pre-S Region Research Articles

Hepatitis B and D virus entry.

Hepatitis B virus (HBV) entry is the initial step of viral infection, leading to the formation of covalently closed circular DNA, which is a molecular reservoir of viral persistence and a key obstacle for HBV cure. The restricted entry of HBV into specific cell types determines the nature of HBV, which has a narrow host range in tissues and species. Hepatitis D virus (HDV) shares viral surface antigens with HBV and thus follows a similar entry mechanism at its early stages. In late 2012, sodium taurocholate cotransporting polypeptide was discovered as an HBV and HDV entry receptor. Since then, the mechanisms of HBV and HDV entry have been extensively analysed. These analyses have expanded our understanding of HBV and HDV host tropism and have provided new strategies for the development of antiviral agents. Notably, the structures of sodium taurocholate cotransporting polypeptide and its interaction with the 2-48 amino acid region of viral preS1 have been recently solved. These findings will stimulate further entry studies. In this Review, we summarize current understanding of HBV and HDV entry and future perspectives.

Ligustilide improves functional constipation by non-covalently activating TRPA1 in colon tissue

Ethnopharmacological relevanceAngelica sinensis (Oliv.) Diels (AS), a medicinal plant renowned for its constipation-relieving properties, lacks comprehensive studies on its active pharmaceutical ingredients (APIs) and underlying mechanisms. In the gastrointestinal tract, TRP channels enhance colonic mucus secretion, expedite intestinal motility, and regulate gastrointestinal hormones; however, few reports have systematically established the relationship between TRPs and ligustilide (Lig), a key API of AS. Aim of the studyThis study aimed to explore the pharmacodynamic properties of AS in alleviating functional constipation, assess the potential of Lig for activating TRPs, and elucidate its mechanism of action. MethodsThe therapeutic efficacy of AS was assessed in a mouse model of loperamide hydrochloride-induced functional constipation. The APIs were screened via integrated activity-based UPLC profiling through periodic acid-Schiff (PAS) staining of the colon and immunofluorescence staining of HT-29 cells. The potential target was identified via target fishing and colocalization imaging via an alkynyl-modified Lig probe (AM-Lig). Molecular docking, microscale thermophoresis (MST), fluorescence quenching (FQ), and Fluo-4/Ca2+ influx assays were employed to reveal the interaction mode between Lig and the target protein. Finally, we assessed the efficacy of Lig in alleviating constipation in an animal model. ResultsThe efficacy of AS in improving functional constipation was demonstrated in a mouse constipation model, with Lig identified as the primary constituent responsible for inducing colon mucus secretion. Lig specifically targets TRPA1 in the colon, leading to calcium influx and subsequent mucus secretion, ultimately ameliorating functional constipation. Furthermore, a binding mode study revealed that Lig attaches to Thr684, located in the pre-S1 region, triggering TRPA1 channel activation. ConclusionsOur findings demonstrate that Lig, the API in AS for constipation treatment, activates TRPA1 through non-covalent interactions, increasing mucus secretion and improving functional constipation. These findings advance our understanding of the therapeutic mechanism of AS and Lig on constipation and suggest a new approach for developing TRPA1 agonists.

Impact of preS1 Evaluation in the Management of Chronic Hepatitis B Virus Infection.

Background and Objectives: The measurement of hepatitis B surface antigen (HBsAg) is essential for managing chronic hepatitis B virus infection (CHB). HBsAg consists of three different surface envelope proteins: large, middle, and small HB surface proteins. However, in clinical practice, it is not common to evaluate each of these HB surface proteins separately. Materials and Methods: In this study, we investigated preS1 expression using seven monoclonal antibodies (mAbs) in 68 CHB patients, as well as examining their antigenicity. Results: Although the seven mAbs had been derived from genotype (Gt) C, they could recognize preS1 with Gts A to D. The epitopes were concentrated within the aa33-47 region of preS1, and their antigenicity was significantly reduced by an aa45F substitution. We found that preS1 expression remained consistent regardless of HBsAg levels and different Gts in CHB patients, in contrast to what was observed in SHBs. Conclusions: These results suggest that the antigenic epitope is preserved among different Gts and that the expression pattern of preS1 is altered during CHB, highlighting its vital role in the HBV infection cycle. Our present results suggest preS1 is a promising therapeutic target in CHB.

The Hepatitis B Virus PreS1/HBsAg Ratio Is a Predictive Marker for the Occurrence of Hepatocellular Carcinoma

The preS1 region of the large hepatitis B virus (HBV) surface protein is a crucial component in HBV infection; however, its impact on the development of hepatocellular carcinoma (HCC) remains unknown. This study investigated the relationship between serum preS1 levels and hepatocarcinogenesis in patients with chronic hepatitis B (CHB). The preS1 levels were measured in 531 patients with CHB without a history of HCC. Among the patients, 293 HBV carriers who had never received nucleotide/nucleoside analog (NA) therapy had their preS1 levels measured at their first visit (non-NA group), and 238 patients who had received NA therapy had their preS1 levels measured at the start of NA administration (NA group). The two groups had no significant differences in hepatitis B surface antigen (HBsAg) levels; however, the NA group’s preS1/HBsAg ratio was significantly higher. The preS1/HBsAg ratio was significantly different between patients with CHB not meeting the NA treatment criteria and patients with chronic hepatitis and cirrhosis who were eligible for NA treatment. The predictors of HCC development were analyzed, and the preS1/HBsAg ratio was identified in both groups. The preS1/HBsAg ratio could predict hepatocarcinogenesis in patients with CHB with or without NA administration.

Establishment of a hepatitis B virus reporter system harboring a HiBiT-tag in the PreS2 region.

Approximately 296 million people suffer from chronic hepatitis B (CHB) caused by hepatitis B virus (HBV). Current standard treatment, nucleos(t)ide analogs, are not efficient enough to eradicate HBV from the hepatocytes. Thus, developing new drugs for CHB is desired to achieve complete cure. Here we established a novel HBV reporter system, HBV-HiBiT-PS2, to screen new drugs for CHB. HBV-HiBiT-PS2 was constructed by introducing a HiBiT-tag at the 5'-end of PreS2 and introduced into HepG2-NTCP cells. Culture supernatant containing HBV-HiBiT-PS2 virions was fractionated by a sucrose density gradient ultracentrifugation to characterize their components. Replication kinetics and reporter function of HBV-HiBiT-PS2 were determined by analyzing the parameters for HBV replication in the presence or absence of HBV inhibitors. HBV-HiBiT-PS2 could be used for monitoring most of the replication cycle of HBV. The effects of well-characterized HBV inhibitors could be evaluated by the HiBiT activity. HBV-HiBiT-PS2 could be specialized for screening secretion inhibitors for hepatitis B surface antigen (HBsAg) because most of the HiBiT activity was derived from subviral particles which are the multimers of HBsAg. We demonstrated that HBV-HiBiT-PS2 would be a robust tool for screening novel drugs, especially HBsAg secretion inhibitors, targeted for CHB.

Ligustilide covalently binds to Cys703 in the pre-S1 helix of TRPA1, blocking the opening of channel and relieving pain in rats with acute soft tissue injury

Ethnopharmacological relevanceThe natural anodyne Ligustilide (Lig), derived from Angelica sinensis (Oliv.) Diels and Ligusticum chuanxiong Hort., has been traditionally employed for its analgesic properties in the treatment of dysmenorrhea and migraine, and rheumatoid arthritis pain. Despite the existing reports on the correlation between TRP channels and the analgesic effects of Lig, a comprehensive understanding of their underlying mechanisms of action remains elusive. Aim of the studyThe objective of this study is to elucidate the mechanism of action of Lig on the analgesic target TRPA1 channel. MethodsThe therapeutic effect of Lig was evaluated in a rat acute soft tissue injury model. The analgesic target was identified through competitive inhibition of TRP channel agonists at the animal level, followed by Fluo-4/Ca2+ imaging on live cells overexpressing TRP proteins. The potential target was verified through in-gel imaging, colocalization using a Lig-derived molecular probe, and a drug affinity response target stability assay. The binding site of Lig was identified through protein spectrometry and further analyzed using molecular docking, site-specific mutation, and multidisciplinary approaches. ResultsThe administration of Lig effectively ameliorated pain and attenuated oxidative stress and inflammatory responses in rats with soft tissue injuries. Moreover, the analgesic effects of Lig were specifically attributed to TRPA1. Mechanistic studies have revealed that Lig directly activates TRPA1 by interacting with the linker domain in the pre-S1 region of TRPA1. Through metabolic transformation, 6,7-epoxyligustilide (EM-Lig) forms a covalent bond with Cys703 of TRPA1 at high concentrations and prolonged exposure time. This irreversible binding prevents endogenous electrophilic products from entering the cysteine active center of ligand-binding pocket of TRPA1, thereby inhibiting Ca2+ influx through the channel opening and ultimately relieving pain. ConclusionsLig selectively modulates the TRPA1 channel in a bimodal manner via non-electrophilic/electrophilic metabolic conversion. The epoxidized metabolic intermediate EM-Lig exerts analgesic effects by irreversibly inhibiting the activation of TRPA1 on sensory neurons. These findings not only highlight the analgesic mechanism of Lig but also offer a novel nucleophilic attack site for the development of TRPA1 antagonists in the pre-S1 region.

PreS1- targeting chimeric antigen receptor T cells diminish HBV infection in liver humanized FRG mice

Current therapies control but rarely achieve a cure for hepatitis B virus (HBV) infection. Restoration of the HBV-specific immunity by cell-based therapy represents a potential approach for a cure. In this study, we generated HBV specific CAR T cells based on an antibody 2H5-A14 targeting a preS1 region of the HBV large envelope protein. We show that the A14 CAR T cell is capable of killing hepatocytes infected by HBV with high specificity; adoptive transfer of A14 CAR T cells to HBV infected humanized FRG mice resulted in reductions of all serum and intrahepatic virological markers to levels below the detection limit. A14 CAR T cells treatment increased the levels of human IFN-γ, GM-CSF, and IL-8/CXCL-8 in the mice. These results show that A14 CAR T cells may be further developed for curative therapy against HBV infection by eliminating HBV-infected hepatocytes and inducing production of pro-inflammatory and antiviral cytokines.

Pre-S1 Mutations as Indicated by Serum Pre-S1 Antigen Negative is Associated with an Increased Risk of Hepatocellular Carcinoma in Chronic Hepatitis B Patients.

Pre-S1 antigen (pre-S1) is a component of hepatitis B virus large surface antigen (L-HBsAg). This study aimed to investigate the association between clinical pre-S1 antigen (pre-S1) status and adverse prognostic events in chronic hepatitis B (CHB) patients. This study retrospectively enrolled 840 CHB patients with comprehensive clinical data, including 144 patients with multiple follow-up of pre-S1 status. All patients were tested for serum pre-S1 and divided into pre-S1 positive and negative groups. Single factor and logistic multiple regression analyses were performed to explore the association between pre-S1 and other HBV biomarkers with the risk of hepatocellular carcinoma (HCC) in CHB patients. The pre-S1 region sequences of HBV DNA were obtained from one pre-S1 positive and two pre-S1 negative treatment-naïve patients using polymerase chain reaction (PCR) amplification followed by Sanger sequencing. The quantitative HBsAg level was significantly higher in the pre-S1 positive group than that in the pre-S1 negative group (Z=-15.983, P<0.001). The positive rate of pre-S1 increased significantly with the increase in HBsAg level (χ 2=317.963, P<0.001) and HBV DNA load (χ 2=15.745, P<0.001). The pre-S1 negative group had a higher HCC risk than the pre-S1 positive group (Z=-2.00, P=0.045, OR=1.61). Moreover, patients in the sustained pre-S1 negative group had a higher HCC risk (Z=-2.56, P=0.011, OR=7.12) than those in the sustained pre-S1 positive group. The sequencing results revealed mutations in the pre-S1 region from samples of pre-S1 negative patients, including frameshift and deletion mutations. Pre-S1 is a biomarker that indicates the presence and replication of HBV. Pre-S1 sustained negativity attributed to pre-S1 mutations in CHB patients may be associated with a higher risk of HCC, which has clinical significance and warrant further investigations.

Neutralization of hepatitis B virus with vaccine-escape mutations by novel hepatitis B vaccine with large-HBs antigen

Although the current hepatitis B (HB) vaccine comprising yeast-derived small hepatitis B surface antigen (HBsAg) is potent and safe and used worldwide, specific concerns should not be ignored, such as the attenuated prophylaxis against hepatitis B virus (HBV) infection with specific amino acid polymorphisms, called vaccine-escape mutations (VEMs). We investigated a novel HB vaccine consisting of large-HBsAg that covers the shortcomings of the current HB vaccine in a nonhuman primate model. The yeast-derived large-HBsAg was mixed with the adjuvant and used to immunize rhesus macaques, and the induction of antibodies to HBsAg was compared with that of the current HB vaccine. The current HB vaccine predominantly induced antibodies to small-HBsAg, whereas immunization with the large-HBsAg vaccine mainly induced antibodies to the preS1 region. Although the antibodies induced by the current HB vaccine could not prevent infection of HBV with VEMs, the large-HBsAg vaccine-induced antibodies neutralized infection of HBV with VEMs at levels similar to those of the wild type. The HBV genotypes that exhibited attenuated neutralization by induced antibodies differed between these vaccines. In conclusion, the novel HB vaccine consisting of large-HBsAg was revealed to be useful to compensate for shortcomings of the current HB vaccine. The combined use of these HB vaccines may be able to induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

Circulating immune complexes and mutations of HBsAg are associated with the undetectable HBsAg in anti-HBs and HBeAg positive occult hepatitis B virus infection.

Occult hepatitis B virus infection (OBI) is an HBsAg negative state in HBV infection with usually inactive HBV replication. However, there were a minority of individuals with positive HBeAg and anti-HBs among OBI blood donors and few studies have focused on this unusual serological pattern. 2022 plasma of blood donors that preliminary screened reactive for HBV DNA and non-reactive for HBsAg were collected from 16 provinces in China from 2015 to 2018. HBV DNA and HBsAg in these samples were retested using the Cobas TaqScreen MPX test and ARCHITECT HBsAg Quantitative II assay. Lumipulse HBsAg-HQ assay and polyethylene glycol (PEG)-double precipitation following HCl and trypsin digestion were performed to detect HBsAg from HBsAg-anti-HBs circulating immune complexes (CICs). 1487 of 2022 samples were positive for Cobas HBV DNA test and non-reactive for ARCHITECT HBsAg assay, while 404 of them were positive using Lumipulse HBsAg-HQ assay. 10 HBsAg-/anti-HBs+/HBeAg+ OBI blood donor samples were further dissociated and HBsAg-CICs were detected in 7 samples. Sequencing analysis showed that D44N, N98T, G73S, Del 56-116, and I161T occurred in the pre-S region, and immune escape mutations such as P127T, F134L, G145R, V168A, and I126T/S in the S region were found. In conclusion, there were a minority of HBsAg-/anti-HBs+/HBeAg+ individuals in OBI blood donors. The undetectable HBsAg in these individuals was mainly due to HBsAg-CICs. Immune escape-associated mutations also happened under the host's selective pressure. HBsAg dissociation methods or Lumipulse HBsAg-HQ assay is recommended to distinguish these individuals.

Identification of neutralizing epitopes in the preS2 domain of the hepatitis B virus

Hepatitis B virus (HBV) infection is a major public health problem. The sodium taurocholate cotransporting polypeptide (NTCP) has been identified as an essential HBV receptor. Human hepatocytes are infected with HBV via binding between the preS1 region of the HBV large envelope protein and the NTCP. However, the role of preS2 in HBV entry is not well understood.In this study, we induced anti-preS2 serum in mice by DNA immunization, and showed that the resulting antiserum neutralized HBV infectivity. Competition assays using overlapping peptides suggested that the neutralizing epitope is located in the N-terminal region of preS2. In addition, monoclonal antibodies targeting the N-terminal region of preS2 neutralized HBV infectivity, indicating that these domains are critical epitopes for viral neutralization. These findings provide new insights into the HBV entry machinery while suggesting a novel modality for the prevention and treatment of HBV infection.

Naturally occurring pre-S mutations promote occult HBV infection by affecting pre-S2/S promoter activity

Occult hepatitis B virus (HBV) infection (OBI) has non-negligible clinical significance, but the mechanism of its occurrence remains unclear. Growing evidence suggests that mutations in the pre-S region of HBV genome may be associated with the occurrence of OBI. However, the role of pre-S mutations in OBI and its molecular mechanism was not fully understand. Here, the pre-S sequences from 307 OBI blood donors and 293 hepatitis B surface protein (HBsAg)-positive blood donors were obtained, and we observed a higher frequency of naturally occurring pre-S mutations in OBI donors infected with genotype B/C HBV than in HBsAg-positive donors, suggesting their potential positive role in OBI. In both genotype B and C, several pre-S mutants resulted in markedly reduced HBsAg production in vitro. In particular, the T68I, S78N and N98T mutants of genotype B were proven to significantly decrease the HBsAg synthesis by affecting the pre-S2/S promoter activity, and thereby promoting the occurrence of OBI.

Hepatitis B virus PreS2 mutants in liver cancer patients

Liver cancer, or Hepatocellular Carcinoma (HCC), is one of the most common cancers in the world. Chronic infection with the hepatitis B virus (HBV) is one of the most critical factors that cause it. During chronic infection with HBV, variants of the virus are created. They may have deletion mutations in the PreS2 region. These variants may play a role in the occurrence of HCC. This study aims to determine the presence of these mutants in patients with liver cancer in China. For this purpose, virus DNA was extracted from the serum of 10 patients with HCC. After amplifying the PreS region and determining the sequence of this region from the genome, the presence of PreS2 mutants in these patients was investigated compared to the database. The results showed that a point mutation was observed at the level of the start codon of PreS2 in two samples. In three of the isolates, several amino acids were deleted at the end of the PreS2 region. In PreS2 deletion mutants, the T-cell and B-cell epitopes on the PreS2 region product are generally deleted. As a result, conditions are created where the virus can escape from the immune system. These mutant PreS2 proteins accumulate in the endoplasmic reticulum (ER) network and cause ER stress. In this way, in addition to creating unstable conditions in the cell genome, the proliferation of hepatocytes is stimulated indirectly. As a result, there is a possibility that the cells will progress toward becoming cancerous.

Genomic Variability of Hepatitis B Virus Circulating in Brazilian Western Amazon.

The emergence of clinically relevant mutations in the hepatitis B virus (HBV) genome has been a matter of great debate because of the possibility of escape from the host’s immune system, the potential to cause more severe progression of liver diseases and the emergence of treatment-resistant variants. Here we characterized the circulating variants of HBV in Rondônia State, in the north of Brazil. Serum samples of 62 chronic HBV carriers were subjected to PCR assays and clinical data were collected. Mutations and genotypes were characterized through direct sequencing. The findings show the presence of subgenotypes A1 (54.83%, 34/62), D3 (16.13%, 10/62), F2 (16.13%, 10/62), A2 (4.84%, 3/62), D2 (3.23%, 2/62), D1 (1.61%, 1/62), D4 (1.61%, 1/62) and F4 (1.61%, 1/62). Deletions in the pre-S2 region were found in 13.79% (8/58) of the samples, mutations in the S gene in 59.68% (37/62) and RT mutations in 48.39% (30/62). We found a variable genotypic distribution in different locations and important mutations related to immune escape and drug resistance in Western Amazonia, which contributed to genetic surveillance and provided important information to help control the disease.

Identification of a novel variant of hepatitis B virus isolated from patient co-infected with hepatitis C virus

Hepatitis B virus (HBV) is a major public health concern worldwide. Co-infection of hepatitis B patients with other pathogens intensifies the severity of the disease. We report a novel variant of hepatitis B virus (HBV) in Bangladesh isolated from a patient co-infected with hepatitis C virus (HCV) who exhibited liver cirrhosis. From 150 collected plasma samples, we sequenced HBV complete genome from one HBV-HCV co-infected patient. The complete genome was analysed using bioinformatics tools, NCBI BLAST, Geno2Pheno, and SnapGene software. The strain belongs to genotype A and subgenotype A1. Upon analysing the complete genome of this strain, we found a frameshift deletion of 54 nucleotides at the pre-S2 region, a functional regulator of HBV surface protein. Furthermore, we observed a Y126H mutation in the polymerase protein of this strain. This is the first report with such an unusual pre-S deletion event of the HBV genome in an HCV-co-infected patient associated with liver cirrhosis. These findings may inform scientists about genomic modifications in the HBV genome associated with HCV co-infection.

Sparse logistic regression revealed the associations between HBV PreS quasispecies and hepatocellular carcinoma

BackgroundChronic infection with hepatitis B virus (HBV) has been proved highly associated with the development of hepatocellular carcinoma (HCC).AimsThe purpose of the study is to investigate the association between HBV preS region quasispecies and HCC development, as well as to develop HCC diagnosis model using HBV preS region quasispecies.MethodsA total of 104 chronic hepatitis B (CHB) patients and 117 HBV-related HCC patients were enrolled. HBV preS region was sequenced using next generation sequencing (NGS) and the nucleotide entropy was calculated for quasispecies evaluation. Sparse logistic regression (SLR) was used to predict HCC development and prediction performances were evaluated using receiver operating characteristic curves.ResultsEntropy of HBV preS1, preS2 regions and several nucleotide points showed significant divergence between CHB and HCC patients. Using SLR, the classification of HCC/CHB groups achieved a mean area under the receiver operating characteristic curve (AUC) of 0.883 in the training data and 0.795 in the test data. The prediction model was also validated by a completely independent dataset from Hong Kong. The 10 selected nucleotide positions showed significantly different entropy between CHB and HCC patients. The HBV quasispecies also classified three clinical parameters, including HBeAg, HBVDNA, and Alkaline phosphatase (ALP) with the AUC value greater than 0.6 in the test data.ConclusionsUsing NGS and SLR, the association between HBV preS region nucleotide entropy and HCC development was validated in our study and this could promote the understanding of HCC progression mechanism.

The Serum Hepatitis B Virus Large Surface Protein as High-Risk Recurrence Biomarker for Hepatoma after Curative Surgery.

Chronic hepatitis B (CHB) virus infection is the most important cause of HCC and is also associated with tumor progression. The development of viral biomarkers for HCC prognosis is critical in evaluating relative risks to recurrence in the CHB HCC patients. We report that the large HBV surface protein (LHBS) expression increased in the tumors, implicating that it played a significant role in tumor development. To detect the LHBS in serum and evaluate its association with HCC progression, we developed a sandwich ELISA method for LHBS. The mouse monoclonal antibodies for the pre-S1, pre-S2, and HBS regions were in-house generated and constructed into a chemiluminescent sandwich ELISA system, which allowed sensitive and quantitative measurement of the protein. Using this ELISA assay, we estimated the expression of LHBS in CHB and HCC patients. We found that the serum LHBS level was correlated with the HBS but not the viral titer in serum, indicating that HBV surface proteins’ expression does not mainly depend on viral replication. Moreover, both serum LHBS and HBS levels were lower in the HCC patients than in the CHB. The liver LHBS signals, detected by immunohistochemical staining, showed significant correlations with the serum LHBS and HBS levels. In addition, the more elevated serum LHBS but not HBS level was significantly associated with cirrhosis and worse disease-free and overall survival rates, based on the multivariate analysis. Conclusion: LHBS plays a specific role in tumor progression and is an independent parameter associated with HCC recurrence. Serum LHBS represents a novel noninvasive biomarker for HCC patients with a worse prognosis after surgery.

Association of human leukocyte antigen-DR-DQ-DP haplotypes with the risk of hepatitis B virus-related hepatocellular carcinoma

Aim: Genetic polymorphisms of human leukocyte antigen (HLA) class II molecules are associated with chronic hepatitis B virus (HBV) infection. We aimed to investigate the impacts of HLA-II haplotypes on viral evolution and the risks of HBV-caused liver diseases. Methods: HLA-DR-DQ-DP haplotypes were estimated in 1210 healthy controls, 296 HBV clearance subjects, 301 asymptomatic hepatitis B surface antigen carriers, 770 chronic hepatitis B patients, 443 HBV-related liver cirrhosis (LC) patients, and 1037 HBV-related hepatocellular carcinoma (HCC) patients. HBV mutations were determined by sequencing. The associations of HLA-DR-DQ-DP haplotypes with viral mutations and the risks of liver diseases were assessed by multivariate logistic regression. Results: Compared to HBV-free subjects, the haplotypes CCAACG, CCGACG, TCAATA, and TCGATA were associated with decreased HCC risk, with an odds ratio (OR) [95% confidence interval (CI)] of 0.62 (0.40-0.95), 0.60 (0.39-0.92), 0.73 (0.54-0.98), and 0.58 (0.42-0.78), respectively. CCAACG, CCGACG, and TCAATA were significantly associated with decreased frequencies of the HCC-risk HBV mutations: preS1 deletion, APOBEC-signature HBV mutations in the core promoter and preS regions, A51C/T, G104C/T, and G146C/T. TCGATA and TTAACG were associated with increased LC risk, with an OR (95%CI) of 1.54 (1.03-2.30) and 2.23 (1.50-3.33), respectively. However, TCGATA and TTAACG were not consistently associated with the cirrhosis-risk HBV mutations. Conclusion: CCAACG, CCGACG, and TCAATA are inversely associated with HCC risk, possibly because they are involved in creating an immune microenvironment attenuating the generation of HCC-risk HBV mutations. TCGATA and TTAACG might predispose the polarity of immunity towards Th17 isotype related to LC.

Construction of an anti-hepatitis B virus preS1 antibody and usefulness of preS1 measurement for chronic hepatitis B patients: Anti-HBV PreS1 antibody

The preS1 region plays an essential role in hepatitis B virus (HBV) infection. We construct an antibody that binds to preS1 and a measurement system for serum preS1 in chronic HBV-infected patients. Hybridoma clones that produce anti-preS1 antibodies were obtained by the iliac lymph node method. Epitope mapping was conducted, and an enzyme-linked immunosorbent assay (ELISA)-based method was developed. Using this ELISA system, serum preS1 levels were measured in 200 chronic HBV-infected patients. Eight types of hybridomas were obtained, of which antibody 3-55 using amino acids 38-47 as the epitope showed high binding affinity to preS1. Serum preS1 levels measured by ELISA using 3-55 antibody were correlated with HBsAg, HBcrAg and HBV DNA levels. Among HBeAg-negative patients without antiviral therapeutic objective (HBV DNA <3.3logIU/mL or alanine aminotransferase ≤30U/L), preS1 was significantly higher in subjects who had progressed to the point of requiring antiviral therapy compared to subjects who had maintained their status for the preceding three years (p<0.01). We constructed an antibody against preS1 and an ELISA system capable of measuring serum preS1 levels. PreS1 may serve as a novel tool to predict the need for antiviral therapy in HBeAg-negative HBV-infected patients.

Complex structural variations in non-human primate hepatitis B virus

BackgroundRecent genome sequence technology has revealed a novel type of genetic rearrangement referred to as complex structural variations (SVs). Previous studies have elucidated the complex SVs in human hepatitis B viruses (HBVs). In this study, we investigated the existence of complex SVs in HBVs from non-human primates (NHPs).MethodsSearches for nucleotide sequences of NHP HBV were conducted using the PubMed, and genetic sequences were retrieved from databases. The candidate genetic sequences harboring complex SVs were analyzed using the CLUSTALW program and MAFFT. Additional bioinformatical analyses were performed to determine strains with complex SVs and to elucidate characteristics of NHP HBV strains.ResultsOne hundred and fifty-four HBV strains from NHPs were identified from databases. SVs and complex SVs were observed in 11 (7.1%) strains. Three gibbon HBV (GiHBV) strains showed complex SVs consisting of an insertion and a deletion in the pre-S1 region. One GiHBV strain possessed a 6-nt insertion, which are normally specific to human HBV genotype A (HBV/A) in the Core region, and further analyses clarified that the 6-nt insertion was not caused by recombination, but rather by simple insertion. Another chimpanzee HBV strain showed complex SVs in the pre-S1 region, which were composed of human HBV/E, G-specific polymorphic SV, and an additional 6-nt insertion.ConclusionsIn this study, complex SVs were observed in HBV strains from NHPs, in addition to human HBV strains, as shown in previous studies. These data suggest that complex SVs could also be found in other members of hepadnaviruses, and may play a role in their genetic diversity.