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. 2020 Jan 15;21(2):546.
doi: 10.3390/ijms21020546.

PreS1 Mutations Alter the Large HBsAg Antigenicity of a Hepatitis B Virus Strain Isolated in Bangladesh

Md Golzar Hossain  1   2 , Md Muket Mahmud  2 , K H M Nazmul Hussain Nazir  2 , Keiji Ueda  1
Affiliations

PreS1 Mutations Alter the Large HBsAg Antigenicity of a Hepatitis B Virus Strain Isolated in Bangladesh

Md Golzar Hossain et al. Int J Mol Sci. .

Abstract

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.

Keywords: Bangladesh; antigenicity; full genome sequence; hepatitis B virus (HBV); preS1 mutations.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Phylogenetic tree analysis of hepatitis B virus (HBV) BD2 (MK628732). The full genome sequences of several HBV strains were extracted from GenBank, and a phylogenetic tree was constructed using CLC Sequence Viewer.
Figure 2
Figure 2
Alignment of HBV full genome sequence with that of reference strains. The HBV complete genome sequence identified in this study was aligned with reference strains MF925359.1 and X01587 and characterized. Mutational analysis was performed by comparing the present sequence with that of the reference HBV genomes.
Figure 2
Figure 2
Alignment of HBV full genome sequence with that of reference strains. The HBV complete genome sequence identified in this study was aligned with reference strains MF925359.1 and X01587 and characterized. Mutational analysis was performed by comparing the present sequence with that of the reference HBV genomes.
Figure 3
Figure 3
Expression analysis of HBV proteins by immunofluorescence analysis. HEK-293T and HepG2 cells were transfected with pCMV-Myc carrying specific HBV genes. Cells were fixed, permeabilized, and stained with Myc tag Ab at 48 h after transfection, followed by an Alexa Fluor 488-conjugated anti-mouse IgG (Green) for HEK-293T cells and Alexa Fluor 546-conjugated anti-mouse IgG (Red) for HepG2 cells. The cell nuclei were stained with DAPI (blue). The experiment was performed at least three independent times, and one representative data set was presented.
Figure 4
Figure 4
PreS1 region amino acid mutations, western blot analysis, and ELISA. (A) Amino acid mutations in the preS1 region compared to the reference HBV strain adr4. Mentioned cells were co-transfected with pCMV-LS carrying a large HBsAg coding sequence and pGFP-Max or pSV-β-Gal and incubated for 48 h. (B,F) Transfection efficiency was tested by detecting GFP signals from the co-transfected cells. Pictures were taken using EVOS FL Auto with 10×/0.30 magnification. (C,H) Cells were lysed and subjected to ELISA. ELISA results were normalized to the β-Gal assay. (D,G) Lysed cells were further subjected to SDS-PAGE followed by western blotting analysis using preS1 and Myc tag antibodies. The β-tubulin used as a loading control. (E) Relative antigenic reactivity of large HBsAg by western blot. Data were generated from the densitometry of western blot band intensities detected by preS1, Myc tag, and β-tubulin antibodies. Relative reactivity of large HBsAg was calculated. Each experiment was performed at least three independent times, and results are shown as mean ± SD. p ≤ 0.05 was considered as statistically significant.
Figure 5
Figure 5
Distribution of silent large HBsAg mutated HBV in Asiatic countries. BLAST searches were performed using 119 amino acids/357 nt of the preS1 region of BD2 genome. A total of 103 amino acid sequences (A) and 60 nucleotide sequences (B) showed 100% sequence identity.
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