Virus Surface Gene Research Articles

Hepatitis B virus surface gene mutants in immunoprophylaxis-failed infants from Southern China.

Hepatitis B virus (HBV) vaccination was considered the most powerful tool for prevention of HBV transmission from mother to infant. In 1992, a universal HBV vaccination program for infants was launched in China. The aim of this study was to investigate the HBV surface antigen (HBsAg) variations in immunoprophylaxis-failed infants, vaccine free infant patients and adult chronic hepatitis B (CHB) patients (without nucleoside analog treatment). Immunoprophylaxis-failed infants were recruited from two centers (Guangdong and Chongqing, representing Southern China). HBV serum markers, including HBsAg, hepatitis e antigen (HBeAg), and core antigen, were detected by the enzyme-linked immunosorbent assay. HBV DNA load was detected by quantitative polymerase chain reaction. Nucleotide sequences encoding HBsAg were amplified and analyzed. Frequencies of amino acid substitutions within the second loop of "a" determinant (region with greater local hydrophilicity) in immunoprophylaxis-failed infants were clearly higher than the unvaccinated infant patients and adult CHB patients (9.6% vs 0% vs 3.8%; χ 2 = 7.454; P = 0.024). More than 50% (52.8%) aa substitutions in immunoprophylaxis-failed infants were located in B cell epitopes, while 64.6% aa substitutions in adult CHB patients were located in CTL cell epitopes. HBeAg negative patients had higher substitution frequency in CTL and Th cell epitopes, and in immunoprophylaxis-failed infant patients with substitution in major hydrophilic region region had a higher alanine aminotransferase level (68.6 ± 111.5 vs 62.1 ± 132.2; P = 0.026), and lower DNA load (7.03 ± 1.72 vs 7.82 ± 1.73; P < 0.001). In conclusion, our results observed vaccine-induced immune selection pressure in vaccine failed infants, substitutions in "a" determinant, especially the G145 substitution was still the most stable and remarkable site of vaccine escape.

Hepatitis B virus surface gene pre‐S2 mutant as a high‐risk serum marker for hepatoma recurrence after curative hepatic resection

Chronic hepatitis B virus (HBV) infection is the major cause of hepatocellular carcinoma (HCC). The pre-S2 mutant large HBV surface antigen (LHBS) is highly associated with HCC. This study analyzed the expression of the large form of surface protein in tumors and evaluated the LHBS with mutations within the pre-S2 region as a high-risk recurrence marker in HCC patients after curative hepatic resection. By analyses using immunohistochemical staining (n = 12) and western blotting (n = 22), the HBV surface protein, which is mainly comprised of the major form of HBV surface antigen, was greatly diminished in the tumors. However, LHBS was not significantly decreased in tumorous regions, suggesting that LHBS maintains its expression in cancer development. A cohort of 175 patients with HBV-related HCC who underwent curative hepatic resection was analyzed for pre-S gene mutations using Pre-S Gene Chip. Results of the multivariate regression analysis showed that the serum pre-S2 mutant level and the American Joint Committee on Cancer stage were the two main independent high-risk factors for recurrence. A Cox proportional hazards analysis also revealed a prediction model, which indicated the recurrence-free survival rate along with the time after surgery; this was developed and further validated in an independent HCC cohort. Receiver operating characteristic curve analysis revealed that the model showed close sensitivities in the main and validation cohorts (area under the curve values, 0.741 and 0.704, respectively). Conclusion: Unlike the major HBV surface antigen, LHBS is mostly expressed in the tumorous regions of HBV-induced HCC, indicating that it plays a unique role in tumor progression; the relative level of pre-S2 mutant in serum is, independently of tumor stage, an important high-risk marker for HCC recurrence after primary hepatic resection. (Hepatology 2018).

Sandwich Immunoassays of Multicomponent Subtrace Pathogenic DNA Based on Magnetic Fluorescent Encoded Nanoparticles

A novel magnetic fluorescent encoded nanoimmunoassay system for multicomponent detection and separation of the subtrace pathogenic DNA (hepatitis B virus surface gene, HBV; hepatitis A virus poly the protein gene, HAV) was established based on new type of magnetic fluorescent encoded nanoparticles and sandwich immunoassay principle. This method combines multifunctional nanoparticles, immunoassay technique, fluorescence labeling, and magnetic separation of multicomponent technology. It has many advantages such as high sensitivity, low detection limit, easy operation, and great potential for development. The results of this work show that, based on nanoimmunoassay system, it could quantitatively detect the multicomponent trace pathogenic HAV and HBV DNA, as well as detection limit up to 0.1 pM and 0.12 pM. Furthermore, with the improvement of the performances of magnetic fluorescent encoded nanoparticles, the sensitivity will be further improved. In this experiment, a new nanoimmunoassay system based on magnetic fluorescent encoded nanoparticles was established, which will provide a new way for the immunoassay and separation of multicomponent biomolecules.

Dysregulation of the TGFBI gene is involved in the oncogenic activity of the nonsense mutation of hepatitis B virus surface gene sW182*

The nonsense mutations of the hepatitis B virus (HBV) surface (S) gene have been reported to have oncogenic potential. We have previously identified several transforming nonsense mutations of the HBV S gene from hepatocarcinoma (HCC) patients. Among them, the sW182* mutant (the stop codon for tryptophan 182) showed the most potent oncogenicity in a mouse xenograft model using stably transfected mouse fibroblast cells. This study is aimed at understanding the molecular mechanisms leading to the oncogenic activity of the sW182* mutant.A gene expression microarray in combination with gene set enrichment analysis (GSEA) revealed differentially expressed gene sets in the sW182* cells, including those related to cell-cycle regulation, deoxyribonucleic acid repair, and genome instability. Of the differentially expressed genes, the transforming growth factor-β-induced (TGFBI) gene was further validated to be dysregulated in the sW182* cells. This dysregulation was accompanied by hypermethylation of the TGFBI promoter.The level of cyclin D1, a negatively regulated TGFBI target, was highly elevated in the sW182* mutant cells, which is consistent with the potent oncogenicity. Furthermore, frequent abnormal mitosis and multinucleation were observed in the mutant cells. Exogenous expression of TGFBI alleviated the oncogenic activity of the sW182* cells. In human HBV-related HCC cancerous tissue, expression of TGFBI was downregulated in 25 of the 55 (45%) patients examined, suggesting that TGFBI dysregulation could occur in HBV-related HCC development in some cases.These results suggest that dysregulation of TGFBI is involved in the oncogenic activity of the sW182* mutant of the hepatitis B virus S gene.

Clinical Spectrum of Hepatitis B Infection in Southern India

© 2011, INASL 8 Results: The IC50 of the monoherbal preparations found out to be 1 mg/mL, 20 uM and 10 uM for Phyllanthus amarus, Andrographis paniculata and Silybum marianum, respectively as obtained from the decline in viral load in the soups collected from HepG2 2.2.15 cell lines. Conclusion: The above monoherbal preparations found to have a significant decline in viral proliferating markers. In comparison with the control, herbal treatment proved to have an efficacious effect and IC50 was calculated for them. Thus the herbs could be beneficial against HBV with least side effects in comparison to synthetic drugs. However it’s a preliminary data and further studies are needed to prove it. Conflict of Interest: None Clinical Spectrum of Hepatitis B Infection in Southern India B Siva Subramaniam, V Arul Selvan, K Arun Kumar, V Jayanthi Department of Gastroenterology and Hepatology, Stanley Medical College and Hospital, Chennai Background: Hepatitis B is a global problem, affecting over 400 million populations. Large numbers of cases are referred to our center. Aim: To determine the spectrum of hepatitis B infection amongst patients attending the Liver Clinic at a tertiary GI and Hepatology center. Method: All patients who were diagnosed as HBsAg positive were included in the study. Baseline evaluation included LFT, ultrasound, alphafetoprotein estimation and virological profile for hepatitis B (HBeAg, quantification of HBsAg, HBV DNA), hepatitis C (anti-HCV antibody) and HIV status. Family members were also screened for the above markers if found to be HBsAg positive. Based on the viral profile, transaminase levels, patients were categorized into immunotolerant, inactive carriers, immune clearance and reactivation phase and risk factors were also looked for. Patients with incomplete virological profile were excluded from the study. Results: One hundred and thirteen patients were fulfilled the criteria. Among the 113 patients 38 (33.6%) patients were in immunotolerant phase, 15 (13.2%) patients in immune clearance phase, 57 (50.4%) patients were inactive carriers and 3 (2.6%) patients were in reactivation phase. Twenty patients had cirrhosis, 2 patients were presented with hepatocellular carcinoma and 7 patients were pregnant. HBV DNA levels are low in patients with cirrhosis and HCC. Table Different phases of HBV infection Variables Immunotolerant Immune clearance Inactive carrier Reactivation phase (n = 38) (33.6%) (n = 15) (13.2%) (n = 57) (50.4%) (n = 3) (2.6%) Mean age 32 (median) 40 (mean) 36 (mean) 55.33 (mean) Sex ratio 24:14 (M:F) 13:2 (M:F) 19:38 (M:F) 3 males ALT 52.4 137.3 34.7 174.6 HBsAg quantification 10905.4 3206.567 5885.958 9368.615 HBV DNA 2672419 856432 313.9963 4491218 Conclusion: The spectrum of hepatitis B infection observed in Southern India was similar to the spectrum seen in West. The immunotolerant phase extends up to 32 years and virus starts clearing in between 32 and 40 years. So far, no similar data is available from South India. Conflict of Interest: None Frequency of Hepatitis B Virus Surface Gene Variant Circulating Among HBsAg Negative Individuals in Coastal Eastern India: A Preliminary Report MK Panigrahi, SK Kar, B Misra, SP Singh, HS Das, C Panda Department of Gastroenterology, SCB Medical College, Cuttack, Orissa, India Background: Genetic mutation might account for the presence of hepatitis B virus (HBV) DNA among HBsAg negative individuals. The aim of the study was to assess the prevalence and significance of surface gene mutations among HBsAg negative chronic liver disease cases (CLD) who attended the Gastroenterology OPD at SCB Medical College, Cuttack. Methods: Twenty HBsAg negative CLD sera of adults (mean age 44.8 ± 12.1; range 28–70 years) were included in this study. Surface gene region were amplified and surface gene was analyzed after direct sequencing. Results: Out of the total 20 CLD cases studied, only 5 samples were found to be HBV DNA positive (5/20; 25.0%). Genotype D was the prevalent genotype found in the study with no prevalence of other genotypes. In all 5 patients, substitutions within as well as outside 03_JCEH-Abstract.indd 8 3/18/2011 11:13:03 AM

Frequency of Hepatitis B Virus Surface Gene Variant Circulating Among HBsAg Negative Individuals in Coastal Eastern India: A Preliminary Report

© 2011, INASL 8 Results: The IC50 of the monoherbal preparations found out to be 1 mg/mL, 20 uM and 10 uM for Phyllanthus amarus, Andrographis paniculata and Silybum marianum, respectively as obtained from the decline in viral load in the soups collected from HepG2 2.2.15 cell lines. Conclusion: The above monoherbal preparations found to have a significant decline in viral proliferating markers. In comparison with the control, herbal treatment proved to have an efficacious effect and IC50 was calculated for them. Thus the herbs could be beneficial against HBV with least side effects in comparison to synthetic drugs. However it’s a preliminary data and further studies are needed to prove it. Conflict of Interest: None Clinical Spectrum of Hepatitis B Infection in Southern India B Siva Subramaniam, V Arul Selvan, K Arun Kumar, V Jayanthi Department of Gastroenterology and Hepatology, Stanley Medical College and Hospital, Chennai Background: Hepatitis B is a global problem, affecting over 400 million populations. Large numbers of cases are referred to our center. Aim: To determine the spectrum of hepatitis B infection amongst patients attending the Liver Clinic at a tertiary GI and Hepatology center. Method: All patients who were diagnosed as HBsAg positive were included in the study. Baseline evaluation included LFT, ultrasound, alphafetoprotein estimation and virological profile for hepatitis B (HBeAg, quantification of HBsAg, HBV DNA), hepatitis C (anti-HCV antibody) and HIV status. Family members were also screened for the above markers if found to be HBsAg positive. Based on the viral profile, transaminase levels, patients were categorized into immunotolerant, inactive carriers, immune clearance and reactivation phase and risk factors were also looked for. Patients with incomplete virological profile were excluded from the study. Results: One hundred and thirteen patients were fulfilled the criteria. Among the 113 patients 38 (33.6%) patients were in immunotolerant phase, 15 (13.2%) patients in immune clearance phase, 57 (50.4%) patients were inactive carriers and 3 (2.6%) patients were in reactivation phase. Twenty patients had cirrhosis, 2 patients were presented with hepatocellular carcinoma and 7 patients were pregnant. HBV DNA levels are low in patients with cirrhosis and HCC. Table Different phases of HBV infection Variables Immunotolerant Immune clearance Inactive carrier Reactivation phase (n = 38) (33.6%) (n = 15) (13.2%) (n = 57) (50.4%) (n = 3) (2.6%) Mean age 32 (median) 40 (mean) 36 (mean) 55.33 (mean) Sex ratio 24:14 (M:F) 13:2 (M:F) 19:38 (M:F) 3 males ALT 52.4 137.3 34.7 174.6 HBsAg quantification 10905.4 3206.567 5885.958 9368.615 HBV DNA 2672419 856432 313.9963 4491218 Conclusion: The spectrum of hepatitis B infection observed in Southern India was similar to the spectrum seen in West. The immunotolerant phase extends up to 32 years and virus starts clearing in between 32 and 40 years. So far, no similar data is available from South India. Conflict of Interest: None Frequency of Hepatitis B Virus Surface Gene Variant Circulating Among HBsAg Negative Individuals in Coastal Eastern India: A Preliminary Report MK Panigrahi, SK Kar, B Misra, SP Singh, HS Das, C Panda Department of Gastroenterology, SCB Medical College, Cuttack, Orissa, India Background: Genetic mutation might account for the presence of hepatitis B virus (HBV) DNA among HBsAg negative individuals. The aim of the study was to assess the prevalence and significance of surface gene mutations among HBsAg negative chronic liver disease cases (CLD) who attended the Gastroenterology OPD at SCB Medical College, Cuttack. Methods: Twenty HBsAg negative CLD sera of adults (mean age 44.8 ± 12.1; range 28–70 years) were included in this study. Surface gene region were amplified and surface gene was analyzed after direct sequencing. Results: Out of the total 20 CLD cases studied, only 5 samples were found to be HBV DNA positive (5/20; 25.0%). Genotype D was the prevalent genotype found in the study with no prevalence of other genotypes. In all 5 patients, substitutions within as well as outside 03_JCEH-Abstract.indd 8 3/18/2011 11:13:03 AM

Frequency and significance of hepatitis B virus surface gene variant circulating among ‘antiHBc only’ individuals in Eastern India

Background Genetic mutation might account for the presence of hepatitis B virus (HBV) DNA among antiHBc only individuals. The aim of the study was to assess the prevalence and significance of surface gene mutations among antiHBc only cases in our population. Methods Three hundred and three antiHBc(+) sera of adults (mean age, 33.7 ± 11.0; range 18–65 years) as well as HBsAg(+)/HBV DNA(+) ( n = 19) controls were included in this study. Surface gene and basal core promoter (BCP)-precore region were amplified and surface gene was analyzed after direct sequencing. Results One hundred and seventy-eight out of 303 (58.8%) was antiHBc only, 39/171 (22.8%) of them was HBV DNA(+). Genotypes A, C, D were found among both HBsAg(+) and antiHBc(+) samples. Single or multiple amino acids substitutions were found in 82% samples, however, G145R vaccine escape mutation was rare. Individuals having substitutions within as well as outside major hydrophilic loop (MHL) region were detected; some of these mutations were in overlapping RT domain of polymerase (Pol) gene. Conclusions The existence of occult HBV infection among antiHBc only individuals could not be explained fully by mutations in the ‘a’ determinant region of surface gene in our population.

P.037 Association of hepatitis B virus surface gene mutations with viral genotypes in patients with occult HBV infection

P.037 Association of hepatitis B virus surface gene mutations with viral genotypes in patients with occult HBV infection

Novel Autoregulatory Function of Hepatitis B Virus M Protein on Surface Gene Expression

The hepatitis B virus surface gene consists of a single open reading frame divided into three coding regions: pre-S1, pre-S2, and S. By alternate translation at each of the three initiation codons, L, M, and S proteins can be synthesized. Studies have shown that M protein is not essential for viral replication, virion morphogenesis, or in vitro infectivity. In this study, we show that native M protein can regulate surface gene expression at the transcriptional level. The regulatory effect of M protein is mediated through the CCAAT box within the S promoter. Deletion mapping analysis indicated that the transactivating effect of M protein is mediated through amino acids 1-57 of M protein (the MHBs(au) domain), although its maximal transactivation activity coincides with that of the pre-S2 domain. This conclusion is supported by the fact that disruption of the putative V8 protease site at the pre-S2/S domain junction not only rendered M protein incapable of transactivating the S promoter but also inactivated its nuclear translocation potential. Immunoprecipitation and immunoblot experiments demonstrated that pre-S2 interacts with the three subunits of the CCAAT box-binding factor/nuclear factor Y, the cognate binding protein of the CCAAT box. These results demonstrate and define a novel regulatory role of M protein, which, under natural conditions, may undergo a proteolytic process to generate an MHBs(au) species that will be translocated inside the nucleus, where it will interact with the CCAAT box-binding factor to regulate surface gene expression. Because the CCAAT box is located at a fixed position within numerous promoters, these observations might provide a plausible explanation for hepatitis B virus-associated hepatocarcinogenesis.

Establishment of a Screening System for Selection of siRNA Target Sites Directed against Hepatitis B Virus Surface Gene

To study the inhibitory effects of plasmid-derived small interfering RNA (siRNA) and synthetic siRNA on the expression of the hepatitis B virus surface (HBs) gene, three plasmid-derived siRNAs and one synthetic siRNA that complement the coding region of the HBs gene were prepared. The HBs expression plasmid pHBs-EGFP was also constructed. HeLa cells were co-transfected with pHBs-EGFP and the above siRNAs. The HBs mRNA quantities were measured by reverse-transcription PCR, and the level of HBs-EGFP fusion protein was quantified by fluorescent microscope. The concentrations of the hepatitis B virus surface antigen (HBsAg) derived from the culture supernatant of transfected HepG2.2.15 cells were measured by an enzyme-linked immunosorbent assay (ELISA) kit. The results showed that the three plasmid-derived siRNAs and the synthetic siRNA can effectively reduce the quantities of HBs mRNA and protein. The plasmid-derived siRNA psiRNA1 was found to be the most effective inhibitor of HBs expression. It can inhibit HBs-EGFP expression by 63.3% and suppress HBs mRNA by 75.6%. To further substantiate the above observations, psiRNA1 was transfected into HepG2.2.15 cells (an HBV secreting cell line). The transfections resulted in almost complete blockage of HBsAg production, whereas control vector-transfected cells secreted high levels of HBsAg 7 days post-transfection. In conclusion, our data suggests that RNA interference (RNAi) is an efficient approach for reducing the level of HBs transcripts and proteins and for suppressing HBsAg production.

Replicating Foamy Virus-Based Vectors Directing High Level Expression of Foreign Genes

Replication-competent retroviral vectors (pFOV-1 to -3 and -7) were constructed on the basis of an infectious human foamy virus molecular clone which has deletions in the U3 region of the long terminal repeat and in the 3′ region of the genome, previously identified to be nonessential for virus replication in vitro, The CAT and luciferase indicator genes were expressed as C-terminal fusion proteins to 215 amino acids of the viral Bet protein in the pFOV-1 vector. Introduction of the foot-and-mouth disease 2A protease sequence between the truncated bet coding sequence and the cloning site for the insertion of foreign genes in the pFOV-7 vector resulted in self-cleaving of the recombinant fusion protein. Alternatively, an internal ribosomal binding site was introduced, allowing expression of authentic foreign protein (pFOV-2 and -3 vectors). DNA fragments derived from the mouse hepatitis virus surface gene up to the length of 1.3 kb were inserted into pFOV-1. The vector constructs gave rise to viruses which were fully infectious in diploid human fibroblasts and recombinant viruses stably expressed high levels of foreign protein indicating that the pFOV vectors may be useful tools to study the effects of proteins of interest at least in tissue culture cells.

Replicating foamy virus based vectors directing high level expression of foreign genes

Replication-competent retroviral vectors (pFOV-1 to -3 and -7) were constructed on the basis of an infectious human foamy virus molecular clone which has deletions in the U3 region of the long terminal repeat and in the 3′ region of the genome, previously identified to be nonessential for virus replication in vitro, The CAT and luciferase indicator genes were expressed as C-terminal fusion proteins to 215 amino acids of the viral Bet protein in the pFOV-1 vector. Introduction of the foot-and-mouth disease 2A protease sequence between the truncated bet coding sequence and the cloning site for the insertion of foreign genes in the pFOV-7 vector resulted in self-cleaving of the recombinant fusion protein. Alternatively, an internal ribosomal binding site was introduced, allowing expression of authentic foreign protein (pFOV-2 and -3 vectors). DNA fragments derived from the mouse hepatitis virus surface gene up to the length of 1.3 kb were inserted into pFOV-1. The vector constructs gave rise to viruses which were fully infectious in diploid human fibroblasts and recombinant viruses stably expressed high levels of foreign protein indicating that the pFOV vectors may be useful tools to study the effects of proteins of interest at least in tissue culture cells.

Differential regulation of the hepatitis B virus surface gene promoters by a second viral enhancer.

The hepatitis B virus surface gene is transcribed from two promoters, and the resulting mRNA species code for three distinct forms of the surface antigen. We show here that the viral transcriptional trans-activator, X protein, has no effect on either promoter. However, a cis-acting element in the downstream half of the X gene, distinct from the previously mapped viral enhancer, selectively activates the major surface gene promoter. Nuclease protection and gel-shift assays reveal that multiple cellular factors bind to two sites within this DNA fragment, both of which are necessary for enhancer activity. Since this region of the viral genome is frequently deleted upon integration into the hsot chromosome in chronic hepatitis B, loss of this second enhancer can alter the relative amounts of the three forms of the surface antigen in infected hepatocytes and thus possibly contribute to cellular damage.