Characteristics of the organism and its antigens

HCV is an enveloped virus containing a single-stranded positive-sense RNA genome of 9.4-9.5 kb. It is classified as a separate genus within the family Flaviviridae. The genome includes a 5' nontranslated (noncoding) region (5'NCR) of 329-341 nucleotides, followed by a large single open reading frame encoding a polyprotein precursor of 3010 amino acids (AA) in length, and a 3' NCR. The open reading frame is translated as a polyprotein containing (from N to C terminus) the core protein (p22), two envelope glycoproteins El (gp35) and E2 (gp70), and a series of nonstructural proteins (NS2-5) (Figure 1). The E2 gene contains a hypervariabie region encoding a domain of 27 amino acids in length. The NS gene products are essential for virus replication, and possess a number of enzymatic properties: protease (NS2, NS3), helicase (NS3), and RNA-dependent RNA polymerase (NS5).

There is considerable sequence variability between isolates, which are classified into six (at present) major genotypes on the basis of 5'NCR and NS5 gene sequence analysis, each of which contains a number of subtypes. The most conserved part of the genome is the 5'NCR (>90%), followed by the core gene (>80%), the least conserved being the 3'NCR (26%), and the El and NS2 genes (55-60%). Genotype distribution around the world is uneven: types 1, 2 and 3 predominate in Europe and the USA, 4 is found in the Middle East, 5 in southern Africa and 6 in China. Studies of potential differences in pathogenicity between the genotypes have been inconclusive. All known genotypes may give rise to life-threatening liver disease. Genotype 1 viruses are less sensitive to I FN a therapy than are the other genotypes.

Diagnostic antibody tests utilize a series of recombinant or synthetic peptide antigens. First generation assays contained only an NS4-derived antigen, clOO. These assays were of low sensitivity and specificity. Second generation tests added core (c22) and NS3 (c33) gene products to the clOO, resulting in considerable improvement in diagnostic accuracy. Current, third-generation, assays also include an NS5 gene product. Commercially available screening assays are in standard sandwich ELISA format. Confirmatory assays, essential in the context of screening of low-risk sera, such as from blood donors, rely on the same antigens, but in immunoblot or 'line assay' format, where the antigens are presented on nitro cellulose strips. The presence of the relatively conserved core and NS3 gene products are essential to avoid false-negative reactivity arising through failure of antibodies raised against one viral genotype to recognize antigens derived from a different genotype. In contrast, the genetic diversity of the NS4 gene is reflected in sufficient antigenic differences between genotypes to allow synthesis of type-specific NS4-derived peptides for use in serotyping assays. Genome detection assays, such as the reverse transcriptase polymerase chain reaction (RT-PCR) using primers from the highly conserved 5'NCR, are useful for determination of carrier status in anti-HCV-posi-tive individuals. Genotyping of the infecting virus may be determined by use of type-specific primers, by restriction fragment length polymorphism analysis of PCR products, or by hybridization of PGR products with type-specific probes.

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