1 Saitama Red Cross Blood Center, 1370-12, Takahagi, Hidaka, Saitama-ken 350-1213, Japan
2 Division of Virology, Department of Infection and Immunity, Jichi Medical School, Tochigi-ken 329-0498, Japan
Correspondence
Akira Yoshikawa
yoshikawa{at}saitama.bc.jrc.or.jp
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence reported in this paper is AB179747.
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MAIN TEXT |
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On July 1 1999, the Japanese Red Cross (JRC) started a nationwide screening by using the nucleic acid amplification test (NAT) of pools of 500 sera. From February 1 2000, the pool size was reduced to 50 (Mine et al., 2003). By March 31 2003, 19 454 693 units in the 500- or 50-sample pools had been tested using NAT screening. During this period, 349 HBV DNA-, 53 hepatitis C virus RNA- and 6 human immunodeficiency virus type 1 RNA-positive donors were found.
The NAT screening system in Japan has been reported previously (Ohtake & Nishioka, 2000; Mine et al., 2003
; Minegishi et al., 2003
). Samples that are serologically positive and have elevated alanine aminotransferase (>60 IU l1) are excluded from NAT screening. All voluntarily donated blood in the JRC is qualified by a questionnaire administered by the JRC blood centres.
HBV DNA loads were calculated from the working curve (107, 106, 105, 104, 103, 102 copies ml1) produced by domestic standard samples that were prepared based on the international standard samples (NIBSC). Quantification was carried out using Sequence Detector version 1.7 (PE Applied Biosystems). The data show the mean of quadruplicate tests. The results have already been reported (Minegishi et al., 2003).
The genotypes of HBV are classified based on an intragroup nucleotide divergence of up to 4·2 % of the S-gene sequences or in some cases up to 8·0 % of complete genomes (Norder et al., 1992, 1993
, 1994
; Okamoto et al., 1988
). Precore mutation (from G to A at nt 1896) or core promoter mutations (from A to T at nt 1762 and/or from G to A at nt 1764) were detected and characterized using the methods of Okamoto et al. (1990
, 1994)
. Out of 349 HBV NAT-positive donors, 17 had precore mutants (genotype B, 7 donors; genotype C, 10 donors), 31 had core promoter mutants (genotype A, 2 donors; genotype C, 29 donors) and 13 had mutants with both precore and core promoter mutations (genotype B, 1 donor; genotype C, 12 donors). Sequencing was carried out directly by using a BigDye Terminator Cycle Sequencing kit and ABI Prism 3100 Genetic Analyser (PE Applied Biosystems). To analyse the sequences, Sequencher Mac version 4.1 (Hitachi Software Engineering) or GENETYX-MAC version 9.0 (Software Development) was used.
In Japan, genotypes C and B have been dominant among HBV-viraemic patients. However, recently genotype A has increased in prevalence (Kobayashi et al., 2002; Koibuchi et al., 2001
; Orito et al., 2001
). Out of 349 HBV NAT-positive donations, there were 40 cases of genotype A (11·5 %), 39 cases of genotype B (11·2 %), 264 cases of genotype C (75·6 %), 5 cases of genotype D (1·4 %) and 1 case of genotype H (0·3 %). Genotypes E, F and G were not detected by NAT.
Genotype H was recognized for the first time in Japan by comparing the full sequence (3215 nt) of NAT-positive sample (02094) against LSA2523 (Arauz-Ruiz et al., 2002). To determine the full sequence of HBV DNA genotype H, three overlapping regions were amplified and subjected to sequence analysis: nt 1668, nt 4791796, nt 16982381, and nt 23323215. The nucleotide sequence identity between 02094 and LSA2523 was found to be 99·3 %. The dendrogram of the strain 02094 was generated using reference strains of genotypes AG and seven strains of genotype H made available in GenBank/EMBL/DDBJ through UPGMA methods (Nei, 1987
) using GENETYX-MAC version 9.0 (Fig. 1
a). Phylogenetic analysis performed on the S gene of genotypes AH, including the 14 genotype H S-gene sequences available in GenBank/EMBL/DDBJ, is shown in Fig. 1(b)
.
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Between the 02094 strain obtained in the present study and the seven other reported strains of the same genotype (genotype H), there were differences ranging from 24 to 84 nt and from 10 to 47 aa in the entire genome. Of note, the 02094 isolate differed from the 1853NIC, 2928NIC, LAS2523, US2065 and US1122 by only 04 aa, but differed from the US10 and US1778 isolates by 13 or 20 aa in the core protein.
Amino acid sequences of the S region (226 aa, 678 nt; 15 strains) were compared between strain 02094 and known strains (Table 1, Fig. 2
.). The characteristic amino acids of the S region in genotype F and H were Val18, Leu61, Glu178, Cys183, Leu193, Ile198, Cys206, Cys220 and Ser225. The remarkable amino acids in genotype H were Val44, Pro45, Gly47 and Ala224 (Fig. 2
). Specific amino acid in strain 02094 was K30.
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ACKNOWLEDGEMENTS |
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Received 28 October 2004;
accepted 9 December 2004.
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