Characterization of mutations in hepatitis B virus DNA isolated from Japanese HBsAg-positive blood donors in 2021 and 2022

Abstract

Missense mutations in certain small envelope proteins diminish the efficacy of antibodies. Consequently, tracking the incidence and types of vaccine-escape mutations (VEMs) was crucial both before and after the introduction of universal hepatitis B vaccination in Japan in 2016. In this study, we isolated hepatitis B virus (HBV) DNA from 58 of 169 hepatitis B surface antigen (HBsAg)-positive blood samples from Japanese blood donors and determined the nucleotide sequence encoding the small envelope protein. DNA from six (10%) of the samples had VEMs, but no missense mutations, such as G145R, were detected. Complete HBV genome sequences were obtained from 29 of the 58 samples; the viral genotype was A1 in one (3%), A2 in three (10%), B1 in nine (31%), B2 in five (17%), B4 in one (3%), and C2 in 10 (34%) samples. Tenofovir-resistance mutations were detected in two (7%) samples. In addition, several core promoter mutations, such as 1762A>T and 1764G>A, and a precore nonsense mutation, 1986G>A, which are risk factors for HBV-related chronic liver disease, were detected. These findings provide a baseline for future research and highlight the importance of ongoing monitoring of VEMs and drug resistance mutations in HBV DNA from HBsAg-positive blood donors without HBV antibodies.

Fig. 1

(a) Summary of the molecular analysis of the 169 samples from HBsAg-positive blood donors. (b) HBV virion. The size of the HBV genome is 3.2 kbp. The HBV genome consists of relaxed circular DNA. HBV genomic DNA is bound to a core protein, which is covered by an envelope protein. The envelope protein is composed of small, middle, and large envelope proteins. The large envelope protein is responsible for binding to hepatocytes. The small envelope protein has hydrophilic amino acids that form the epitope targeted by the hepatitis B vaccine. HBeAg derived from mRNA encoding precore-core protein circulates in the blood. HBeAg is not detected in the presence of a stop codon mutation (1896G>A) in the precore region. Vesicles composed of surface protein called "filaments" or "spheres" are observed in the blood of individuals with HBV infection.

Fig. 2

(a) Phylogenetic analysis based on HBV whole-genome sequences. (b) Phylogenetic analysis of HBV partial sequences encoding the envelope protein

Fig. 3

Amino acid mutations observed in the preS1/preS2/S coding region. The diagram shown above the alignment represents the amino acid sequence of the preS1/preS2/S region. The left side of the box represents the N-terminus of the amino acid sequence, and the right side represents the C-terminus. (a) Alignment of amino acid sequences of the small envelope protein. Green arrowheads indicate the major vaccine-escape mutations (VEMs). The green symbols indicate amino acid mutations that are known to be VEMs. (b) Alignment of amino acid sequences of the preS1 region. The HBV receptor recognition region is composed of amino acids 2–48 at the N-terminus; these amino acids are colored blue. "/" indicates an amino acid deletion. (c) Alignment of amino acid sequence of preS2. The purple arrowheads and symbols represent significant amino acid mutations.

Fig. 3

Amino acid mutations observed in the preS1/preS2/S coding region. The diagram shown above the alignment represents the amino acid sequence of the preS1/preS2/S region. The left side of the box represents the N-terminus of the amino acid sequence, and the right side represents the C-terminus. (a) Alignment of amino acid sequences of the small envelope protein. Green arrowheads indicate the major vaccine-escape mutations (VEMs). The green symbols indicate amino acid mutations that are known to be VEMs. (b) Alignment of amino acid sequences of the preS1 region. The HBV receptor recognition region is composed of amino acids 2–48 at the N-terminus; these amino acids are colored blue. "/" indicates an amino acid deletion. (c) Alignment of amino acid sequence of preS2. The purple arrowheads and symbols represent significant amino acid mutations.

Fig. 4

Schematic diagram of the HBV polymerase showing the positions of drug resistance mutations in blood samples from HBsAg-positive donors.. The reverse transcriptase domain comprises amino acids 349–692 (rt1 through rt344) of HBV polymerase. The rtA194T mutation is a tenofovir (TFV)-resistance mutation. The blue symbols indicate TFV-resistance mutations.

Fig. 5

Schematic diagram showing the main nucleic acid mutations in the core promoter (1575–1849, arrow) and precore coding (1814–) regions in viruses from blood samples obtained from HBsAg-positive donors. The blue boxes indicate amino acids from 1610 to 1660 on the left and 1749 to 1780 on the right. The green box indicates amino acids from 1850 to 1901. The blue arrowheads at the top of the nucleotide sequence alignment indicate the positions of the nucleotide mutations. The purple, dark blue, and light blue arrowheads to the left side of the alignment indicate samples with core promoter mutations. The green arrowheads indicate samples with precore mutations. Symbols in the same color as the arrowheads indicate nucleic acid mutations.

Fig. 6

Schematic diagram showing the amino acid sequence of the X protein, comprising the X-box binding domain, BH-3-like motif, and zinc-finger motif indicating the positions of amino acid mutations observed in viruses from blood samples obtained from HBsAg-positive blood donors. The boxes at the top indicate amino acids, and the arrows below indicate nucleotides. The purple symbols indicate amino acid mutations. The purple arrowheads at the top of the amino acid alignment indicate the H94Y, I127D/T/N, I127M, and K130M/V131I mutations. The purple arrowheads to the left of the amino acid alignment indicate samples with amino acid mutations.