Evolution of the Predominant Sequence of the Hypervariable Region in the Putative Envelope Gene E2/NS1 of Hepatitis C Virus in Patients on Haemodialysis

Abstract

Follow-up studies of patients with acute HCV infection indicate that up to 50% of them develop a chronic infection. Hypervariability of the putative envelope protein E2/NS1 of HCV suggests that one mechanism by which the virus may persist in the host is by the generation of viral escape mutants. By this theory, a predominant population would evolve to which antibody is made which neutralises that viral population allowing the emergence of a new antigenically different predominant population. A characteristic clinical feature of HCV infection is the episodic fluctuating pattern of alanine transaminase (ALT) levels in which periods of elevated ALT levels are interspersed with periods of normal levels. The emergence of a new genetic variant may be expected to coincide with a period of liver dysfunction. Patients on long term renal dialysis are more likely to have elevated ALT levels than uninfected patients. To characterise the emergence of genetic variants of the hypervariable region (HVR1) of the E2/NS1 protein and correlate their appearance with periods of liver dysfunction, a retrospective study of the HVR1 amplified from multiple serum samples taken over several years from three renal dialysis patients was performed. A total of 36 serum samples serially obtained from three patients with chronic hepatitis C were studied. HCV RNA was extracted from patient serum and the E2/NS1 HVR1 was amplified by reverse transcription and nested polymerase chain reaction. The amplified cDNA was analysed by direct chain termination nucleotide sequencing using the Sanger technique. The data indicate nucleotide sequence variation of HVR1 over time in each of the three renal dialysis patients but the emergence of a new sequence variant could not be linked with to a specific peak in ALT level. However, persistently raised ALT levels did appear to be associated with increased rate of change of the HVR1. The second study presented in this thesis was undertaken to investigate the effect serum storage conditions had on the stability of HCV RNA and its detection by RT-PCR. Serum taken from a chronically infected patient was stored at