1 Division of Epidemiology, Department of Infectious Diseases, Oita Medical University, Hasama-machi, Oita 879-5593, Japan
4 Research Center for Asian and Caribbean Diseases, Oita Medical University, Hasama-machi, Oita 879-5593, Japan
2 First Department of Internal Medicine, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan
3 Department of Virology, Faculty of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan
Correspondence
Shao-Ping Ma (at Oita Medical University)
masp{at}oita-med.ac.jp
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ABSTRACT |
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The nucleotide sequence data reported in this paper were deposited in DDBJ/EMBL and GenBank under the accession number AB088679.
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MAIN TEXT |
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Based on geographical origin and genomic divergence, HDV strains are divided into at least three genotypes: I, II and III (Casey et al., 1993; Lai, 1995
). Recently, Wu et al. (1998)
reported a novel strain of genotype II in Taiwan and proposed genotypes IIa and IIb. The Miyako Islands, which are located in the southernmost part of Japan, about 390 km east of Taiwan, are one of the areas in Japan that is endemic for HDV (Sakugawa et al., 1995
, 1997a
, b
, 1999
; Nakasone et al., 1998
; Arakawa et al., 2000
). The majority of patients with chronic HDV-related liver disease in the Miyako Islands exhibit a relatively mild course of infection that evolves to a quiescent cirrhotic condition (Sakugawa et al., 1997a
). Sakugawa et al. (1999)
sequenced the 645 nt of HDAg (position 9401584; numbering according to Wang et al., 1987
) of six strains of genotype II, collected from patients with HDV-related chronic liver disease in the Miyako Islands, and reported the presence of genotype IIb. However, the entire genome sequence of the Miyako isolate has not been reported. Hence, we describe the entire nucleotide sequence of HDV isolated in the Miyako Islands.
The serum sample L215 was chosen from a panel of sera collected in 1992 from cirrhotic patients and stored at the University of the Ryukyus, Okinawa, Japan. L215 serum was positive for HBsAg and negative for hepatitis B e antigen (HBeAg), while it was positive for anti-HBe antibodies and HDV RNA. RNA was extracted from the serum using the acid guanidinium/phenol extraction method (Chomczynski & Sacchi, 1987) and was dissolved in 30 µl DEPC-treated distilled water. The RNA was denatured at 94 °C for 2 min and then reverse-transcribed at 42 °C for 1 h in a mixture containing RNA template, 50 mM Tris/HCl (pH 8·3), 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 250 nM each of four dNTPs, 100 pmol antigenomic-sense primer (Fig. 1
), 10 units RNaseout (GibcoBRL) and 100 units RiverTraAce reverse transcriptase (Toyobo). PCR was carried out in a buffer containing 10 mM Tris/HCl (pH 8·9), 0·1 M KCl, 1·5 mM MgCl2, 250 nM each of four dNTPs, 100 pmol genomic-sense primer and 4 units Tth DNA polymerase (Boehringer Mannheim), and performed for 40 cycles; each cycle consisted of 95 °C for 1 min, 42 °C for 1·5 min and 72 °C for 1 min. The PCR product was analysed by electrophoresis in 1·5 % agarose gels and visualized by staining with ethidium bromide. The specific PCR products were cut out from the agarose gels and cleaned using the QIAquick Gel Extraction kit (Qiagen). Cleaned products were then cloned into the pCR2.1 TA cloning vector (Invitrogen), according to the manufacturer's instructions. Nucleotide sequencing was performed using the Dye Terminator Cycle Sequencing FS Ready Reaction kit (Applied Biosystems), according to the manufacturer's instructions. Three clones of each PCR product were sequenced. Sequencing data were analysed using DNASIS, version 3.6 (Hitachi Software Engineering).
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Phylogenetic analysis was performed using the UPGMA method, both on the entire HDV nucleotide sequences of L215 and five previously reported strains, including three genotypes (Fig. 2a). The L215 strain showed the highest homology (80·9 %) to the Taiwan genotype IIb strain (TWD62), while it was distantly related (69·2 %) to genotype IIa and more distantly related to genotypes I and III (65·5 and 54·8 %, respectively). A more inclusive phylogenetic analysis using the available six partial HDV sequences of Miyako isolates (nt 9401584) was performed also (Fig. 2b
). Results demonstrated that all of the Miyako isolates clustered into genotype IIb.
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The RNA-editing event is an important control point in the HDV life cycle because it results both in packaging of viral RNA and in inhibiting HDV replication (Casey et al., 1992). Thus, the predicted secondary structures of the nucleotide pairs between positions 578 and 583, and 1022 and 1017 (nucleotide positions are according to the L215 strain) of three different genotypes were compared. There were at least three WatsonCrick pairs on either side of the RNA-editing site within genotype I and III sequences, although the secondary structure of this region was significantly different between these two strains (Fig. 3
). Our predicted secondary structures also agreed with previous reports (Casey et al., 1992
; Nakano et al., 2001
). The published genotype II sequences have four conserved WatsonCrick pairs on the right-hand side of the RNA-editing site. However, there were only two GC pairs on the left-hand side. Ivaniushina et al. (2001)
reported that genotype II(a) isolates from Yakutia, Russia, have two conserved GC pairs on the left-hand side of the RNA-editing site. However, the L215 strain (genotype IIb) had only one GC pair in this position, which might form a less strong secondary structure. Hsu et al. (2002)
reported that the editing efficiency of HDV genotype I is higher than that of genotype II. They reported also that the nucleotide and structural changes surrounding the RNA-editing site might be responsible for the lower RNA-editing efficiency of genotype II strains. Further study is needed to evaluate the RNA-editing efficiency of the Miyako isolate L215 strain.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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---|
Bergmann, K. F. & Gerin, J. L. (1986). Antigens of hepatitis delta virus in the liver and serum of humans and animals. J Infect Dis 154, 702706.[Medline]
Bonino, F., Heermann, K. H., Rizzetto, M. & Gerlich, W. H. (1986). Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope. J Virol 58, 945950.[Medline]
Casey, J. L., Bergmann, K. F., Brown, T. L. & Gerin, J. L. (1992). Structural requirements for RNA editing in hepatitis delta virus: evidence for a uridine-to-cytidine editing mechanism. Proc Natl Acad Sci U S A 89, 71497153.[Abstract]
Casey, J. L., Brown, T. L., Colan, E. J., Wingnall, F. S. & Gerin, J. L. (1993). A genotype of hepatitis D virus that occurs in northern South America. Proc Natl Acad Sci U S A 90, 90169020.[Abstract]
Chomczynski, P. & Sacchi, N. (1987). Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162, 156159.[CrossRef][Medline]
Gerin, J. L., Casey, J. L. & Purcell, R. H. (2001). Hepatitis delta virus. In Fields Virology, 4th edn, vol. II, pp. 30373050. Edited by D. M. Knipe & P. M. Howley. Philadelphia: Lippincott Williams & Wilkins.
Hsu, S.-C., Syu, W.-J., Sheen, I.-J., Liu, H.-T., Jeng, K.-S. & Wu, J.-C. (2002). Varied assembly and RNA editing efficiencies between genotype I and II hepatitis D virus and their implications. Hepatology 35, 665672.[CrossRef][Medline]
Imazeki, F., Omata, M. & Ohto, M. (1991). Complete nucleotide sequence of hepatitis delta virus RNA in Japan. Nucleic Acids Res 19, 5439.[Medline]
Ivaniushina, V., Radjef, N., Alexeeva, M., Gault, E., Semenov, S., Salhi, M., Kiselev, O. & Deny, P. (2001). Hepatitis delta virus genotypes I and II cocirculate in an endemic area of Yakutia, Russia. J Gen Virol 82, 27092718.
Lai, M. M. C. (1995). The molecular biology of hepatitis delta virus. Annu Rev Biochem 64, 259286.[CrossRef][Medline]
Lee, C.-M., Changchien, C.-S., Chung, J.-C. & Liaw, Y.-F. (1996). Characterization of a new genotype II hepatitis delta virus from Taiwan. J Med Virol 49, 145154.[CrossRef][Medline]
Makino, S., Chang, M.-F., Shieh, C.-K., Kamahora, T., Vannier, D. M., Govindarajan, S. & Lai, M. M. C. (1987). Molecular cloning and sequencing of a human hepatitis delta () virus RNA. Nature 329, 343346.[CrossRef][Medline]
Monjardino, J. (1996). Replication of hepatitis delta virus. J Viral Hepat 3, 163166.[Medline]
Nakano, T., Shapiro, C. N., Hadler, S. C., Casey, J. L., Mizokami, M., Orito, E. & Robertson, B. H. (2001). Characterization of hepatitis D virus genotype III among Yucpa Indians in Venezuela. J Gen Virol 82, 21832189.
Nakasone, H., Sakugawa, H., Shokita, H. & 8 other authors (1998). Prevalence and clinical features of hepatitis delta virus infection in the Miyako Islands, Okinawa, Japan. J Gastroenterol 33, 850854.[CrossRef][Medline]
Rizzetto, M., Canese, M. G., Arico, S., Crivelli, O., Trepo, C., Bonino, F. & Verme, G. (1977). Immunofluorescence detection of new antigen-antibody system (/anti-
) associated to hepatitis B virus in liver and in serum of HBsAg carriers. Gut 18, 9971003.[Abstract]
Sakugawa, H., Nakasone, H., Shokita, H., Nakayoshi, T., Kinjo, F., Saito, A., Yamashiro, A. & Miyagi, Y. (1995). Seroepidemiological study of hepatitis delta virus infection in Okinawa, Japan. J Med Virol 45, 312315.[Medline]
Sakugawa, H., Nakasone, H., Shokita, H. & 10 other authors (1997a). Seroepidemiological study on hepatitis delta virus infection in the Irabu Islands, Okinawa, Japan. J Gastroenterol Hepatol 12, 299304.[Medline]
Sakugawa, H., Nakasone, H., Kawakami, Y. & 12 other authors (1997b). Determination of hepatitis delta virus (HDV)-RNA in asymptomatic cases of HDV infection. Am J Gastroenterol 92, 22322236.[Medline]
Sakugawa, H., Nakasone, H., Nakayoshi, T. & 7 other authors (1999). Hepatitis delta virus genotype IIb predominates in an endemic area, Okinawa, Japan. J Med Virol 58, 366372.[CrossRef][Medline]
Saldanha, J. A., Thomas, H. C. & Monjardino, J. P. (1990). Cloning and sequencing of RNA of hepatitis delta virus isolated from human serum. J Gen Virol 71, 16031606.[Abstract]
Wang, K.-S., Choo, Q.-L., Weiner, A. J. & 7 other authors (1986). Structure, sequence and expression of the hepatitis delta () viral genome. Nature 323, 508514.[Medline]
Wang, K.-S., Choo, Q.-L., Weiner, A. J. & 7 other authors (1987). Structure, sequence and expression of the hepatitis delta () virus genome. Nature 328, 456.
Weiner, A. J., Choo, Q.-L., Wang, K.-S., Govindarajan, S., Redeker, A. G., Gerin, J. L. & Houghton, M. (1988). A single antigenomic open reading frame of the hepatitis delta virus encodes the epitope(s) of both hepatitis delta antigen polypeptides p24 and p27
. J Virol 62, 594599.[Medline]
Wu, J.-C., Chiang, T.-Z. & Sheen, I.-J. (1998). Characterization and phylogenetic analysis of a novel hepatitis D virus strain discovered by restriction fragment length polymorphism analysis. J Gen Virol 79, 11051113.[Abstract]
Yang, A., Papaioannou, C., Hadzyannis, S., Thomas, H. & Monjardino, J. (1995). Base changes at positions 1014 and 578 of delta virus RNA in Greek isolates maintain base pair in rod conformation with efficient RNA editing. J Med Virol 47, 113119.[Medline]
Received 7 August 2002;
accepted 23 September 2002.