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We analyzed hepatitis C virus (HCV) genotype 4 isolates circulating in the Alexandria District (Egypt) in terms of genetic divergence and the presence of different subtypes. Hypervariable region 1 (HVR1) and the NH2 region of the E2 protein were characterized, and the heterogeneity of subtype 4a isolates was evaluated by analyzing epitope frequencies, immunoproteasome prediction, and possible glycosylation patterns. The heterogeneity of the nucleotide sequences was greater than that found in previous studies, which reported only subtype 4a. Subtype 4a was most common (78% of cases), yet four new subtypes were found, with subtype 4m representing 11% of the cases and the other three subtypes representing another 11%. Substantial heterogeneity was also found when the intrasubtype 4a sequences were analyzed. Differences in the probability of glycosylation and in the positions of the different sites were also observed. The analysis of the predicted cytotoxic-T-lymphocyte epitopes showed differences in both the potential proteosome cleavage and the prediction score. The Egyptian isolates in our study also showed high variability in terms of the HVR1 neutralization epitope. Five of these isolates showed amino acid substitutions never previously observed (a total of six positions). Four of these residues (in four different isolates) were in positions involved in anchoring to the E2 glycoprotein core and in maintaining the HVR1 conformation. The results of this study indicate that HCV genotype 4 in Egypt is extremely variable, not only in terms of sequence, but also in terms of functional and immunological determinants. These data should be taken into account in planning the development of vaccine trials in Egypt.

The subtype distribution of HCV genotype 4 was studied in two different African countries, Egypt and Tanzania. The HCV isolates were obtained from epidemiological studies involving, respectively, 135 hepatopatic patients and 1043 pregnant women and outpatients. Sequence comparison and phylogenetic analysis of the NS5b genome region (nt 8327-8499) were performed. Fourteen out of 18 isolates from Egypt, but only 3 out of 6 isolates from Tanzania clustered in the same branch of subtype 4a. Three new proposed subtypes have been identified. The first includes 1 isolate from Egypt (EGY15); the second, 2 isolates from Egypt (EGY193 and EGY44) and 2 isolates from Tanzania (D776, D61); and the third, 1 isolate from Egypt (EGY47) and 1 isolate from Tanzania (D70). These isolates cluster in branches different from any other, corresponding to a known subtype of genotype 4. In conclusion, remarkable genetic heterogeneity has been found among genotype 4 isolates simultaneously circulating in a restricted area. This was particularly observed in the study performed in Tanzania. Potential concern about the sensitivity of diagnostic assays and possible implications in the development of future vaccines have been stressed.