Department of Pathology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan1
Division of Gastroenterology, International Medical Center of Japan, Tokyo, Japan2
Author for correspondence: Kenji Abe. Fax +81 3 5285 1189. e-mail kenjiabe{at}nih.go.jp
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Abstract |
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Two different HGV isolates, recovered from serum specimens of a Myanmarese subject [29-year-old female, infected with HCV and human immunodeficiency virus (HIV)] and a Vietnamese subject (17-year-old female, infected with hepatitis B virus and HIV), respectively, were cloned and sequenced. These isolates were designated HGV-MY14 and HGV-VT48, respectively. The methods for RNA extraction from serum, amplification of HGV cDNA and cloning strategy have been described previously (Abe et al., 1997 ; Kaneko et al., 1998
). The sequence of PCR primers is listed in Table 1
. The terminal sequence of the 3'-UTR of the HGV genome was isolated by a rapid amplification of the cDNA ends (RACE) kit (5'/3' RACE kit; Boehringer Mannheim). This method is based on a one-sided PCR procedure. For the terminal sequence of the 3'-UTR, a poly(A) tail, which is lacking in HGV/GBV-C, was added to RNA extracted from serum by poly(A) polymerase (TaKaRa Biochemicals) before the RACE step. Briefly, RNA extracted by SepaGene RV-R (Sanko Junyaku) was dissolved in 10 µl RNase-free H2O and mixed with 40 µl buffer containing 50 mM TrisHCl, pH 8·0, 10 mM MgCl2, 2·5 mM MnCl2, 250 mM NaCl, 1 mM DTT, 0·5 mg/ml BSA, 0·1 mM ATP and 1 U poly(A) polymerase. After incubation at 37 °C for 1 h, poly(A)-tailed RNA was ethanol-precipitated and subjected to the 3' RACE technique as directed by the manufacturer. Amplification conditions included pre-incubation at 95 °C, 10 min activation of AmpliTaq Gold DNA polymerase (Perkin Elmer), followed by 35 cycles of PCR (94 °C, 20 s; 60 °C, 20 s; 72 °C, 60 s; with a final extension for 10 min, 72 °C). The recovered PCR products were re-amplified with internal HGV-specific primers. PCR products were separated by 12% agarose gel electrophoresis and purified using the QIAquick gel extraction kit (Qiagen). Recovered PCR products were subcloned using a pBluescript II SK(-) vector (Stratagene) via the EcoRV site. Alternatively, purified PCR products were subjected to direct sequencing from both directions using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer). Sequences of amplified cDNA were determined using an ABI model 373A sequencer (Applied Biosystems).
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The full-length genome sequences of HGV-MY14 and HGV-VT48 were obtained from serum samples from Myanmarese and Vietnamese subjects, respectively. Both isolates were composed of 9228 nt and contained a long open reading frame spanning 8529 nt and encoding 2843 aa flanked by putative 5'-UTR (nt 1389 for MY14 and nt 1387 for VT48) and 3'-UTR (nt 89199228 for MY14 and nt 89179228 for VT48) without a poly(U) stretch or poly(A) tail. Based on the predicted cleavage sites indicated by Mushahwar and colleagues (Muerhoff et al., 1995 ; Leary et al., 1996
), we obtained the size of each region of the polyprotein in the isolate as follows: core=48 nt, 16 aa; E1=564 nt, 188 aa; E2=1161 nt, 387 aa; NS2=843 nt, 281 aa; NS3=2031 nt, 677 aa; NS4=945 nt, 315 aa; NS5a=1245 nt, 415 aa; and NS5b=1692 nt, 564 aa. The genomic organization was similar to that of previously reported isolates. As in prototypes of HGV and GBV-C isolates, the putative core region in MY14 and VT48 isolates could not be clearly defined as it consisted of only 16 aa. As shown in Table 2
, when compared to other previously reported HGV/GBV-C isolates with a full-length genome sequence, HGV-VT48 showed overall identities of 8587% at the nucleotide level and 9496% at the amino acid level, thereby indicating that they were the same virus. Among the coding regions, there was a tendency for the putative core region to show the highest similarity in nucleotide sequence with an identity of 8896%. In contrast, lower similarities were observed in the E2 region. The 5'- and 3'-UTR were also highly conserved among the HGV/GBV-C genomes. HGV/GBV-C isolates with fully sequenced genomes, including database-derived sequences, were grouped into four major genotypes by phylogenetic analysis (Fig. 1
). A similar finding was observed by phylogenetic analysis of the 5'-UTR sequence of the same isolates as reported previously (Naito et al., 1999
). Based on these results, HGV-MY14 and HGV-VT48 isolates can be classified as a novel genotype, designated type 4.
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In conclusion, we have cloned the entire nucleotide sequence of two different HGV genomes recovered from the serum of Myanmarese and Vietnamese subjects, respectively. Both isolates (HGV-MY14 and HGV-VT48) were classified as a novel genotype, named type 4. It seems likely that HGV genotype 4 is rather common in south-eastern parts of the Asian continent. Additional studies, including analyses of sequences from other geographic regions, are required for the further classification of HGV/GBV-C.
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References |
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Received 6 July 1999;
accepted 22 September 1999.