Ultradeep Sequencing for Detection of Quasispecies Variants in the Major Hydrophilic Region of Hepatitis B Virus in Indonesian Patients

Abstract

Quasispecies of hepatitis B virus (HBV) with variations in the major hydrophilic region (MHR) of the HBV surface antigen (HBsAg) can evolve during infection, allowing HBV to evade neutralizing antibodies. These escape variants may contribute to chronic infections. In this study, we looked for MHR variants in HBV quasispecies using ultradeep sequencing and evaluated the relationship between these variants and clinical manifestations in infected patients. We enrolled 30 Indonesian patients with hepatitis B infection (11 with chronic hepatitis and 19 with advanced liver disease). The most common subgenotype/subtype of HBV was B3/adw (97%). The HBsAg titer was lower in patients with advanced liver disease than that in patients with chronic hepatitis. The MHR variants were grouped based on the percentage of the viral population affected: major, ≥20% of the total population; intermediate, 5% to <20%; and minor, 1% to <5%. The rates of MHR variation that were present in the major and intermediate viral population were significantly greater in patients with advanced liver disease than those in chronic patients. The most frequent MHR variants related to immune evasion in the major and intermediate populations were P120Q/T, T123A, P127T, Q129H/R, M133L/T, and G145R. The major population of MHR variants causing impaired of HBsAg secretion (e.g., G119R, Q129R, T140I, and G145R) was detected only in advanced liver disease patients. This is the first study to use ultradeep sequencing for the detection of MHR variants of HBV quasispecies in Indonesian patients. We found that a greater number of MHR variations was related to disease severity and reduced likelihood of HBsAg titer.

FIG 1

Relationship between the rate of variation in the major hydrophilic region (MHR) and the α-determinant region in different viral populations and severity of liver disease (chronic hepatitis and advanced liver disease). MHR variations in HBV quasispecies were detected by ultradeep sequencing and were divided into the groups according to the percentage of the total variant population: major (≥20% of the total population), intermediate (5% to <20% of the total population), and minor viral population (1% to <5% of the total population). The rate of variation was calculated as (number of changes in the region of each viral population/total number of sites in the region) × 100%. For example, in one patient with 10 amino acid substitutions in the 70-amino-acid MHR existing in the minor viral population, the rate of variation, the calculation was (10/70) × 100% = 14.29%. *, P < 0.05; **, P > 0.05.

FIG 2

Distribution of the MHR variations in the major viral population. The amino acid sequences of the MHR (amino acids 100 to 169) in samples from patients with chronic hepatitis (n = 11) and advanced liver disease (n = 19) were compared with the consensus sequences of genotype B and other genotypes (genotypes A, C, D, E, F, and G). The amino acid substitutions were distinguished based on the percentage of the variant population in a quasispecies. Substitutions were categorized as being of the major viral population if they were present in ≥20% of the total population. The major viral population was subdivided into the mixed population (20% to <80% of the total population) or the dominant population (≥80% of the total population [letters in bold type]). CH, chronic hepatitis; LC, liver cirrhosis; HCC, hepatocellular carcinoma. The numbers in parentheses are the % of the variant population.

FIG 3

Frequency of MHR variants in the mixed population (present in 20% to <80% of the total viral population). The number of MHR variants that exist in a mixed population was compared between samples from patients with chronic hepatitis (n = 11) and advanced liver disease group (n = 19). MHR, major hydrophilic region. *, P < 0.05.

FIG 4

Distribution of the MHR variations in the intermediate viral population. The amino acid sequences of the MHR (amino acids 100 to 169) in samples from patients with chronic hepatitis (n = 11) and advanced liver disease (n = 19) were compared with the consensus sequences of genotype B and other genotypes (genotypes A, C, D, E, F, and G). The amino acid substitutions were distinguished based on the percentage of the variant population in a quasispecies. Substitutions were categorized as being of the intermediate viral population if they were present in 5% to <20% of the total population. CH, chronic hepatitis; LC, liver cirrhosis; HCC, hepatocellular carcinoma. The numbers in parentheses are the % of the variant population.