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Genotyping of hepatitis B virus in Malaysia based on the nucleotide sequence of preS and S genes

Ong HT, Duraisamy G, Kee Peng N, Wen Siang T, Seow HF

Microbes Infect., 2005 Mar;7(3):494-500.
PMID: 15792534

Hepatitis B virus (HBV) has been classified into eight genotypes, designated A-H. These genotypes are known to have distinct geographic distributions. The clinical importance of genotype-related differences in the pathogenicity of HBV has been revealed recently. In Malaysia, the current distribution of HBV remains unclear. The aim of this study was to determine the genotypes and subtypes of HBV by using PCR, followed by DNA sequencing, as well as to analyse the mutations in the immunodominant region of preS and S proteins. The S gene sequence was determined from HBV DNA of four apparently healthy blood donors' sera and three sera from asymptomatic chronic hepatitis B carriers. Of this batch of sera, the preS gene sequence was obtained from HBV DNA from three out of the four blood donors and two out of the three chronic carriers. Due to insufficient sera, we had to resort to using sera from another blood donor to make up for the sixth DNA sequence of the preS gene. Based on the comparative analysis of the preS sequences with the reported sequences in the GenBank database, HBV DNA from two normal carriers was classified as genotype C. Genotype B was assigned to HBV from one blood donor and two hepatitis B chronic carriers, whereas HBV of one chronic carrier was of genotype D. Based on the S gene sequences, HBV from three blood donors was of genotype C, that of one blood donor and one chronic carrier was of genotype B, and the remaining, of genotype D. In the five cases where both preS and S gene sequences were determined, the genotypes assigned based on either the preS or S gene sequences were in concordance. The nature of the deduced amino acid (aa) sequences at positions 125, 127, 134, 143, 159, 161 and 168 of the S gene enabled the classification of these sequences into subtypes, namely, adrq+, adw2 and ayw2. The clustering of our DNA sequences into genotype groups corresponded to their respective subtype, that is, adw2 in genotype B, adrq in genotype C and ayw in genotype D. Analysis of the point mutations revealed that five of the sequences contained aa substitutions at immunodominant epitopes involved in B or/and T cell recognition. In conclusion, despite the low numbers of samples studied, due to budget constraints, these data are still worthwhile reporting, as it is important for the control of HBV infections. In addition, the genotype and mutational data obtained in this study may be useful for designing new treatment regimes for HBV patients.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics*
Cloning, expression and display of the PreS domain of hepatitis B virus on filamentous bacteriophage M13

Kok WL, Yusoff K, Nathan S, Tan WS

J. Biochem. Mol. Biol. Biophys., 2002 Feb;6(1):55-8.
PMID: 12186783

The PreS domain of hepatitis B virus (HBV) is believed to be involved in virion assembly and attachment to a hepatocyte receptor during infection. In order to study the functions of this region, we fused it to the g3p protein of bacteriophage M13 that allows the fusion protein to be displayed at the tip of the filament. The fusion protein was detected by the anti-E tag antibody on a Western blot. The polypeptide in a soluble form was produced by transfecting a non-suppressor E. coli host cell with the recombinant phagemid. The soluble protein was detected in cytoplasm, in the periplasmic space and also in the medium. The functional display of the PreS domain would provide an alternative means to study its interactions with the nuleocapsid and hepatocytes.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics*
The full-length clone of cucumber green mottle mosaic virus and its application as an expression system for Hepatitis B surface antigen

Ooi A, Tan S, Mohamed R, Rahman NA, Othman RY

J Biotechnol, 2006 Feb 24;121(4):471-81.
PMID: 16271415

A cucumber green mosaic mottle virus (CGMMV) full-length clone was developed for the expression of Hepatitis B surface antigen (HBsAg). The expression of the surface displayed HBsAg by the chimeric virus was confirmed through a double antibody sandwich ELISA. Assessment of the coat protein composition of the chimeric virus particles by SDS-PAGE analysis showed that 50% of the coat proteins were fused to the HBsAg. Biological activity of the expressed HBsAg was assessed through the stimulation of in vitro antibody production by cultured peripheral blood mononuclear cells (PBMC). PBMC that were cultured in the presence of the chimeric virus showed up to an approximately three-fold increase in the level of anti HBsAg immunoglobulin thus suggesting the possible use of this new chimeric virus as an effective Hepatitis B vaccine.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics
Molecular and serological detection of occult hepatitis B virus among healthy hepatitis B surface antigen-negative blood donors in Malaysia

Hudu SA, Harmal NS, Saeed MI, Alshrari AS, Malik YA, Niazlin MT, et al.

Afr Health Sci, 2016 Sep;16(3):677-683.
PMID: 27917199

BACKGROUND: Occult hepatitis B infections are becoming a major global threat, but the available data on its prevalence in various parts of the world are often divergent.
OBJECTIVE: This study aimed to detect occult hepatitis B virus in hepatitis B surface antigen-negative serum using anti-HBc as a marker of previous infection.
PATIENT AND METHODS: A total of 1000 randomly selected hepatitis B surface antigen-negative sera from blood donors were tested for hepatitis B core antibody and hepatitis B surface antibody using an ELISA and nested polymerase chain reaction was done using primers specific to the surface gene (S-gene).
RESULTS: Of the 1000 samples 55 (5.5%) were found to be reactive, of which 87.3% (48/55) were positive for hepatitis B surface antibody, indicating immunity as a result of previous infection however, that does not exclude active infection with escaped mutant HBV. Nested PCR results showed the presence of hepatitis B viral DNA in all the 55 samples that were positive for core protein, which is in agreement with the hepatitis B surface antibody result.
CONCLUSION: This study reveals the 5.5% prevalence of occult hepatitis B among Malaysian blood donors as well as the reliability of using hepatitis B core antibody in screening for occult hepatitis B infection in low endemic, low socioeconomic settings.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics
Fulltext Enhanced cell disruption strategy in the release of recombinant hepatitis B surface antigen from Pichia pastoris using response surface methodology

Tam YJ, Allaudin ZN, Lila MA, Bahaman AR, Tan JS, Rezaei MA

BMC Biotechnol, 2012 Oct 05;12:70.
PMID: 23039947 DOI: 10.1186/1472-6750-12-70

BACKGROUND: Cell disruption strategies by high pressure homogenizer for the release of recombinant Hepatitis B surface antigen (HBsAg) from Pichia pastoris expression cells were optimized using response surface methodology (RSM) based on the central composite design (CCD). The factors studied include number of passes, biomass concentration and pulse pressure. Polynomial models were used to correlate the above mentioned factors to project the cell disruption capability and specific protein release of HBsAg from P. pastoris cells.
RESULTS: The proposed cell disruption strategy consisted of a number of passes set at 20 times, biomass concentration of 7.70 g/L of dry cell weight (DCW) and pulse pressure at 1,029 bar. The optimized cell disruption strategy was shown to increase cell disruption efficiency by 2-fold and 4-fold for specific protein release of HBsAg when compared to glass bead method yielding 75.68% cell disruption rate (CDR) and HBsAg concentration of 29.20 mg/L respectively.
CONCLUSIONS: The model equation generated from RSM on cell disruption of P. pastoris was found adequate to determine the significant factors and its interactions among the process variables and the optimum conditions in releasing HBsAg when validated against a glass bead cell disruption method. The findings from the study can open up a promising strategy for better recovery of HBsAg recombinant protein during downstream processing.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics*
Display of hepatitis B virus PreS1 peptide on bacteriophage T7 and its potential in gene delivery into HepG2 cells

Tang KH, Yusoff K, Tan WS

J Virol Methods, 2009 Aug;159(2):194-9.
PMID: 19490973 DOI: 10.1016/j.jviromet.2009.03.015

Hepatitis B is a major public health problem worldwide which may lead to chronic liver diseases, cirrhosis and hepatocellular carcinoma. An interaction between hepatitis B virus (HBV) envelope protein, particularly the PreS1 region, and a specific cell surface receptor is believed to be the initial step of HBV infection through attachment to hepatocytes. In order to develop a gene delivery system, bacteriophage T7 was modified genetically to display polypeptides of the PreS1 region. A recombinant T7 phage displaying amino acids 60-108 of the PreS1 region (PreS1(60-108)) was demonstrated to be most effective in transfecting HepG2 cells in a dose- and time-dependant manner. The phage genome was recovered from the cell lysate and confirmed by PCR whereas the infectious form of the internalized phage was measured by a plaque-forming assay. The internalized phage exhibited the appearance of green fluorescent dots when examined by immunofluorescence microscopy. Surface modification, particularly by displaying the PreS1(60-108) enhanced phage uptake, resulting in more efficient in vitro gene transfer. The ability of the recombinant phage to transfect HepG2 cells demonstrates the potential of the phage display system as a gene therapy for liver cancer.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics
Antigenicity and immunogenicity of the immunodominant region of hepatitis B surface antigen displayed on bacteriophage T7

Tan GH, Yusoff K, Seow HF, Tan WS

J Med Virol, 2005 Dec;77(4):475-80.
PMID: 16254965

The immunodominant region of hepatitis B virus (HBV) located in the viral small surface antigen (S-HBsAg) elicits virus-neutralizing and protective antibodies. In order to develop an easy and inexpensive method to produce this region without the need for extensive purification, amino acid residues 111-156 of S-HBsAg were fused to the C-terminal end of the 10B capsid protein of T7 phage. Western blotting and ELISA confirmed the expression of the recombinant protein on the surface of the phage particles. The recombinant phage exhibited the antigenic and immunogenic characteristics of HBsAg, illustrating its potential as an immunological reagent and vaccine.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics
Fulltext Naturally occurring hepatitis B virus surface antigen mutant variants in Malaysian blood donors and vaccinees

Hudu SA, Harmal NS, Saeed MI, Alshrari AS, Malik YA, Niazlin MT, et al.

Eur J Clin Microbiol Infect Dis, 2015 Jul;34(7):1349-59.
PMID: 25792010 DOI: 10.1007/s10096-015-2358-1

Hepatitis B virus surface mutants are of enormous importance because they are capable of escaping detection by serology and can infect both vaccinated and unvaccinated populations, thus putting the whole population at risk. This study aimed to detect and characterise hepatitis B-escaped mutants among blood donors and vaccinees. One thousand serum samples were collected for this study from blood donors and vaccinees. Hepatitis B surface antigen, antibodies and core antibodies were tested using a commercial enzyme-linked immunosorbent assay (ELISA) kit. DNA detection was performed via nested polymerase chain reaction (PCR), and the S gene was sequenced and analysed using bioinformatics. Of the 1,000 samples that were screened, 5.5% (55/1,000) were found to be HBsAg-negative and anti-HBc- and HBV DNA-positive. All 55 isolates were found to belong to genotype B. Several mutations were found across all the sequences from synonymous and non-synonymous mutations, with the most nucleotide mutations occurring at position 342, where adenine was replaced by guanine, and cytosine at position 46 was replaced by adenine in 96.4% and 98% of the isolates, respectively. Mutation at position 16 of the amino acid sequence was found to be common to all the Malaysian isolates, with 85.7% of the mutations occurring outside the major hydrophilic region. This study revealed a prevalence of 5.5% for hepatitis B-escaped mutations among blood donors and vaccinated undergraduates, with the most common mutation being found at position 16, where glutamine was substituted with lysine.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics*
Fulltext PreS1 Mutations Alter the Large HBsAg Antigenicity of a Hepatitis B Virus Strain Isolated in Bangladesh

Hossain MG, Mahmud MM, Nazir KHMNH, Ueda K

Int J Mol Sci, 2020 Jan 15;21(2).
PMID: 31952213 DOI: 10.3390/ijms21020546

Mutations in the hepatitis B virus (HBV) genome can potentially lead to vaccination failure, diagnostic escape, and disease progression. However, there are no reports on viral gene expression and large hepatitis B surface antigen (HBsAg) antigenicity alterations due to mutations in HBV isolated from a Bangladeshi population. Here, we sequenced the full genome of the HBV isolated from a clinically infected patient in Bangladesh. The open reading frames (ORFs) (P, S, C, and X) of the isolated HBV strain were successfully amplified and cloned into a mammalian expression vector. The HBV isolate was identified as genotype C (sub-genotype C2), serotype adr, and evolutionarily related to strains isolated in Indonesia, Malaysia, and China. Clinically significant mutations, such as preS1 C2964A, reverse transcriptase domain I91L, and small HBsAg N3S, were identified. The viral P, S, C, and X genes were expressed in HEK-293T and HepG2 cells by transient transfection with a native subcellular distribution pattern analyzed by immunofluorescence assay. Western blotting of large HBsAg using preS1 antibody showed no staining, and preS1 ELISA showed a significant reduction in reactivity due to amino acid mutations. This mutated preS1 sequence has been identified in several Asian countries. To our knowledge, this is the first report investigating changes in large HBsAg antigenicity due to preS1 mutations.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics
Occult hepatitis B infection in blood donors from South East Asia: molecular characterisation and potential mechanisms of occurrence

Candotti D, Lin CK, Belkhiri D, Sakuldamrongpanich T, Biswas S, Lin S, et al.

Gut, 2012 Dec;61(12):1744-53.
PMID: 22267593 DOI: 10.1136/gutjnl-2011-301281

To investigate the molecular basis of occult hepatitis B virus (HBV) infection (OBI) in Asian blood donors.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics
An Overview of Hepatitis B Virus Surface Antigen Mutant in the Asia Pacific

Hudu SA, Malik YA, Niazlin MT, Harmal NS, Sekawi Z

Curr Issues Mol Biol, 2014;16:69-78.
PMID: 24014801

Hepatitis B virus infection is a serious health problem worldwide, and more than 350 million people are chronic carriers, constituting a major global threat. Southeast Asia and the Western Pacific have the highest levels of endemicity in the world, with an estimated seroprevalence ranging between 2% and 31%. Mutations in the hepatitis B surface antigen (HBsAg) have been reported in many parts of the world but are most common in Asian infants; such mutants have several clinical effects, such as the development of hepatocellular carcinoma. Diagnostic failures by commercial assays have reduced the diagnostic effectiveness of HBsAg detection. For example the substitution of an amino acid in the major hydrophilic region of the S gene reduces the binding of hepatitis B surface antibodies leading to immune escape. The safety of blood transfusion may be compromised by current screening tests due to escape from being neutralised by antibodies induced by HBsAg mutants, and undetectable levels of viral surface protein. Data on the epidemiology of HBsAg mutation in Asia Pacific are scant; however, this manuscript has reviewed the available information on the epidemiology of HBsAg mutation in Asia Pacific.

Matched MeSH terms: Hepatitis B Surface Antigens/genetics*