Hepatitis C.

Hepatitis C is a major cause of liver related morbidity and mortality worldwide and represents a major public health problem (Alberti and Benvegnu, 2003). Hepatitis C virus (HCV) is a parenterally transmitted RNA virus that causes chronic hepatitis and liver disease. In contrast to infections with other hepatitis viruses, the onset of HCV infection is clinically in apparent in the majority of cases, and the diagnosis of hepatitis C is therefore typically made only after persistent infection is established (Mizukoshi and Rehermann, 2001).

            HCV persistence occurs in 70% of patients and fewer than half of those are likely to respond to the current antiviral therapy of interferon and ribavirin. Because there are so many HCV-infected patients, it is estimated that the incidence of complications from chronic hepatitis, namely, liver cirrhosis and hepatocellular carcinoma, will increase in the next few decades (Mizukoshi and Rehermann, 2001).

            Many studies published to date have demonstrated that HCV-specific humoral and cellular immune responses are readily detectable in most, if not all, infected patients. It is, however, still unknown if natural sterilizing and protective immunity against HCV exists and if it could be therapeutically induced (Mizukoshi and Rehermann, 2001).

2.2.2. Hepatitis C in Egypt.

            The World Health Organization has declared hepatitis C a global health problem, with approximately 3% of the world^,s population (roughly 170-200 million people) infected with HCV. In the USA approximately 3 million people are chronically infected, many of whom are still undiagnosed. In Egypt the situation is quite worse with a high prevalence rate (about 24.5%) among Egyptians (Abdel-Aziz et al., 2000 and Mangoud et al., 2004).

            HCV infection is a major public health problem in Egypt (Hibbs et al., 1993; Abdel-wahab  et al., 1994 and El-sayed et al., 1996). Egypt has a population of 62 million and contains the highest prevalence of hepatitis C in the world. Blood bank and community-based surveys conducted in Egypt have reported sero-prevalence rates of HCV to be as high as 40% in some parts of the country (Darwish et al., 1993). These rates are substantially higher in the Nile Delta region compared with the rest of the country (Frank et al., 2000).

2.2.3. Molecular Virology.

2.2.3.1. Molecular analysis of the HCV genome.

The genomic organization of HCV is illustrated schematically in (Fig. i). Comparative analysis of the genomes of several HCV strains (Kato et al., 1990; Choo et al., 1991; Okamoto et al., 1991 and Takamizawa et al., 1991) indicated the virus to be closely related to both the Pestivirus and Flavivirus genera within the family Flaviviridae. The viral genome is a single-stranded RNA molecule approximately 9.5 Kb in length which is positive sense and possesses a unique open reading frame, coding for a single polyprotein, flanked by untranslated regions at both its 5^/  and 3^/ ends.

Expressed Polyprotein. HCV, a single-stranded RNA virus of 9.5 kb, consists of a single open reading frame and two untranslated regions (UTRs). It encodes a polyprotein of approximately 3010 amino acids, which is cleaved into single proteins by a host signal peptidase in the structural region and the HCV-encoded proteases in the nonstructural (NS) region. The structural region contains the core protein and two envelope proteins (E1 and E2). Two regions in E2, called hypervariable regions 1 and 2 (HVR 1 and HVR 2), show extreme sequence variability, which is thought to be the result of selective pressure by virus-specific antibodies. E2 also contains the binding site for CD81, the putative HCV receptor or coreceptor. The nonstructural proteins have been assigned functions as proteases (in the case of NS2, NS3, and NS4A), helicase (in the case of NS3), and RNA-dependent RNA polymerase (NS5B). Although the crystal structure of NS3 and NS5 is known, 31 the function and properties of the other proteins are less well characterized. A region in NS5A has been linked to the response to interferon alfa therapy and is therefore called the interferon-sensitivity–determining region (ISDR). However, the relevance and importance of this region are still unclear (Pileri et al., 1998).

The length of the polyprotein-encoding region varies according to the isolate and genotype of the virus from 3008 to 3037 amino acids (Chamberlain et al., 1997). Apart from differences in length HCV genotypes show diversities of around 30% in the nucleotide sequences of their whole genomes and comprehensive analysis of these sequences has revealed the existence of at least 6 genotypes throughout the world (Bukh et al., 1995 and Simmonds, 1995). The importance of the genomic heterogeneity lies in the fact that some genotypes appear to be associated with more severe pathology and are more refractory to treatment by current therapies (Bukh et al., 1995). However, other studies show no significant association between genotype and disease progression (Benvegnu et al., 1997).

2.2.3.2.    5^/ Untranslated region (5^/ UTR).

An obvious characteristic of HCV is the presence of a long untranslated region (UTR) at the 5^/ end of the genome and detailed molecular analysis indicates that polyprotein synthesis is initiated at nucleotide 342 (Han et al., 1991). This region of the genome was predicated to be capable of forming extensive secondary structures (Tsukiyama-Kohara et al., 1992), which were biochemically confirmed by Brown et al., (1992). Furthermore, a survey of the sequences within the 5^/ UTR from 39 different isolates and genotypes of HCV showed that there was remarkable sequence conserved region of the whole genome, a characteristic which has allowed it to be used as a diagnostic marker for HCV by PCR (Smith et al., 1995)

2.2.3.3. Virus-encoded proteins.

The nascent viral polyprotein is processed by a combination of host and viral proteinases into the mature viral proteins (Grakoui et al., 1993a and Hijikata et al., 1993). At least 10 distinct viral proteins have so far been identified:

a) Core: It is released from the viral polyprotein by nascent proteolytic cleavage at amino acid 191 by host proteases (Ralston et al., 1993). The full length protein known as P21, has been identified by both in vitro and in vivo expression (Hijikata et al., 1991 and Lo et al., 1996).

b) E1/E2: The major viral structural proteins are the glcoproteins E1 and E2, which are released from the viral polyprotein by the action of host cell signal peptidases. Analysis of the amino acids 383 and 746 respectively (Mizushima et al., 1994).

c) P7: E2 is sometimes found extended at its carboxy terminus to include a smaller protein known as P7. Numerous studies have reported interactions between the viral structural and non-structural proteins and it is clear that complex protein-protein association is a critical part of virus replication. Although E1 has been shown to associate through its carboxy terminus with the core protein (Lo et al., 1996) and E2 with NS2 (Matsuura et al., 1994 and Selby et al., 1994).

d) NS2: The NS2 protein has been shown to be a trans-membrane protein with its carboxy terminus translocated into the lumen of the ER while its amino terminus lies in the cytosol (Santolini et al., 1995). Although immuno-precipitation studies (Grakoui et al., 1993b and Matsuura et al., 1994) have shown that NS2 is closely associated with the structural proteins, the biological function of the majority of the NS2 protein is still unclear.

e) NS3: The region coding for the NS3 protein has been the most extensively studied of the whole genome. The NS3 protein has been shown in numerous studies to be a protein of approximately 70 KD in size and to posses several diverse biochemical functions.

f) NS4: The NS4 region of the polyprotein comprises two proteins, namely NS4a and NS4b. Both of these are released from the viral polyprotein by the NS3 by cis cleavage at the NS3/NS4 and transcleavage at the NS4A/NS4B and NS4B/NS5A junctions. NS4A is a small protein, approximately 6 KD in size, and appear to have diverse functions such as anchorage of replication complexes and as a cofactor for the NS3 proteases. Currently, there is no ascribed function for the NS4B protein but it is likely that it plays an integral role within HCV replication complexes (Clark, 1997).

g) NS5A and NS5B: The NS5 region of the polyprotein is composed of two major proteins, NS5a and NS5b, which are released as mature products by the action of the NS3 protease in conjunction with NS4A. A part from the probable role of NS5A in the replication cycle, recent evidence has suggested that it may be a critical factor in determining the susceptibility of the virus to treatment with interferon. It was initially reported that IFN sensitivity correlated with mutations within a discrete region of NS5a which was subsequently named the IFN sensitivity determining region (Enomoto et al., 1995, 1996).

            The sequence of the NS5B protein is highly conserved, not only between different strains of HCV but also in Pestiviruses, Flaviviruses and even in other RNA viruses. The function of NS5B in HCV has been speculated to be the viral polymerase (Behrens et al., 1996).

h) 3^/ Untranslated region (3^/ UTR): A  significant recent finding in the HCV area has been the revelation that the considered 3^/ terminal region of the genome was incorrect. A number of studies had suggested that the 3^/ terminus of the genome terminated in a poly (U) tract (Kato et al., 1991; Takamizawa et al., 1991 and Okamoto et al., 1992a).