A genetic variant of hepatitis B virus divergent from known human and ape genotypes isolated from a Japanese patient and provisionally assigned to new genotype J

Abstract

Hepatitis B virus (HBV) of a novel genotype (J) was recovered from an 88-year-old Japanese patient with hepatocellular carcinoma who had a history of residing in Borneo during the World War II. It was divergent from eight human (A to H) and four ape (chimpanzee, gorilla, gibbon, and orangutan) HBV genotypes, as well as from a recently proposed ninth human genotype I, by 9.9 to 16.5% of the entire genomic sequence and did not have evidence of recombination with any of the nine human genotypes and four nonhuman genotypes. Based on a comparison of the entire nucleotide sequence against 1,440 HBV isolates reported, HBV/J was nearest to the gibbon and orangutan genotypes (mean divergences of 10.9 and 10.7%, respectively). Based on a comparison of four open reading frames, HBV/J was closer to gibbon/orangutan genotypes than to human genotypes in the P and large S genes and closest to Australian aboriginal strains (HBV/C4) and orangutan-derived strains in the S gene, whereas it was closer to human than ape genotypes in the C gene. HBV/J shared a deletion of 33 nucleotides at the start of preS1 region with C4 and gibbon genotypes, had an S-gene sequence similar to that of C4, and expressed the ayw subtype. Efficient infection, replication, and antigen expression by HBV/J were experimentally established in two chimeric mice with the liver repopulated for human hepatocytes. The HBV DNA sequence recovered from infected mice was identical to that in the inoculum. Since HBV/J is positioned phylogenetically in between human and ape genotypes, it may help to trace the origin of HBV and merits further epidemiological surveys.

FIG. 1.

Phylogenetic tree constructed on the entire genome sequences of 44 HBV isolates representing four ape and eight human genotypes. A woolly monkey HBV isolate serves as an outgroup. The HBV/J isolate (JRB34) is indicated by an arrowhead, and the genetic distance is indicated by a bar below.

FIG. 2.

Complete genome scanning carried by PHYLIP, the phylogeny inference package implemented in the Simmonic software, for the JRB34 strain versus 228 selected nonrecombinant HBV genotypes (HBV/Ba and HBV/I not included) reference strains grouped by genotype. Kimura two-parameter distance model (A) and grouping scan (B) were determined with a 300-nt size window sliding by an increment of 15 nucleotides. The x axis indicates the genome position (corresponding to the midpoint of the scanning fragment), and the y axis indicates the mean distances between JRB34 and reference groups (A). Phylogenetic association (y axis) was evaluated throughout entire HBV genome (x axis) with the same window and step size parameters (B). The association value below 0.5 was considered to represent an outgroup. The open reading frame map is shown schematically at the top of the figure.

FIG. 3.

Phylogenetic tree constructed on the preS/S gene (A) and C gene (B) sequences of 44 HBV isolates representing four ape and eight human genotypes. A woolly monkey HBV isolate serves as an outgroup. The HBV/J isolate (JRB34) is indicated by an arrowhead, and an HBVC4 isolate from Australian aborigine is indicated by an open triangle. The genetic distance is indicated by a bar below.

FIG. 4.

Comparison of the amino acid sequence in the preC gene and carboxy-terminal amino acid sequences in the C gene of HBV isolates of various genotypes. The sequence of the HBV/J isolate (JRB34) is indicated at the top. Dots represent amino acids shared by JRB34, and a dash indicates the deletion of an amino acid. The sequence of the arginine-rich domain bearing the binding site with HBV DNA is boxed.

FIG. 5.

Comparison of amino acid sequences of the preS/S gene among HBV isolates of various genotypes. The sequence of the HBV/J isolate (JRB34) is indicated at the top. Dots represent amino acids shared by JRB34, and a dash indicates the deletion of an amino acid. The sequence from positions 101 to 156 forming loops, bearing the common antigenic determinants of HBsAg, is boxed.

FIG. 6.

Markers of HBV infection in two chimeric mice inoculated with the HBV/J isolate (JRB34). The levels of HBV DNA are illustrated in panel A, and those of HBsAg and HBcrAg are illustrated in panel B. Values represent the means for two mice.

FIG. 7.

(A and B) Immunofluorescent staining of a frozen liver section of a chimera mouse inoculated with the HBV/J isolate (JRB34). HBcAg is stained in panel A, and human albumin is stained in panel B. (C) Colocalization of HBcAg and human albumin is revealed by double staining. (D to F) HBV-uninfected mouse liver shows that only human albumin is stained.