HCV Quasispecies Dynamics and Liver Transplant

DESCRIPTION (provided by applicant): HCV related cirrhosis is the leading cause of liver transplantation in this country and in Europe. A significant proportion of patients have a rapid recurrence of hepatitis that leads to graft loss and mortality. The dynamic nature of HCV populations could underlie its biology, including immune evasion and persistence. We propose to study the transmission and evolution of HCV quasispecies 65 liver transplantation recipients. Aim 1: Is there a selection of viral variants within quasispecies at the time of transmission? We hypothesize that not all viral variants present in infectious blood are able to establish infection in a susceptible host. Therefore, we will compare the composition of viral quasispecies in all 65 transplant recipients using single-strand conformation polymorphism (SSCP), cloning and sequencing to evaluate changes that correlate with the clinical course. This analysis will be conducted in the highly variable E2 and conserved 5' untranslated (5'UTR) regions. Aim 2: How does the HCV quasispecies distribution evolve over time in the natural course of infection and does it have a prognostic value with respect to outcome? We hypothesize that in OLT recipients infected with HCV, the quasispecies composition evolves in the E2 and 5'UTR regions and these changes correlate with the clinical outcome. Therefore, we will analyze quasispecies complexity, divergence and genetic drift longitudinally using SSCP and heteroduplex mobility assays in all 65 infected recipients. We will correlate quasispecies behavior with clinical outcome of the infection. Aim 3: What is the biological basis of selective transmission and evolution of quasispecies in the E2 and 5'UTR regions? We hypothesize that the selection and evolution of variants within the 5'UTR quasispecies is related to differences in viral fitness associated to its competence to direct translation. We will clone representative 5'UTR fragments into bicistronic dual luciferase reporter plasmid and we will measure and compare their translation efficiency in vitro. We hypothesize that E2 region quasispecies variants bound by antibodies are less efficient in establishing infection in the graft. Furthermore, we postulate that new viral variants appearing during the infection represent mutants which escape immune detection. We will separate viral variants bound in immunocomplexes from "free" virus and compare the variants using SSCP, cloning and sequencing.