A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes A through F of hepatitis B virus (HBV). This assay system is considered to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys.Hepatitis B virus (HBV) is a well-known agent of acute and chronic hepatitis, with an estimated 350 million chronic carriers around the world. HBV has a circular and partially doublestranded DNA genome of 3.2 kb containing four overlapping open reading frames. HBV strains isolated worldwide have been classified into six genomic groups deduced from genome comparisons and designated genotypes A to F (3, 6, 7). The HBV genotypes have a characteristic geographic distribution. HBV genotyping by phylogenetic analysis based on nucleotide sequences produces the most reliable and certain genotyping results. However, this is not an appropriate method for largescale genotyping. On the other hand, several groups have reported the genotyping of HBV by the restriction fragment length polymorphism method (1, 2, 4, 8). However, their methods were not so sensitive and specific compared with our PCR genotyping method. In this paper, we report a simpler, more rapid, and more specific genotyping system for HBV involving PCR using type-specific primers.A total of 55 HBV DNA-positive serum samples obtained from individuals in six different countries, including Japan, Vietnam, the United States, Egypt, Ghana, and Bolivia, were used for the evaluation of our genotyping system. We selected the HBV DNA-positive samples by nested PCR. The sequences of PCR primers used in this study are shown in Table 1. The first-round PCR primers (outer primer pairs) and second-round PCR primers (inner primer pairs) were designed on the basis of the conserved nature of nucleotide sequences in regions of the pre-S1 through S genes, irrespective of the six HBV genotypes. P1 (sense) and S1-2 (antisense) were universal outer primers (1,063 bases). B2 was used as the inner primer (sense) with a combination called mix A for genotypes A, B, and C. Mix A consisted of antisense primers BA1R (type A specific), BB1R (type B specific), and BC1R (type C specific). B2R was used as the inner primer (antisense) with a combination called mix B for genotypes D, E, and F. Mix B consisted of sense primers BD1 (type D specific), BE1 (type E specific), and BF1 (type F specific). These primer combinations for second-round PCR were designed on the basis of the differences in the sizes of the genotype-specific bands. The type-specific primers were designed on the basis of the conserved nature of those sequences within a genotype and on the basis of their poor homology with the sequences derived from other HBV genotypes. The strategy for HBV genotyping is illustrated in Fig.

Transfusion-transmissible infections among 808 blood donors in Ghana were investigated in 1999. Antibody seroprevalences of 3.8, 0.7, 8.4, and 13.5%, respectively, for human immunodeficiency virus, human T-cell lymphotrophic virus type 1, hepatitis C virus (HCV), and Treponema pallidum were obtained. The seroprevalence of HCV infection was confirmed to be 0.9% after supplementary testing, and the transfusion risk potential of these pathogens was demonstrated.

Hepatitis G virus (HGV) isolates obtained from 20 Myanmarese and 10 Vietnamese subjects were analyzed. A cluster of isolates not belonging to any known genotype of HGV was found in five Myanmarese subjects and three Vietnamese subjects by phylogenetic analysis, and we classified this new genotype as type 4. These results revealed that the HGV genome can be classified into at least four major genotypes.

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