Molecular and phylogenetic analyses suggest an additional hepatitis B virus genotype "I"

Abstract

A novel hepatitis B virus (HBV) strain (W29) was isolated from serum samples in the northwest of China. Phylogenetic and distance analyses indicate that this strain is grouped with a series of distinct strains discovered in Vietnam and Laos that have been proposed to be a new genotype I. TreeOrderScan and GroupScan methods were used to study the intergenotype recombination of this special group. Recombination plots and tree maps of W29 and these putative genotype I strains exhibit distinct characteristics that are unexpected in typical genotype C strains of HBV. The amino acids of P gene, S gene, X gene, and C gene of all genotypes (including subtypes) were compared, and eight unique sites were found in genotype I. In vitro and in vivo experiments were also conducted to determine phenotypic characteristics between W29 and other representative strains of different genotypes obtained from China. Secretion of HBsAg in Huh7 cells is uniformly abundant among genotypes A, B, C, and I (W29), but not genotype D. HBeAg secretion is low in genotype I (W29), whose level is close to genotype A and much lower than genotypes B, C, and D. Results from the acute hydrodynamic injection mouse model also exhibit a similar pattern. From an overview of the results, the viral markers of W29 (I1) in Huh7 cells and mice had a more similar level to genotype A than genotype C, although the latter was closer to W29 in distance analysis. All evidence suggests that W29, together with other related strains found in Vietnam and Laos, should be classified into a new genotype.

Figure 1. Phylogenetic analysis of complete genomes (left) and P gene products (right).

The sequences include all the reference sequences, representative Vietnam and Laos' sequence and W29 strains. The putative genotype I strains are separated as an independent clade. Sequences of Genotype A-H have been collapsed into black triangles.

Figure 2. Mean distances between full-length sequence of genotype A-H, genotype chimpanzee (Chi) and genotype I, generated by MEGA4 package.

Values on the diagonal are the mean distances within each genotype. The calculation is based on the Maximum Composite Likelihood distance correction method.

Figure 3. Group Scan Analysis.

Eight HBV strains were submitted to Group Scan program and calculated against the reference groups of nonrecombinant HBV sequences from human genotypes A to H and nonhuman ape-derived variants (n = 322) .

Figure 4. TreeOrder Scan of nonrecombinant HBV sequences.

It shows positions of individual sequences (y axis) in phylogenetic trees generated from sequential 250-base sequence fragments, incrementing by 25 bases (midpoints indicated in x axis). Changes in sequence orders resulting from changes in phylogeny at the 70% bootstrap level are shown. Sequences are color coded by genotype, as indicated by labels in left margins: genotype A, red; B, green; C, yellow; D, blue; E, purple; F, pink; G, pale yellow; H, brown; chimpanzee (Ch), light blue; woolly monkey (outgroup on line 1), black. The boundaries of sequence groupings with 70% or greater bootstrap support are indicated with black horizontal lines; for clarity, only clades between genotypes (except for group I) and with six or more members were demarcated. For comparison, the TreeOrder Scan was aligned with a scale genome diagram of HBV (upper panel).

Figure 5. Consensus amino acid sequences of 4 gene products of HBV (P, S, X and Pre-core/core) were arrayed according to genotypes and subtypes, with identical columns excluded.

Consensus sequences were derived from the 322 reference sequences. For page view limited, unique sites in subtype were also omitted and the sequence number of amino acids was indicated alternately on the top and bottom of the aligned matrix. Genotype-specific amino acids were highlighted with different colors and subtype-specific amino acids were only displayed in corresponding colored letters.

Figure 6. Comparison of expression levels of HBV markers between different genotypes in vitro and in vivo.

Fig. a, b, c, d: HBsAg, HBeAg, HBV DNA and HBcAg levels in Huh7 cell culture supernatant. SEAP reporter plasmid was co-transfected with each HBV plasmid as an internal control. The SEAP activity of the first group (transfected with N10 (Ae) plasmid) was set as 1.0 and served as a reference, and the other groups were calculated as relative SEAP activity. The presented data are a ratio of actual value to the relative SEAP activity value. Fig. e, f: Dynamic curve of HBsAg and HBeAg expression levels in serums of hydrodynamic-injected mice. For each HBV plasmid, five mice were repeated. Due to the limited serum resources, each sample was diluted 10-fold for mensuration.