, primarily within mitochondrial DNA [ 57 ]. Furthermore, the occurrence of integration in acute hepatitis B also suggests its early onset following infection [ 58 ]. Taken together, these findings strongly indicate that integration may take place within the host genome during the initial stages of hepadnaviral infection. Considering that HBV DNA integration predominantly occurs during viral replication, it becomes crucial to hinder replication at the early stage of infection in order to prevent integration.

The integration of HBV DNA and Hepatitis promotes each other

Liver damage caused by HBV infection is characterized by persistent necrotizing inflammation accompanied by immune regulation [ 59 ]. Integrations, as an early event in HBV infection, are closely associated with ongoing immune-mediated inflammatory responses. The oxidative damage to hepatocellular DNA acts as breakpoints for dslDNA integration [ 60 ]. Multiple studies consistently report a positive correlation between the extent of hepadnavirus integration and oxidative damage [ 61 , 62 ]. From another perspective, HBV-specific cytotoxic T lymphocytes (CTLs) selectively target and eliminate hepatocytes replicating HBV, leading to the preferential clonal expansion of HBV DNA-integrated hepatocytes that may evade the immune response mediated by HBV-specific CTLs [ 47 ]. Moreover, the integration of HBV DNA can trigger an inflammatory response. Integrated HBV DNA is considered a potential source of HBsAg, which is derived from both a 2.1Kb transcript and mRNA transcribed from integrated HBV DNA. The presence of HBsAg plays a crucial role in the pathogenesis of hepatitis [ 63 ]. It is widely recognized that elevated production of HBsAg contributes to T cell exhaustion, resulting in restricted or impaired T cell responses and even the elimination of T cells recognizing specific epitopes [ 64 , 65 ]. Additionally, CD205 has recently been identified as a pivotal receptor involved in the capture of CpG-oligodeoxynucleotides in vivo. The enhanced expression of CD205 on Kupffer cells in HBsAg-transgenic mice may be attributed to mild inflammation associated with HBsAg [ 66 , 67 ]. In previous consensus, cccDNA has been acknowledged as the primary transcriptional template for HBsAg production [ 68 , 69 ]. This hypothesis is further supported by evidence documented in chimpanzees with chronic HBV infection [ 70 ]. A dynamic observation using liver biopsy specimens from CHB patients revealed that individuals with HBV S gene integration experienced a slower decline in serum HBsAg levels compared to those without such integration following prolonged therapy [ 71 ]. This finding highlights the diverse origin of HBsAg. Researchers from Switzerland, based on liver biopsies obtained from HBe(-) patients, discovered that transcriptionally active integrated HBV DNA can autonomously generate HBsAg without relying on HBV replication [ 72 ]. This result may explain why serum HBsAg level is much less correlation with HBV DNA in HBe(-) patients with a very low HBV replication state.

The interaction between HBV DNA integration and hepatitis is complex, as they mutually reinforce each other through immune responses starting from the early stages of HBV infection, ultimately leading to the development of HCC.

Examination and research models for HBV integration

With the progress of high-throughput sequencing technologies, different strategies have been implemented to enable a more precise investigation into the implications of the integration process. These strategies aid in the detection of integrated viral DNA within the host genome. The unique features of each strategy are summarized in Table  1 .

Examination methods based on DNA hybridization

The integration of HBV was initially detected in HCC patient tissue and the PLC/PRF/5 cell line in 1980 through Southern Blot hybridization using HBV as a probe 31 . It was observed that most integration events took place at the nicked cohesive end region of HBV DNA. Moreover, Northern blot analysis revealed the presence of specific transcripts of HBsAg even in the absence of HBcAg [ 73 ]. Following these discoveries, the Southern Blot hybridization technique was employed to detect viral integration within the host cell genome [ 2 , 74 ]. Subsequent investigations progressively unveiled the integration of HBV DNA in liver tissues of patients with HBV-related conditions such as HCC, acute HBV hepatitis, chronic HBV infection, and HBV-related liver cirrhosis [ 2 ]. These findings highlight use of Southern Blot hybridization as a valuable tool in studying viral integration. In addition to this approach, in situ hybridization based on the same principle as Southern Blot hybridization was utilized to identify the chromosomal sites of HBV DNA integration [ 75 ]. Subsequently, Fluorescence In Situ Hybridization (FISH) emerged as a more sensitive and specific method for detecting integrated HBV DNA, replacing the previous techniques [ 76 , 77 ].

Examination methods based on PCR amplification

Recombinant plasmid vectors were utilized for the direct cloning of virus-cell junctions, allowing for a comprehensive examination of integrated HBV DNA fragments [ 78 ]. However, the presence of diverse virus-cell junctions poses significant challenges in achieving accurate and sensitive detection of HBV integration.

The detection of virus-cell junctions has been made possible through the development of various PCR-based strategies, including the Arthrobacter luteus-PCR(Alu-PCR) [ 95 ]. Alu elements, which are short interspersed nuclear elements (SINEs), are widely distributed throughout primate genomes and can be found in approximately 1,000,000 copies per human genome [ 96 ]. By utilizing a combination of HBV and Alu repetitive element primers, it is possible to amplify and sequence fragments of virus-cell DNA junctions [ 71 , 97 , 98 ]. However, Alu-PCR has limitations in detecting HBV integrated fragments that are located far from the Alu repeat sequence or accurately quantifying the integration junctions.

In addition to Alu-PCR, another technique called inverse nested PCR (invPCR) can be employed for amplifying virus-cell DNA junctions. This method provides an alternative approach to detect and analyze these junction fragments. In 1995, Gong et al. successfully detected DR-related integrations of wild-type DHBV in LMH-D2 cells using inv PCR, which introduced a novel protocol for detecting and characterizing integrations of DHBV derived from episomal viral DNAs [ 99 ]. This strategy was primarily designed to selectively amplify virus-cell DNA junctions near the DR sequences, as these DR sequences are recognized as preferred integration sites for hepadnaviral DNA [ 23 , 45 , 55 , 99 , 100 ]. To detect integrations in or near hypothetical sites, high-molecular-weight liver DNA was cleaved by restriction endonucleases specifically targeting and cleaving HBV DNA and host DNA at unknown sites. Subsequently, the DNA was circularized using T4 DNA ligase and further cleaved by another restriction endonuclease, resulting in the generation of linear strands. Within these strands, the viral-cellular DNA junctions were located internally, with viral fragments present at both termini. These fragments were then amplified through nested PCR utilizing virus-specific primers [ 16 , 84 ]. This technique has been widely employed for the detection of integrated HBV DNA due to its high sensitivity and specificity [ 101 , 102 , 103 ]. However, it is important to note that this method can only detect DNA sequences in close proximity to the junctions and is heavily reliant on restriction endonucleases [ 47 ].

Examination methods based on high-throughput sequencing technology

Whole-genome sequencing (WGS) and whole-exome sequencing (WES) are two widely used next-generation sequencing (NGS) methods that have found extensive applications in various areas of virology research. NGS offers several advantages, including the elimination of the need for prior viral DNA information and improved sensitivity in detection. WGS allows comprehensive coverage of host genomes, enabling the identification of viral sequences [ 104 ]. On the other hand, WES provides greater depth than WGS Nanopore sequencing, but it focuses solely on coding regions 91 . However, deep sequencing with significant insertions or deletions remains challenging due to the intrinsic error-prone nature and limited length of the generated sequence reads [ 105 ]. In recent years, the field of third-generation sequencing technology has witnessed a remarkable advancement, offering inherent advantages in exploring complex genomic rearrangements [ 106 ]. This technology allows the generation of complete HBV genomes in a single sequencing read, facilitating the investigation of intricate and diverse distribution patterns of rapidly mutating viral genomes [ 107 ]. By combining third-generation sequencing with the analysis of biological information, a deeper understanding of HBV integration can be achieved [ 106 ].

Research models for HBV DNA integration

Comprehensive investigations into HBV integration face challenges due to the limited availability of human non-tumor liver tissues at all stages of HBV infection, especially compared to HCC tissues. Additionally, the scarcity of suitable models for studying HBV infection further hampers research on HBV integrations. To overcome these limitations, several in vitro studies have utilized PHH, HepaRG-NTCP, HepG2-NTCP, and Huh7-NTCP cells to investigate the mechanisms and timing of HBV DNA integration [ 35 , 57 , 77 ]. These cell-based models offer valuable insights into HBV integration. Furthermore, other hepadnavirus-infected animal models have also contributed significantly. For example, studies using ducklings infected with DHBV and woodchucks infected with WHV have provided important contributions to our understanding of HBV integration [ 45 , 55 , 56 ]. These animal models offer insights that complement the in vitro studies and enhance our overall understanding of HBV integration. Since HBV integration in both genomic DNA and RNA transcripts was observed in various cell lines including HepG2.2.15, HepAD38, PLC/PRF/5, DE19, MHCC97H, MHCC97L, MHCCLM3 cells as well as Huh1 and Hep3B cells, [ 88 , 92 , 93 ] therefore, HBV-related HCC cell lines could also be utilized as the cell model for HBV integration. Animal infection models, including chimpanzees, human liver chimeric mice, Tupaia, and hNTCP-expressing macaques, have been utilized to study HBV infection. These models demonstrate susceptibility to chronic HBV infection and can generate clonally expanded hepatocytes that contain integrated viral DNA [ 17 , 108 , 109 , 110 ].

HBV DNA integration induces HCC

Previously, it was suggested that integrated HBV DNA had no discernible function due to its random distribution and lack of requirement in HBV replication. However, over the past decade, numerous studies have shown the significant impact of HBV DNA integrations on both HBV infection and carcinogenesis (Fig.  3 ) [ 5 , 6 , 32 , 36 , 46 , 52 , 101 , 99 , 111 , 112 , 113 , 114 , 115 , 116 , 117 , 118 , 119 , 120 , 121 ]. Therefore, conducting more research to understand the relationships between integration translocations in host genes, fragments of HBV genome, and carcinogenetic mechanism of integration are of great clinical significance [ 112 ]. The integration of HBV DNA into the host genome is an early event that precedes clonal tumor expansion [ 122 , 123 ] and the presence of integration events indicates their potential role as precursors to tumor development in patients with chronic hepatitis and during the acute infection stage [ 35 , 58 , 124 , 125 ]. HBV DNA integration primarily contributes to HCC through three mechanisms: (1) modulation of the expression or function of proto-oncogenes and tumor suppressor genes, (2) induction of chromosomal instability, and (3) expression of integrated mutant HBV proteins [ 54 ].

Schematic diagram of HBV DNA integration from chronic HBV infection to hepatocellular carcinoma. (A) Initially HBV DNA randomly integrates into host genome. (B) The infected hepatocytes are eliminated by host immune response. Infected hepatocytes with favorable integrations survive and clonally expand. (C) Hepatocytes with integrations expand. When integration happens near/into HCC-related genes, HCC initiating cells may occur. (D) HCC initiating cells expand and HCC cells with carcinogenetic integrations appear. (E) HCC cells with carcinogenetic integrations expand leading to the development of HCC

HBV DNA integration modulates cancer-related genes

The integration of HBV in the human genome was observed to have a distinct distribution pattern in tumors compared to non-tumor tissues, with a tendency for enrichment around cancer driver genes [ 118 ]. The chromosomal locus 11q13.3 has a significant tendency to serve as a recurring site for HBV integration [ 126 ]. This specific genomic region contains crucial oncogenic driver genes, namely CCND1 and FGF19 , which are frequently amplified in HCC [ 127 ]. Additionally, the expression levels of cancer-associated genes, such as hTERT , KMT2B , MLL4 , CCNE1 and PAK3 , were found to be up-regulated in tumor tissues compared to their corresponding normal counterparts [ 44 , 128 , 129 ]. Telomeres, which enhance telomerase activity, play a crucial role in maintaining genome stability. Additionally, the upregulation of hTERT has been extensively reported [ 130 ]. Furthermore, studies investigating hTERT integration sites have shown that HBV DNA integration at the hTERT promoter is pivotal in the overexpression of the hTERT gene [ 131 , 132 ].

Integration events involving mitochondrial DNA have been identified in tissue samples obtained from both tumor and non-tumor areas of HCC patients. This new finding highlights mitochondrial DNA as a newly recognized target of HBV integration, causing mitochondrial instability and dysfunction. Consequently, this contributes to the development and progression of HCC [ 133 ]. In a recent study, the coexistence of two distinct HCC subtypes was observed in a patient with HBV infection, with no identical integration sites detected. This finding suggests that the multicentric occurrence of HCC may be attributed to diverse HBV DNA integration events [ 120 ].

HBV DNA integration induces chromosomal instability

Chromosomal instability is a fundamental characteristic of human cancer, and it is closely associated with unfavorable prognosis, metastasis, and resistance to therapeutic interventions [ 134 ]. The breakpoints of HBV integration have been found to be correlated with an increased level of copy-number variation [ 44 ]. This observation highlights the potential contribution of HBV integration to the chromosomal instability observed in the HCC genome [ 38 , 53 ].

One study suggests that HBV has a preferential integration site in the human genome, particularly fragile sites and CpG islands [ 38 ]. These are regions of the genome that are prone to rearrangements and genetic alterations, which can lead to the development of cancer. HBV integration into these regions can also lead to epigenetic instability, which can further contribute to the development of HCC [ 135 ]. Additionally, HBV integration events were observed to be enriched in the proximity of telomeres, which play a crucial role in maintaining genome stability. Dysfunction of telomeres can lead to extensive DNA rearrangements, deletions, and amplification, all of which are commonly associated with the development of cancer [ 136 ].

HBV DNA integration expresses truncated HBV proteins

Truncated HBs and HBx proteins, derived from integration fragments of HBV DNA, are recognized as significant contributors to the development of HCC [ 137 ]. Truncated preS2/S sequences within hepatocytes, commonly observed in integrated HBV DNA, have been implicated in promoting HCC progression through multiple pathways [ 138 , 139 , 140 ]. The accumulation of truncated mutant HBsAg induces endoplasmic reticulum stress, leading to the generation of reactive oxygen species, oxidative stress, and DNA damage [ 141 ]. Moreover, the down-regulated expression of TGFBI induced by truncated HBsAg in the TGF-β/Smad signaling pathway also contributes to carcinogenesis [ 142 ]. Additionally, the truncated S protein impedes the G1/S phase cell cycle checkpoint by suppressing the expression of the p53-p21 axis [ 143 ]. Multiple truncated HBx proteins, particularly those with C-terminal truncation (ct-HBx), have been identified to exert diverse functions in HCC, including the induction of stem cell-like characteristics, inhibition of apoptosis, and promotion of HCC invasion and metastasis [ 144 , 145 , 146 , 147 , 148 , 149 , 150 ].

The incidence of HCC is significantly higher in males compared to females, with a ratio of approximately 4:1 but the reasons for the gender bias are unclear. Some certain integration sites of HBV can be identified as human somatic risk loci for HBV integration (VIMs). The enriched transcription factors in VIMs are involved in DNA repair and the androgen receptor (AR) signaling pathway. There are significant interactions between the AR pathway and the complement system. These interactions, along with the X-linked ZXDB regulon that includes albumin (ALB), may contribute to the male predominance observed in HCC [ 151 ]. However, additional research is required to confirm the association between HBV integration and male predominance in HCC. Furthermore, studying the underlying mechanisms of integrated HBV DNA in promoting HCC can aid in the development of more targeted therapeutic strategies for HCC and provide novel biomarkers for monitoring its occurrence.

The quest for non-invasive biomarkers in HBV DNA integration during HCC development

Until now, the quantification of integrations has predominantly been conducted in liver tissues. However, liver biopsy is an invasive procedure associated with inherent risks. Alternative biomarkers such as serum levels of HBV core-related antigen (HBcrAg) and HBV RNA may serve as indicators of transcriptional activity specific to cccDNA, as they are expected to be exclusively generated from cccDNA rather than integrated HBV DNA due to the absence of a promoter that initiates core RNA transcription [ 22 , 152 , 153 , 154 ]. Therefore, further investigation is needed to identify specific serum biomarkers for HBV DNA integration. Cell-free DNA (cfDNA) is an emerging noninvasive blood biomarker that is used to assess tumor progression, evaluate prognosis, diagnose diseases, and monitor response to treatment [ 155 ]. Recent studies have reported the detection of HBV integration in circulating cfDNA from both HCC and liver cirrhosis patients’ plasma [ 89 ]. Since cfDNA primarily originates from dying tumor cells, the release of cfDNAs from non-HCC liver tissues is considerably lower compared to HCC liver tissues [ 156 ]. As a result, cfDNA is more suitable for monitoring HBV integration in HCC development. In a study on the early recurrence of HCC after surgical resection, researchers found that plasma virus-host chimera DNA (vh-DNA) could serve as a biomarker for detecting residual tumor cells and predicting recurrence [ 94 ]. Detecting sequence-unknown vh-DNA directly from cfDNA requires a sensitive NGS approach with a standardized workflow and appropriate cutoff values, along with a population study to ensure sensitivity and specificity, incorporating known tumor-related somatic mutations [ 157 ].

The importance of early intervention

HBV DNA integration has the potential to generate a portion of HBsAg and contribute to HCC development, making early intervention for HBV infection crucial. While NAs may not eradicate integrated HBV DNA, initiating treatment at an early stage can reduce the occurrence of integrations, potentially reducing oncogenic mutations. The continuous suppression of the virus through effective treatment significantly lowers the risk of oncogenic mutations. Functional cure, achieved through sustained virus suppression, greatly diminishes the likelihood of carcinogenic mutations. The elimination of cccDNA, the viral reservoir in hepatocytes, is essential in preventing HBV reactivation and relapse. The ultimate objective in managing HBV infection is to achieve a sterilizing cure, which involves the complete eradication of cccDNA and integrated HBV DNA from the host genome. Strategies aimed at accomplishing this goal include utilizing antiviral agents that specifically target and eliminate integrated HBV DNA from the host cells.

The slow decline, or no decline of serum HBsAg levels during NAs treatment may be due to the ongoing production of HBsAg from integrated HBV DNA, particularly in HBeAg-negative patients [ 158 ]. Recent studies have confirmed the presence of integrant-derived RNAs (id-RNAs) and 5’-human-HBV-3’ transcripts originating from integrated HBV DNA in serum [ 159 ]. Initiating treatment at an early stage may enhance the likelihood of achieving a functional cure by reducing HBV DNA integration. Additionally, quantifying integrations in these patients can help identify factors that contribute to the slow clearance of HBsAg.

Exploring strategies for HBV DNA integration inhibition and elimination: current progress and future directions

The integration of HBV DNA can contribute to both neoplasia and a portion of HBsAg production. The elimination of integrated HBV DNA is also regarded as a critical measure for achieving complete eradication of HBV [ 1 ]. Inhibiting HBV DNA integration at an early stage holds immense importance and has consistently garnered significant attention from researchers in this field [ 160 , 161 ]. The efficacies of current strategies to eliminate HBV DNA integration are concluded in Table  2 .

The commonly used treatment, such as NAs, has been recognized for its efficacy in inhibiting the production of integrated HBV DNA [ 71 ]. NAs are effective in suppressing HBV replication, thereby reducing the generation of integrated viral DNA resulting from viral replication. After entecavir (ETV) treatment, the pattern of HBV integration appears to be more random and irregular, potentially contributing to a decreased risk of HCC [ 162 ]. In a recent study, researchers procured liver tissue specimens from individuals diagnosed with chronic hepatitis B before the initiation of NAs treatment. Subsequently, they obtained liver tissue samples from the same individuals after five and ten years of continuous NAs treatment. This longitudinal analysis revealed a gradual reduction in the frequency of HBV integration events within the liver tissue over the specified treatment durations [ 167 ]. A plausible mechanism underlying this phenomenon is that NAs effectively suppress viral replication, while concomitant normal hepatocyte regeneration results in the gradual dilution of the frequency of viral integration events. However, it is important to note that NAs do not have any effect on eliminating cccDNA or integrated DNA [ 79 , 168 ].

In comparison to NAs treatment, therapies that target innate immunity, such as IFN-α, are more likely to possess the potency to eliminate cccDNA [ 192 ]. This therapeutic approach has shown success in inducing a functional cure among a minority of patients with CHB in clinical settings [ 193 ]. Reports indicate that patients who are functionally cured and exhibit intrahepatic HBsAg possess higher levels of integrated HBV DNA than those without intrahepatic HBsAg. Interestingly, a certain subset of these patients maintains transcriptional activity of the integrated viral DNA [ 170 , 171 ]. Utilizing spatial transcriptome sequencing, it was found that transcriptionally active HBV integration is relatively low in patients who have cleared HBsAg. In addition, there’s a close correlation between the level of intrahepatic cccDNA and virus integration events [ 194 ]. IFN-α has been shown to indirectly reduce the synthesis of pgRNA, which is vital for HBV DNA integration. Nevertheless, research is currently scant on whether IFN-α can completely eliminate integration. Thus, further exploration in this field is warranted.

The utilization of CRISPR/Cas9 has shown effectiveness in eliminating both HBV cccDNA and integrated HBV DNA [ 15 , 176 ]. When selecting target sequences, it is important to optimize them to maximize the elimination of viral genes while minimizing potential damage to the human genome [ 195 ]. Following this principle, sequence design could focus on targeting the full-length 3,175-bp HBV DNA sequence [ 174 ]. Additionally, studies have explored targeting specific open reading frames of HBV, such as the S and X regions [ 175 ]. Both approaches hold promising potential for achieving a radical cure. However, the clinical application of CRISPR/Cas9 technology is currently limited due to factors like off-target cleavage and the risk of inducing genome instability when cutting integrated HBV DNA. A recently devised technique, involving the concurrent administration of Cas9 mRNA and guide RNAs, demonstrates its efficacy in modifying HBV integration DNA in mouse and tree shrew models, exhibiting a notable absence of liver enzyme elevation and minimal off-target effects [ 177 ]. A separate study put forth the hypothesis that pre-existing viral integrations within clonal HBV-infected hepatocytes could be eliminated during liver damage in patients with CHB. The researchers observed a negative correlation between the types and frequencies of breakpoints and the grade score for liver inflammation activity, providing support for this hypothesis [ 196 ].

It is intriguing to note that the majority of HBV transcripts show consistent termination sites within the viral genome, creating a unique opportunity to leverage RNA silencing mechanisms [ 70 , 197 ]. RNA interference agents have emerged as a novel strategy for eradicating integrated DNA, with the potential to comprehensively influence the viral life cycle by downregulating all virus-generated mRNA [ 158 ]. One such agent, ARC-520, has shown promising results in reducing viral proteins, RNA, and DNA, leading to a surprising decrease in integrated HBV DNA in both chimpanzees and patients. However, it does not directly affect cccDNA [ 182 ].

Zinc-finger nucleases (ZFNs) or transcription activator-like effector nucleases (TALENs) have shown potential in manipulating HBV cccDNA in cellular models, which may help attenuate integration events [ 15 , 161 ]. As our knowledge of cccDNA formation continues to grow, future therapeutic strategies could target nuclear enzymes, histones, and other essential components that play a crucial role in cccDNA generation [ 22 ].

HBV integration refers to the insertion of DNA fragments derived from HBV into the human genome [ 119 ]. The integration of HBV DNA into the human genome has been extensively studied, revealing its confirmed carcinogenic potential in various experimental models. While integration events occur early during HBV infection, their exact role in the development of HCC is yet to be fully verified.

In the future, the establishment of comprehensive animal models that encapsulate the entire HBV infection process is pivotal. Such models will afford a more nuanced exploration of the ramifications of integrated HBV DNA on the hepatocytic transformation into a carcinogenic phenotype. Moreover, the development of pragmatic and cost-efficient methodologies for detecting integrations, coupled with the identification of pertinent serological markers denoting their presence, will significantly augment our capacity to appraise the potential for attaining a functional cure.

Furthermore, given that integrated HBV DNA contributes to the production of HBsAg and may impede the realization of a functional cure, prospective research should concentrate on discerning novel serological markers that more accurately signify the presence of integrations. This is particularly imperative in patients subjected to NAs treatment, where the absence of correlation between HBsAg levels and serum HBV DNA poses challenges in monitoring the efficacy of antiviral therapy.

Moreover, for the advancement of the field, the prioritization of clinical trials assessing the efficacy of diverse treatments in expediting the clearance of HBV integrations is essential. The exploration of innovative therapeutic modalities tailored specifically to target integrated HBV DNA will be instrumental in achieving comprehensive elimination. Thus, forthcoming research endeavors should be strategically oriented toward these pivotal domains to unravel the intricacies of HBV DNA integration and pave the way for more efficacious therapeutic interventions.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

• Hepatitis B virus

Chronic hepatitis B

• Hepatocellular carcinoma

Covalently closed circular DNA

Nucleos(t)ide analogues

Hepatitis surface antigen

Hepatitis B core protein

Double-stranded DNA

Open reading frames

HBV core antigen

HBV e antigen

HBV X protein

Relaxed circular DNA

Pregenomic RNA

Double-stranded linear DNA

Direct repeat 1

Direct repeat 2

Non-homologous end joining

Microhomology-mediated end joining

Ribonucleoprotein

Terminal protein

Duck hepatitis B virus

Woodchuck hepatitis virus

Primary human hepatocytes

Cytotoxic T lymphocytes

Fluorescence In Situ Hybridization

Arthrobacter luteus-PCR

Short interspersed nuclear elements

Inverse nested PCR

Whole-genome sequencing

Whole-exome sequencing

Next-generation sequencing

c-terminal truncation HBV X protein

Androgen receptor

HBV core-related antigen

Cell-free DNA

Integrant-derived RNAs

Zinc-finger nucleases

Transcription activator-like effector nucleases

Cornberg M, Lok AS, Terrault NA, Zoulim F, Faculty E-AHTEC. Mar. Guidance for design and endpoints of clinical trials in chronic hepatitis B - Report from the 2019 EASL-AASLD HBV Treatment Endpoints Conference(double dagger). J Hepatol . 2020;72(3):539–557.

Shafritz DASD, Sherman HI, Hadziyannis SJ, Kew MC. Integration of hepatitis B virus DNA into the genome of liver cells in chronic liver disease and hepatocellular carcinoma. Studies in percutaneous liver biopsies and post-mortem tissue specimens. N Engl J Med. 1981;305:1067–73.

Article   CAS   PubMed   Google Scholar  

Meyer M, Wiedorn KH, Hofschneider PH, Koshy R, Caselmann WH. A chromosome 17:7 translocation is associated with a hepatitis B virus DNA integration in human hepatocellular carcinoma DNA. Hepatology. 1992;15(4):665–71.

Wang Y, Lau SH, Sham JS-T, Wu M-C, Wang T, Guan X-Y. Characterization of HBV integrants in 14 hepatocellular carcinomas: association of truncated X gene and hepatocellular carcinogenesis. Oncogene. 2004;23(1):142–8.

Levrero M, Zucman-Rossi J. Mechanisms of HBV-induced hepatocellular carcinoma. J Hepatol Apr. 2016;64(1 Suppl):S84–101.

Article   CAS   Google Scholar  

Hai H, Tamori A, Kawada N. Role of hepatitis B virus DNA integration in human hepatocarcinogenesis. World J Gastroenterol May. 2014;28(20):6236–43.

Article   Google Scholar  

Lok AS, McMahon BJ, Brown RS Jr., et al. Antiviral therapy for chronic hepatitis B viral infection in adults: a systematic review and meta-analysis. Hepatol Jan. 2016;63(1):284–306.

Lee HW, Lee JS, Ahn SH. Hepatitis B Virus Cure: targets and future therapies. Int J Mol Sci Dec 28 2020;22(1).

Wang M, Qian M, Fu R, et al. The Impact of Nucleos(t)ide analogs off-Therapy among Chronic Hepatitis B patients: a systematic review and Meta-analysis. Front Public Health. 2021;9:709220.

Article   PubMed   PubMed Central   Google Scholar  

Sorrell MF, Belongia EA, Costa J, et al. National Institutes of Health consensus development conference statement: management of hepatitis B. Hepatology. 2009;49(S5):S4–12.

Ghany MG, Buti M, Lampertico P, Lee HM, Faculty A-EH-HTEC. Guidance on treatment endpoints and study design for clinical trials a iming to achieve cure in chronic hepatitis B and D: Report from the 20 22 AASLD-EASL HBV-HDV Treatment Endpoints Conference. J Hepatol . 2023/11// 2023;79(5):1254–1269.

Asselah T, Loureiro D, Boyer N, Mansouri A. Targets and future direct-acting antiviral approaches to achieve hepatitis B virus cure. Lancet Gastroenterol Hepatol. 2019;4(11):883–92.

Article   PubMed   Google Scholar  

Liaw YF, Kao JH, Piratvisuth T, et al. Asian-pacific consensus statement on the management of chronic hepatitis B: a 2012 update. Hepatol Int Jun. 2012;6(3):531–61.

Moreno-Cubero E, Del Arco RTS, Pena-Asensio J, de Villalobos ES, Miquel J, Larrubia JR. Is it possible to stop nucleos(t)ide analogue treatment in chronic hepatitis B patients? World J Gastroenterol May. 2018;7(17):1825–38.

Fanning GC, Zoulim F, Hou J, Bertoletti A. Therapeutic strategies for hepatitis B virus infection: towards a cure. Nat Rev Drug Discovery. 2019;18(11):827–44.

Yang W, Summers J. Integration of Hepadnavirus DNA in Infected Liver: Evidence for a Linear Precursor. 1999, 73, 9710–9717. J Virol . 1999;73:9710–9717.

Allweiss L, Volz T, Giersch K, et al. Proliferation of primary human hepatocytes and prevention of hepatitis B virus reinfection efficiently deplete nuclear cccDNA in vivo. Gut. 2018;67(3):542–52.

Tsukuda S, Watashi K. Hepatitis B virus biology and life cycle. Antiviral Res. 2020;(1872–9096 (Electronic)).

Königer C, Wingert I, Marsmann M, Rösler C, Beck J, Nassal M. Involvement of the host DNA-repair enzyme TDP2 in formation of the covalently closed circular DNA persistence reservoir of hepatitis B viruses. Proceedings of the National Academy of Sciences . 2014;111(40).

Hu J, Qi Y, Gao Z et al. DNA polymerase κ is a Key Cellular factor for the formation of covalently closed circular DNA of Hepatitis B Virus. PLoS Pathog. 2016;12(10).

Siddiqui A, Kitamura K, Que L et al. Flap endonuclease 1 is involved in cccDNA formation in the hepatitis B virus. PLoS Pathog. 2018;14(6).

Hu QLRY. J ea. The role of host DNA ligases in hepadnavirus covalently closed circular DNA formation. PLoS Pathogens . 2017;13(12):e1006784.

Bill CA, Summers J, Genomic DNA. Double-strand breaks are targets for Hepadnaviral DNA Integration. Proc Natl Acad Sci USA. 2004;101:11135–40.

Article   CAS   PubMed   PubMed Central   Google Scholar  

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 May. 2014;88(10):5742–54.

Blondot ML, Bruss V, Kann M. Intracellular transport and egress of hepatitis B virus. J Hepatol Apr. 2016;64(1 Suppl):S49–59.

S S, DD L, D G. Mutations affecting hepadnavirus plus-strand DNA synthesis dissociate primer cleavage from translocation and reveal the origin of linear viral DNA. J Virol. 1991;65(3):1255–62.

KM H DDL. The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus. J Mol Biol. 2007;370(3):471–80.

Lee J, Shin MK, Lee HJ, Yoon G, Ryu WS. Three novel cis-acting elements required for efficient plus-strand DNA synthesis of the hepatitis B virus genome. J Virol Jul. 2004;78(14):7455–64.

Lewellyn EB, Loeb DD. Base pairing between cis-acting sequences contributes to template switching during plus-strand DNA synthesis in human hepatitis B virus. J Virol Jun. 2007;81(12):6207–15.

Shafritz DA, Shouval D, Sherman HI, Hadziyannis SJ, Kew MC. Integration of Hepatitis B Virus DNA into the genome of liver cells in Chronic Liver Disease and Hepatocellular Carcinoma. Studies in Percutaneous Liver biopsies and Post-mortem tissue specimens. N Engl J Med. 1981;305:1067–73.

C B, C P, B R AL. Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature. 1980;286:533–5.

Bousali M, Karamitros T, Hepatitis B. Virus integration into transcriptionally active loci and HBV-Associated Hepatocellular Carcinoma. Microorganisms Jan 24 2022;10(2).

Bousali M, Papatheodoridis G, Paraskevis D, Karamitros T, Hepatitis B, Virus DNA. Integration, chronic infections and Hepatocellular Carcinoma. Microorganisms. 2021;9(8).

Tu T, Urban S. Virus entry and its inhibition to prevent and treat hepatitis B and hepatitis D virus infections. Curr Opin Virol Jun. 2018;30:68–79.

Tu T, Budzinska MA, Vondran FWR, Shackel NA, Urban S, Hepatitis B, Virus DNA. Integration occurs early in the viral life cycle in an in vitro infection model via Sodium Taurocholate Cotransporting polypeptide-dependent uptake of enveloped virus particles. J Virol Jun 1 2018;92(11).

Cui X, Wei W, Wang C et al. Studies on the correlation between mutation and integration of HBV in hepatocellular carcinoma. Biosci Rep Aug 28 2020;40(8).

Tu T, Budzinska MA, Shackel NA, Urban S. HBV DNA integration: Molecular mechanisms and clinical implications. Viruses Apr 10 2017;9(4).

Zhao LH, Liu X, Yan HX, et al. Genomic and oncogenic preference of HBV integration in hepatocellular carcinoma. Nat Commun Oct. 2016;5:7:12992.

Lau C-C, Sun T, Ching Arthur KK, et al. Viral-human chimeric transcript predisposes risk to Liver Cancer Development and Progression. Cancer Cell. 2014;25(3):335–49.

Lieber MR. The mechanism of human nonhomologous DNA end joining. J Biol Chem. 2008;283(1):1–5.

Ma J-L, Kim EM, Haber JE, Lee SE. Yeast Mre11 and Rad1 proteins define a Ku-Independent mechanism to repair double-strand breaks lacking overlapping end sequences. Mol Cell Biol. 2003;23(23):8820–8.

Dh HVB. e. Microhomology-mediated mechanisms underlie non-recurrent disease-causing microdeletions of the FOXL2 gene or its regulatory domain. PLoS Genet. 2013;9(3):e1003358.

Nassal M. HBV cccDNA: viral persistence reservoir and key obstacle for a cure of chronic hepatitis B. Gut Dec. 2015;64(12):1972–84.

Sung WK, Zheng H, Li S, et al. Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma. Nat Genet May. 2012;27(7):765–9.

Chauhan R, Churchill ND, Mulrooney-Cousins PM, Michalak TI. Initial sites of hepadnavirus integration into host genome in human hepatocytes and in the woodchuck model of hepatitis B-associated hepatocellular carcinoma. Oncog Apr. 2017;17(4):e317.

Google Scholar  

Yang L, Ye S, Zhao X, et al. Molecular characterization of HBV DNA integration in patients with Hepatitis and Hepatocellular Carcinoma. J Cancer. 2018;9(18):3225–35.

Mason WS, Liu C, Aldrich CE, Litwin S, Yeh MM. Clonal expansion of normal-appearing human hepatocytes during chronic hepatitis B virus infection. J Virol Aug. 2010;84(16):8308–15.

Li X, Zhang J, Yang Z, et al. The function of targeted host genes determines the oncogenicity of HBV integration in hepatocellular carcinoma. J Hepatol May. 2014;60(5):975–84.

Horikawa I, Barrett JC. Transcriptional regulation of the telomerase hTERT gene as a target for cellular and viral oncogenic mechanisms. Carcinogenesis. 2003;24(7):1167–76.

Paterlini-Bréchot P, Saigo K, Murakami Y, et al. Hepatitis B virus-related insertional mutagenesis occurs frequently in human liver cancers and recurrently targets human telomerase gene. Oncogene. 2003;22(25):3911–6.

Saigo K, Yoshida K, Ikeda R, et al. Integration of hepatitis B virus DNA into the myeloid/lymphoid or mixed-lineage leukemia (MLL4) gene and rearrangements of MLL4 in human hepatocellular carcinoma. Hum Mutat. 2008;29(5):703–8.

Murakami Y, Saigo K, Takashima H, et al. Large scaled analysis of hepatitis B virus (HBV) DNA integration in HBV related hepatocellular carcinomas. Gut Aug. 2005;54(8):1162–8.

Jiang Z, Jhunjhunwala S, Liu J, et al. The effects of hepatitis B virus integration into the genomes of hepatocellular carcinoma patients. Genome Res. 2012;22(4):593–601.

Budzinska MA, Shackel NA, Urban S, Tu T. Cellular genomic sites of Hepatitis B Virus DNA integration. Genes (Basel) . Jul 20 2018;9(7).

W Y. Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor. J Virol. 1999;73:9710–7.

Chauhan R, Shimizu Y, Watashi K, Wakita T, Fukasawa M, Michalak TI. Retrotransposon elements among initial sites of hepatitis B virus integration into human genome in the HepG2-NTCP cell infection model. Cancer Genet Jun. 2019;235–236:39–56.

Mayuko Furuta HT, Yuichi Shiraishi. Characterization of HBV integration patterns and timing in liver cancer and HBV-infected livers. Oncotarget. 2018;9(38):25075–88.

Kimbi GCKA, Kew MC. Integration of hepatitis B virus DNA into chromosomal DNA during acute hepatitis B. World J Gastroenterol. 2005;11:6416–21.

Tsukuda S, Watashi K. Hepatitis B virus biology and life cycle. Antiviral Res. 2020;182.

Hagen TM, Huang S, Curnutte J et al. Extensive oxidative DNA damage in hepatocytes of transgenic mice with chronic active hepatitis destined to develop hepatocellular carcinoma. Proc Natl Acad Sci U S A . 1994/12/20/ 91(26):12808-12.

Dandri M, Burda MR, Bürkle A et al. Increase in de novo HBV DNA integrations in response to oxidative DNA damage or inhibition of poly(ADP-ribosyl)ation. Hepatology (Baltimore, Md) . 2002/1// 35(1):217 – 23.

Petersen J, Dandri M, Bürkle A, Zhang L, Rogler CE. Increase in the frequency of hepadnavirus DNA integrations by oxidativ e DNA damage and inhibition of DNA repair. Journal of virology . 1997/7// 1997;71(7):5455-63.

Yuan T, Jiang Y, Li M, Li W. Chronic hepatitis B surface antigen seroclearance-related immune factors. Hepatol Res Jan. 2017;47(1):49–59.

Boni C, Fisicaro P, Valdatta C, et al. Characterization of hepatitis B virus (HBV)-specific T-cell dysfunction in chronic HBV infection. J Virol Apr. 2007;81(8):4215–25.

Wu S, Yi W, Gao Y, et al. Immune mechanisms underlying Hepatitis B Surface Antigen Seroclearance in Chronic Hepatitis B patients with viral coinfection. Front Immunol. 2022;13:893512.

Yong L, Li M, Gao Y et al. Identification of pro-inflammatory CD205 + macrophages in livers of hepatitis B virus transgenic mice and patients with chronic hepatitis B. Sci Rep. 2017;7(1).

Hou X, Hao X, Zheng M, et al. CD205-TLR9-IL-12 axis contributes to CpG-induced oversensitive liver injury in HBsAg transgenic mice by promoting the interaction of NKT cells with Kupffer cells. Cell Mol Immunol. 2016;14(8):675–84.

N M, H S. Hepatitis B virus replication. Trends Microbiol. 1993;1(6):221–8.

Omata M, Yokosuka O, Imazeki F, et al. Correlation of hepatitis B virus DNA and antigens in the liver. A study in chronic liver disease. Gastroenterol Jan. 1987;92(1):192–6.

CAS   Google Scholar  

Wooddell CI, Yuen M-F, Chan HL-Y et al. RNAi-based treatment of chronically infected patients and chimpanzees reveals that integrated hepatitis B virus DNA is a source of HBsAg. Sci Transl Med. 2017;9(eaan0241.).

Hu B, Wang R, Fu J, et al. Integration of hepatitis B virus S gene impacts on hepatitis B surface antigen levels in patients with antiviral therapy. J Gastroenterol Hepatol Jul. 2018;33(7):1389–96.

Meier MA, Calabrese D, Suslov A, Terracciano LM, Heim MH, Wieland S. Ubiquitous expression of HBsAg from integrated HBV DNA in patients with low viral load. J Hepatol Oct. 2021;75(4):840–7.

JC E, LB PGPV, WJ R. Integration of hepatitis B virus sequences and their expression in a human hepatoma cell. Nature. 1980;286:535–8.

Yaginuma KKH, Kobayashi M, Morishima T, Matsuyama K, Koike K. Multiple integration site of hepatitis B virus DNA in hepatocellular carcinoma and chronic active hepatitis tissues from children. J Virol. 1987;61:1808–13.

Tokino T, Matsubara K. Chromosomal sites for hepatitis B virus integration in human hepatocellular carcinoma. J Virol. 1991;65:6761–4.

MM AG. Fluorescence in situ hybridization: uses and limitations. Semin Hematol. 2000;37(4):0037–1963. (Print).

TH H, QJ Z, QD X, LP Z. Presence and integration of HBV DNA in mouse oocytes. World J Gastroenterol. 2005;11(Print):1007–9327.

HINO O, OHTAKE K, ROGLER CE. Features of two Hepatitis B Virus (HBV) DNA integrations suggest mechanisms of HBV Integration. J Virol. 1989;63:2638–43.

B C, H M, Scotto Jea. State of hepatitis B virus DNA in hepatocytes of patients with hepatitis B surface antigen-positive and -negative liver diseases. Proc Natl Acad Sci U S A. 1981;78(6):3906–10.

PJ B, SW S. Detection of integration during active replication of hepatitis B virus in the liver. J Med Virol. 1985;16(1):47–54.

O Y, F I SK. State of HBV DNA in HBsAg-negative, anti-HCV-positive hepatocellular carcinoma: existence of HBV DNA possibly as nonintegrated form with analysis by Alu-HBV DNA PCR and conventional HBV PCR. Med Virol. 2001;64(4):410–8.

Murakami Y, Minami M, Daimon Y, Okanoue T. Hepatitis B virus DNA in liver, serum, and peripheral blood mononuclear cells after the clearance of serum hepatitis B virus surface antigen. J Med Virol. 2004;72(2):203–14.

Tu H. Gao Hf Fau - Ma G-h, Ma Gh Fau - Liu Y, Liu Y. [Identification of hepatitis B virus integration sites in hepatocellular carcinoma tissues from patients with chronic hepatitis B]. (0376–2491 (Print)).

Tu T, Jilbert AR. Detection of hepatocyte clones containing Integrated Hepatitis B Virus DNA using Inverse Nested PCR. Methods Mol Biol. 2017;1540:97–118.

Mason WS, Low HC, Xu C, et al. Detection of clonally expanded hepatocytes in chimpanzees with chronic hepatitis B virus infection. J Virol Sep. 2009;83(17):8396–408.

Ruan P, Dai X, Sun J, et al. Integration of hepatitis B virus DNA into p21-activated kinase 3 (PAK3) gene in HepG2.2.15 cells. Virus Genes Apr. 2020;56(2):168–73.

Choo KB, Liu MS, Chang PC, et al. Analysis of six distinct Integrated Hepatitis B virus sequences cloned from the Cellular DNA of a human Hepatocellular Carcinoma. Virology. 1986;154:405–8.

Ramirez R, van Buuren N, Gamelin L, et al. Targeted long-read sequencing reveals Comprehensive Architecture, Burden, and Transcriptional signatures from Hepatitis B Virus-Associated Integrations and translocations in Hepatocellular Carcinoma Cell lines. J Virol Sep. 2021;9(19):e0029921.

Zheng B, Liu X-L, Fan R, et al. The Landscape of cell-free HBV Integrations and mutations in cirrhosis and Hepatocellular Carcinoma patients. Clin Cancer Res. 2021;27(13):3772–83.

Wang A, Wu L, Lin J et al. Whole-exome sequencing reveals the origin and evolution of hepato-cholangiocarcinoma. Nat Commun. 2018;9(1).

Svicher V, Salpini R, Piermatteo L, et al. Whole exome HBV DNA integration is independent of the intrahepatic HBV reservoir in HBeAg-negative chronic hepatitis B. Gut Dec. 2021;70(12):2337–48.

Hu X, Jiang J, Ni C, et al. HBV Integration-mediated cell apoptosis in HepG2.2.15. J Cancer. 2019;10(17):4142–50.

Ishii T, Tamura A, Shibata T et al. Analysis of HBV Genomes Integrated into the Genomes of Human Hepatoma PLC/PRF/5 cells by HBV sequence capture-based next-generation sequencing. Genes (Basel) Jun 18 2020;11(6).

Li CL, Ho MC, Lin YY, et al. Cell-free virus-host chimera DNA from Hepatitis B Virus Integration sites as a circulating biomarker of Hepatocellular Cancer. Hepatol Dec. 2020;72(6):2063–76.

Razavi-Shearer D, Gamkrelidze I, Nguyen MH, et al. Global prevalence, treatment, and prevention of hepatitis B virus infection in 2016: a modelling study. Lancet Gastroenterol Hepatol. 2018;3(6):383–403.

Lander ESLL, Birren B, Nusbaum C, et al. Initial sequencing and analysis of the human genome. Nature. 2001;409:860–921.

Erken R, Loukachov V, van Dort K, et al. Quantified integrated hepatitis B virus is related to viral activity in patients with chronic hepatitis B. Hepatol Jul. 2022;76(1):196–206.

Larsson SB, Tripodi G, Raimondo G, et al. Integration of hepatitis B virus DNA in chronically infected patients assessed by Alu-PCR. J Med Virol Oct. 2018;90(10):1568–75.

Gong SS, Jensen AD, Wang H. Rogler duck hepatitis B virus integrations in LMH chicken hepatoma cells: identification and characterization of new episomally derived integrations. J Virol. 1995;69:8102–8.

Yan H, Yang Y, Zhang L, et al. Characterization of the genotype and integration patterns of hepatitis B virus in early- and late‐onset hepatocellular carcinoma. Hepatology. 2015;61(6):1821–31.

Budzinska MA, Shackel NA, Urban S, Tu T. Sequence analysis of integrated hepatitis B virus DNA during HBeAg-seroconversion. Emerg Microbes Infect Aug. 2018;8(1):142.

Mason WS, Gill US, Litwin S, et al. HBV DNA integration and clonal hepatocyte expansion in Chronic Hepatitis B patients considered Immune Tolerant. Gastroenterol Nov. 2016;151(5):986–e9984.

Tu T, Mason WS, Clouston AD, et al. Clonal expansion of hepatocytes with a selective advantage occurs during all stages of chronic hepatitis B virus infection. J Viral Hepat Sep. 2015;22(9):737–53.

Li W, Qi Y, Xu H, Wei W, Cui X. Profile of different Hepatitis B virus integration frequency in hepatocellular carcinoma patients. Biochem Biophys Res Commun May. 2021;14:553:160–4.

Beerenwinkel N, Gunthard HF, Roth V, Metzner KJ. Challenges and opportunities in estimating viral genetic diversity from next-generation sequencing data. Front Microbiol. 2012;3:329.

Li W, Wei W, Hou F, Xu H, Cui X. The integration model of hepatitis B virus genome in hepatocellular carcinoma cells based on high-throughput long-read sequencing. Genomics Jan. 2022;114(1):23–30.

Betz-Stablein BD, Topfer A, Littlejohn M, et al. Single-molecule sequencing reveals Complex Genome Variation of Hepatitis B Virus during 15 years of chronic infection following liver transplantation. J Virol Aug. 2016;15(16):7171–83.

Yang C, Ruan P, Ou C et al. Chronic hepatitis B virus infection and occurrence of hepatocellular carcinoma in tree shrews (Tupaia belangeri chinensis). Virol J. 2015;12(1).

Walter E, Keist R, Niederöst B, Pult I, Blum HE. Hepatitis B virus infection of tupaia hepatocytesin vitroandin vivo. Hepatology. 1996;24(1):1–5.

CAS   PubMed   Google Scholar  

Burwitz BJ, Wettengel JM, Mück-Häusl MA et al. Hepatocytic expression of human sodium-taurocholate cotransporting polypeptide enables hepatitis B virus infection of macaques. Nat Commun. 2017;8(1).

Tan AT, Yang N, Lee Krishnamoorthy T, et al. Use of expression profiles of HBV-DNA Integrated into Genomes of Hepatocellular Carcinoma Cells to select T cells for Immunotherapy. Gastroenterol May. 2019;156(6):1862–e18769.

Wang SH, Yeh SH, Chen PJ. Unique Features of Hepatitis B Virus-Related Hepatocellular Carcinoma in Pathogenesis and clinical significance. Cancers (Basel) May 18 2021;13(10).

Kuang XJ, Jia RR, Huo RR, et al. Systematic review of risk factors of hepatocellular carcinoma after hepatitis B surface antigen seroclearance. J Viral Hepat Sep. 2018;25(9):1026–37.

Zhao XL, Yang JR, Lin SZ, et al. Serum viral duplex-linear DNA proportion increases with the progression of liver disease in patients infected with HBV. Gut Mar. 2016;65(3):502–11.

Jang JW, Kim JS, Kim HS, et al. Persistence of intrahepatic hepatitis B virus DNA integration in patients developing hepatocellular carcinoma after hepatitis B surface antigen seroclearance. Clin Mol Hepatol Jan. 2021;27(1):207–18.

Lau KC, Joshi SS, Gao S, et al. Oncogenic HBV variants and integration are present in hepatic and lymphoid cells derived from chronic HBV patients. Cancer Lett Jun. 2020;28:480:39–47.

Amaddeo G, Cao Q, Ladeiro Y, et al. Integration of tumour and viral genomic characterizations in HBV-related hepatocellular carcinomas. Gut May. 2015;64(5):820–9.

Peneau C, Imbeaud S, La Bella T, et al. Hepatitis B virus integrations promote local and distant oncogenic driver alterations in hepatocellular carcinoma. Gut Mar. 2022;71(3):616–26.

Yeh SH, Li CL, Lin YY, et al. Hepatitis B Virus DNA Integration drives carcinogenesis and provides a New Biomarker for HBV-related HCC. Cell Mol Gastroenterol Hepatol Jan. 2023;20(4):921–9.

Lu HZYW. L ea. HBV-integrated local genomic alterations reveal multicentric independent occurrences of multifocal HCC. Clin Transl Med 2023;13(6):e1313.

Alvarez EG, Demeulemeester J, Otero P, et al. Aberrant integration of Hepatitis B virus DNA promotes major restructuring of human hepatocellular carcinoma genome architecture. Nat Commun Nov. 2021;25(1):6910.

Brunner SF, Roberts ND, Wylie LA, et al. Somatic mutations and clonal dynamics in healthy and cirrhotic human liver. Nature. 2019;574(7779):538–42.

Saitta C, Tripodi G, Barbera A, et al. Hepatitis B virus (HBV) DNA integration in patients with occult HBV infection and hepatocellular carcinoma. Liver Int Oct. 2015;35(10):2311–7.

Kremsdorf D, Soussan P, Paterlini-Brechot P, Brechot C. Hepatitis B virus-related hepatocellular carcinoma: paradigms for viral-related human carcinogenesis. Oncogene Jun. 2006;26(27):3823–33.

Ruan P, Dai X, Sun J, et al. Different types of viral–host junction found in HBV integration breakpoints in HBV–infected patients. Mol Med Rep Feb. 2019;19(2):1410–6.

Kojima R, Nakamoto S, Kogure T et al. Re-analysis of hepatitis B virus integration sites reveals potential new loci associated with oncogenesis in hepatocellular carcinoma. World J Virol . 2023/06/25/ 2023;12(3):209–220.

Kang Hyo J, Haq F, Sung Chang O, et al. Characterization of Hepatocellular Carcinoma patients with FGF19 amplification assessed by fluorescence in situ hybridization: a large cohort study. Liver Cancer. 2019;8(1):12–23.

Cui X, Li Y, Xu H, Sun Y, Jiang S, Li W. Characteristics of Hepatitis B virus integration and mechanism of inducing chromosome translocation. NPJ Genom Med . 2023/06/02/ 2023;8(1):11.

Ruan P, Zhou R, He C et al. Two fragments of HBV DNA integrated into chrX: 11009033 and its genetic regulation in HepG2.2.15. Molecular Medicine Reports . 2023/05// 2023;27(5):98.

Deng Y, Chang S. Role of telomeres and telomerase in genomic instability, senescence and cancer. Lab Invest. 2007;87(11):1071–6.

Maciejowski J, de Lange T. Telomeres in cancer: tumour suppression and genome instability. Nat Rev Mol Cell Biol Mar. 2017;18(3):175–86.

Sze KMF, Ho DWH, Chiu YT, et al. Hepatitis B virus–telomerase reverse transcriptase promoter integration harnesses host ELF4, resulting in Telomerase Reverse transcriptase gene transcription in Hepatocellular Carcinoma. Hepatology. 2020;73(1):23–40.

Giosa D, Lombardo D, Musolino C et al. Mitochondrial DNA is a target of HBV integration. Commun Biol . 2023/07/03/ 2023;6(1):684.

Bakhoum SF, Cantley LC. The multifaceted role of chromosomal instability in Cancer and its Microenvironment. Cell. 2018;174(6):1347–60.

Katoh H, Shibata T, Kokubu A, et al. Epigenetic instability and chromosomal instability in Hepatocellular Carcinoma. Am J Pathol. 2006;168(4):1375–84.

Bailey SM. Telomeres, chromosome instability and cancer. Nucleic Acids Res. 2006;34(8):2408–17.

Rosato RNDB. HBV infection and host interactions: the role in viral persistence and oncogenesis. Int J Mol Sci. 2023;24(8):7651.

Tavis JE, Na B, Huang Z et al. Transgenic expression of entire Hepatitis B Virus in mice induces Hepatocarcinogenesis Independent of Chronic Liver Injury. PLoS ONE. 2011;6(10).

Barone M, Spano D, D’Apolito M, et al. Gene Expression Analysis in HBV Transgenic Mouse Liver: a model to study early events related to Hepatocarcinogenesis. Mol Med. 2006;12(4–6):115–23.

Wang ML, Tang H. Nucleos(t)ide analogues causes HBV S gene mutations and carcinogenesis. (1499–3872 (Print)).

Choi Y-M, Lee S-Y, Kim B-J. Naturally occurring Hepatitis B virus mutations leading to endoplasmic reticulum stress and their contribution to the Progression of Hepatocellular Carcinoma. Int J Mol Sci. 2019;20(3).

ML W, DB W, YC T, et al. The truncated mutant HBsAg expression increases the tumorigenesis of hepatitis B. Virol J. 2018;15(1):1743–61.

SA L, K K. Nucleotide change of codon 182 in the surface gene of hepatitis B virus genotype. J Hepatol. 2012;56(1):63–9. (1600 – 0641 (Electronic)).

Ma N-F, Lau SH, Hu L, et al. COOH-Terminal truncated HBV X protein plays key role in Hepatocarcinogenesis. Clin Cancer Res. 2008;14(16):5061–8.

Wu X, Ni Z, Song T et al. C-Terminal truncated HBx facilitates oncogenesis by modulating cell cycle and glucose metabolism in FXR-Deficient Hepatocellular Carcinoma. Int J Mol Sci. 2023;24(6).

Sze KMF, Chu GKY, Lee JMF, Ng IOL. C-terminal truncated hepatitis B virus x protein is associated with metastasis and enhances invasiveness by c-jun/matrix metalloproteinase protein 10 activation in hepatocellular carcinoma. Hepatology. 2013;57(1):131–9.

Zhang Y, Yan Q, Gong L, et al. C-terminal truncated HBx initiates hepatocarcinogenesis by downregulating TXNIP and reprogramming glucose metabolism. Oncogene. 2020;40(6):1147–61.

Quetier I, Brezillon N, Revaud J, et al. C-terminal-truncated hepatitis B virus X protein enhances the development of diethylnitrosamine-induced hepatocellular carcinogenesis. J Gen Virol. 2015;96(3):614–25.

Zhang C, Xiao C, Ren G et al. C-terminal-truncated hepatitis B virus X protein promotes hepatocarcinogenesis by activating the MAPK pathway. Microb Pathog. 2021;159.

KY N. C-terminal truncated hepatitis B virus X protein promotes hepatocellular. Oncotarget. 2016;26(7):1949–2553. (Electronic)).

Guo M, Zhao L, Jiang C et al. Multiomics analyses reveal pathological mechanisms of HBV infection and integration in liver cancer. 2023;(1096–9071 (Electronic)).

Pollicino T, Caminiti G. HBV-Integration studies in the clinic: role in the natural history of infection. Viruses Feb 26 2021;13(3).

Luo M, Zhou B, Hou J, Jiang D. Biomarkers for predicting nucleos(t)ide analogs discontinuation and hepatitis B virus recurrence after drug withdrawal in chronic hepatitis B patients. Hepatol Res Apr. 2022;52(4):337–51.

Xie Y, Li M, Ou X, et al. HBeAg-positive patients with HBsAg < 100 IU/mL and negative HBV RNA have lower risk of virological relapse after nucleos(t)ide analogues cessation. J Gastroenterol Sep. 2021;56(9):856–67.

DM M. Diagnostic and prognostic value of circulating tumor-related DNA in cancer patients. Expert Rev Mol Diagn. 2013;13(Electronic):1744–8352.

Chen W, Zhang K, Dong P, et al. Noninvasive chimeric DNA profiling identifies tumor-originated HBV integrants contributing to viral antigen expression in liver cancer. Hepatol Int May. 2020;14(3):326–37.

Li C-L, Yeh S-H, Chen P-J. Circulating virus–host chimera DNAs in the clinical monitoring of Virus-related cancers. Cancers. 2022;14(10).

Lindh M, Rydell GE, Larsson SB. Impact of integrated viral DNA on the goal to clear hepatitis B surface antigen with different therapeutic strategies. Curr Opin Virol Jun. 2018;30:24–31.

Zaiets I, Gunewardena S, Menne S, Weinman SA, Gudima SO. Sera of Individuals Chronically Infected with Hepatitis B Virus (HBV) Contain Diverse RNA Types Produced by HBV Replication or Derived from Integrated HBV DNA. Journal of Virology . 2023/03/30/ 2023;97(3):e01950-22.

Gehring AJ, Protzer U. Targeting Innate and Adaptive Immune responses to Cure Chronic HBV infection. Gastroenterology. 2019;156(2):325–37.

Maepa M, Roelofse I, Ely A, Arbuthnot P. Progress and prospects of Anti-HBV Gene Therapy Development. Int J Mol Sci. 2015;16(8):17589–610.

Zhang M, Zhang H, Cheng X, et al. Liver biopsy of chronic hepatitis B patients indicates HBV integration profile may complicate the endpoint and effect of entecavir treatment. Antiviral Res Aug. 2022;204:105363.

Chen H, Mu M, Liu Q, et al. Hepatocyte endoplasmic reticulum stress inhibits Hepatitis B Virus Secretion and Delays Intracellular Hepatitis B Virus Clearance after Entecavir Treatment. Front Med (Lausanne). 2020;7:589040.

Lai CL, Wong DK, Wong GT, Seto WK, Fung J, Yuen MF. Rebound of HBV DNA after cessation of nucleos/tide analogues in chronic hepatitis B patients with undetectable covalently closed. JHEP Rep Jun. 2020;2(3):100112.

Hsu Y-C, Suri V, Nguyen MH et al. Inhibition of Viral Replication Reduces Transcriptionally Active Distinct Hepatitis B Virus Integrations With Implications on Host Gene Dysregulation. Gastroenterology . 2022/04// 2022;162(4):1160–1170.e1.

Yip TC, Wong GL, Chan HL, et al. HBsAg seroclearance further reduces hepatocellular carcinoma risk after complete viral suppression with nucleos(t)ide analogues. J Hepatol Mar. 2019;70(3):361–70.

Chow N, Wong D, Lai C-L et al. Effect of Antiviral Treatment on Hepatitis B Virus Integration and Hepatocyte Clonal Expansion. Clinical Infectious Diseases: An Official Publication of the Infectious Diseases Society of America . 2023/02/08/ 2023;76(3):e801-e809.

Baran B. Nucleos(t)ide analogs in the prevention of hepatitis B virus related hepatocellular carcinoma. World J Hepatol Jul. 2015;8(13):1742–54.

Wen X, Wu X, Sun Y et al. Long-term antiviral therapy is associated with changes in the profile of transcriptionally active HBV integration in the livers of patients with CHB. J Med Virol. 2024;96(6).

Gan W, Gao N, Gu L et al. Reduction in Intrahepatic cccDNA and Integration of HBV in Chronic Hepatitis B Patients with a Functional Cure. Journal of Clinical and Translational Hepatology . 2023/04/28/ 2023;11(2):314–322.

Gao N, Guan G, Xu G et al. Integrated HBV DNA and cccDNA maintain transcriptional activity in intrahepatic HBsAg-positive patients with functional cure following PEG-IFN-based therapy. Alimentary Pharmacology & Therapeutics . 2023/08/29/ 2023:apt.17670.

Zhang J, Zhang Y, Khanal S et al. Synthetic gRNA/Cas9 ribonucleoprotein targeting HBV DNA inhibits viral replication. Journal of Medical Virology . 2023/07// 2023;95(7):e28952.

Seeger C, Sohn JA, Targeting Hepatitis B. Virus with CRISPR/Cas9. Mol Ther Nucleic Acids Dec. 2014;16(12):e216.

Li H, Sheng C, Wang S, et al. Removal of Integrated Hepatitis B Virus DNA using CRISPR-Cas9. Front Cell Infect Microbiol. 2017;7:91.

Karimova M, Beschorner N, Dammermann W, et al. CRISPR/Cas9 nickase-mediated disruption of hepatitis B virus open reading frame S and X. Sci Rep. Sep 2015;3:5:13734.

Ramanan V, Shlomai A, Cox DB, et al. CRISPR/Cas9 cleavage of viral DNA efficiently suppresses hepatitis B virus. Sci Rep Jun. 2015;2:5:10833.

Yi J, Lei X, Guo F et al. Co-delivery of Cas9 mRNA and guide RNAs edits hepatitis B virus episomal and integration DNA in mouse and tree shrew models. 2023;(1872–9096 (Electronic)).

Gao L, Yang J, Feng J, et al. PreS/2-21-Guided siRNA nanoparticles target to Inhibit Hepatitis B Virus infection and replication. Front Immunol. 2022;13:856463.

Hui RW, Mak LY, Seto WK, Yuen MF. RNA interference as a novel treatment strategy for chronic hepatitis B infection. Clin Mol Hepatol Jul. 2022;28(3):408–24.

Teng Y, Xu Z, Zhao K, et al. Novel function of SART1 in HNF4alpha transcriptional regulation contributes to its antiviral role during HBV infection. J Hepatol Nov. 2021;75(5):1072–82.

Wang J, Chen R, Zhang R, et al. The gRNA-miRNA-gRNA Ternary Cassette combining CRISPR/Cas9 with RNAi Approach strongly inhibits Hepatitis B Virus Replication. Theranostics. 2017;7(12):3090–105.

Wooddell CI, Gehring AJ, Yuen MF, Given BD. RNA interference therapy for chronic Hepatitis B predicts the importance of addressing viral integration when developing Novel cure strategies. Viruses Mar 30 2021;13(4).

Yuen MF, Schiefke I, Yoon JH, et al. RNA interference therapy with ARC-520 results in prolonged Hepatitis B Surface Antigen response in patients with chronic Hepatitis B infection. Hepatol Jul. 2020;72(1):19–31.

T D, S N, A E, P A, K B. Improved antiviral efficacy using TALEN-mediated homology directed recombination to introduce artificial primary miRNAs into DNA of hepatitis B virus. Biochem Biophys Res Commun. 2016;478(4):1563–8.

Bloom K, Ely A, Mussolino C, Cathomen T, Arbuthnot P. Inactivation of hepatitis B virus replication in cultured cells and in vivo with engineered transcription activator-like effector nucleases. Mol Ther Oct. 2013;21(10):1889–97.

Chen J, Zhang W, Lin J, et al. An efficient antiviral strategy for targeting hepatitis B virus genome using transcription activator-like effector nucleases. Mol Ther Feb. 2014;22(2):303–11.

Cradick TJ, Keck K, Bradshaw S, Jamieson AC, McCaffrey AP. Zinc-finger nucleases as a novel therapeutic strategy for targeting hepatitis B virus DNAs. Mol Ther May. 2010;18(5):947–54.

Kasianchuk N, Dobrowolska K, Harkava S et al. Gene-editing and RNA interference in treating Hepatitis B: a review. Viruses Dec 8 2023;15(12).

Schiffer JT, Swan DA, Stone D, Jerome KR. Predictors of hepatitis B cure using gene therapy to deliver DNA cleavage enzymes: a mathematical modeling approach. PLoS Comput Biol. 2013;9(7):e1003131.

Weber ND, Stone D, Jerome KR. TALENs targeting HBV: designer endonuclease therapies for viral infections. Mol Ther Oct. 2013;21(10):1819–20.

Smith T, Singh P, Chmielewski KO et al. Improved specificity and safety of Anti-hepatitis B Virus TALENs using Obligate Heterodimeric FokI nuclease domains. Viruses Jul 12 2021;13(7).

F Z SL. Targeting innate immunity: a new step in the development of combination therapy for chronic hepatitis B. Durantel DGastroenterology. 2013;144(7):1342–4.

Li Q, Sun B, Zhuo Y et al. Interferon and interferon-stimulated genes in HBV treatment. Front Immunol. 2022;13.

Yu X, Gong Q, Yu DA-O et al. Spatial transcriptomics reveals a low extent of transcriptionally active hepatitis B virus integration in patients with HBsAg loss. Gut. 2023;(1468–3288 (Electronic)):Published online November 15, 2023.

Horodecka K, Düchler M. CRISPR/Cas9: Principle, Applications, and delivery through Extracellular vesicles. Int J Mol Sci. 2021;22(11).

Hu G, Huang MX, Li WY, Gan CJ, Dong WX, Peng XM. Liver damage favors the eliminations of HBV integration and clonal hepatocytes in chronic hepatitis B. Hepatol Int Feb. 2021;15(1):60–70.

Hu B, Zhong L, Weng Y et al. Therapeutic siRNA: state of the art. Signal Transduct Target Therapy. 2020;5(1).

Download references

Acknowledgements

Not applicable.

This work is supported by the National Key Research and Development Program of China (No.2022YFC2304800) and 1.3.5 project for disciplines of excellence, West China Hospital, Sichuan University (No.ZYGD23030).

Author information

Mingming Zhang and Han Chen contributed equally to this work.

Authors and Affiliations

Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China

Mingming Zhang, Han Chen, Huan Liu & Hong Tang

Laboratory of Infectious and Liver Diseases, Institute of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, 610041, China

You can also search for this author in PubMed   Google Scholar

Contributions

MZ and HC conceived the manuscript; MZ, HC and HT searched for the papers and made the outline; MZ and HC wrote the initial draft; MZ, HC and HL designed and drew the figures; MZ, HC and HL checked all references and formatting; MZ and HC contributed equally to this work. All authors revised and contributed to the final version of the manuscript.

Corresponding author

Correspondence to Hong Tang .

Ethics declarations

Ethics approval and consent to participate, consent for publication, competing interests.

The authors declare no competing interests.

Additional information

Publisher’s note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Cite this article.

Zhang, M., Chen, H., Liu, H. et al. The impact of integrated hepatitis B virus DNA on oncogenesis and antiviral therapy. Biomark Res 12 , 84 (2024). https://doi.org/10.1186/s40364-024-00611-y

Download citation

Received : 22 May 2024

Accepted : 29 June 2024

Published : 15 August 2024

DOI : https://doi.org/10.1186/s40364-024-00611-y

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Virus DNA integration
• Antiviral therapy

Biomarker Research

ISSN: 2050-7771

• General enquiries: [email protected]

• Open access
• Published: 16 August 2024

MicroRNA levels in patients with chronic hepatitis B virus and HIV coinfection in a high-prevalence setting; KwaZulu-Natal, South Africa

• Lulama Mthethwa 1 ,
• Raveen Parboosing 1 , 2 &
• Nokukhanya Msomi 1  

BMC Infectious Diseases volume  24 , Article number:  833 ( 2024 ) Cite this article

482 Accesses

7 Altmetric

Metrics details

Hepatitis B virus (HBV) and human immunodeficiency virus (HIV) co-infection are significant public health issues, despite the availability of an effective HBV vaccine for nearly three decades and the great progress that has been made in preventing and treating HIV. HBV and HIV both modulate micro-ribonucleic acids (microRNA) expression to support viral replication. The aim of this study was to describe the pattern of microRNA expression in patients coinfected with chronic HBV and HIV with varying disease severity, as indicated by Hepatitis B e antigen (HBeAg) status, HBV viral load, alanine transaminase (ALT) levels, and HIV viral load.

Plasma microRNAs, specific to HBV, were measured by quantitative real-time polymerase chain reaction (qRT-PCR) in HBV and HIV-negative healthy controls ( n  = 23) and patients coinfected with chronic HBV-HIV ( n  = 50). MicroRNA expression levels were compared between patients with high vs low HBV viral load, HBeAg positive vs HBeAg negative, high vs low ALT levels, and high vs low HIV viral load. Additionally, HBV viral load, ALT levels, and HIV viral load were correlated with microRNA expression levels.

Significantly higher expression levels of selected microRNAs were observed in chronic HBV-HIV coinfected patients compared to healthy controls. Significantly higher expression levels of hsa-miR-122-5p, hsa-miR-192-5p, and hsa-miR-193b-3p were observed in patients with high HBV viral load compared with low HBV viral load patients, and the levels of these microRNAs were correlated with HBV viral load levels. Significantly higher levels of hsa-miR-15b-5p and hsa-miR-181b-5p were observed in HBeAg-negative patients.

This study demonstrates the potential use of hsa-miR-15b-5p, hsa-miR-122-5p, hsa-miR-181b-5p, hsa-miR-192-5p and hsa-miR-193b-3p as additional diagnostic biomarkers in chronic HBV disease progression.

Peer Review reports

Introduction

Elimination of Hepatitis B virus (HBV) infection is still a global health challenge despite the availability of an effective prophylactic vaccine [ 1 , 2 ]. HBV infection causes acute and chronic hepatitis and complications such as liver cirrhosis and hepatocellular carcinoma (HCC) [ 3 , 4 ]. Even though there is limited information about HBV distribution and prevalence in some populations and regions, it is estimated to be the 7 th major cause of morbidity and death globally [ 5 , 6 ]. HBV affects 296 million people globally and about 81 million of these are in sub-Saharan Africa, where 990 000 new infections occurred in 2019 and 80 000 individuals died from HBV infection-related complications [ 7 ]. In 2019, there were 1.1 million deaths globally due to viral hepatitis, of which 96% were due to HBV and Hepatitis C Virus (HCV), which is greater than HIV mortality [ 7 ]. Most of the viral hepatitis deaths were due to liver cirrhosis and HCC [ 7 ]. Approximately 3.5 million people are infected with HBV in South Africa [ 8 , 9 ]. Globally, about 39 million people were living with HIV in 2022, and treatment options are more available worldwide for HIV compared to HBV or HCV [ 10 ]. About 2.7 million people are estimated to be co-infected with HBV and HIV worldwide. South Africa is an endemic setting for HBV and HIV infections [ 11 , 12 ].

MicroRNAs’ role in HBV pathogenesis and prognosis has been previously investigated [ 13 ]. MicroRNAs are small non-coding, single-stranded RNAs that inhibit or degrade messenger ribonucleic acids (mRNAs) during post-transcriptional gene expression by binding to the 3’-untranslated region (3’-UTR) of the target mRNA [ 14 , 15 , 16 ]. They were first discovered in Caenorhabditis elegans (C. elegans) but have now been found in some viruses and in all multi-cellular eukaryotes [ 17 ]. Numerous studies have been conducted to investigate the role of microRNAs in cellular response regulation such as proliferation, protein synthesis, differentiation, energy production, and apoptosis [ 18 , 19 ].

Through direct interactions with viruses or viral components, cellular microRNAs can negatively or positively affect viral replication. Certain microRNAs have been identified that directly target viral transcripts, affecting HBV replication such as hsa-miR-122 [ 20 ]. Altered expression of specific microRNAs has been associated with different stages of HBV infection and the progression of liver diseases, including cirrhosis and HCC [ 21 ]. MicroRNAs have been investigated for role as diagnostic biomarkers for monitoring chronic HBV disease progression [ 22 ]. Numerous studies have investigated the role of microRNAs in HBV mono-infection [ 23 , 24 , 25 , 26 ], However, there is a paucity of studies investigating microRNA profiles in chronic HBV-HIV coinfected individuals, especially in sub-Saharan Africa, which is endemic for both infections [ 27 ]. Therefore, we sought to describe the pattern of microRNA expression in chronic HBV and HIV coinfected patients with varying disease severity, as indicated by HBeAg status, HBV viral load, ALT levels, and HIV viral load.

Materials and methods

Study design and study samples.

In this retrospective case–control study, microRNA profile was compared in samples stratified according to serological markers, viral load, and ALT levels. The 10 candidate microRNAs included hsa-miR-15b-5p, hsa-miR-20a-5p, hsa-miR-29a-3p, hsa-miR-122-5p, hsa-miR-125b-5p, hsa-miR-181b-5p, hsa-miR-192-5p, hsa-miR-193b-3p, hsa-miR-194-5p, and U6 snRNA which had been identified as being potentially specific for Hepatitis B viral infection on a miRTarBase database ( http://miRTarBase.cuhk.edu.cn/ ). Samples stored at the Department of Virology, Inkosi Albert Luthuli Central Hospital (IALCH), Durban from 50 chronically infected with sub-genotype A1 HBV patients were used in this study. All samples were hepatitis B surface antigen (HBsAg)-positive for at least 6 months. HBV viral load, ALT, HBeAg, and HIV viral load results were available for these samples in the database from the previous study “Hepatitis B virus variants in HBV mono-infected and HIV/HBV co-infected samples in a high dual infection setting” and were downloaded anonymously together with demographic (age/gender) and other relevant clinical data (e.g., antiretroviral treatment, co-infections, co-morbidities, history of liver disease). Some patients were in the immune-active chronic HBV phase and the others were in the inactive chronic HBV clinical phase. This study cohort included 29 males (18–52 years) and 21 females (23–61 years). Samples were grouped according to HBeAg status (positive or negative), and ALT levels (≤ 35 or > 35 U/L); HBV viral load (≤ 1000 or > 1000 IU/ml); and HIV viral load (≤ 1000 or > 1000 IU/ml). The cut-off criteria for ALT and HBV viral load was based on that normal ALT levels for males are ≤ 35 U/L and ≤ 25 U/L for females therefore, ALT ≤ 35 U/L was taken as an inclusive cut-off value for low levels [ 28 ] and patients with HBV viral load of 1000 IU/ml identify as inactive carriers which are accompanied by normal or low ALT levels. 23 subjects, negative for the HBV core antigen, HBsAg and HIV stored at the Department of Virology, IALCH, Durban were used as the healthy control group. The control cohort included 7 males (18–47 years) and 16 females (13–62 years). This study was approved by University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC 00002418/2021).

RNA extraction and quantification

Plasma samples stored at –80 °C were thawed and mixed using a vortex mixer and RNA was extracted from 500 µl plasma using the NucliSens easyMAG system (Biomeriux, Marcy I’Etoile, France) following the manufacturer’s instructions. The ribonucleic acid (RNA) concentration was measured at an absorbance of 260 nm in a NanoDrop 1000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, United States) using nuclease-free water as a blank. RNA extracts were stored at -80 °C.

Reverse transcription (RT) or complementary DNA (cDNA) synthesis

The TaqMan microRNA reverse transcription kit (Applied Biosystems, Vilnius, Lithuania) and the custom RT primer pool (Applied Biosystems, Pleasanton, United States) were used to reverse-transcribe the total RNA to cDNA following the manufacturer’s instructions. The custom primer pool was prepared by combining 10 μl of each 5X RT stem-loop specific microRNA primer in 1.5 ml microcentrifuge, and 1X Tris–EDTA (TE) buffer was added to bring the final volume to 1000 μl. The RT reactions were performed in 8-strip tubes with 4 µl of RNA sample, 8 µl RT primer pool (containing 0.05X of each stem-loop microRNA-specific RT primer), 2 µl of 10X RT buffer, 0.4 µl of 2 mM of dNTPs with dTTP, 4 µl of 10 U/μl MultiScribe Reverse Transcriptase, 0.25 µl of 0.25 U/μl RNase inhibitor and 1.35 µl nuclease-free water. The stem-loop microRNA-specific RT primers were designed by Thermo Fisher Scientific, Pleasanton, United States. The reaction tubes were sealed, inverted to mix, and centrifuged for 10 s then incubated on ice for 5 min. The reaction tubes were placed on the ProFlex 96-well PCR System (Applied Biosystems, Foster City, United States) thermal cycler, following cycling parameters on Table S1. The RT products were used immediately for the cDNA preamplification step or stored at -20 °C for up to one week.

cDNA preamplification

To ensure that there would be sufficient cDNA product to amplify, a preamplification step was added. The custom preamplification primer pool was prepared by combining 10 μl of each 20X TaqMan MicroRNA assay (Applied Biosystems, Pleasanton, United States) in a 1.5 ml microcentrifuge. The reaction components were prepared following Table S2-A and the cycling conditions were set following Table S2-B. The reaction tubes were removed from the thermal cycler, briefly centrifuged and 175 μl of 0.1X TE (pH 8.0) was added to dilute the preamplification reaction product. The reaction tubes were sealed, inverted to mix, and briefly centrifuged. The products were stored at -20 °C for use in real-time quantitative PCR for up to one week.

Real-time quantitative PCR

Real-time quantitative PCR was used to assess the expression of microRNAs in preamplified products. A PCR reaction master mix was prepared for each microRNA assay containing 1 µl of 20X TaqMan microRNA Assay (Applied Biosystems, Pleasanton, United States), 10 µl of 2X TaqMan Universal Master mix II, No AmpErase UNG (Applied Biosystems, Vilnius, Lithuania) and 8.8 µl of nuclease-free water. The 20X TaqMan microRNA Assay contained specific reverse and forward primers and a TaqMan probe dye-labelled (FAM) (Applied Biosystems, Pleasanton, United States). The mixture was vortexed and centrifuged briefly to mix and collect contents properly. The 19 µl of the PCR reaction master mix was transferred to each well of the optical 96-well reaction plate, and 1 µl of the preamplified product was added. The plate was sealed with MicroAmp Optical Adhesive Film, vortexed briefly and centrifuged for 30 s to collect the contents to the bottom of the wells. The plate was placed on a QuantStudio 7 Flex Real-time PCR system (Applied Biosystems, Foster City, United States) for amplification. The cycling parameters were set following Table S3. Normalisation in microRNA expression analysis was performed using the U6 snRNA TaqMan microRNA assay (Applied Biosystems, Pleasanton, United States) as an endogenous control.

Data analyses and statistical analyses

Quantitative PCR threshold cycles (Ct) represent the number of cycles required for the fluorescent signal to cross the threshold. Each target was quantified in duplicates per sample. Among the 10 microRNA panels chosen, the U6 snRNA microRNA was used as an endogenous control for the normalization of the nine targets in the panel. The amplification plots of microRNAs were analysed using Design and Analysis software version 2.6 (Applied Biosystems, Foster City, United States) for analysis of the Ct values. The cut-off Ct value for microRNAs expression was 35: Ct values ≥ 35 were regarded as undetectable and were substituted with a Ct value of 35 for further analysis [ 29 ]. The microRNA expression relative to small RNA U6 was reported as delta Ct (∆Ct), calculated by subtracting the average Ct of U6 RNA from the average Ct of microRNA target. To interpret the results, the relative change in expression was assessed using a comparative Ct method. Relative microRNA expression levels were presented as equal to 2 −∆∆Ct and delta-delta Ct (∆∆Ct) was calculated by subtracting ∆Ct average for the control group from ∆Ct of the target microRNA [ 30 ]. The normality tests were performed to assess whether the data was normally distributed or not using Kolmogorov–Smirnov test, the Shapiro–Wilk test, D'Agostino & Pearson test, and the Anderson Darling test (Table S4). The levels of expression for the microRNA targets were assessed in five subgroupings, chronic HBV vs healthy control samples, High vs low HBV viral load, HBeAg status (positive and negative), ALT levels, and high vs low HIV viral load. Expression levels of microRNAs in different groups were compared using an unpaired Mann–Whitney U test, and a Spearman’s correlation coefficient (rho) analysis was used to evaluate the association of clinical parameters with microRNAs. Possible confounding variables were tested using a linear regression model. The Benjamini–Hochberg FDR method was used to correct false discovery rate [ 31 ]. All statistical analyses were performed using GraphPad prism version 7.0 (GraphPad Software, San Diego, United States) and IBM SPSS Statistics version 28.0 (IBM SPSS, New York, United States). A two-tailed p value of < 0.05 was regarded as statistically significant.

Sample characteristics

This study analysed samples from 50 patients with chronic HBV and 23 controls without serological evidence of HBV. Characteristics of female chronic HBV samples (42%), healthy female controls (69.6%), male chronic HBV samples (58%), and healthy male control samples (30.4%) are summarised in Table  1 . A significant difference in age and gender distribution was observed between chronic HBV samples and healthy controls (Table  1 ). However, there was no significant difference in microRNA expressions between age groups and gender groups in chronic HBV and healthy control samples (Table S5).

Expression levels of microRNA panel in chronic HBV samples vs healthy controls

Compared to healthy control groups, patients with chronic HBV infection showed significantly higher expression levels of all studied microRNAs (Fig.  1 ). The linear regression analysis revealed that age and gender did not influence the differences in the levels of microRNA expression, but these differences were due to HBsAg seropositivity (Table S7).

MicroRNA panel expression levels in chronic HBV samples compared to healthy controls. Relative microRNA level is shown as 2 ^ – (∆Ct of target microRNA– arithmetic mean of ∆Ct for the control group). Relative expressions are expressed as median and interquartile range. Statistical comparisons were performed using an unpaired Mann–Whitney U test. Significant differences are shown by an (*) system (**** P  < 0.0001). CHBV, chronic hepatitis B virus; IQR, Interquartile range

Different microRNA expression signatures in chronic HBV samples with high vs low HBV DNA

Low HBV viral load (< 3 log 10 IU/ml) is whereby partial treatment response is generally considered and high HBV viral load (> 3 log 10 IU/ml) is whereby a virological breakthrough is defined [ 24 , 28 , 32 , 33 ]. Samples with high HBV viral load had significantly higher median expression levels of specific microRNAs compared to samples with low HBV viral load – 3.74 vs 2.56 for hsa-miR-122-5p ( p  = 0.0001), 3.07 vs 1.94 for hsa-miR-192-5p ( p  = 0.0003) and 2.14 vs 0.82 for hsa-miR-193b-3p ( p  = 0.0002) (Table S6, Fig.  2 ). The expression levels of hsa-miR-20a-5p, hsa-miR-29a-5p, and hsa-miR-194-5p were slightly higher in patients with high HBV viral load compared to patients with low HBV viral load, but the difference was not statistically significant, while there was no significant difference in expression levels of microRNAs, hsa-miR-15b-5p, hsa-miR-125b-5p, and hsa-miR-181b-5p between high HBV and low HBV viral load samples. In samples with high HBV viral load hsa-miR-15b-5p had the lowest expression level followed by hsa-miR-194-5p (Table S6, Fig.  2 ), while hsa-miR-122-5p (median (IQR), 3.74 (1.18); p  = 0.0001) had the highest expression level. A receiver operating characteristic (ROC) curve analysis was done to assess performance of hsa-miR-122-5p, hsa-miR-192-5p and hsa-miR-193b-3p in differentiating patients with low vs high HBV viral load (Fig.  3 ). The ROC curve revealed that the AUC was 0.82 (95% CI: 0.70 to 0.94, p  = 0.0002) for hsa-miR-122-5p, 0.80 (95% CI: 0.68 to 0.92, p  = 0.0005) for hsa-miR-192-5p and 0.81 (95% CI: 0.69 to 0.93, P  = 0.0003) for hsa-miR-193b-3p for differentiating patients with low HBV viral load from patients with high HBV viral load.

Different microRNA expression levels in chronic HBV samples with high and low HBV viral load. Relative microRNA level is shown as 2 ^ – (∆Ct of target microRNA– arithmetic mean of ∆Ct for the control group). Relative microRNA expressions are expressed as median and interquartile range. Statistical comparisons were performed using an unpaired Mann–Whitney U test. Significant differences are shown by an (*) system (*** P  < 0.001, ns P  > 0.05). HBVVL, Hepatitis B virus viral load; IQR, Interquartile range; IU/ml, international units per millilitre

Differentiating power of hsa-miR-122-5p, hsa-miR-192-5p & hsa-miR-193b-3p in patients with low and high HBV viral load. Receiver operating characteristic curves and area under the curves (AUC) are presented for hsa-miR-122-5p, hsa-miR-192-5p & hsa-miR-193b-3p for differentiating patients with low HBV viral load from patients with high HBV viral load

Expression levels of microRNAs associated with HBeAg status

Plasma microRNA levels were compared between chronic HBV samples who are positive and negative for the HBeAg. HBeAg-negative samples had significantly higher median levels of the following microRNAs compared to HBeAg-positive samples: hsa-miR-15b-5p (2.45 vs 1.76; p  = 0.0054) and hsa-miR-181b-5p (2.58 vs 2.22; p  = 0.03) (Table S6, Fig.  4 ). Slightly higher median expression levels were observed for hsa-miR-20a-5p, hsa-miR-29a-3p, hsa-miR-125b-5p, hsa-miR-192-5p, hsa-miR-193b-3p and hsa-miR-194-5p in HBeAg negative samples even though the differences were not significant compared to HBeAg positive samples (Table S6, Fig.  4 ). HBeAg positive samples had higher expression levels of hsa-miR-122-5p compared to HBeAg negative samples but the difference was not significant (Table S6, Fig.  4 ).

Plasma microRNA levels in chronic HBV samples from HBeAg ( +) and HBeAg (-) groups. Relative microRNA level is shown as 2 ^ – (∆Ct of target microRNA– arithmetic mean of ∆Ct for the control group). Relative microRNA expressions are expressed as median and interquartile range. Statistical comparisons were performed using an unpaired Mann–Whitney U test. Significant differences are shown by an (*) system (** P  < 0.01, * P  < 0.05, ns P  > 0.05). HBeAg, Hepatitis B e Antigen; IQR, Interquartile range

Correlation of microRNA expression levels with markers of disease severity

A significant moderate positive correlation was observed between HBV viral load and hsa-miR-122-5p, hsa-miR-192-5p, and hsa-miR-193-3p (Fig.  5 ). To further confirm the association between HBV viral load and the abovementioned microRNA levels and to avoid any potential confounding effects of other variables such as HIV viral load, ALT levels, gender and age, a Benjamini–Hochberg correction test was performed. The significant positive correlation between HBV viral load with hsa-miR-122-5p and hsa-miR-193-3p was confirmed while the positive correlation between hsa-miR-192-5p and HBV viral load was found to be not significant. A weak positive correlation was observed between HBV viral load and hsa-miR-194-5p, and a weak negative correlation was observed between HBV viral load with hsa-miR15b-5p and hsa-miR-181b-5p, but these associations were not significant. No correlation was observed between HBV viral load and expression levels of hsa-miR-20a-5p, hsa-miR-29a-5p, and hsa-miR-125b-5p. No significant correlations were observed between our studied microRNAs and ALT levels, and HIV viral load. Our studied microRNAs were not able to differentiate elevated ALT levels from normal ALT levels as well as low vs high HIV viral load.

Heat map of the correlation of plasma relative microRNA level with HIV viral load, HBV viral load, and ALT level. Correlation coefficient (rho) was calculated using a Spearman correlation test. Significant relationships are shown by ** P  < 0.01. ALT, alanine transaminase; HBVVL, Hepatitis B virus viral load; HIVVL, human immunodeficiency virus viral load; IU/ml, international units per millilitre; U/l, Units per litre

Discussion and conclusion

Our study described the level of microRNA expression in samples from patients with chronic hepatitis B infection and furthermore compared microRNA expression in terms of various disease severity biomarkers (HBV viral load, HBeAg status, ALT levels, and HIV viral load). In our study, chronic HBV samples were compared with healthy samples and, all nine microRNA targets (hsa-miR-15b-5p, hsa-miR-20a-5p, hsa-miR-29a-3p, hsa-miR-122-5p, hsa-miR-125b-5p, hsa-miR-181b-5p, hsa-miR-192-5p, hsa-miR-193b-3p & hsa-miR-194-5p) revealed significantly higher levels in chronic HBV samples. The results from our study are congruent with the results from previous studies that investigated the expression pattern of the microRNAs in our panel in patients with chronic HBV [ 24 , 34 , 35 , 36 , 37 ]. Chronic HBV infection is associated with significant morbidity and mortality. Chronic HBV infection is associated with a 15% to 40% risk of cirrhosis, HCC, and/or liver failure, as well as a 15% to 25% mortality risk from HBV-associated liver diseases [ 38 ]. It is essential to predict chronic HBV infection progression in patients so that antiviral therapy can be initiated at the right time. The current diagnostic markers for HBV infection can be used as indicators of specific infection phases, monitor the progress of infection and guide treatment decisions [ 39 , 40 ]. Because of their non-invasive nature, serum or plasma microRNAs have attracted significant research attention as potential diagnostic and prognostic markers for chronic hepatitis B [ 41 ]. Several studies suggest that the use of microRNA panels in serum or plasma could improve the specificity of HBV diagnostics [ 13 , 26 , 42 , 43 , 44 ].

Patients with high HBV viral load expressed significantly higher levels of microRNA (hsa-miR-122-5p, hsa-miR-192-5p & hsa-miR-193b-3p), since HBV viral load levels are associated with disease progression, the levels of these microRNAs were also shown to be positively correlated with HBV viral load, these results suggest the potential use of these microRNAs as additional biomarkers for chronic hepatitis B disease progression. MicroRNAs may contribute to defining the phase of chronic hepatitis B infection by being involved in modulating the host immune response or contributing to inflammation and immune activation and/or assist in regulating viral gene expression and host immune responses, and treatment indication and may even allow assessment of antiviral therapy effectiveness [ 45 ]. Li et al . (2016) studied the expression levels of hsa-miR-125b-5p, and hsa-miR-122-5p and patients with high HBV viral loads demonstrated high expression levels of these microRNAs compared to patients with low HBV viral loads. In addition, elevated levels of hsa-miR-125b-5p were observed in patients in the immune-tolerant phase and at different stages of chronic hepatitis B infection. Lower levels of hsa-miR-125b-5p were observed in immune-tolerant phase compared to immune-reactive phase patients, which indicated that microRNAs may not only be influenced by HBV replication but by other factors such as liver necroinflammation [ 46 ]. These results may be relevant in the potential use of hsa-miR-122-5p, hsa-miR-192-5p, and hsa-miR-193b-3p as additional biomarkers for chronic HBV disease progression in our clinical setting.

In South Africa, the HBV genotype A is predominant with subtype A1 occurring in about 97% of rural Africans [ 47 , 48 , 49 ]. Infection with HBV genotype A is associated with chronicity, low HBeAg-positivity, horizontal transmission, and increased liver damage [ 50 ]. In this study expression levels of hsa-miR-15b-5p and hsa-miR-181b-5p were found to be significantly higher in HBeAg negative samples, in contrast to our findings, a study by Yu et al. (2015) found significantly high expression levels of hsa-miR-181b-5p in HBeAg-positive patients. Several previous studies based on microRNA profiling showed high expression levels of our studied microRNAs in HBeAg-positive patients when compared to HBeAg-negative patients [ 34 , 51 , 52 ]. This discordance between the findings in the current study and the abovementioned previous studies can be explained by the difference in specific geographical HBV genotypes and our cohort has sub-genotype A1 chronic HBV, most of the previous studies were done in settings predominated with genotype B, C & E chronic HBV. Different HBV genotype carriers are prone to different clinical outcomes or severity of the infection [ 49 ]. The evidence suggests that HBV genotypes may affect HBV endemicity, mutation patterns in the core and pre-core promoter regions, HBeAg seroconversion rates, clinical outcomes, and treatment response, however, the biological characteristics responsible for these differences have not yet been established [ 53 , 54 ]. A study investigating HBV infection persistence found high persistence of HBV infection in patients with sub-genotype A1 when compared to non-A genotypes [ 55 ]. In chronic HBV patients without HBeAg, due to a mutation in the pre-core or core promoter region of the genome, HBV has a naturally occurring mutant that does not produce HBeAg [ 56 , 57 ]. HBeAg-negative chronic HBV individuals may remain at risk for liver-related complications, especially if there is significant liver fibrosis or cirrhosis [ 38 ]. Therefore, non-invasive methods to monitor disease progression are needed. Hsa-miR-15b-5p and hsa-miR-181b-5p may serve as potential markers in HBeAg-negative chronic HBV disease progression. The assumption is that most of the study participants would have been infected as children and remained for a long period in the immune-tolerant phase, which is mediated by HBeAg status. Our results suggest that microRNA expressions of hsa-miR-15b-5p and hsa-miR-181b-5p may be associated with HBeAg production in our clinical setting and can be potentially used as biomarkers of HBV replication. The association of these microRNAs with HBeAg-negative status while the panel has correlation with HBV viral load could be explained by the following possible mechanisms such as these microRNAs may be associated with immune control mechanisms, influencing viral replication and clearance in the absence of HBeAg. The hsa-miR-15b-5p and hsa-miR-181b-5p microRNAs may have additional roles in influencing the clinical presentation, progression, or treatment response in HBeAg-negative patients. HBeAg-negative patients may have differences in the severity of liver disease, fibrosis, or risk of complications. These microRNAs might capture specific aspects related to disease progression or severity that are not solely reflected in HBV DNA levels such as co-infection with HIV.

This study also investigated the microRNA expression levels in high vs low HIV viral load and high vs low ALT levels but no significant differences were observed in the respective groups (Figure S1, Figure S2). This study investigated potential relationships between circulating microRNAs and HBV viral load, HIV viral load, as well as ALT levels, and a significant positive correlation was found between hsa-miR-122-5p and HBV viral load as well as between hsa-miR-192-5p and HBV viral load. We have similar results to previous studies that found hsa-miR-122-5p and hsa-miR-192-5p expression levels to be significantly correlated with HBV viral load [ 25 , 34 , 36 ]. The studies by van der Ree et al. (2017) and Wu et al. (2019) were conducted on patients that were undergoing antiviral treatment and the study by Winther et al. (2013) was conducted on patients’ mono-infected with chronic HBV. Our study was able to confirm these findings in samples with chronic HBV-HIV coinfection. According to these results, HBV viral replication may be influenced by miR-122-5p and miR-192-5p which could be assessed using cell culture, functional studies and other methods.

Other studies reported a significant negative correlation between HIV viral load and hsa-miR-29a-5p expression levels [ 37 , 58 , 59 ]. Our results did not show the potential role that our studied microRNAs may play in viral replication, and their potential to be utilized as a prognostic marker for HIV disease progression. Therefore, there is a need for more similar studies that will validate our findings or reveal the potential relationship that may exist between HIV viral load and our studied microRNAs. Our study showed no significant correlation between ALT levels and expression levels of our studied microRNAs. Wu et al. (2019) reported a negative correlation between hsa-miR-122-5p levels and ALT, however, another study found a positive correlation between ALT and hsa-miR-122-5p. In agreement with our findings, other studies found no correlation between hsa-miR-122-5p, hsa-miR-20a-5p, and hsa-miR181b-5p and ALT levels.

This is one of the few studies investigating microRNA profiling in a South African setting, a country where HBV genotype A1 predominates [ 60 ]. Hence, there is a need for more studies to be conducted in this setting to understand the role of microRNAs in the pathogenesis of chronic HBV infection. This study was able to show that the selected panel of microRNAs can differentiate between healthy control samples and chronic HBV samples, and that hsa-miR-122-5p, hsa-miR-192-5p & hsa-miR-193b-3p can potentially be used to differentiate between low versus high HBV viral load samples with over 80% accuracy which were supported by previous studies. However, further studies are required in this clinical setting to validate our findings. This study could not differentiate between low versus high ALT levels using microRNA profiling, which was supported by previous studies, therefore more future studies should investigate the association of microRNA expression with ALT levels, since ALT is considered to be a marker of active disease. The association of HBeAg status with specific microRNAs should be validated by more studies in our clinical setting to better understand the role of chronic HBV sub-genotype A1 on HBeAg seropositivity and disease progression and the role that microRNAs may play in viral replication. This study was able to correlate the hsa-miR-122-5p and hsa-miR-192-5p expression levels with HBV viral load which measures the virus titre in the bloodstream implying that these microRNAs have promising use as biomarkers for disease progression. Functional studies are needed to investigate the role of microRNAs in chronic HBV pathogenesis [ 61 ].

The sample size is one of the constraints of this study and the number of replicates used which were due to the costs of reagents used for testing. Future studies should increase the sample size and the number of replicates. Our microRNA profiles were not assessed according to clinical stage of HBV infection which should covered in further studies to better understand their role. Our studied microRNAs may have a function in the replication of HBV by targeting specific cellular factors or HBV transcripts. This microRNA panel can be utilised to differentiate patients with chronic HBV from healthy individuals in future studies, however, further validation studies are needed to establish the clinical utility of these microRNAs. Future studies should investigate the relationship between microRNAs and their presumed targets and HBV replication. It is possible that microRNAs may serve as markers for chronic HBV disease stages or prognostic markers for antiviral treatment response if they are found to play a direct or indirect role in regulating HBV replication. Furthermore, future research should include broader microRNA profiling to capture a more comprehensive picture of microRNA involvement in this intricate interplay between viruses and host responses.

Issues of chronic HBV infection such as HCC and cirrhosis often develop over decades, and HCC is often diagnosed much too late, leaving patients with poor prognoses and limited treatment options. For early detection of individuals at increased risk, sensitive and non-invasive methods are required that can detect subtle changes in the disease state. Through the monitoring of gene and microRNA expression in chronic HBV and liver disease, plasma or serum microRNA may be able to improve early detection. Therefore, our study was able to demonstrate the potential role of hsa-miR-15b-5p, hsa-miR-122-5p, hsa-miR-181b-5p, hsa-miR-192-5p and hsa-miR-193b-3p as biomarkers that could add to existing biomarkers with further studies, preferably prospective studies or retrospective where these markers are measured over time and associated/correlated to other clinical markers/signs for chronic HBV disease progression monitoring.

Availability of data and materials

Supplementary data can be found in the Supplementary Figures and Tables. Other data is available upon reasonable request from the corresponding author.

Abbreviations

Alanine transaminase

Biomedical research ethics committee

Complementary deoxyribonucleic acid

Threshold cycle

Hepatitis B e antigen

Hepatitis B surface antigen

Hepatitis B virus

Hepatocellular carcinoma

Hepatitis C virus

Human immunodeficiency virus

Inkosi Albert Luthuli central hospital

Micro-ribonucleic acids

Messenger ribonucleic acid

Quantitative real-time polymerase chain reaction

Ribonucleic acid

Reverse transcription

Untranslated region

Alter HJ. To have B or not to have B: vaccine and the potential eradication of hepatitis B. J Hepatol. 2012;57(4):715–7.

Article   PubMed   Google Scholar  

Amponsah-Dacosta E. Hepatitis B virus infection and hepatocellular carcinoma in sub-Saharan Africa: Implications for elimination of viral hepatitis by 2030? World J Gastroenterol. 2021;27(36):6025–38.

Article   PubMed   PubMed Central   Google Scholar  

Liaw YF, Chu CM. Hepatitis B virus infection. Lancet (London, England). 2009;373(9663):582–92.

Article   CAS   PubMed   Google Scholar  

Wu B, Yeh MM. Pathology of Hepatitis B Virus (HBV) Infection and HBV-Related Hepatocellular Carcinoma. In: Kao J-H, editor. Hepatitis B Virus and Liver Disease. Singapore: Springer Singapore; 2021. p. 99–122.

Chapter   Google Scholar  

Stanaway JD, Flaxman AD, Naghavi M, Fitzmaurice C, Vos T, Abubakar I, et al. The global burden of viral hepatitis from 1990 to 2013: findings from the Global Burden of Disease Study 2013. Lancet (London, England). 2016;388(10049):1081–8.

Sheena BS, Hiebert L, Han H, Ippolito H, Abbasi-Kangevari M, Abbasi-Kangevari Z, et al. Global, regional, and national burden of hepatitis B, 1990–2019: a systematic analysis for the Global Burden of Disease Study 2019. Lancet Gastroenterol Hepatol. 2022;7(9):796–829.

Article   Google Scholar  

WHO. Global progress report on HIV, viral hepatitis and sexually transmitted infections, 2021: accountability for the global health sector strategies 2016–2021: actions for impact. 2021.

Google Scholar  

Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet (London, England). 2015;386(10003):1546–55.

Samsunder N, Ngcapu S, Lewis L, Baxter C, Cawood C, Khanyile D, et al. Seroprevalence of hepatitis B virus: Findings from a population-based household survey in KwaZulu-Natal, South Africa. Int J Infect Dis. 2019;85:150–7.

UNAIDS Global HIV & AIDS statistics – 2022 fact sheet. Accessed April 26, 2023 at http://www.unaids.org/en/resources/fact-sheet .

Mphahlele MJ, Lukhwareni A, Burnett RJ, Moropeng LM, Ngobeni JM. High risk of occult hepatitis B virus infection in HIV-positive patients from South Africa. J Clin Virol. 2006;35(1):14–20.

Msomi N, Naidoo K, Yende-Zuma N, Padayatchi N, Govender K, Singh JA, et al. High incidence and persistence of hepatitis B virus infection in individuals receiving HIV care in KwaZulu-Natal, South Africa. BMC Infect Dis. 2020;20(1):847.

Jin X, Cai C, Qiu Y. Diagnostic value of circulating microRNAs in hepatitis B virus-related hepatocellular carcinoma: a systematic review and meta-analysis. J Cancer. 2019;10(20):4754–64.

Article   CAS   PubMed   PubMed Central   Google Scholar  

Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell. 2004;116(2):281–97.

Bartel DP. MicroRNAs: target recognition and regulatory functions. Cell. 2009;136(2):215–33.

Yoshida T, Asano Y, Ui-Tei K. Modulation of MicroRNA processing by dicer via its associated dsRNA binding proteins. Non-Coding RNA. 2021;7(3):57.

Lee RC, Feinbaum RL, Ambros V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell. 1993;75(5):843–54.

Taganov KD, Boldin MP, Baltimore D. MicroRNAs and immunity: tiny players in a big field. Immunity. 2007;26(2):133–7.

O’Brien J, Hayder H, Zayed Y, Peng C. Overview of MicroRNA biogenesis, mechanisms of actions, and circulation. Front Endocrinol. 2018;9:402.

Article   CAS   Google Scholar  

Lamontagne J, Steel LF, Bouchard MJ. Hepatitis B virus and microRNAs: Complex interactions affecting hepatitis B virus replication and hepatitis B virus-associated diseases. World J Gastroenterol. 2015;21(24):7375–99.

Liu WH, Yeh SH, Chen PJ. Role of microRNAs in hepatitis B virus replication and pathogenesis. Biochim Biophys Acta. 2011;1809(11):678–85.

Sagnelli E, Potenza N, Onorato L, Sagnelli C, Coppola N, Russo A. Micro-RNAs in hepatitis B virus-related chronic liver diseases and hepatocellular carcinoma. World J Hepatol. 2018;10(9):558–70.

Naito Y, Hamada-Tsutsumi S, Yamamoto Y, Kogure A, Yoshioka Y, Watashi K, et al. Screening of microRNAs for a repressor of hepatitis B virus replication. Oncotarget. 2018;9(52):29857–68.

Yu F, Zhou G, Li G, Chen B, Dong P, Zheng J. Serum miR-181b is correlated with hepatitis B virus replication and disease progression in chronic hepatitis B patients. Dig Dis Sci. 2015;60(8):2346–52.

Wu Y, Gao C, Cai S, Xia M, Liao G, Zhang X, et al. Circulating miR-122 is a predictor for virological response in CHB patients with high viral load treated with nucleos(t)ide analogs. Front Genet. 2019;10:243.

Tan B, Liu M, Wang L, Wang J, Xiong F, Bao X, et al. Serum microRNAs predict response of patients with chronic hepatitis B to antiviral therapy. Int J Infect Dis. 2021;108:37–44.

Maepa MB, Ely A, Kramvis A, Bloom K, Naidoo K, Simani OE, et al. Hepatitis B virus research in South Africa. Viruses. 2022;14(9):1939.

Terrault NA, Lok ASF, McMahon BJ, Chang KM, Hwang JP, Jonas MM, et al. Update on prevention, diagnosis, and treatment of chronic hepatitis B: AASLD 2018 hepatitis B guidance. Hepatology. 2018;67(4):1560–99.

Chen C, Tan R, Wong L, Fekete R, Halsey J. Quantitation of microRNAs by real-time RT-qPCR. Methods Mol Biol. 2011;687:113–34.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402–8.

Thissen D, Steinberg L, Kuang D. Quick and Easy Implementation of the Benjamini-Hochberg Procedure for Controlling the False Positive Rate in Multiple Comparisons. J Educ Behav Stat. 2002;27(1):77–83.

Zoulim F, Locarnini S. Hepatitis B virus resistance to nucleos(t)ide analogues. Gastroenterology. 2009;137(5):1593-608.e1-2.

Orrell C, Kaplan R, Wood R, Bekker LG. Virological breakthrough: a risk factor for loss to followup in a large community-based cohort on antiretroviral therapy. AIDS Res Treat. 2011;2011:469127.

PubMed   PubMed Central   Google Scholar  

Winther TN, Bang-Berthelsen CH, Heiberg IL, Pociot F, Hogh B. Differential plasma microRNA profiles in HBeAg positive and HBeAg negative children with chronic hepatitis B. PLoS One. 2013;8(3):e58236.

Huang C, Zheng JM, Cheng Q, Yu KK, Ling QX, Chen MQ, et al. Serum microRNA-29 levels correlate with disease progression in patients with chronic hepatitis B virus infection. J Dig Dis. 2014;15(11):614–21.

van der Ree MH, Jansen L, Kruize Z, van Nuenen AC, van Dort KA, Takkenberg RB, et al. Plasma MicroRNA levels are associated with hepatitis B e antigen status and treatment response in chronic hepatitis B patients. J Infect Dis. 2017;215(9):1421–9.

Yousefpouran S, Mostafaei S, Manesh PV, Iranifar E, Bokharaei-Salim F, Nahand JS, et al. The assessment of selected MiRNAs profile in HIV, HBV, HCV, HIV/HCV, HIV/HBV Co-infection and elite controllers for determination of biomarker. Microb Pathog. 2020;147:104355.

National Department of Health. National guidelines for the management of viral hepatitis. South Africa: NDOH; 2020. Available from: https://knowledgehub.health.gov.za/elibrary/national-guidelines-management-viral-hepatitis .

Coffin CS, Zhou K, Terrault NA. New and Old Biomarkers for Diagnosis and Management of Chronic Hepatitis B Virus Infection. Gastroenterology. 2019;156(2):355-68.e3.

Kramvis A, Chang K-M, Dandri M, Farci P, Glebe D, Hu J, et al. A roadmap for serum biomarkers for hepatitis B virus: current status and future outlook. Nat Rev Gastroenterol Hepatol. 2022;19(11):727–45.

Wang TZ, Lin DD, Jin BX, Sun XY, Li N. Plasma microRNA: A novel non-invasive biomarker for HBV-associated liver fibrosis staging. Exp Ther Med. 2019;17(3):1919–29.

CAS   PubMed   Google Scholar  

Bonino F, Piratvisuth T, Brunetto MR, Liaw YF. Diagnostic markers of chronic hepatitis B infection and disease. Antivir Ther. 2010;15(Suppl 3):35–44.

Gümüşay O, Ozenirler S, Atak A, Sönmez C, Ozkan S, Tuncel AF, et al. Diagnostic potential of serum direct markers and non-invasive fibrosis models in patients with chronic hepatitis B. Hepatol Res. 2013;43(3):228–37.

Peng C, Li Z, Xie Z, Wang Z, Ye Y, Li B, et al. The role of circulating microRNAs for the diagnosis of hepatitis B virus-associated hepatocellular carcinoma with low alpha-fetoprotein level: a systematic review and meta-analysis. BMC Gastroenterol. 2020;20(1):249.

Sartorius K, Makarova J, Sartorius B, An P, Winkler C, Chuturgoon A, et al. The Regulatory Role of MicroRNA in Hepatitis-B Virus-Associated Hepatocellular Carcinoma (HBV-HCC) Pathogenesis. Cells. 2019;8:1504. https://doi.org/10.3390/cells8121504.(12) .

Li F, Zhou P, Deng W, Wang J, Mao R, Zhang Y, et al. Serum microRNA-125b correlates with hepatitis B viral replication and liver necroinflammation. Clin Microbiol Infect. 2016;22(4):384.e1-.e10.

Firnhaber C, Reyneke A, Schulze D, Malope B, Maskew M, MacPhail P, et al. The prevalence of hepatitis B co-infection in a South African urban government HIV clinic. S Afr Med J. 2008;98(7):541–4.

Boyles TH, Cohen K. The prevalence of hepatitis B infection in a rural South African HIV clinic. S Afr Med J. 2011;101(7):470–1.

PubMed   Google Scholar  

Kramvis A. Molecular characteristics and clinical relevance of African genotypes and subgenotypes of hepatitis B virus. S Afr Med J. 2018;108(8b):17–21.

Kew MC, Kramvis A, Yu MC, Arakawa K, Hodkinson J. Increased hepatocarcinogenic potential of hepatitis B virus genotype A in Bantu-speaking sub-saharan Africans. J Med Virol. 2005;75(4):513–21.

Zhou J, Yu L, Gao X, Hu J, Wang J, Dai Z, et al. Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol. 2011;29(36):4781–8.

Zhang X, Chen C, Wu M, Chen L, Zhang J, Zhang X, et al. Plasma microRNA profile as a predictor of early virological response to interferon treatment in chronic hepatitis B patients. Antivir Ther. 2012;17(7):1243–53.

Liu CJ, Kao JH. Global perspective on the natural history of chronic hepatitis B: role of hepatitis B virus genotypes A to. J Seminars Liver Dis. 2013;33(2):97–102.

Elizalde MM, Tadey L, Mammana L, Quarleri JF, Campos RH, Flichman DM. Biological characterization of hepatitis B virus genotypes: their role in viral replication and antigen expression. Front Microbiol. 2021;12:758613.

Ito K, Yotsuyanagi H, Yatsuhashi H, Karino Y, Takikawa Y, Saito T, et al. Risk factors for long-term persistence of serum hepatitis B surface antigen following acute hepatitis B virus infection in Japanese adults. Hepatology. 2014;59(1):89–97.

Carman W, Hadziyannis S, McGarvey M, Jacyna M, Karayiannis P, Makris A, et al. Mutation preventing formation of hepatitis B e antigen in patients with chronic hepatitis B infection. Lancet. 1989;334(8663):588–91.

Zhang J, Xu WJ, Wang Q, Zhang Y, Shi M. Prevalence of the precore G1896A mutation in Chinese patients with e antigen negative hepatitis B virus infection and its relationship to pre-S1 antigen. Bras J Microbiol. 2009;40(4):965–71.

Witwer KW, Watson AK, Blankson JN, Clements JE. Relationships of PBMC microRNA expression, plasma viral load, and CD4+ T-cell count in HIV-1-infected elite suppressors and viremic patients. Retrovirology. 2012;9:5.

Moghoofei M, Bokharaei-Salim F, Esghaei M, Keyvani H, Honardoost M, Mostafaei S, et al. microRNAs 29, 150, 155, 223 level and their relation to viral and immunological markers in HIV-1 infected naive patients. Futur Virol. 2018;13(9):637–45.

Sartorius K, Sartorius B, Kramvis A, Singh E, Turchinovich A, Burwinkel B, et al. Circulating microRNA’s as a diagnostic tool for hepatocellular carcinoma in a hyper endemic HIV setting, KwaZulu-Natal, South Africa: a case control study protocol focusing on viral etiology. BMC Cancer. 2017;17(1):894.

Sartorius K, Makarova J, Sartorius B, An P, Winkler C, Chuturgoon A, et al. The Regulatory Role of MicroRNA in Hepatitis-B Virus-Associated Hepatocellular Carcinoma (HBV-HCC) Pathogenesis. Cells. 2019;8(12):1504.

Download references

Acknowledgements

We acknowledge support from the Department of Virology at the University of KwaZulu-Natal and National Health Laboratory Service.

This work was financially supported by the National Health Laboratory Services Research Trust (NHLS-RT), GRANT004_ 94829; National Research Foundation (NRF), Ref: MND190812465492; and Poliomyelitis Research Foundation (PRF), Grant 20/18.

Author information

Authors and affiliations.

Discipline of Virology, School of Laboratory Medicine and Medical Sciences, University of KwaZulu-Natal and National Health Laboratory Service, 800 Vusi Mzimela Road, Durban, 4058, South Africa

Lulama Mthethwa, Raveen Parboosing & Nokukhanya Msomi

Department of Virology, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, and National Health Laboratory Service (NHLS), Johannesburg, South Africa

Raveen Parboosing

You can also search for this author in PubMed   Google Scholar

Contributions

NM conceptualised the study and collected samples. LM designed the study. NM and RP reviewed study design. LM, NM, and RP acquired funds. LM performed laboratory experiments. LM performed data and statistical analysis. RP reviewed statistical and data analysis. RP and NM supervised the study. LM drafted manuscript, and NM and RP reviewed the manuscript.

Corresponding author

Correspondence to Lulama Mthethwa .

Ethics declarations

Ethics approval and consent to participate.

We obtained ethics approval from the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (BREC 00002418/2021). Ethics approval was also obtained from the Biomedical Research Ethics Committee of the University of KwaZulu-Natal (BE 324/16) for variant study. All participants gave written informed consent to participate in the variant study and gave written informed consent for sample storage and re-use.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary material 1., rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

Reprints and permissions

About this article

Cite this article.

Mthethwa, L., Parboosing, R. & Msomi, N. MicroRNA levels in patients with chronic hepatitis B virus and HIV coinfection in a high-prevalence setting; KwaZulu-Natal, South Africa. BMC Infect Dis 24 , 833 (2024). https://doi.org/10.1186/s12879-024-09715-0

Download citation

Received : 06 October 2023

Accepted : 02 August 2024

Published : 16 August 2024

DOI : https://doi.org/10.1186/s12879-024-09715-0

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Chronic HBV
• HBV-HIV coinfection
• HBV viral load

BMC Infectious Diseases

ISSN: 1471-2334

• General enquiries: [email protected]

A .gov website belongs to an official government organization in the United States.

A lock ( ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

• Safety Information by Vaccine
• Common Vaccine Safety Questions and Concerns
• Vaccine Recalls: FAQs
• Historical Concerns
• Information for Healthcare Providers

Related Topics:

• View All Home
• Vaccine Safety Systems

Hepatitis B Vaccine Safety

• Hepatitis B is a liver infection caused by the hepatitis B virus.
• There are vaccines that can protect against hepatitis B.

Hepatitis B is a vaccine-preventable liver infection caused by HBV. Hepatitis B can range from a mild, short-term, acute illness lasting a few weeks to a serious, long-term, chronic infection.

Available vaccines & manufacturer package inserts

Vaccines against hepatitis b and manufacturer inserts.

Contain only hepatitis B vaccine.

• The FDA approved Engerix-B in 1989 for use in people from birth through adulthood, although the dose varies by age group.
• FDA approved Heplisav-B in 2017 for use in people 18 years and older.
• FDA approved PREHEVBRIO in 2021 for use in people 18 years and older.
• FDA approved Recombivax HB in 1986 for use from birth through adulthood, although the dose varies by age group.

Vaccine against Hepatitis A and B and manufacturer insert

• FDA approved Twinrix in 2001 for use in people 18 years and older. It protects against hepatitis A and hepatitis B.

Childhood vaccines that include Hepatitis B and manufacturer inserts

Contain hepatitis B vaccine plus other vaccines.

• FDA approved Pediarix in 2002 for use in infants and children 6 weeks through 6 years old. It protects against hepatitis B, diphtheria, tetanus, pertussis, and polio.
• FDA approved VAXELIS in 2018 for use in children 6 weeks through 4 years of age. It protects against hepatitis B, diphtheria, tetanus, pertussis, and polio.

Who should & should not get the vaccine

• All infants within 24 hours of birth (usually 3 doses completed over a 6-month period)
• Children and adolescents younger than 19 years of age who have not yet gotten the vaccine
• People who are at increased risk of hepatitis B due to travel to certain countries, exposure to blood in the workplace, household or sexual exposure to an infected person, injection drug use or certain medical conditions
• Anyone who wants protection against hepatitis B

Common side effects

• Pain, soreness, redness, or swelling in the arm where the shot was given.
• Irritability, diarrhea, loss of appetite in healthy infants and children who received (Recombivax, Vaxelis, Pediarix).
• Vomiting, crying, drowsiness in children (Vaxelis, Pediarix).

When to call 911‎

Vaccines, like any medicine, can have side effects. Many people who get a hepatitis B vaccine have no side effects at all. The most common side effects include injection site pain, soreness, or redness, headache, and fatigue, and are usually mild lasting 1-2 days.

Report possible adverse events to VAERS‎ ‎

A closer look at the safety data.

• A Vaccine Safety Datalink (VSD) study compared deaths among newborns vaccinated with hepatitis B and unvaccinated newborns. The study found no differences between vaccinated and unvaccinated newborns. 1
• CDC reviewed VAERS reports of adverse events following hepatitis B vaccination from 2005 through 2015. During that time, 20,231 reports following hepatitis B or hepatitis B-containing vaccines, were submitted to VAERS. Over half of reports were in persons younger than 2 years of age; the majority of reports (78%) were following hepatitis B-containing vaccines in combination with other vaccines at the same visit. The most frequently reported adverse events for vaccines given in combination were fever, injection site redness, and vomiting. This review of the hepatitis B vaccine did not detect any new or unexpected safety concerns. These findings are consistent with pre-licensure clinical trials and other post-licensure monitoring and research. 2
• A separate review of VAERS studied reports made following the administration of hepatitis A (inactivated) and hepatitis B (recombinant) vaccines combined from May 2001 to September 2003. There were no unexpected health problems. 3
• In the early 1990s, CDC conducted a study of healthy full-term newborns to determine whether hepatitis B vaccination of newborns increases the risk of fever and/or suspected sepsis. The study found no evidence of increased fevers, sepsis evaluations, allergy or brain problems or medical procedures after to newborn hepatitis B vaccination. 4
• In a 4-year case series review of hepatitis B vaccine reports among newborns, there were no serious health problems linked to the hepatitis B vaccine. This was the largest case series review of hepatitis B vaccination reports among newborn babies and infants. Several studies have evaluated a possible link between hepatitis B vaccination and multiple sclerosis or optic neuritis. The studies did not show any link. 5

Hepatitis B and multiple sclerosis

Key points‎, hepatitis b vaccination does not cause ms.

Hundreds of millions of people worldwide have received hepatitis B vaccine without developing MS or any other autoimmune disease. As with all vaccines and any disease, due to the large number of vaccinations administered worldwide, surveillance systems that monitor health concerns after vaccination do expect to receive reports of MS occurring after vaccination that happen by chance alone.

To further explore any possible connection between hepatitis B vaccination and MS, many scientific studies have been conducted, and have concluded that hepatitis B vaccination does not cause MS. 6 7 8 9 10 11

Research on vaccines and other autoimmune diseases

CDC takes concerns about vaccines and immune system diseases and disorders very seriously. Researchers at CDC and elsewhere have conducted studies to examine the possible link between vaccines and autoimmune conditions like MS, diabetes, and asthma. These studies have been reassuring, providing no evidence to suggest a link between vaccines and autoimmune conditions.

As part of ongoing vaccine safety surveillance , CDC continues to conduct research to examine the effects vaccines may have on the immune system.

How CDC monitors vaccine safety

CDC and the Food and Drug Administration (FDA) are committed to ensuring that vaccines provided to the public are safe and effective. Once vaccines are licensed or authorized for emergency use in the United States, CDC and FDA continuously monitor them through several safety systems.

• Eriksen, E. M., Perlman, J. A., Miller, A., Marcy, S. M., Lee, H., Vadheim, C., Zangwill, K. M., Chen, R. T., DeStefano, F., Lewis, E., Black, S., Shinefield, H., & Ward, J. I. (2004). Lack of association between hepatitis B birth immunization and neonatal death: a population-based study from the vaccine safety datalink project. The Pediatric infectious disease journal , 23 (7), 656–662. https://doi.org/10.1097/01.inf.0000130953.08946.d0
• Haber, P., Moro, P. L., Ng, C., Lewis, P. W., Hibbs, B., Schillie, S. F., Nelson, N. P., Li, R., Stewart, B., & Cano, M. V. (2018). Safety of currently licensed hepatitis B surface antigen vaccines in the United States, Vaccine Adverse Event Reporting System (VAERS), 2005-2015. Vaccine , 36 (4), 559–564. https://doi.org/10.1016/j.vaccine.2017.11.079
• Woo, E. J., Miller, N. B., Ball, R., & VAERS Working Group (2006). Adverse events after hepatitis A B combination vaccine. Vaccine , 24 (14), 2685–2691. https://doi.org/10.1016/j.vaccine.2005.10.049
• Lewis, E., Shinefield, H. R., Woodruff, B. A., Black, S. B., Destefano, F., Chen, R. T., Ensor, R., & Vaccine Safety Datalink Workgroup (2001). Safety of neonatal hepatitis B vaccine administration. The Pediatric infectious disease journal , 20 (11), 1049–1054. https://doi.org/10.1097/00006454-200111000-00009
• Mikaeloff, Y., Caridade, G., Rossier, M., Suissa, S., & Tardieu, M. (2007). Hepatitis B vaccination and the risk of childhood-onset multiple sclerosis. Archives of pediatrics & adolescent medicine , 161 (12), 1176–1182. https://doi.org/10.1001/archpedi.161.12.1176
• Sadovnick, A. D., & Scheifele, D. W. (2000). School-based hepatitis B vaccination programme and adolescent multiple sclerosis. Lancet (London, England) , 355 (9203), 549–550. https://doi.org/10.1016/S0140-6736(99) 02991-8
• Institute of Medicine (US) Immunization Safety Review Committee, Stratton, K., Almario, D., & McCormick, M. C. (Eds.). (2002). Immunization Safety Review: Hepatitis B Vaccine and Demyelinating Neurological Disorders . National Academies Press (US).
• Confavreux, C., Suissa, S., Saddier, P., Bourdès, V., Vukusic, S., & Vaccines in Multiple Sclerosis Study Group (2001). Vaccinations and the risk of relapse in multiple sclerosis. Vaccines in Multiple Sclerosis Study Group. The New England journal of medicine , 344 (5), 319–326. https://doi.org/10.1056/NEJM200102013440501
• Ascherio, A., Zhang, S. M., Hernán, M. A., Olek, M. J., Coplan, P. M., Brodovicz, K., & Walker, A. M. (2001). Hepatitis B vaccination and the risk of multiple sclerosis. The New England journal of medicine , 344 (5), 327–332. https://doi.org/10.1056/NEJM200102013440502
• DeStefano, F., Verstraeten, T., Jackson, L. A., Okoro, C. A., Benson, P., Black, S. B., Shinefield, H. R., Mullooly, J. P., Likosky, W., Chen, R. T., & Vaccine Safety Datalink Research Group, National Immunization Program, Centers for Disease Control and Prevention (2003). Vaccinations and risk of central nervous system demyelinating diseases in adults. Archives of neurology , 60 (4), 504–509. https://doi.org/10.1001/archneur.60.4.504
• Groom HC, Irving SA, Koppolu P, Smith N, Vazquez-Benitez G, Kharbanda EO, Daley MF, Donahue JG, Getahun D, Jackson LA, Tse Kawai A, Klein NP, Nordin JD, Sukumaran L, Naleway AL. Uptake and safety of Hepatitis B vaccination during pregnancy: A Vaccine Safety Datalink study. Vaccine. 2018 Oct 1; 26(41): 6111-6116. Epub 2018 Sep 5.
• Schillie S, Vellozzi C, Reingold A, Harris A, Haber P, Ward JW, Nelson NP. Prevention of Hepatitis B Virus Infection in the United States: Recommendations of the Advisory Committee on Immunization Practices. MMWR Recomm Rep 2018 Jan 12: 67(No. RR-1): 1-31.
• Haber P, Moro PL, Ng C, Lewis PW, Hibbs B, Schillie SF, Nelson NP, Li R, Stewart B, Cano MV. Safety of currently licensed hepatitis B surface antigen vaccines in the United States, Vaccine Adverse Event Reporting System (VAERS), 2005-2015. Vaccine . 2018 Jan 25; (4): 559-564. Epub 2017 Dec 11.
• Moro PL, Zheteyeva Y, Barash F, Lewis P, Cano M. Assessing the safety of hepatitis B vaccination during pregnancy in the Vaccine Adverse Event Reporting System (VAERS), 1990-2016. Vaccine. 2018 Jan 2; 36(1): 50-54. Epub 2017 Nov 27.
• Schillie S, Murphy TV, Sawyer M, Ly K, Hughes E, Jiles R, de Perio MA, Reilly M, Byrd K, Ward JW. CDC Guidance for Evaluating Health-Care Personnel for Hepatitis B Virus Protection and for Administering Postexposure Management. MMWR Recomm Rep. 2013 Dec 20: 62(RR10); 1-19.
• Sawyer MH, Hoerger TJ, Murphy TV, Schillie SF, Hu D, Spradling PR, Byrd KK, Xing J, Reilly ML, Tohme RA, Moorman A, Smith EA, Baack BN, Jiles RB, Klevens M, Ward JW, Kahn HS, Zhou F. Use of Hepatitis B Vaccination for Adults with Diabetes Mellitus: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR. 2011 Dec 23: 60(50); 1709-1711.
• Yu O, Bohlke K, Hanson CA, Delaney K, Rees TG, Zavitkovsky A, Ray P, Mullooly J, Black SB, Benson P, Thompson WW, Davis RL, Jackson LA. Hepatitis B vaccine and risk of autoimmune thyroid disease: A Vaccine Safety Datalink study . Pharmacoepidemiol Drug Saf . 2007 Jul;16(7):736-45.
• Hocine MN, Farrington CP, Touze E, Whitaker HJ, Fourrier A, Moreau T, Tubert-Bitter P. Hepatitis B vaccination and first central nervous system demyelinating event: Reanalysis of a case-control study using the self-controlled case series method . Vaccine . 2007;25(31):5938-5943.
• Woo EJ, Miller NB, Ball R. Adverse events after hepatitis A B combination vaccine . Vaccine . 2006 Mar 24;24(14):2685-91.
• DiMiceli L, Pool V, Kelso JM, Shadomy SV, Iskander J; VAERS Team. Vaccination of yeast sensitive individuals: review of safety date in the US vaccine adverse event reporting system (VAERS) . Vaccine . 2006 Feb 6;24(6):703-7.
• Eriksen EM, Perlman JA, Miller A, Marcy SM, Lee H, Vadheim C, Zangwill KM, Chen RT, Destefano F, Lewis E, Black S, Shinefield H, Ward JI. Lack of association between hepatitis B birth immunization and neonatal death: A population-based study from the Vaccine Safety Datalink project . Pediatr Infect Dis J . 2004 Jul;23(7):656-62.
• DeStefano F, Verstraeten T, Jackson LA, Okoro CA, Benson P, Black SB, Shinefield HR, Mullooly JP, Likosky W, Chen RT, Vaccine Safety Datalink Research Group, National Immunization Program, CDC. Vaccinations and risk of central nervous system demyelinating diseases in adults . Arch Neurol . 2003 Apr;60(4):504-9.
• Lewis E, Shinefield HR, Woodruff BA, Black SB, Destefano F, Chen RT, Ensor E, Vaccine Safety Datalink Workgroup. Safety of neonatal hepatitis B vaccine administration . Pediatr Infect Dis J . 2001 Nov;20(11):1049-54.
• DeStefano F, Verstraeten T, Chen RT. Hepatitis B vaccine and risk of multiple sclerosis . Expert Rev Vaccines . 2002 Dec;1(4):461-6.
• Niu MT, Rhodes P, Salive M, Lively T, Davis DM, Black S, Shinefield H, Chen RT, Ellenberg SS. Comparative safety of two recombinant hepatitis B vaccines in children: Data from the Vaccine Adverse Event Reporting System (VAERS) and Vaccine Safety Datalink (VSD) . J Clin Epidemiol . 1998 Jun;51(6):503-10.
• Niu MT, Davis DM, Ellenberg S. Recombinant hepatitis B vaccination of neonates and infants: emerging safety data from the Vaccine Adverse Event Reporting System . Pediatr Infect Dis J . 1996 Sep;15(9):771-6.
• PREHEVBRIO vaccine is the only hepatitis B vaccine that does not contain yeast, making it safe for people who are allergic to yeast.

Vaccine Safety

Information about vaccine side effects, answers to common vaccine safety questions, and specific information for healthcare professionals and parents.

For Everyone

Health care providers.

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings

Preview improvements coming to the PMC website in October 2024. Learn More or Try it out now .

• Advanced Search
• Journal List
• HHS Author Manuscripts

Hepatitis B: The Virus and Disease

T. jake liang.

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), National Institutes of Health (NIH), Bethesda, MD

Hepatitis B virus (HBV) infects more than 300 million people worldwide and is a common cause of liver disease and liver cancer. HBV, a member of the Hepadnaviridae family, is a small DNA virus with unusual features similar to retroviruses. HBV replicates through an RNA intermediate and can integrate into the host genome. The unique features of the HBV replication cycle confer a distinct ability of the virus to persist in infected cells. Virological and serological assays have been developed for diagnosis of various forms of HBV-associated disease and for treatment of chronic hepatitis B infection. HBV infection leads to a wide spectrum of liver disease ranging from acute (including fulminant hepatic failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Acute HBV infection can be either asymptomatic or present with symptomatic acute hepatitis. Most adults infected with the virus recover, but 5%–10% are unable to clear the virus and become chronically infected. Many chronically infected persons have mild liver disease with little or no long-term morbidity or mortality. Other individuals with chronic HBV infection develop active disease, which can progress to cirrhosis and liver cancer. These patients require careful monitoring and warrant therapeutic intervention. Extrahepatic manifestations of HBV infection are rare but can be difficult to diagnose and manage. The challenges in the area of HBV-associated disease are the lack of knowledge in predicting outcome and progression of HBV infection and an unmet need to understand the molecular, cellular, immunological, and genetic basis of various disease manifestations associated with HBV infection.

The hepatitis B virus (HBV) is a small DNA virus with unusual features similar to retroviruses. 1 , 2 It is a prototype virus of the Hepadnaviridae family. Related viruses are found in woodchucks, ground squirrels, tree squirrels, Peking ducks, and herons. Based on sequence comparison, HBV is classified into eight genotypes, A to H. Each genotype has a distinct geographic distribution. Three types of viral particles are visualized in infectious serum by electron microscopy. Two of the viral particles are smaller spherical structures with a diameter of 20 nm and filaments of variable lengths with a width of 22 nm ( Fig. 1 ). The spheres and filaments are composed of hepatitis B surface antigen (HBsAg) and host-derived lipids without viral nucleic acids and are therefore noninfectious. 3 The infectious HBV virion (Dane particle) has a spherical, double-shelled structure 42 nm in diameter, consisting of a lipid envelope containing HBsAg that surrounds an inner nucleocapsid composed of hepatitis B core antigen (HBcAg) complexed with virally encoded polymerase and the viral DNA genome. The genome of HBV is a partially double-stranded circular DNA of about 3.2 kilobase (kb) pairs. The viral polymerase is covalently attached to the 5′ end of the minus strand. 4

Electron micrograph of circulating forms of HBV particles in the blood is shown at the top and a schematic drawing of Dane particle, the infectious HBV particle, is shown at the bottom with various structural features.

The viral genome encodes four overlapping open reading frames (ORFs: S , C , P , and X ) ( Fig. 2A ). 1 , 2 The S ORF encodes the viral surface envelope proteins, the HBsAg, and can be structurally and functionally divided into the pre-S1, pre-S2, and S regions. The core or C gene has the precore and core regions. Multiple in-frame translation initiation codons are a feature of the S and C genes, which give rise to related but functionally distinct proteins. The C ORF encodes either the viral nucleocapsid HBcAg or hepatitis B e antigen (HBeAg) depending on whether translation is initiated from the core or precore regions, respectively ( Fig. 2B ). The core protein has the intrinsic property to self-assemble into a capsid-like structure and contains a highly basic cluster of amino acids at its C-terminus with RNA-binding activity. 5 The precore ORF codes for a signal peptide that directs the translation product to the endoplasmic reticulum, where the protein is further processed to form the secreted HBeAg. The function of HBeAg remains largely undefined, although it has been implicated as an immune tolerogen, whose function is to promote persistent infection. 6 The polymerase (pol) is a large protein (about 800 amino acids) encoded by the P ORF and is functionally divided into three domains: the terminal protein domain, which is involved in encapsidation and initiation of minus-strand synthesis; the reverse transcriptase (RT) domain, which catalyzes genome synthesis; and the ribonuclease H domain, which degrades pregenomic RNA and facilitates replication. The HBV X ORF encodes a 16.5-kd protein (HBxAg) with multiple functions, including signal transduction, transcriptional activation, DNA repair, and inhibition of protein degradation. 7 – 10 The mechanism of this activity and the biologic function of HBxAg in the viral life-cycle remain largely unknown. However, it is well established that HBxAg is necessary for productive HBV infection in vivo and may contribute to the oncogenic potential of HBV.

The HBV genome. (A) The genomic organization, RNA transcripts and gene products are shown with several key regulatory elements. (B) The transcription start sites of various HBV transcripts and the proteins they encode (see text for details).

Other functionally important elements within the HBV genome include two direct repeats (DR1 and DR2) in the 5′ ends of the plus strand, which are required for strand-specific DNA synthesis during replication. 11 Two enhancer elements, designated as En1 and En2, confer liver-specific expression of viral gene products. 12 A glucocorticoid-responsive element (GRE) sequence within the S domain, 13 a polyadenylation signal within the core gene, and a posttranscriptional regulatory element overlapping En1 and part of HBxAg ORF have also been described. 14

The HBV replication pathway has been studied in great detail and is summarized in Fig. 3 . The initial phase of HBV infection involves the attachment of mature virions to host cell membranes, likely involving the pre-S domain of the surface protein. 15 Various cellular factors have been proposed to be the viral receptors, but only carboxypeptidase D has been shown to play an essential role in viral entry for the duck HBV. 16 Mechanisms of viral disassembly and intracellular transport of the viral genome into the nucleus are not well understood and probably involve modification of the nucleocapsid core protein. 17 After entry of the viral genome into the nucleus, the single-stranded gap region in the viral genome is repaired by the viral pol protein, and the viral DNA is circularized to the covalently closed circular (cccDNA) form. 18 This form of HBV DNA serves as the template for transcription of several species of genomic and sub-genomic RNAs and is the stable component of the replication cycle that is relatively resistant to antiviral action and immune clearance ( Fig. 2B ).

The replication cycle of HBV (see text for details).

The transcripts from the cccDNA are unspliced, polyadenylated, and possess a 5′ cap structure. The 3.5-kb genomic transcripts consist of two species with different 5′ ends: the pregenomic and the precore RNAs. The pregenomic RNA (pgRNA) serves as the template for reverse transcription and the messenger RNA for core and polymerase; the precore RNA directs the translation of the precore gene product. The polymerase translation is initiated at the pol start codon of the pgRNA, probably as a result of a ribosomal scanning mechanism. 19 The large HBsAg (L-HBsAg) protein is translated from the 2.4-kb subgenomic RNA, the middle (M-HBsAg) and small HBsAg (S-HBsAg) proteins from the various forms of 2.1-kb RNAs, and the HBxAg protein from the 0.7-kb RNA.

The S-HBsAg is the major S gene product and the L and M proteins are the minor species. Each surface protein has a glycosylation site in the S domain. Additional modifications of the L and M proteins occur at the pre-S2 domain with an N-linked oligosaccharide and a myristic acid at the amino-terminal glycine residue of the pre-S1 domain. 20 The distribution of the three envelope glycoproteins varies among the types of viral particles, with little or no L and M protein in the 20-nm particles but relatively more L protein in the Dane particles.

Replication of HBV begins with encapsidation of the genome. The packaging signal is a cis -acting element referred to as epsilon, which contains a stem-loop structure. 21 The terminal protein of the pol interacts with the epsilon and in concert with the core protein forms the nucleocapsid. After encapsidation, the pol mediates the reverse transcription of the pgRNA to minus-strand DNA and subsequent positive-strand synthesis. The circular form of the DNA is completed through several complicated steps of strand transfer. 22 The nucleocapsid then interacts with the envelope proteins in the endoplasmic reticulum to assemble into mature virions, which are then secreted into the extra-cellular milieu.

Diagnosis and Serology

HBV infection leads to a wide spectrum of liver disease ranging from acute hepatitis (including fulminant hepatic failure) to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). 2 The diagnosis of HBV infection and its associated disease is based on a constellation of clinical, biochemical, histological, and serologic findings. A number of viral antigens and their respective antibodies can be detected in serum after infection with HBV, and proper interpretation of the results is essential for the correct diagnosis of the various clinical forms of HBV infection ( Table 1 ).

Hepatitis B Virus Serological and Virological Markers

The typical course of acute hepatitis B is shown in Fig. 4A . HBV DNA followed shortly afterward by HBsAg and HBeAg are the first viral markers detected in serum. 23 HBsAg may be detected as early as 1–2 weeks or as late as 11–12 weeks after exposure, and its persistence is a marker of chronicity. HBeAg correlates with the presence of high levels of HBV replication and infectivity. 24 Within a few weeks of appearance of viral markers, serum alanine and aspartate aminotransferase (ALT, AST) levels begin to rise and jaundice may appear. HBeAg is usually cleared early, at the peak of clinical illness, whereas HBsAg and HBV DNA usually persist in the serum for the duration of clinical symptoms and are cleared with recovery. Antibodies to the HBV proteins arise in different patterns during acute hepatitis B. Antibody to HBcAg (anti-HBc) generally appears shortly before onset of clinical illness, the initial antibody being mostly immunoglobulin M (IgM) class, which then declines in titer as levels of IgG anti-HBc arise. Antibody to HBeAg (anti-HBe) usually appears shortly after clearance of HBeAg, often at the peak of clinical illness. Thus, loss of HBeAg and appearance of anti-HBe is a favorable serological marker during acute hepatitis B, indicating the initiation of recovery. Antibody to HBsAg arises late during infection, usually during recovery or convalescence after clearance of HB-sAg. Anti-HBs persists after recovery, being the antibody associated with immunity against HBV. However, between 10% and 15% of patients who recover from hepatitis B do not develop detectable anti-HBs and have anti-HBc alone as a marker of previous infection. For this reason, anti-HBc testing is the most reliable means of assessing previous infection with HBV, whereas anti-HBs testing is used to assess immunity and response to HBV vaccine. 25

The clinical course and serologic profiles of (A) acute and (B) chronic hepatitis B.

Patients who develop chronic hepatitis B ( Fig. 4B ) have a similar initial pattern of serological markers with appearance of HBV DNA, HBsAg, HBeAg, and anti-HBc. In these persons, however, viral replication persists and HBsAg, HBeAg, and HBV DNA continue to be detectable in serum, often in high titers. The subsequent course of chronic hepatitis B is quite variable. Most persons remain HBsAg-positive for years if not for life and have some degree of chronic liver injury (chronic hepatitis) that can lead to significant fibrosis and cirrhosis. Persons with chronic HBV infection are also at high risk to ultimately develop HCC.

The diagnosis of acute hepatitis B is reliably made by the finding of IgM anti-HBc in serum, particularly in a patient with HBsAg and signs, symptoms, or laboratory features of acute hepatitis. Nevertheless, in some instances, HBsAg is cleared rapidly from the serum, and IgM anti-HBc is the only marker detectable when the patient presents with hepatitis. Testing for anti-HBc (total) and anti-HBs are not useful in diagnosis, and testing for HBeAg and anti-HBe should be reserved for persons who test positive for HBsAg. The finding of HBsAg without IgM anti-HBc suggests the presence of chronic hepatitis B, but this diagnosis generally also rests upon finding of persistence of HBsAg for at least 6 months. 23 , 26 HBV DNA testing can also be helpful in the assessment of level of viral replication and possibly helpful in assessing prognosis and need for antiviral therapy. Assays for HBV DNA level have improved substantially over the years. 27 The current real-time polymerase chain reaction–based assay (TaqMan) has a lower limit of detection of 5–10 HBV DNA copies/mL and can accurately quantify a wide range of levels ( Fig. 5 ). With this degree of sensitivity, HBV DNA can be detected early during the course of infection, arising before the appearance of other serological markers, such as HBsAg or anti-HBc. As a consequence, testing for HBV DNA has emerged as a primary approach in the diagnosis and management of HBV infection. HBV DNA testing has now become routinely used in blood product screening (nucleic acid testing) 28 and monitoring of patients with HBV during treatment. 29 Persistently high levels of HBV DNA following resolution of hepatitis may be indicative of a failure to control the infection and an evolution into chronic infection.

The detection limits and dynamic ranges of assays for HBV DNA.

Acute Hepatitis B

About two-thirds of patients with acute HBV infection have a mild, asymptomatic and subclinical illness that usually goes undetected. 30 Approximately one-third of adults with acute HBV infection develop clinical symptoms and signs of hepatitis, which range fom mild constitutional symptoms of fatigue and nausea, to more marked symptoms and jaundice, and rarely to acute liver failure. The clinical incubation period of acute hepatitis B averages 2–3 months and can range from 1–6 months after exposure, the length of the incubation period correlating, to some extent, with the level of virus exposure. 31 The incubation period is followed by a short preicteric or prodromal period of constitutional symptoms such as fever, fatigue, anorexia, nausea, and body aches. During this phase, serum ALT levels rise and high levels of HBsAg and HBV DNA are detectable. The preicteric phase lasts a few days to as long as a week and is followed by onset of jaundice or dark urine. The icteric phase of hepatitis B lasts for a variable period averaging 1–2 weeks, during which viral levels decrease. In convalescence, jaundice resolves but constitutional symptoms may last for weeks or even months. During this phase, HBsAg is cleared followed by the disappearance of detectable HBV DNA from serum.

Acute liver failure occurs in approximately 1% of patients with acute hepatitis B and jaundice. 32 The onset of fulminant hepatitis is typically marked by the sudden appearance of fever, abdominal pain, vomiting, and jaundice, followed by disorientation, confusion, and coma. HBsAg and HBV DNA levels generally fall rapidly as liver failure develops, and some patients are HBsAg-negative by the time of onset of hepatic coma. Patients with acute liver failure due to hepatitis B require careful management and monitoring and should be referred rapidly to a tertiary medical center with the availability of liver transplantation. 33

Chronic Hepatitis B

Chronic hepatitis B has a variable and dynamic course. Early during infection, HBeAg, HBsAg, and HBV DNA are usually present in high titers, and there are mild to moderate elevations in serum aminotransferase levels ( Fig. 4B ). With time, however, the disease activity can resolve either with persistence of high levels of HBeAg and HBV DNA (the “immune tolerance phase”) or with loss of HBeAg and fall of HBV DNA to low or undetectable levels (“inactive carrier state”). Other patients continue to have chronic hepatitis B, although some lose HBeAg and develop anti-HBe (HBeAg-negative chronic hepatitis B). The course and natural history of hepatitis B are discussed in detail elsewhere in these proceedings. 34

The overall prognosis of patients with chronic hepatitis is directly related to the severity of disease. For those with severe chronic hepatitis and cirrhosis, the 5-year survival rate is about 50%. 35 – 37 Among patients with evidence of chronic hepatitis (elevated ALT and inflammation and/or fibrosis on liver biopsy), many are asymptomatic or have nonspecific symptoms, such as fatigue and mild right upper quadrant discomfort. Patients with more severe disease or cirrhosis may have significant constitutional symptoms, jaundice, and peripheral stigmata of end-stage liver disease including spider angiomata, palmar erythema, splenomegaly, gynecomastia, and fetor hepaticus. Ascites, peripheral edema, encephalopathy, and gastrointestinal bleeding are seen in patients with more advanced cirrhosis. ALT and AST are often elevated but may not correlate well with severity of liver disease. Bilirubin, prothrombin time, and albumin often become abnormal with progressive disease. Decreasing platelet count is often a poor prognostic sign.

Patients with chronic hepatitis may develop acute exacerbations with markedly elevated serum ALT. This scenario is more frequently described in those with HBeAg-negative chronic hepatitis B. 6 To distinguish between acute hepatitis B and chronic hepatitis B with a flare, anti-HBc IgM is a useful marker, as described in the previous section. However anti-HBc of the IgM class can be detected occasionally in patients with chronic hepatitis B with exacerbation. Alpha-fetoprotein (AFP), used as a marker for HCC, is often elevated in parallel with ALT during acute exacerbation. 38 However, it is unlikely to exceed 400 ng/mL. In patients with AFP much greater than this level, development of HCC should be suspected. 39

An estimated one-third of persons with chronic HBV infection will ultimately develop a long-term consequence of the disease, such as cirrhosis, end-stage liver disease, or HCC. The determinants of outcome of chronic hepatitis B appear to be both viral (HBV DNA levels, HBV genotype, some HBV mutation patterns) and host-specific (age, gender, genetic background, immune status).

Extrahepatic Manifestations of Hepatitis B

Extrahepatic manifestations of hepatitis B are present in 1–10% of HBV-infected patients and include serum-sickness–like syndrome, acute necrotizing vasculitis (polyarteritis nodosa), membranous glomerulonephritis, and papular acrodermatitis of childhood (Gianotti-Crosti syndrome). 40 , 41 Although the pathogenesis of these disorders is unclear, immune complex–mediated injury related to high level of HBV antigenemia is thought to be the cause.

The serum-sickness–like syndrome occurs in the setting of acute hepatitis B, often preceding the onset of jaundice. 42 The clinical features are fever, skin rash, and polyarteritis. The symptoms often subside shortly after the onset of jaundice, but can persist throughout the duration of acute hepatitis B. The course of this syndrome often parallels the duration and level of HBV viremia: rapid clearance of the virus leads to rapid resolution of the illness. This disorder resembles experimental serum sickness, in which immune complexes activate the complement pathways leading to complement-mediated injury. Patients with this syndrome have low complement levels and high-level circulating immune complexes containing HBV antigens and complement components.

About 30%–50% of patients with acute necrotizing vasculitis (polyarteritis nodosa) are HBV carriers. 41 , 43 This entity is more commonly seen in patients with recent exposure to HBV. Immune-mediated vascular injury can involve large, medium, and small vessels. Early clinical features are marked constitutional symptoms, high fever, anemia, and leukocytosis. Multisystem involvement is common, including arthritis, renal disease (proteinuria and hematuria), heart disease (pericarditis and congestive heart failure), hypertension, gastrointestinal disease (acute abdominal pain and bleeding), skin involvement (vasculitic lesions), and neurological disorders (mononeuritis multiplex and central nervous system abnormalities). The disease is highly variable and has a mortality rate of 30% within 5 years if not treated.

HBV-associated nephropathy has been described in adults but is more common in children. 44 , 45 Membranous glomerulonephritis is the most common form. Liver disease may be mild or absent in many of these patients. This disorder is frequently observed in countries with high prevalence of HBV infection. About 30%–60% of children with this disorder experience spontaneous remission, especially with HBeAg seroconversion. However, about 30% of adults with this condition can progress to renal failure with as many as 10% requiring dialysis or renal transplant. 46

Papular acrodermatitis (Gianotti-Crosti syndrome) is a distinct skin manifestation of acute HBV infection in childhood. 47 Skin lesions are maculopapular, erythematous, and nonpruritic, and involve the face and extremities. The syndrome lasts about 15–20 days and can either precede or follow the onset of jaundice in acute hepatitis B. Generalized lymphadenopathy and hepatomegaly have been described.

Other immune-mediated hematological disorders, such as essential mixed cryoglobulinemia and aplastic anemia have been described as part of the extrahepatic manifestations of HBV infection, but their association is not as well-defined; therefore, they probably should not be considered etiologically linked to HBV.

Occult or Latent HBV Infection

Other atypical HBV infections include seronegative occult or latent HBV infections. This heterogeneous group consists of patients who are HBsAg-negative who are either seronegative for all HBV markers or positive for anti-HBc and/or anti-HBs. 48 – 52 Many of these patients are positive for HBV DNA by polymerase chain reaction either in the liver or serum or both. Some of these patients have underlying liver disease, suggestive of ongoing hepatocellular injury from persistent HBV infection. Studies in animal models have demonstrated long-term persistence of viral genomes in the serum and/or liver of animals that have biochemical and serologic evidence of viral clearance and recovery from infection. 49 , 53 The important question is whether this observation represents ongoing viral replication and therefore clinically significant infection in terms of liver disease and transmission. Existing evidence supports the notion that it indeed indicates low-level viral replication, capable of transmission. 49 , 54 Studies in liver transplantation revealed transmission of HBV infection to recipients if the donors carried the anti-HBc marker. 55 In addition, reactivation of HBV infection in patients with serologic evidence of recovery undergoing immunosuppression or chemotherapy has been reported. 56 – 59 These observations, together with the immunologic studies described above, provide compelling evidence that one may not be able to completely eliminate HBV infection. Patients with serologic evidence of recovery probably have low-level viral replication that is effectively controlled by an active immune response. The possibility that these occult infections are caused by HBV mutants has been proposed. Although mutations have been reported in various regions of the viral genome, 60 – 63 definitive evidence in support of a pathogenic role of these mutants is lacking. Furthermore, whether liver disease can indeed result from these occult HBV infections is controversial. At present, there are no convincing studies in support of a causal relationship. Therefore, these occult HBV infections, other than the special situations described above, may not be clinically important.

Important Questions and Needs for Future Research

• How does HBV establish productive infection in vivo and what is the host response early during the infection? Despite well-described information on the clinical manifestations and natural history of acute HBV infection, detailed knowledge of the virus-host interaction during this stage remains poorly defined. Advances in this area would offer a better understanding of the pathogenesis of HBV infection and its associated disease.
• What is the immunologic basis of chronic infection and hepatocellular injury? There have been great strides in understanding the virology and immune response of HBV infection, but the molecular mechanisms whereby the host fails to clear the virus and develops chronic infection remain largely unknown. In addition, the adaptive evolution of virus under host immune pressure remains to be elucidated. Finally, the pathogenesis of various extra-hepatic manifestations associated with HBV infection is poorly understood. Further research in these areas is crucial not only in better understanding the natural history and disease progression but also in improving treatment for chronic hepatitis B.
• What is the genetic basis of the diverse clinical manifestations and disease outcomes of HBV infection? With the recent advances in genetic and genomic medicine, there are increasing opportunities to elucidate the genetic basis for variations in expression and susceptibility to HBV-associated diseases. Genome-wide association studies and other genomic technological advances would provide crucial information to identify useful genetic markers for disease outcome, clinical manifestations, and treatment response of HBV-associated disease.

Acknowledgments

Supported by the NIDDK Intramural Research Program, NIH.

Abbreviations

Potential conflict of interest: Nothing to report.

• Open access
• Published: 20 August 2024

Patient perspective on the elimination mother-to-child transmission of HIV, syphilis, and hepatitis B in Bali, Indonesia: a qualitative study

• Luh Nik Armini 1 , 2 ,
• Elsa Pudji Setiawati 3 ,
• Nita Arisanti 3 &
• Dany Hilmanto 4  

BMC Public Health volume  24 , Article number:  2258 ( 2024 ) Cite this article

47 Accesses

1 Altmetric

Metrics details

This study aimed to explore the facilitators and barriers to the elimination of human immunodeficiency virus (HIV), syphilis, and hepatitis B transmission based on the perspectives of mothers living with HIV, syphilis, and hepatitis B.

This study employed a descriptive, qualitative design. Semi-structured interviews were conducted with mothers living with HIV, syphilis, and/or hepatitis B virus. A total of 25 participants were included in the study. This study used a triangulation method conducted by members to enhance the validity and dependability of the findings. The study was conducted at referral hospitals and community health centers between September 2022 and February 2023. Data analysis utilized deductive content analysis and categorized themes based on a socio-ecological framework.

The findings revealed facilitators and barriers across five levels of the socio-ecological framework and 21 subcategories. The findings included the following: (1) At the policy level, facilitators were mandatory testing programs, and barriers were separating testing services from antenatal care facilities. (2) At the community level, facilitators included the involvement of non-governmental organizations (NGOs) and cross-sector support. Barriers included challenges faced by non-residents and fear of stigma and discrimination. (3) At the healthcare system level, facilitators included tracking and follow-up by midwives, positive relationships with healthcare providers, and satisfaction with healthcare services. Barriers included prolonged waiting times, insufficient information from healthcare providers, and administrative limitations. (4) At the interpersonal level, facilitators included partner and family support, open communication, and absence of stigma. Barriers included the reluctance of sexual partners to undergo screening. (5) At the individual level, facilitators included the desire for a healthy baby, adequate knowledge, self-acceptance, and commitment to a healthy lifestyle; barriers included the lack of administrative discipline.

Mothers living with HIV, syphilis, or hepatitis B require tailored healthcare approaches. Healthcare professionals must understand and meet the needs of mothers within a comprehensive care continuum. The findings of this study advocate for the development and implementation of integrated care models that are responsive to the specific challenges and preferences of affected mothers, aiming to improve health outcomes for both mothers and their children.

Peer Review reports

The triple elimination program for mother-to-child transmission (EMTCT) of human immunodeficiency virus (HIV), syphilis, and hepatitis B in Indonesia began in 2018 through a policy issued by the Ministry of Health of the Republic of Indonesia in 2017. EMTCT programs are integrated into antenatal care (ANC) [ 1 ]. Although hepatitis B may not be sexually acquired, these three diseases have the same mode of transmission from mother to child. Hence, efforts toward elimination can be carried out together to provide benefits and efficiency in its implementation [ 2 , 3 ]. Although policies and strategies to achieve triple elimination have been established and implemented, pregnant women living with HIV, syphilis, and/or hepatitis B face challenges in preventing transmission of infection from mother to child; many pregnant women are screened late, even before delivery, and do not receive treatment [ 4 , 5 ]. In Bali Province, EMTCT achievements in three districts have not achieved the target set by the World Health Organization (WHO) and the Ministry of Health and have faced challenges [ 6 ]. Treatment of pregnant women living with HIV, syphilis, and/or hepatitis B was far below the target despite screening of pregnant women who exceeded WHO targets [ 7 ]. A study in Indonesia revealed that one-third of pregnant women living with HIV never initiated ART treatment, only half of those who initiated treatment remained on ART, and ART treatment retention among pregnant women was greater in tertiary and secondary hospitals [ 8 ].

The WHO advocates for EMTCT through a comprehensive approach spanning prevention, diagnosis, treatment, and care [ 9 ]. Health systems and programs need to ensure that all pregnant women living with HIV, syphilis, and hepatitis B, along with their infants, are effectively treated and that their sexual partners also undergo examination and treatment [ 10 ]. The set The WHO global target for EMTCT validation at 95% for all infections, 50 per 100,000 live births for HIV- and syphilis-infected children, and ≤ 2% for the hepatitis B MTCT rate. The national targets to end the transmission of HIV, syphilis, and hepatitis B have not been achieved by 2022. In Indonesia, only approximately 54% of the mothers are screened for HIV infection. The number of babies born to HIV-positive mothers was 10%, the number of children who received prophylaxis was 103 (76.87%), and the number of children who received ART was 26 (19.4%) [ 11 ]. Among 35,757 children born to mothers with hepatitis B infection in 2022, only 27% (9,239 children) were screened, and approximately 1.46% (135 children) were hepatitis B-positive. In 2016, there were approximately 661,000 children with congenital syphilis in 2016. The decline in congenital syphilis is slow, and there is a gap between hepatitis B screening and the management of HBsAg-positive patients [ 12 ]. Therefore, antenatal testing among pregnant women and Indonesia's impact target (case rate among children) is still below the expected global target.

The WHO also expects each country to tailor its actions to achieve EMTCT to local conditions while ensuring fair access and respecting human rights, with care centered on individuals, especially pregnant or postpartum mothers [ 4 ]. This study aimed to explore the facilitators of and barriers to the elimination of HIV, syphilis, and/or hepatitis B transmission from mother to child from the perspective of infected mothers. The findings of this study may help to characterize mothers and provide insights into improving EMTCT services. This study may also be utilized in providing women-centered care services, enabling precise interventions for infected mothers based on the challenges they face, and facilitating the success of preventing this infection from mother to child. Moreover, this study may provide insights into gender equality in efforts to eliminate mother-to-child transmission of HIV, syphilis, and hepatitis B by providing mothers with an enabling environment and social support. This highlights the need for a holistic approach to address ongoing gender inequalities. Public health programs should specifically target these issues in order to achieve effective elimination.

Study design

This study was designed as a descriptive qualitative study. [ 13 ] Semi-structured qualitative interviews were conducted with postpartum mothers living with HIV, syphilis, and/or hepatitis B to explore the facilitators of and barriers to the elimination of infection transmission from mother to child.

Study setting

The study was conducted from September 2022 to February 2023 at two referral hospitals and five public health centers. This study was conducted over a period to capture a diverse range of experiences and responses from the participants, thereby enhancing the reliability and depth of the study findings.

Sample size

A purposive sampling strategy was used to select the participants. The sample in this study comprised 25 participants, including mothers living with HIV, Syphilis, and Hepatitis B. This sample was reached according to saturation.

Inclusion and exclusion criteria

This study included postpartum mothers who were newly diagnosed with HIV, syphilis, and hepatitis B and had experience in receiving EMTCT services, including screening, treatment during pregnancy, and prevention for the baby. Furthermore, the study exclusively included mothers who provided informed consent to participate. Newly diagnosed mothers were defined as those who were diagnosed at the recent stage of pregnancy and induced the EMTCT program. The accuracy of the testing history, maternal and infant treatment, and immunization status were cross-checked through the participants’ medical records and maternal and child health books. Additionally, mothers with severe mental health conditions that may affect their ability to participate were excluded.

Recruitment participants

The participants in this study included only mothers who met the inclusion criteria. Face-to-face interviews were conducted with participants at locations agreed upon by the interviewer. Participants were recruited until they reached a high level of saturation. Before receiving EMTCT services, primary maternal care can be provided in both private and public settings. However, patients who test positive are referred to public health services, whereas those who test negative continue antenatal care at their original primary care provider. Therefore, the sample selection was conducted only for public health services. The participant recruitment scheme is shown in Fig.  1 .

Rectruitment of partcipant’s diagram

Data collection

This study was conducted after obtaining approval from the heads of the public referral hospitals and public health centers. Nineteen interviews were conducted in hospitals, and six were conducted at public health centers. The interviews were facilitated by the author (LNA) and an independent facilitator from a healthcare professional who had received interview and/or qualitative study training. This study was initiated by developing an interview guide designed to facilitate data collection using a series of semi-structured questions. These interview guidelines were developed through research (see Supplementary File 1). The development of the question guide was based on the social-ecological framework model and a review of the literature on factors influencing the elimination of the transmission of three infectious diseases, HIV, syphilis, and hepatitis B, from mother to child [ 14 , 15 , 16 ]. Separate interview guides were created for mothers living with HIV, syphilis, and hepatitis B, taking into consideration different contexts and experiences. Recognizing the potential for co-infections among participants, we used only one of them in the interview guide. The interview guide was designed for Indonesia. The interviewers were fluent in Indonesian and Balinese and had a qualitative interview experience. The interview sessions lasted approximately 30–60 min. The data collectors were midwives who assisted in identifying respondents who met the study criteria and had received comprehensive training. This training focused on the nuances of using the interview guide, ethical considerations, and techniques for capturing context and nuances beyond audio recordings such as supplementary notes. After ensuring that all data collectors were adequately prepared, interviews were conducted with the approval of the heads of the public referral hospitals and public health centers. The interviews were audio-recorded to ensure accuracy and supplementary notes were taken to capture any contextual details or nonverbal cues that were not evident in the audio recordings. Throughout this process, quality assurance measures were applied rigorously to maintain the integrity and reliability of the collected data.

Data analysis

Descriptive statistical analysis was used to calculate the frequency and percentage of participants' sociodemographic data, which provided information on their characteristics. The study conducted deductive content analysis to identify emerging themes and used existing theories or literature to guide and identify variables or concepts of interest [ 17 , 18 ]. Data saturation was reached with a total of 25 participants [ 15 ]. The interviews were transcribed verbatim, and the transcripts were coded using NVivo 12.0 software. The transcripts were coded line-by-line for data analysis, emphasizing the patient’s barriers and facilitator factors of continuity care toward triple EMTCT. The researchers independently coded and integrated the codes, when necessary. Potential main themes were derived by fusing similar codes into subthemes. Categories were grouped based on overarching themes and mapped onto [ 19 ]. The socioecological health model, which is also known as the socioecological model, a framework developed to understand the multifaceted influences on human behavior. This model considers factors at various levels, from the individual to the broader community and policy contexts [ 19 ]. The use of socioecological models in the context of infectious disease is well established. Public health studies have previously utilized this framework to explore barriers and facilitators for mothers living with HIV in care retention and HIV-exposed infant testing [ 20 , 21 ]. This study identified five categories and 21 subcategories. This category refers to the socio-ecological framework. Facilitators and barriers that influence treatment adherence were identified in this subcategory.

Trustworthiness

This reliability was evaluated using four criteria: credibility, dependability, transferability, and confirmability [ 22 ]. Two researchers established credibility by initially categorizing the data, which were reviewed and restructured by a third researcher. In addition, specific interview transcripts were returned to participants for clarification. At this stage, the author ensured that the participants and peer researchers had potentially stigmatizing narratives. Participants concurred with the content and interpretation of the transcripts. Continuous evaluation and assessment of the significance and completeness of data ensured dependability. Confirmability was attained through the categorization of coding into distinct groups, and all researchers collectively deliberated on the main groups. A single researcher with expertise in qualitative research and auditing oversaw the entire auditing process. Concurrently, deepening of the data was made possible through semi-structured interviews. A comprehensive exposition was provided regarding all stages of data acquisition and analysis as well as the participants to enhance transferability. Moreover, this study was conducted by members (PhD qualifications), and data checking using the triangulation method enhanced the validity and dependability of the findings. The researchers assessed their role in the data collection and analysis in a reflective manner. The research team engaged in discussions about the data, codes, and themes. It comprised of a midwife, pediatrician, family physician, and public health practitioner. The interviews were followed by debriefing sessions for the researchers.

Characteristics of the participants

A total of 25 participants were interviewed, comprising mothers infected with HIV (30.33%), mothers infected with syphilis (33.33%), and mothers infected with hepatitis B (36.37%). Sociodemographic characteristics including age, education, occupation, parity, marital status, pre-existing conditions, partner infection status, and place of residence are presented in Table  1 .

Patient perspective on their care of triple EMTCT

This study revealed five categories following a socio-ecological framework: policy, community, healthcare system, intrapersonal, and individual. There were 21 subcategories encompassing facilitators and barriers. The categories and subcategories are presented in Fig.  2 and Table  2 , respectively. Facilitator factors include (1) mandatory programs, (2) the involvement of nongovernmental organizations (NGOs), and (3) cross-sectoral involvement. (4) Tracking and follow-up by the assigned midwife in the area; (5) positive relationship between patients and healthcare providers; (6) satisfaction with hospital facilities and healthcare services; (7) support from the partner or family; (8) open communication with the partner and/or family members; (9) freedom from stigma from the partner and family; (10) desire to have a healthy baby; (11) sufficient knowledge; (12) self-acceptance; and (13) desire for a healthy lifestyle. Moreover, the barrier factors include (1) testing services separate from the ANC facility, (2) challenges for nonresidents, (3) fear of stigma and discrimination, (4) long waiting times for hospital visits, (5) insufficient information from healthcare providers regarding continuous care for mothers and infants, (6) administrative limitations in healthcare services for insurance users, (7) the sexual partner or spouse not yet willing to undergo screening, and (8) lack of administrative discipline.

Barriers and Facilitators of EMTCT in Bali Province, Indonesia

Policy-level factor

Facilitators at the policy level are related to adherence to mandatory programs from healthcare professionals for testing. All mothers reported testing for HIV, syphilis, and hepatitis B on the recommendations of their midwives and doctors, and none reported testing on their own (Quotation 1/Q1).

Barriers at the policy level are related to testing services that are separate from ANC facilities. All mothers were tested at public health centers while performing antenatal check-ups at the midwives’ or doctors’ private practices (Q2).

Community-level factors

Facilitators at the community level are involved in Non-Governmental (NGOs) and cross-sector involvement (Q3-Q4). Several NGOs played a crucial role in Bali. They have field outreach workers in hospitals and public health centers. Mothers infected with HIV mentioned having companions " from NGOs (Q3). This support is not only for mothers living with HIV but also for their partners, families, and newborns (Q4). This assistance is instrumental because it provides information on HIV infection, accompanies patients during treatment or examinations during pregnancy, and ensures regular medication intake (Q4).

Barriers at the community level included challenges for non-residents and fear of stigma and discrimination from the community (Q5-Q7). Non-residents of mothers who did not have any health insurance or could not afford medical care, childbirth, or other examinations could receive free health insurance from the local government, provided that they had a valid ID (KTP in Indonesia) in that area and a letter of inability to pay from the village head (Q6). On the other hand, mothers who are not native residents and do not have a valid ID with the same domicile face difficulties obtaining health insurance, where the payments are covered by the local government (known as the Healthy Indonesia Card/KIS) (Q5-Q6). They felt a significant financial burden when using health insurance that was paid independently every month (Q6). Some mothers expressed embarrassment about seeking care at public health centers or hospitals, particularly if the people around their community know who those seeking care are because of being infected with HIV and syphilis (Q7). However, mothers living with hepatitis B rarely express shame for seeking care at hospitals.

Healthcare system or institutional-level factors

Facilitators in the healthcare system were related to tracking and follow-up by the assigned midwife in the area, good relationships between patients and healthcare providers, and satisfaction with hospital facilities and healthcare services (Q8-Q9). All pregnant mothers living with the virus mentioned receiving home visits from midwives once during pregnancy and after giving birth, both during pregnancy and postpartum, and visits to health facilities (Q8 and Q9). Mothers who did not undergo testing until the third trimester were visited by village midwives (Q8 and Q9). All pregnant mothers stated that they always followed and trusted all the information provided by the midwives and doctors conducting the examinations (Q10). Some mothers expressed comfort in giving birth at referral hospitals (public sector), even though they were far from their homes, owing to the quick response from healthcare professionals and air-conditioned rooms, even when using free health insurance (Q11).

Barriers to the healthcare system are related to long waiting times for hospital visits, insufficient information from healthcare providers regarding continuous care for mothers and infants, and administrative limitations in healthcare services for insurance users (Q12-Q15). Long waiting times, especially at the ticket counter, became inhibiting factors, as mothers had to spend the entire day at the hospital (Q12). Almost all participants mentioned that even if they arrived early, they still had to wait at the registration counter until noon, especially when seeking treatment and undergoing examinations at the hospital's healthcare services (Q12). However, almost all mothers expressed that during pregnancy checkups with midwives or specialist doctors, they did not receive clear information about the examinations or types of testing to be performed at public health centers, leading them to postpone testing because they were not provided with complete information regarding the types of testing and their purposes (Q13). Almost all mothers mentioned not receiving information that their child would be retested to determine whether the child was infected (Q14). The separation of ANC services from treatment services for mothers living with HIV and hepatitis B has prevented them from simultaneously accessing pregnancy examination services and taking medications. This was also influenced by private health insurance, which participants paid per month ( Badan Penyelenggara Jaminan Sosial Kesehatan /BPJS), or public health insurance provided by the government (Kartu Indonesia Sehat/KIS) administration (Q15).

Interpersonal-level factors

Facilitators at the interpersonal level included support, open communication, and free stigma from the partner or family (Q16-Q18). All mothers living with HIV, syphilis, and/or hepatitis B who had successfully undergone adequate treatment received support from their husbands and families (Q16). Husbands remind them to take medication and accompany them for prenatal checkups or treatment. (Q16). Families with financial and decision-making powers, such as fathers or siblings, play a crucial role in ensuring that mothers receive treatment according to the doctors' instructions. (Q17). Families also serve as substitutes for husbands in terms of collecting medication from public health centers for pregnant mothers or accompanying them for prenatal checkups and treatment when husbands cannot take leave at work or face other barriers. (Q17). Almost all pregnant mothers who had successfully received adequate treatment and had taken ARV medication until the time of the interview demonstrated openness regarding their condition to their sexual partners, husbands, and/or family members (Q18). Almost all mothers were open to family members who were more economically stable than economically unstable.

A significant barrier at the personal level, highlighted by the experiences of the participants, was the reluctance of sexual partners to undergo health screening (Q19). This fear of potential diagnoses can deter partners from participating in essential health checks, thereby not only compromising their own health but also impacting the effectiveness of programs aimed at eliminating mother-to-child transmission of infections such as HIV, syphilis, and hepatitis B.

Individual-level factors

Facilitators at the individual level are related to mothers who desire a healthy baby and healthy lifestyle (Q20-Q21). Sufficient knowledge and self-acceptance were facilitators at the individual level (Q22-Q24). All mothers said that they wanted their children to be healthy and did not suffer from diseases like themselves. (Q20). All infected mothers had a desire to recover, so they always followed the advice of doctors and midwives, sought treatment regularly, and gave birth at the hospital according to the doctor's advice (Q21). All mothers received counseling from health providers and knew that the disease could be transmitted to the baby if they did not seek treatment (Q22 and Q23). However, not all mothers had good knowledge of the causes of maternal infection and comprehensive care for mother-baby-husband pairs (Q22 and Q23). Acceptance of the mother's illness affects her openness to her partner and/or family, and routine treatment (Q24).

Barriers at the individual level are related to a lack of administrative discipline. Some mothers did not have ID cards; therefore, they did not have KIS or national health coverage from the local government (Q25). The lack of an administrative order is a barrier for mothers to receive treatment immediately because it takes approximately one month to take care of the population administration and activate their KIS after receiving an ID card (Q25). Mothers who do not have ID cards or health insurance take approximately 4–10 weeks to start treatment for positive detection (Q25).

This study explored the barriers and facilitators of EMTCT. The findings of this study reveal that both Indonesia and Uganda face significant challenges in EMTCT, including healthcare system limitations and the need for effective community engagement [ 23 ]. However, Indonesia's struggle with bureaucratic barriers and financial constraints is in stark contrast to Uganda, where community mobilization and healthcare worker training have become more central issues [ 23 ]. Moreover, Nepal's approach to EMTCT, focusing on small-scale, community-focused interventions, offers a different perspective, emphasizing the importance of localized strategies [ 24 ]. These comparisons highlight the need for tailored approaches to EMTCT that consider each country's unique healthcare infrastructure, cultural dynamics, and public health literacy levels.

The Ministry of Health Regulation No. 57 of 2017 and Integrated ANC Guidelines in Indonesia are important for EMTCTs for HIV, syphilis, and hepatitis B [ 1 , 9 ]. These regulations mandate early pregnancy screening as a crucial step toward managing infections in mothers, aligning with the WHO and MOH standards for universal testing in pregnant women [ 1 , 4 ]. The success of EMTCT programs hinges on comprehensive support through human resources, finance, infrastructure, and supplies, as well as free screening and treatment policies, as demonstrated by countries such as Cuba and Thailand [ 16 , 25 ]. However, challenges such as policy-induced testing barriers and the need for high screening coverage remain. At the community level, cross-sectoral involvement and community engagement facilitated the EMTCT. Community involvement can be achieved through mentorship programs or organizations created to promote treatment adherence [ 26 , 27 ]. This is also supported by research results showing that peer group intervention by community volunteers increases HIV prevention knowledge [ 28 ]. However, challenges at The WHO community level, especially those faced by migrant mothers, include a greater likelihood of not receiving adequate treatment [ 5 , 29 , 30 ]. The WHO's efforts to validate EMTCT in each country, considering the stigma and human rights of mothers, still face obstacles, as revealed by research indicating that stigma, not only from partners, families, and society but also from healthcare providers and gender-related factors remain barriers to accessing preventive mother-to-child transmission services [ 31 ].

At the healthcare system or institutional level, tracking and follow-up by healthcare professionals, especially through home visits by midwives, trust in healthcare professionals, and satisfaction with healthcare services and hospitals, contribute to the success of EMTCT. The preparedness and adequacy of resources, facilities, and healthcare professionals' knowledge are critical factors in achieving EMTCT [ 8 , 16 , 32 ]. Inadequate counseling, insufficient information from healthcare providers, long waiting times, and stigma from healthcare professionals inhibit elimination [ 33 ].

At the interpersonal level, support from partners and families is a strong source of support, especially for treatment. This finding aligns with research findings that mothers with hepatitis B have strong knowledge and understanding of the disease and its transmission from mother to child. Motivated by ensuring their children’s health, they receive support from their partners and parents [ 20 , 34 ]. Partner support in breaking the chain of transmission is crucial, especially for sexually transmitted infections [ 35 , 36 ]. Both parties are expected to undergo treatment to prevent horizontal transmission. Despite partner support during pregnancy and childbirth, some mothers reported that their partners were unwilling to undergo testing, contrary to research indicating that male partners' involvement in HIV testing and counseling is influenced by their presence in antenatal care spaces with their wives [ 36 ].

At the individual level, the motivation to have a healthy child, recovery from illness, knowledge of preventing disease transmission from mother to child, and self-acceptance are facilitator factors in the success of EMTCT for HIV, syphilis, and hepatitis B. This finding is consistent with studies suggesting that positive perceptions about the benefits of testing for oneself and the baby influence testing and treatment continuity during pregnancy [ 16 ]. Individual experiences and the desire to maintain the baby's health contribute to mothers' adherence to treatment during pregnancy and breastfeeding [ 20 ]. A lack of comprehensive knowledge is an inhibiting factor in achieving EMTCT [ 37 , 38 ]. Not having a national identity card complicates access to health insurance, leading to self-stigmatization and a lack of health insurance (costs), hindering access to EMTCT services. These challenges are consistent with various studies indicating that citizenship status, costs, stigma, and lack of information about sexual health are inhibiting factors in the screening and treatment of mothers living with any of the three infectious diseases [ 30 ].

The International Community of Women Living with HIV has submitted a report to the UN Working Group on Discrimination against Women, highlighting discrimination and abuse faced by women in healthcare [ 39 ]. Fear of mistreatment in maternal care deters women from seeking skilled care more significantly than factors such as cost or distance, especially in regions with high maternal mortality rates [ 39 ]. In this study, we found a different finding, where barriers to cost and distance to services related to bureaucratic issues were more vocally articulated by mothers than was fear of discrimination. Moreover, stigma, especially from health care providers, prevents mothers from accessing care. However, this study revealed that families and health workers provided support to mothers, and that mothers did not disclose social stigma. Hence, access to antenatal care in Bali Province shows that two out of three cities reached the coverage target (> 95%) [ 6 ]. Therefore, the problem landscape faced in EMTCT in Bali Province, Indonesia, is slightly different from the global problem, where bureaucracy is the main concern in accessing services compared to discrimination against women.

Implications for practice

The implications of these studies include several key points: improving mothers’ knowledge remains crucial, even when adherence rates are satisfactory. Therefore, better and more continuous education programs are needed to provide in-depth information on prevention and treatment, which can strengthen public understanding, establish a solid foundation for long-term adherence, and reduce the stigma and discrimination against mothers. In this study, sufficient knowledge and self-acceptance were identified as facilitators of progress toward EMTCT. Besides receiving information from healthcare professionals, mothers also sought information about their condition online using their mobile phones. To ensure that mothers living with HIV, syphilis, and hepatitis B receive accurate and reliable information, it is essential to develop digital health literacy programs. Additionally, the involvement of NGOs and cross-sectoral collaboration (e.g., village heads and social services) played a crucial role in supporting these efforts. There is a strong correlation between digital health literacy and efforts to eradicate EMTCT. Digital health literacy strengthens community engagement in EMTCT efforts, facilitates the search for services, and improves treatment adherence, access, and comprehension of health information [ 40 , 41 , 42 ]. Multidisciplinary collaboration between multidisciplinary healthcare professionals and NGOs is essential for addressing the gap in limited sources. Moreover, further studies are needed to develop integrated care with technology based on mothers’ values and preferences in Bali, as a natural setting.

Considering the significant bureaucratic barriers to treatment access identified in our study, it is imperative to devise a comprehensive action plan to overcome these obstacles. This plan should prioritize the simplification of administrative processes to enhance the accessibility of EMTCT services. Key actions could include advocating policy reforms to reduce unnecessary red tape and facilitating collaboration between various governmental and nongovernmental entities to ensure a streamlined healthcare delivery system. Drawing inspiration from successful models in other Southeast Asian regions, including Thailand and Malaysia [ 10 ], these reforms must be supported by strong political commitment and leadership to foster a more efficient and responsive healthcare framework, ultimately improving outcomes for mother-to-child transmission prevention efforts. Moreover, the Healthy Indonesian Card for National Health Insurance Premium Assistance Recipients should be strengthened as an alternative option for individuals struggling to afford health insurance to reduce financial constraints and improve access to health care services.

Further studies with a cultural focus are necessary to understand why adherence rates remain high despite the potentially low levels of knowledge. By revealing the cultural and contextual elements that may elude detection through knowledge parameters in isolation, this study can serve as a foundation for developing more comprehensive and culturally appropriate strategies to address the requirements of the local population.

Strengths and Limitations

Acknowledging the limitations in the accuracy of policies and healthcare practices from a patient's perspective is critical for ensuring patient-centered care. Patients often experience the impact of policies and healthcare decisions directly, and understanding their perspectives sheds light on the real-world effectiveness of these measures. The key limitation lies in the potential gap between policy intention and practical implementation. Patients’ perspectives also highlight the importance of their involvement in the decision-making process. Policies crafted without considering patient input may overlook crucial aspects of the patient experience, leading to potential inaccuracies in the relevance and effectiveness of the policy. Engaging patients in policymaking dialogue fosters a sense of ownership and ensures that policies align more closely with patients’ needs and preferences. However, acknowledging these limitations is crucial to maintaining transparency and managing patient expectations. Moreover, a patient's health literacy can influence the accuracy of healthcare information and the effectiveness of policies. Patients with varying levels of health literacy may interpret and apply healthcare information differently, affecting the outcomes of policy implementation. Financial insecurity is closely linked to lower health literacy, indicating that individuals struggling financially may benefit significantly from programs aimed at enhancing health literacy among people living with HIV [ 43 ]. Such programs can improve health outcomes by building confidence and resourcefulness, thus enabling individuals to better engage with health information. This approach not only educates, but also empowers, addressing both informational and financial barriers to effective health management [ 43 ].

Additionally, the study did not deeply explore the types, frequencies, or durations of support provided by NGOs and peer groups, thus limiting the understanding of effective support mechanisms. In addition, the findings are specific to the Indonesian context, and may not be generalizable to other regions with different cultural or health system dynamics. Therefore, future studies should conduct a detailed analysis of the different types of support provided by NGOs and peer groups to understand how variations affect the outcomes. Comparative studies across diverse geographical and cultural settings can enhance the generalizability of findings.

This study identified key facilitators and barriers to healthcare access for mothers living with HIV, syphilis, and hepatitis B, emphasizing the importance of policy-driven screening, cross-sectoral support for free health insurance, positive healthcare relationships, partner involvement, and patient willingness for a healthy baby. Challenges include inadequate information from healthcare providers, complex health insurance processes, and self-stigmatization. This study suggests collaborative care interventions and integrated digital solutions to overcome these barriers, aiming to eliminate the mother-to-child transmission of these diseases by 2030.

Availability of data and materials

The data presented in this study are available in the manuscript.

Kemenkes. Peraturan Menteri Kesehatan Republik Indonesia Nomor 52 Tahun 2017 Tentang Eliminasi Penularan Human Deficiency Virus, Sifilis Dan Hepatitis B Dari Ibu Ke Anak. Prog Phys Geogr. 2017;14(7):450. https://tel.archives-ouvertes.fr/tel-01514176

Bell L, van Gemert C, Allard N, et al. Progress towards triple elimination of mother-to-child transmission of HIV, hepatitis B and syphilis in Pacific Island Countries and Territories: a systematic review. Lancet Reg Heal - West Pacific. 2023;35: 100740. https://doi.org/10.1016/j.lanwpc.2023.100740 .

Article   Google Scholar  

Zhang L, Tao Y, Woodring J, et al. Integrated approach for triple elimination of mother-to-child transmission of HIV, hepatitis B and syphilis is highly effective and cost-effective: An economic evaluation. Int J Epidemiol. 2019;48(4):1327–39. https://doi.org/10.1093/ije/dyz037 .

Article   PubMed   Google Scholar  

WHO. Global Health Sector Strategies on, Respectively, HIV, Viral Hepatitis and Sexually Transmitted Infections for the Period 2022–2030. Vol 33.; 2022. https://www.who.int/teams/global-hiv-hepatitis-and-stis-programmes/strategies/global-health-sector-strategies

Liu H, Chen N, Tang W, et al. Factors influencing treatment status of syphilis among pregnant women: a retrospective cohort study in Guangzhou. China Int J Equity Health. 2023;22(1):1–11. https://doi.org/10.1186/s12939-023-01866-x .

Armini LN, Setiawati EP, Arisanti N. Evaluation of Process Indicators and Challenges of the Elimination of Mother-to-Child Transmission of HIV , Syphilis , and Hepatitis B in Bali Province , Indonesia ( 2019 – 2022 ): A Mixed Methods Study. Published online 2023:1–15.

Prabawa A, Sudarsana P, Hendra Setiawan K. Preventing mother-to-child transmission of human immunodeficiency virus (HIV), syphilis and hepatitis B: a secondary hospital approach in North Bali, Indonesia. Intisari Sains Medis | Intisari Sains Medis. 2023;14(2):641–644. https://doi.org/10.15562/ism.v14i2.1752

Lumbantoruan C, Kelaher M, Kermode M, Budihastuti E. Pregnant women’s retention and associated health facility characteristics in the prevention of mother-to-child HIV transmission in Indonesia: Cross-sectional study. BMJ Open. 2020;10(9):1–6. https://doi.org/10.1136/bmjopen-2019-034418 .

WHO. The Triple Elimination of Mother-to-Child Transmission of HIV, Hepatitis B and Syphilis in Asia and the Pacific, 2018–2030. Worls Heal Organ West Pacific Reg. Published online 2018:44. https://www.who.int/publications/i/item/9789290618553

Elgalib A, Lau R, Al-Habsi Z, et al. Elimination of mother-to-child transmission of HIV, syphilis and viral hepatitis B: A call for renewed global focus. Int J Infect Dis. 2023;127:33–5. https://doi.org/10.1016/j.ijid.2022.11.031 .

Kemenkes. HIV AIDS 2022.; 2022. https://p2p.kemkes.go.id/laporan-tahunan-hiv-aids-2022-versi-english/%0D

Pham TTH, Maria N, Cheng V, et al. Gaps in Prenatal Hepatitis B Screening and Management of HBsAg Positive Pregnant Persons in the U.S., 2015–2020. Am J Prev Med. 2023;65(1):52–59. https://doi.org/10.1016/j.amepre.2023.01.041

Kim H, Sefcik JS, Bradway C. Characteristics of Qualitative Descriptive Studies: A Systematic Review. Res Nurs Heal. 2017;40(1):23–42. https://doi.org/10.1002/nur.21768 .

Article   CAS   Google Scholar  

Cohn J, Owiredu MN, Taylor MM, et al. Eliminating mother-to-child transmission of human immunodeficiency virus, syphilis and hepatitis b in sub-saharan africa. Bull World Health Organ. 2021;99(4):287–95. https://doi.org/10.2471/BLT.20.272559 .

Article   PubMed   PubMed Central   Google Scholar  

Malterud K, Siersma VD, Guassora AD. Sample Size in Qualitative Interview Studies: Guided by Information Power. Qual Health Res. 2016;26(13):1753–60. https://doi.org/10.1177/1049732315617444 .

Zhang Y, Guy R, Camara H, et al. Barriers and facilitators to HIV and syphilis rapid diagnostic testing in antenatal care settings in low-income and middle-income countries: A systematic review. BMJ Glob Heal. 2022;7(11). https://doi.org/10.1136/bmjgh-2022-009408

Elo S, Kyngäs H. The qualitative content analysis process. J Adv Nurs. 2008;62(1):107–15. https://doi.org/10.1111/j.1365-2648.2007.04569.x .

Hsieh HF, Shannon SE. Three approaches to qualitative content analysis. Qual Health Res. 2005;15(9):1277–88. https://doi.org/10.1177/1049732305276687 .

Mcleroy KR, Bibeau D, Steckler A, Glanz K. An Ecological Perspective on Health Promotion Programs. Heal Educ Behav. 1988;15(4):351–77. https://doi.org/10.1177/109019818801500401 .

Humphrey J, Alera M, Kipchumba B, et al. A qualitative study of the barriers and enhancers to retention in care for pregnant and postpartum women living with HIV. Khan MS, ed. PLOS Glob Public Heal. 2021;1(10):e0000004. https://doi.org/10.1371/journal.pgph.0000004

Karugaba G, Simpson J, Mathuba B, et al. The barriers and facilitators of HIV-exposed infant testing as perceived by HIV-positive mothers in Botswana: A qualitative study. PLoS One. 2022;17(8 August). https://doi.org/10.1371/journal.pone.0273777

Lincoln YS, Guba EG. But Is It Rigorous? Authenticity in Trustworthiness and Naturalistic Evaluation. Willey online Libr. 1986;30:73–84. https://doi.org/10.1002/ev.1427 .

Alege JB, Oyore JP, Nanyonga RC. Barriers and facilitators of integrated hepatitis B, C, and HIV screening among pregnant mothers and their newborns attending maternal and newborn clinics in Koboko District. Uganda: A qualitative inquiry of providers ’ perspective. Published online; 2023. p. 1–19.

Google Scholar  

Sabin L, Saville N, Dixit Devkota M, Haghparast-Bidgoli H. Factors affecting antenatal screening for HIV in Nepal: results from Nepal Demographic and Health Surveys 2016 and 2022. BMJ Open. 2023;13(12): e076733. https://doi.org/10.1136/bmjopen-2023-076733 .

Mwanza J, Kawonga M, Kumwenda A, Gray GE, Mutale W, Doherty T. Health system response to preventing mother-to-child transmission of HIV policy changes in Zambia: a health system dynamics analysis of primary health care facilities. Glob Health Action. 2022;15(1). https://doi.org/10.1080/16549716.2022.2126269

Hill LM, Saidi F, Freeborn K, et al. Tonse Pamodzi: Developing a combination strategy to support adherence to antiretroviral therapy and HIV pre-exposure prophylaxis during pregnancy and breastfeeding. PLoS One. 2021;16(6 June). https://doi.org/10.1371/journal.pone.0253280

Igumbor JO, Ouma J, Otwombe K, et al. Effect of a Mentor Mother Programme on retention of mother-baby pairs in HIV care: A secondary analysis of programme data in Uganda. PLoS ONE. 2019;14(10):1–12. https://doi.org/10.1371/journal.pone.0223332 .

Kumbani LC, Jere DL, Banda CK, et al. A peer group intervention implemented by community volunteers increased HIV prevention knowledge. BMC Public Health. 2023;23(1):1–15. https://doi.org/10.1186/s12889-022-14715-3 .

Bisnauth MA, Coovadia A, Kawonga M, Vearey J. Providing HIV Prevention of Mother to Child Transmission (PMTCT) Services to Migrants During the COVID-19 Pandemic in South Africa: Insights of Healthcare Providers. Heal Serv Insights. 2022;15. https://doi.org/10.1177/11786329211073386

Chan EYL, Smullin C, Clavijo S, et al. A qualitative assessment of structural barriers to prenatal care and congenital syphilis prevention in Kern County, California. PLoS One. 2021;16(4 April):1–12. https://doi.org/10.1371/journal.pone.0249419

Najmah, Andajani S, Davies SG. Perceptions of and barriers to HIV testing of women in Indonesia. Sex Reprod Heal Matters. 2020;28(2). https://doi.org/10.1080/26410397.2020.1848003

Baker C, Limato R, Tumbelaka P, et al. Antenatal testing for anaemia, HIV and syphilis in Indonesia - A health systems analysis of low coverage. BMC Pregnancy Childbirth. 2020;20(1):1–11. https://doi.org/10.1186/s12884-020-02993-x .

Teshome GS, Modiba LM. Strategies to eliminate mother-to-child transmission of hiv in Addis Ababa, Ethiopia (Qualitative study). HIV/AIDS - Res Palliat Care. 2020;12:821–37. https://doi.org/10.2147/HIV.S277461 .

Ahad M, Wallace J, Xiao Y, et al. Hepatitis B and pregnancy: understanding the experiences of care among pregnant women and recent mothers in metropolitan Melbourne. BMC Public Health. 2022;22(1):1–8. https://doi.org/10.1186/s12889-022-13112-0 .

Jones DL, Rodriguez VJ, Soni Parrish M, et al. Maternal and infant antiretroviral therapy adherence among women living with HIV in rural South Africa: a cluster randomised trial of the role of male partner participation on adherence and PMTCT uptake. Sahara J. 2021;18(1):17–25. https://doi.org/10.1080/17290376.2020.1863854 .

Gessesse NA, Gela GB, Aweke AM, Balcha WF. Male partners involvement in human immune deficiency virus testing and counseling during prenatal care visits in Bichena town Westcentral Ethiopia: a cross-sectional study. BMC Res Notes. 2022;15(1):1–7. https://doi.org/10.1186/s13104-022-06215-9 .

Chotta NAS, Msuya SE, Mgongo M, Hashim TH, Stray-Pedersen A. Mother’s Knowledge on HIV, Syphilis, Rubella, and Associated Factors in Northern Tanzania: Implications for MTCT Elimination Strategies. Int J Pediatr (United Kingdom). 2020;2020. https://doi.org/10.1155/2020/7546954

Yeshaneh A, Abebe H, Tafese FE, Workineh A. Knowledge, attitude, and practice towards prevention of mother-to-child transmission of HIV among antenatal care attendees in Ethiopia, 2020. PLoS One. 2023;18(2 February):1–15. https://doi.org/10.1371/journal.pone.0277178

ICW. International Community of Women Living with HIV Submission to the UN Working Group on the Issue of Discrimination against Women in Law and in Practice. Published online 2013:1–7. https://www.ohchr.org/Documents/Issues/Women/WG/righthealth/WS/ICW_HIV.pdf

Odeny TA, Hughes JP, Bukusi EA, et al. Text messaging for maternal and infant retention in prevention of mother-to-child HIV transmission services: A pragmatic stepped-wedge cluster-randomized trial in Kenya. PLoS Med. 2019;16(10): e1002924. https://doi.org/10.1371/journal.pmed.1002924 .

Finocchario-Kessler S, Brown M, Maloba M, et al. A Pilot Study to Evaluate the Impact of the HIV Infant Tracking System (HITSystem 2.0) on Priority Prevention of Mother-to-Child Transmission (PMTCT) Outcomes. AIDS Behav. 2021;25(8):2419–2429. https://doi.org/10.1007/s10461-021-03204-0

Dryden-Peterson S, Bennett K, Hughes MD, et al. An augmented SMS intervention to improve access to antenatal CD4 testing and ART initiation in HIV-infected pregnant women: a cluster randomized trial. PLoS ONE. 2015;10(2): e0117181. https://doi.org/10.1371/journal.pone.0117181 .

Article   CAS   PubMed   PubMed Central   Google Scholar  

Power J, Lea T, Melendez-Torres GJ, et al. Health literacy, financial insecurity and health outcomes among people living with HIV in Australia. Health Promot Int. 2022;37(6):daac161. https://doi.org/10.1093/heapro/daac161

Download references

Acknowledgements

We would like to express our heartfelt gratitude to Dinas Kesehatan (Health Office) in Denpasar, Buleleng, and Badung for their invaluable support and cooperation throughout this study. We also sincerely appreciate the dedicated staff and healthcare professionals at the public health centers in the three districts.

Open access funding provided by University of Padjadjaran This research and APC were funded and supported by the Center for Higher Education Funding (BPPT), Indonesia Endowment Fund for Education (LPDP), and the Directorate of Research and Community Engagement, Universitas Padjadjaran, Bandung, Indonesia.

Author information

Authors and affiliations.

Doctoral Study Program, Faculty of Medicine, Universitas Padjadjaran, Sumedang, 45363, Indonesia

Luh Nik Armini

Midwifery Science Program, Faculty of Medicine, Universitas Pendidikan Ganesha, Bali, 81116, Indonesia

Department of Public Health, Faculty of Medicine, Universitas Padjadjaran, Sumedang, West Java, 45363, Indonesia

Elsa Pudji Setiawati & Nita Arisanti

Department of Child Health, Faculty of Medicine, Universitas Padjadjaran, Sumedang, 45363, Indonesia

Dany Hilmanto

You can also search for this author in PubMed   Google Scholar

Contributions

Conceptualization, L.N.A., E.P.S., N.A., and D.H.; methodology, L.N.A., E.P.S., N.A., and D.H.; software, L.N.A.; validation, E.P.S., N.A., and D.H.; formal analysis, L.N.A.; investigation, L.N.A.; resources, L.N.A.; data curation, E.P.S., N.A., and D.H.; writing—original draft preparation, L.N.A.; writing—review and editing, L.N.A.; visualization, L.N.A.; supervision, L.N.A., E.P.S., N.A., and D.H.; project administration, L.N.A.; funding acquisition, E.P.S., N.A., and D.H. All authors read and agreed to the published version of the manuscript.

Corresponding author

Correspondence to Elsa Pudji Setiawati .

Ethics declarations

Ethics approval and consent to participate.

Research ethics approval was granted by the Universitas Padjadjaran (number 798/UN6). KEP/EC/2022. Written consent was obtained from the mothers living with HIV, syphilis, and/or hepatitis B who participated in the interviews. All participants were informed that the interview results had been published. Nevertheless, all data related to personal information or any information that could put participants at risk were protected, and data confidentiality was maintained. The interviews were conducted in a dedicated room within the voluntary counseling and testing (VCT) area of the hospital and public health center to ensure privacy and comfort of the participants.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Additional information

Publisher's note.

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Supplementary material 1., rights and permissions.

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ . The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/ ) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Cite this article.

Armini, L.N., Setiawati, E.P., Arisanti, N. et al. Patient perspective on the elimination mother-to-child transmission of HIV, syphilis, and hepatitis B in Bali, Indonesia: a qualitative study. BMC Public Health 24 , 2258 (2024). https://doi.org/10.1186/s12889-024-19692-3

Download citation

Received : 12 April 2024

Accepted : 05 August 2024

Published : 20 August 2024

DOI : https://doi.org/10.1186/s12889-024-19692-3

Share this article

Anyone you share the following link with will be able to read this content:

Sorry, a shareable link is not currently available for this article.

Provided by the Springer Nature SharedIt content-sharing initiative

• Hepatitis B
• Facilitators

BMC Public Health

ISSN: 1471-2458

• General enquiries: [email protected]

An official website of the United States government

The .gov means it’s official. Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

• Publications
• Account settings
• My Bibliography
• Collections
• Citation manager

Save citation to file

Email citation, add to collections.

• Create a new collection
• Add to an existing collection

Add to My Bibliography

Your saved search, create a file for external citation management software, your rss feed.

• Search in PubMed
• Search in NLM Catalog
• Add to Search

Hepatitis B virus infection and the risk of gynecologic cancers: a systematic review and meta-analysis

Affiliations.

• 1 Department of Radiation Oncology, The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Yunnan Cancer Center), 519 Kunzhou Road, Kunming, 650118, People's Republic of China.
• 2 Department of Medical Administration, The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Yunnan Cancer Center), 519 Kunzhou Road, Kunming, 650118, China.
• 3 Department of Gynecologic Oncology, The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Yunnan Cancer Center), 519 Kunzhou Road, Kunming, 650118, China.
• 4 Department of Radiation Oncology, The Third Affiliated Hospital of Kunming Medical University (Yunnan Cancer Hospital, Yunnan Cancer Center), 519 Kunzhou Road, Kunming, 650118, People's Republic of China. [email protected].
• PMID: 39120631
• PMCID: PMC11315852
• DOI: 10.1007/s12672-024-01213-8

Objectives: The relationship between hepatitis B virus (HBV) infection and gynecologic cancers is controversial. We aimed to evaluate the risk of gynecologic cancers associated with HBV infection using a meta-analysis.

Methods: Two independent reviewers identified publications in the PubMed, Embase and Cochrane Library databases that reported an association between HBV and the risk of gynecologic malignancy from inception to December 31, 2022. The Newcastle-Ottawa Scale (NOS) was used to evaluate the quality of the included articles. Pooled odds ratios (ORs) and 95% corresponding confidence intervals (CIs) were calculated using a fixed effects model or random effects model.

Results: We collected data from 7 studies that met the inclusion criteria, including 2 cohort studies and 5 case-control studies. HBV was significantly associated with the risk of cervical cancer in the general population (OR 1.22, 95% CI 1.09-1.38, P = 0.001), although the same trend was not found in endometrial cancer (OR 1.30, 95% CI 0.95-1.77, P = 0.105) and ovarian cancer (OR 1.03, 95% CI 0.79-1.35, P = 0.813). Subgroup analysis showed that HBV infection was positively associated with the risk of cervical cancer (OR 1.27, 95% CI 1.13-1.44, P = 0.000) in case-control studies. Asian women infected with HBV have a significantly increased risk of cervical cancer (OR 1.24, 95% CI 1.10-1.40, P = 0.001) and endometrial cancer (OR 1.46, 95% CI 1.07-1.99, P = 0.018). Hospital-based studies were found to be associated with an increased risk of cervical cancer (OR 1.30, 95% CI 1.14-1.47, P = 0.000) and endometrial cancer (OR 1.61, 95% CI 1.04-2.49, P = 0.032). The results of Begg's and Egger's tests showed no publication bias.

Conclusions: This meta-analysis shows a positive association between HBV infection and cervical cancer. HBV is positively correlated with the risk of cervical cancer and endometrial cancer in Asian women and hospital-based populations. More multicenter prospective studies are required to confirm the findings.

Keywords: Gynecologic cancers; Hepatitis B virus (HBV); Meta-analysis; Systematic review.

© 2024. The Author(s).

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interests.

Flow diagram of the literature…

Flow diagram of the literature search and selection process for the meta-analysis

Association between HBV and cervical…

Association between HBV and cervical cancer. Forest plot showing A the relationship between…

Association between HBV and endometrial…

Association between HBV and endometrial cancer. Forest plot showing A the relationship between…

Association between HBV and ovarian…

Association between HBV and ovarian cancer. Forest plot showing A the relationship between…

Publication bias for studies included.…

Publication bias for studies included. Begg’s funnel plots and Egger’s publication bias plots…

Similar articles

• Association between hepatitis B virus/hepatitis C virus infection and primary hepatocellular carcinoma risk: A meta-analysis based on Chinese population. Li L, Lan X. Li L, et al. J Cancer Res Ther. 2016 Dec;12(Supplement):C284-C287. doi: 10.4103/0973-1482.200763. J Cancer Res Ther. 2016. PMID: 28230038
• Folic acid supplementation and malaria susceptibility and severity among people taking antifolate antimalarial drugs in endemic areas. Crider K, Williams J, Qi YP, Gutman J, Yeung L, Mai C, Finkelstain J, Mehta S, Pons-Duran C, Menéndez C, Moraleda C, Rogers L, Daniels K, Green P. Crider K, et al. Cochrane Database Syst Rev. 2022 Feb 1;2(2022):CD014217. doi: 10.1002/14651858.CD014217. Cochrane Database Syst Rev. 2022. PMID: 36321557 Free PMC article.
• Hepatitis B virus infection increases the risk of pancreatic cancer: a meta-analysis. Liu X, Zhang ZH, Jiang F. Liu X, et al. Scand J Gastroenterol. 2021 Mar;56(3):252-258. doi: 10.1080/00365521.2020.1868568. Epub 2021 Jan 5. Scand J Gastroenterol. 2021. PMID: 33399501
• Hepatitis B or C viral infection and risk of pancreatic cancer: a meta-analysis of observational studies. Xu JH, Fu JJ, Wang XL, Zhu JY, Ye XH, Chen SD. Xu JH, et al. World J Gastroenterol. 2013 Jul 14;19(26):4234-41. doi: 10.3748/wjg.v19.i26.4234. World J Gastroenterol. 2013. PMID: 23864789 Free PMC article. Review.
• Hepatitis B and C virus infections and the risk of biliary tract cancers: a meta-analysis of observational studies. Wang Y, Yuan Y, Gu D. Wang Y, et al. Infect Agent Cancer. 2022 Aug 27;17(1):45. doi: 10.1186/s13027-022-00457-9. Infect Agent Cancer. 2022. PMID: 36030232 Free PMC article. Review.
• Torre LA, Islami F, Siegel RL, Ward EM, Jemal A. Global cancer in women: burden and trends. Cancer Epidemiol Biomark Prev. 2017;26(4):444–57.10.1158/1055-9965.EPI-16-0858 - DOI - PubMed
• Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. Global Cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. Cancer J Clin. 2021;71(3):209–49.10.3322/caac.21660 - DOI - PubMed
• Ferrari F, Giannini A. Approaches to prevention of gynecological malignancies. BMC Womens Health. 2024;24(1):254. 10.1186/s12905-024-03100-4 - DOI - PMC - PubMed
• Hassan MM, Hwang LY, Hatten CJ, Swaim M, Li D, Abbruzzese JL, Beasley P, Patt YZ. Risk factors for hepatocellular carcinoma: synergism of alcohol with viral hepatitis and diabetes mellitus. Hepatology. 2002;36(5):1206–13. 10.1053/jhep.2002.36780 - DOI - PubMed
• Engels EA, Cho ER, Jee SH. Hepatitis B virus infection and risk of non-hodgkin lymphoma in South Korea: a cohort study. Lancet Oncol. 2010;11(9):827–34. 10.1016/S1470-2045(10)70167-4 - DOI - PMC - PubMed

Related information

Grants and funding.

• 202305AS350020/Innovative Research Team of Yunnan Province
• 82160562/National Natural Science Foundation of China

LinkOut - more resources

Full text sources.

• PubMed Central

Miscellaneous

• NCI CPTAC Assay Portal
• Citation Manager

NCBI Literature Resources

MeSH PMC Bookshelf Disclaimer

The PubMed wordmark and PubMed logo are registered trademarks of the U.S. Department of Health and Human Services (HHS). Unauthorized use of these marks is strictly prohibited.