Isolates of a virus are usually distinguished by their genetic relatedness (genotype) such as bacteria, and sometimes viruses, are often separated by antibody reactivity (serotypes). The striking genetic heterogeneity of HCV suggested that the virus might have different genotypes. The validity of this reasoning was supported by a combination of molecular biology and statistical techniques including pair wise distance determination and phylogenetic tree construction. Different isolates could indeed be classified by their nucleotide variability into genotypes or subtypes (Bukh et al., 1995 and Simmonds, 1995).
Isolates of the same genotype have an average sequence homology of 95%, with a range of 88% to 100% based on sequencing of relatively well conserved regions of E1, NS4 or NS5. Subtypes within the same genotype have an average sequence homology of about 86%. By contrast, different genotypes have sequence similarity of only about 65%. The distribution of divergence is discontinuous, making the genotypes distinctive relatives rather than a spectrum of variants resulting from random genetic drift (Simmonds, 1995).
Based on sequence similarity, HCV isolates can be classified into genotypes I, II, III, IV, V (Mori et al., 1992 and Okamoto et al., 1992a, 1993) or into three major types designated 1,2 and 3, with each type divided into two subtypes, a and b (Chan et al., 1992; McOmish et al., 1993; Simmonds et al., 1993 and Stuyver et al., 1993). Genotype I corresponds to 1a, II to 1b, III to 2a, IV to 3a and VI to 3b. There are many methods for determining viral genotype. Although the most accurate is undoubtedly sequencing of the complete 9,500 nucleotide prohibitively expensive and time consuming (Ohno and Lau, 1996). Furthermore, multiple clones would need to be examined to exclude mixed genotype infections. Thus, subgenomic genotyping methods have evolved, which are usually based on relatively well conserved regions of the genome such as the 5/ UTR, core, E1, and NS5b. The nucleotide sequences within these relatively conserved regions are genotype-specific, and therefore isolates can be accurately typed regardless of which of these regions is used for analysis (Lau et al., 1994).
Subgenomic genotypic methods include amplification and region sequencing (Bukh et al., 1992), Polymerase Chain Reaction (PCR) with genotype specific primers (Okamoto et al., 1992a), restriction fragment length polymorphism of PCR amplicon (Davidson et al., 1995), differential hybridization including the reverse hybridization line probe assay (LIPA, Innogenetics, Ghent, Belgium) (Stuyver et al., 1993), and serologic genotyping (Machida et al., 1992).
2.2.4. Relationship between HCV and the flaviridae family:
The discovery of the hepatitis C virus (HCV) in 1989 was a major breakthrough. Prior to that point, it had been clear that a major cause of acute hepatitis after a blood transfusion was neither related to hepatitis A nor to hepatitis B (hence the early name for this disease, non-A, non-B hepatitis). The virus was cloned using molecular biology techniques after extensive testing of serum from experimentally infected animals, and found to be an RNA virus that belongs the Flaviviridae family and genus Hepacivirus. The genome of the HCV comprises a positive-stranded RNA molecule of about 9,500 nucleotides (nt) containing a long translational open reading frame (ORF) that encodes a large polypeptide of approximately 3,010 amino acids (aa) beginning with the first inframe methionine codon (Choo et al., 1989,1991b; Kato et al., 1990 and Takamizawa et al., 1991).
The 5/ terminus of the RNA genome has substantial primary sequence identity with the corresponding region of the pestivirus genomes (Takeuchi et al., 1989; Choo et al., 1991b and Han et al., 1991) and a region of the encoded polypeptide exhibits significant sequence identity with nucleoside triphosphate (NTP) binding helicases encoded by the pestiviruses and to a lesser extent, the flaviviruses (Miller and Purcell, 1990 and Choo et al., 1991a,b). Protease and replicase sequence motifs conserved among the pestiviruses and flaviviruses are also present within the HCV-encoded polyprotien, which, along with the more extensively conserved helicase sequence, are all similarly collinear among the three types of viral polyprotiens (Choo et al., 1991b).
Although these are the only regions of HCV exhibiting significant primary sequence identity with the pestiviruses and flaviviruses, the hydropathicity of the HCV-encoded polypeptide is remarkably similar to that of the flaviviruses and, to a lesser extent, the pestiviruses, thus indicating similarities in their basic structures and functions (Choo et al., 1991b). Three new flavi-like viruses, GBV-A, GBV-B, and GBV-C (also known as HGV), have been identified (Simons et al., 1995a). Nucleotides and protein sequence analyses show that these three viruses are more closely related to each other and HCV than to the other members of the Flaviviridae family. HCV and GBV-A, -B, and –C appear to form a discrete cluster of related viruses within the larger genus of Flavivirus (Simons et al., 1995b).
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