Distinct phylogeographic distributions and frequencies of precore and basal core promoter mutations between HBV subgenotype C1 rt269L and rt269I types

Abstract

Hepatitis B virus (HBV) genotype C exhibits two distinct polymorphisms in its viral polymerase: rt269I and rt269L. Recently, we reported that there are distinct virological and clinical profiles between chronic patients with subgenotype C2 with the rt269I polymorphism and those with the rt269L polymorphism, with the latter being more closely related to liver disease severity. This study explored the phylogenetic and geographic distributions, as well as the mutation frequencies, of precore (T1858C/G1896A) and basal core promoter (BCP) (A1762T/G1764A) mutations between these two types within the HBV subgenotype C1. Analysis of 408 HBV/C1 full-genome sequences from GenBank revealed clear phylogenetic separation between rt269L and rt269I in subgenotype C1. Geographically, rt269I strains within subgenotype C1 are predominant in Southwest Asia (e.g., Thailand and Bangladesh), whereas rt269L strains are more common in East Asia and Southeast Asia (e.g., Vietnam, China, and Hong Kong). Notably, compared with rt269L in subgenotype C2, rt269I presented higher frequencies of the C1858 and BCP mutations but lower frequencies of the G1896A mutation. These findings suggest significantly distinct phylogeographic and mutational characteristics of the rt269L and rt269I types of subgenotype C1, impacting clinical outcomes and evolutionary trajectories.

Keywords: Genotype C; Hepatitis B virus (HBV); Sub-genotype C1; r269L Type; rt269I type.

Fig. 1

Phylogenetic distribution of rt269L and rt269I in various subgenotypes of HBV genotype C. This phylogenetic tree was constructed utilizing the whole genome sequences (3215 bp) of 1,402 HBV genotype C with three genotype A outgroups. This figure illustrates the distribution of leucine (L type, blue) and isoleucine (I type, red) at the amino acid of rt269 site across HBV genotype C in the inner ring. The outer ring represents different subgenotypes: C1, C2(1), C2(2), C2(3), C4, C5, C8, C10, and C11, each distinguished by a unique color as indicated in the legend. A FastTree support value is listed beneath the name of each main clade. This illustration demonstrates the prevalence and evolutionary relationships of rt269 variants within and between these HBV genotypes.

Fig. 2

Genetic diversity comparison of HBV subgenotype C according to nucleotide variants of rt269 Site. Violin plots illustrating the genetic diversity of the rt269L and rt269I variants within HBV sub-genotypes C1 and C2. The p-values indicate statistically significant differences in genetic diversity between the variants (**: p < 0.01, ****: p < 0.0001).

Fig. 3

Phylogenetic distribution of quasispecies of HBV subgenotype C2 in patients. This phylogenetic tree was based on 30 subgenotype C2 and 15 serum-derived reverse transcriptase regions (1032 bp), with three genotype A outgroups. illustrates the distribution of leucine (L type, blue) and isoleucine (I type, red) at the rt269 amino acid position. Ten sequences each of subgenotypes C2(1), C2(2), and C2(3) have been added, as well as HBV reverse transcriptase sequences from patients (B182, B184) with coexisting HBV rt269L type and rt269I type.

Fig. 4

Phylogenetic divergence of rt269L and rt269I in subgenotype C1. This phylogenetic tree was constructed with 408 HBV subgenotype C1 and 3 genotype A as outgroups. This figure shows that rt269L and rt269I in subgenotype C1 have diverged since the beginning of evolution. The inner ring represents nucleotide triplets, color-coded according to the legend on the right (e.g., TTG, TTA, CTC). The outer ring categorizes these sequences into two primary types: the L type, depicted in blue, and the I type, depicted in red. The support value is listed beneath the name of each main clade.

Fig. 5

Geographical distribution of the rt269L and rt269I types in HBV subgenotype C1. This figure illustrates the geographical distribution of leucine (L) and isoleucine (I) types at the rt269 site within the HBV subgenotype C1 across Asia. In the left panel, darker blue indicates higher frequencies of the L variant in Thailand, Cambodia, and Bangladesh. Similarly, the right panel uses darker blue to denote higher frequencies of the I variant in Vietnam, Hong Kong, and China.