A cDNA fragment of hepatitis C virus isolated from an implicated donor of post-transfusion non-A, non-B hepatitis in Japan

Kubo, Yusuke; Takeuchi, Kenji; Boonmar, Sumalee; Katayama, Tohru; Choo, Qui‐Lim; Kuo, George; Weiner, Amy J.; Bradley, Daniel W.; Houghton, Michael; Saito, Izumu; Miyamura, Tatsuo

doi:10.1093/nar/17.24.10367

Cited by 158 publications

(80 citation statements)

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“…HCV cDNA fragments were obtained from the blood by RT-PCR. 20,21 Blood collected 6 months later was injected into chimpanzees and was infectious. The infectivity was 10 5.5 chimpanzee infectious doses/mL.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, it is impossible to assess the authenticity of the cDNA clones of HCV at the moment. However, the complete cDNA clone of HCV constructed in this study is believed to originate from infectious HCV clones for the following reasons: 1) the blood sample from the donor was infectious to both chimpanzees and humans 20,22 ; 2) from the sample, we isolated 12 overlapping clones covering the entire region of HCV that were major clones in each region, and their overlapping sequences were almost identical to the adjacent clones; and 3) the virus clones with sequences in HVR identical to those of the complete cDNA clone were detected in both recipient and chimpanzee after infection. Recently, Kolykhalov et al showed that in vitro synthesized full-length HCV RNA derived from the consensus cDNA clone was infectious to a chimpanzee after direct injection into the liver.…”

Section: Discussionmentioning

confidence: 99%

“…We selected a donor blood sample that had been shown to be infectious to both human and chimpanzees. [20][21][22][23] This plasma was obtained from a healthy blood donor who was implicated in the transmission of HCV. The donor blood was later found to be positive for anti-HCV antibody.…”

Section: Methodsmentioning

confidence: 99%

“…18,19 To construct a complete cDNA clone of the HCV genome, we selected a blood donor sample which was shown to be infectious to both humans and chimpanzees. [20][21][22][23] From that sample we have cloned several fragmental cDNAs and constructed a full-length cDNA containing the 98-base pair (bp)-3ЈX sequence. We also compared the sequence in the hypervariable region (HVR) of HCV in the donor from whom the cDNA was derived with those derived from the recipient and the experimentally infected chimpanzee.…”

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Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

et al. 1998

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We constructed a full-length complementary DNA (cDNA) clone of hepatitis C virus (HCV) from a blood sample of an HCV carrier. The blood from the carrier was eventually transfused to a patient who later developed typical posttransfusion hepatitis C. It was also shown to be infectious to chimpanzees. We obtained 12 overlapping cDNA fragments altogether, covering the entire HCV genome. By subcloning and sequencing, clones considered to constitute the major population were selected. We could also detect 98 base pairs of extra sequences at the 3Ј end of the genome. After confirming the overlapping sequences, we combined the fragments to make a full-length cDNA. The HCV population in the donor was heterogeneous, as determined by their nucleotide sequences of the hypervariable region in envelope protein, but a few virus clones were selected in the recipient after transmission. The similar convergence of the virus population was previously observed when the same blood sample was injected into a chimpanzee. Interestingly, virus clones isolated during the acute phase in the recipient and the chimpanzee had sequences in the hypervariable region identical to that of the full-length cDNA clone. The full-length cDNA clone of HCV constructed in this study may originate from infectious virus clones.(HEPATOLOGY 1998;27:621-627.)Comparative study of the amino acid sequence of hepatitis C virus (HCV) with those of flaviviruses and pestiviruses and gene expression experiments in bacteria, yeast, and animal cells have revealed that the proteins of HCV are processed by a host-derived signalase and cleaved by virus-coded proteases. [1][2][3][4][5] In vitro culture systems that support partial replication of this virus have been developed from human T-and B-cell lines, 6,7 human fetal hepatocytes, 8 chimpanzee hepatocytes, 9 and human primary hepatocytes. 10 However, fully efficient, long-term viral replication has not yet been established. To study the biology of HCV and its pathogenesis, it is essential to establish an efficient in vitro cell culture system or an infectious complementary DNA (cDNA) clone to support the complete replication of HCV.As a first step to construct an infectious cDNA clone of HCV, it is important to select an adequate sample containing infectious HCV. To date, a considerable number of ''complete'' clones of the HCV genome have been reported. However, it is not certain that those clones have truly originated from infectious HCV, because the materials used usually were pooled plasma samples. Furthermore, the plasma of HCV carriers or patients is generally composed of quasispecies of HCV population. 11 To construct an infectious cDNA clone of RNA viruses, it is crucial to retain both 5Ј and 3Ј ends of the sequence, which are highly conserved and are considered to play essential roles for RNA synthesis, transcription, and translation. 12 Many have reported that the 3Ј untranslated region (UTR) of HCV consisted of poly(U) [13][14][15][16] or poly(A) 17 homopolymer tracts. A novel 98-nucleotide (nt) sequence down...

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample†

et al. 1998

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“…An aliquot of cDNA reaction solution (5 pl) was directly amplified by polymerase chain reaction (PCR) with a pair of primers 464 S (5'-GGCTATACCGGCGACTTCGA-3') and 526 A (5'-GACATGCATGTC-ATGATGTA-3'), encompassing the 623 base pairs (bp) (18). Amplification was done in a 100-pl reaction volume and on a DNA Thermal Cycler (Perkin Elmer Cetus, Norwalk, CN) for 5 min at 94 C, 1 min at 50 C and 1 min at 72 C for 10 min.…”

Section: Methodsmentioning

confidence: 99%