Recently, by using the long reverse transcriptase–PCR (RT-PCR) method we succeeded in the synthesis of 7.5-kb DNA amplicons of hepatitis A virus from which infectious RNA was transcribed ( 24). Therefore, it is important to devise a strategy for efficiently constructing such clones. Given the extensive genotype diversity of HCV, it also may be important to have infectious clones representative of more than one genotype or variant. An infectious molecular clone of HCV would be an important tool for better understanding of its molecular biology and pathogenesis. In the Flaviviridae family, infectious transcripts of full-length cDNAs have been described for flaviviruses ( 14– 18) and pestiviruses ( 19– 23). Thus, genomic RNA, as well as RNA transcripts from full-length cDNA clones, should be infectious. The genome of positive-strand RNA viruses functions as mRNA from which all viral proteins necessary for virus propagation are translated. This stable infectious molecular clone should be an important tool for developing a better understanding of the molecular biology and pathogenesis of HCV. The sequence of the virus recovered from the chimpanzee was identical to that of the injected RNA transcripts. Injection of RNA transcripts from this consensus clone into the liver of a chimpanzee resulted in viral replication. Next, we constructed a consensus chimera from four of the full-length cDNA clones with just two ligation steps. Sequence analysis of these and 16 additional full-length clones revealed that seven clones were defective for polyprotein synthesis, and the remaining nine clones had 6–28 amino acid mutations in the predicted polyprotein compared with the consensus sequence of H77. However, we found no evidence for HCV replication. The infectivity of two complete full-length cDNA clones was tested by the direct intrahepatic injection of a chimpanzee with RNA transcripts. We devised a cassette vector with fixed 5′ and 3′ termini and constructed multiple full-length cDNA clones of H77 in a single step by cloning of the entire ORF, which was amplified by long reverse transcriptase–PCR, directly into this vector. We have succeeded in constructing a stable full-length cDNA clone of strain H77 (genotype 1a) of hepatitis C virus (HCV).
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