Hepatitis C virus, the major causative agent of blood-borne non-A, non-B hepatitis in the world, has been the subject of considerable nucleic acid sequence analysis. Although all reported hepatitis C sequences from the United States have been represented by the prototype hepatitis C virus type 1 sequence, two groups of variant sequences have been reported in Japan. However, we have noted five distinct, but related, genotypes (I-V) throughout the world, based on detailed sequence determination and analysis of the first 1700 nucleotides and part of the nonstructural region 5 at the C terminus of the open reading frme. The nucleotide sequence for a large number of hepaiis C virus islates sanning six continents was obtained by direct sequence analysis of PCR products after reverse transcription. Genotype was clafied by using several distinct sequence motifs. We observed that most genotypes coexist in several geographic regions, iuding the United States, Japan, Germany, and Italy. So far, genotype V has been found only in South Africa. Interestingly, each distinct genotype seems to be maintained throughout the genome in the segments tudied. These genotype dictions should be considered when d ing specific diagosic tests, developing potential vaccines, and studying viral transmio.Hepatitis C virus (HCV) was isolated from a chimpanzee chronically infected with a contaminated human factor VIII concentrate in 1989 (1). An immunoassay for circulating antibody to HCV was developed by using a yeast-derived recombinant antigen encoded by a segment of the cloned HCV genome (2). Results indicated HCV to be the major causative agent of the blood-borne non-A, non-B hepatitis (1-3).The genomic organization and characterization of HCV has been reported (4, 5). The HCV genome is a positivestrand RNA of -9.4 kilobases and contains a large open reading frame that encodes a polyprotein precursor of 3011 amino acids. Based on comparative sequence analysis of the genome and encoded polyprotein, HCV is thought to be distantly related to the flaviviruses and the pestiviruses. The large open reading frame appears to encode colinearly structural and nonstructural proteins, with the structural proteins located at the 5'-end portion of the genome. Putative boundaries are assigned that separate the 5'-untranslated region (5UT), the core protein (C), the glycoproteins envelope 1 (El) and nonstructural protein l/envelope 2 (NS1/E2), the nonstructural proteins 2-5 (NS2-NS5), and the 3'-untranslated region (3UT). The prototype HCV nucleotide sequence reported is termed HCV1 (refs. 4 and 5; see ref. 6 for review).Using pooled plasma samples from human non-A, non-B hepatitis patients and potential HCV carriers, Kato et al. (7), Takamizawa et al. (8), and Okamoto et al. (9) have reported entire genomic sequences of Japanese HCV strains. Some partial 5'-end HCV sequences from individual Japanese samples were reported by Takeuchi et al. (10,11) and Okamoto et al. (12). In addition, partial sequences in the NS3, NS4, and NS5 regions have been r...

The nucleic acid sequence of the putative 5'-untranslated (5PUT) region of hepatitis C virus (HCV), determined for samples obtained from a variety of geographic origins, was found to be over 98% conserved among all isolates. On the basis of this signature sequence for HCV, a viral RNA assay was developed by using cDNA synthesis with reverse transcriptase, followed by polymerase chain reaction (PCR). The new assay was compared with the Ortho-Chiron C100-3 HCV enzyme-linked immunosorbent assay to research radioimmunoassays for antibodies to the C33c and C22 HCV antigens and to the first reported set of HCV PCR primers designed from the NS3 domain. Plasma samples from 16 Japanese patients with non-A, non-B hepatitis (NANBH) and 16 immunoassay-positive blood donors from the United States were investigated. The 5PUT PCR primers were found to be superior to the NS3 primers in sensitivity and specificity (15 of 25 versus 3 of 25 of the C100 enzyme-linked immunosorbent assay-positive samples, respectively). Samples from two C100-negative patients with acute NANBH were found to react with the 5PUT primers but not with the NS3 primers. Also, two of three patients with chronic NANBH converted from reverse transcriptase PCR positive to negative after interferon treatment. Although the clinical significance of the presence or absence of HCV RNA in samples from patients is not fully understood, the use of probes and primers from the 5PUT region (as opposed to primers from other segments) should not lead to false-negative results due to nucleic acid sequence variations in viral isolates.