Typing Hepatitis B Virus by Homology in Nucleotide Sequence: Comparison of Surface Antigen Subtypes

Abstract

SUMMARYThe complete nucleotide sequences of the DNA of three hepatitis B virus (HBV) genomes of subtype adw, cloned from plasma samples of asymptomatic carriers living in the mainland and Okinawa Prefecture of Japan and Indonesia were determined. All three comprised 3215 bp and differed in sequence by only 3.9 to 5.6%. When these isolates were compared with the reported sequences of two HBV genomes of the same subtype derived from American carriers, however, the differences were greater (8.3 to 9.3%) to an extent comparable with the nucleotide divergence between an HBV genome of subtype adw and that of a heterotypic subtype, such as adr, ayw or ayr. A total of 18 HBV genomes of various subtypes, including the three described here, 10 reported previously and five unpublished ones, were classified into four groups based on an inter-group divergence in nucleotide sequence of 8 ~o or greater: group A (two adw genomes), group B (four adw), group C (three adw, four adr and one ayr) and group D (four ayw). Thus, the nine genomes of HBV subtype adw were distributed into three groups with considerably different sequences. These results indicate that the four major antigenically defined subtypes of envelope polypeptide do not reflect true genotypic variation of HBV. The fact that dtoy, as well as w to r, subtypic change can be induced by an A --, G point mutation at nucleotides 365 and 479 in the S gene, respectively, supports this view.

Reactivity of 13 in vitro expressed hepatitis B surface antigen variants in 7 commercial diagnostic assays

“…Five microliters of extracted DNA was amplified in a 50-µL solution containing 1 U Taq polymerase (Life Technologies, Paisley, UK), 1.4 µmol/L TaqStart antibody (Clontech Laboratories Inc., Palo Alto, CA), 0.25 mmol/L dNTPs (Pharmacia, St. Albans, UK), 2.5 mmol/L MgCl 2 , 5 µL of 10 ϫ PCR buffer, and 25 pmol/L of primers S1 (56F, 5Ј-CCTGCTGGTGGCTCCAGTTC-3Ј) and S2Na (1003R, 5Ј-CCACAATTCKTTGACATACTTTCCA-3Ј, where Kϭ G or T), for 5 cycles of 95°C for 60 seconds, 55°C for 60 seconds, and 72°C for 90 seconds followed by 35 cycles with the denaturation temperature reduced to 90°C. All primers are numbered according to the system used by Okamoto et al 22 One microliter of first round PCR product was reamplified as described above with the nested primers, S6C (129F, 5Ј-GCACACGGAATTCCGAGGACTGGGGACCCTG-3Ј) and S7D (842R, 5Ј-GACACCAAGCTTGGTTAGGGTTTAAATGTATACC-3Ј) for 5 cycles of 95°C for 60 seconds, 55°C for 75 seconds, and 72°C for 90 seconds followed by 25 cycles, with the denaturation temperature reduced to 90°C. DNA fragments of expected size, were extracted from 1% agarose gel using Geneclean II kit (Bio 101, La Jolla, CA).…”

Tracing the history of hepatitis B virus genotype D in western Japan

“…HBV has approximately 3,200 bases that can be divided into several genotypes by sequence divergence, with 8 genotypes (A-H) reported [Okamoto et al, 1988;Norder et al, 1994;Stuyver et al, 2000;Arauz-Ruiz et al, 2002]. These genotypes have a distinct geographical distribution, with genotypes A (HBV/A) and HBV/ D predominant in Europe, Middle East, Central Asia, Siberia, and America, HBV/B and HBV/C in East Asia, and HBV/E in Africa.…”

[No Title Available]

“…HBV isolates have been classified into eight genotypes (A to H), considering a divergence of more than 8% in the entire virus genomic sequence (Okamoto et al 1988, Norder et al 1994, Stuyver et al 2000, Arauz-Ruiz et al 2002. HCV is a positive-sense, single-stranded RNA virus that presents a high mutation rate (10 -3 substitutions/site/year).…”