Molecular cloning and characterization of the common 1b subtype of HCV from Turkey

Abstract

Hepatitis C Virus is a major cause of acute and chronic hepatitis worldwide. 80-90% of Hepatitis C Virus infections become chronic and 75% of these cases lead to liver disease, including cirrhosis, liver failure and hepatocellular carcinoma. Hepatitis C Virus was first identified by molecular cloning of the viral genome in 1989. Hepatitis C Virus is an enveloped virus containing a positive stranded RNA genome with a size of around 9.5 kilobases. In terms of genomic organization, it was accepted as a member of Flaviviridae family as a new genus named Hepaciviruses. The single-stranded RNA genome encodes a single open reading frame, which is transcribed into a single polypeptide of 3010 or 3030 amino acids and cleaved into viral proteins Core, E l, E2/p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B by host and viral proteases. Sequencing, serotyping and RFLP studies indicate that Hepatitis C Virus genome is highly variable. There are six distinct genotypes and at least 74 subtypes with different distributions between the geographic areas. Variability is not equally distributed throughout the genome. 5' UTR, some parts of the 3' UTR and capsid protein are the most conserved regions. The predominant genotype in the Turkish population was found to be lb by sequencing of the 5-UTR. In this study, the entire sequence encompassing the complete coding region and partial non coding regions of the genotype lb obtained from a HCV-infected Turkish patient was cloned to investigate its evolutionary relationship with other genotypes and to study its overall genome organization,. In order to characterize the viral genome, viral RNA was extracted from the serum, cDNA was synthesized, the HCV genome was amplified by PCR in 7 overlapping fragments, PCR fragments were cloned into bacterial vectors and cloned inserts were sequenced by automated sequencing methodology. The partial sequence data covering 70% of the cloned HCV genome indicate that the Turkish lb genotype displays high homology to other lb genotypes, but differs from others by distinct amino acid changes. To our knowledge, this is the first report about the HCV genome structure from Turkey. The HCV subgenomic fragments obtained here will serve to further molecular and immunologic studies on this dominant form of HCV found in Turkish patients.