Chronic Multiple-genotype HCV infection exhibits genotype-specific evolution patterns.

CULASSO ANDRÉS; BARÉ PATRICIA; CAMPOS RODOLFO

Congreso; 150 years of Darwin’s Evolutionary Theory. A South American Celebration; 2009

Facultad de Ciencias. Universidad de la República y otros – URUGUAY

Chronic infections with fast-evolving RNA viruses like HCV provide a unique model for evolution studies. Patients are usually followed up for long periods. Regular samples provides heterocrhonus data for evolution rate and phylogenetic estimations. Samples Eleven blood samples were obtained from a patient with hemophilia between 1993 and 2006. The sample from 2004 correspond to the end of PEG-INF treatment. Materials and methods RNA was extracted with QIAGEN kit. Retrotrascritpion was performed with Random Hexamer primers using MMLV-RT. Genotype specific PCR (E1-E2 for Genotypes 1a, 2a and 3a) were performed for direct sequencing and cloning. Genotype 2a E1-E2 PCR products were cloned into E. coli using pGEM-T Easy Cloning Vector. Draft phylogeny and clones distances were computed with MEGA4. Best Maximum Likelihood trees were searched with PAUP* using parameters estimated by ModelTest 3.7. Substitutions rates were inferred by Montecarlo Coupled Marcov Chains with BEAST software analyzed by Bayesian Methods with Tracer Software. Results HCV genotype 1a, 2a and 3a were detected trough the follow-up period. Genotype specific E1-E2 PCR allowed to direct sequence samples from 1993 to 2002 for genotype 3a, samples from 1995 to 1998 for genotype 1a and samples from 1999 to 2006 for genotype 2a. Continuous evolution of sequences were observed in ML-trees for each genotype. Genotype 2a showed larger branches lengths than observed in genotype 3a or 1a. Substitutions rates (subs./sites/year) estimated with BEAST were 3.31E-3, 1.31E-2 and 4.41E-3 for genotypes 1a, 2a and 3a respectively. Most of substitution in E1-E2 for genotype 2 were located at hyper variable region 1. The genotype 2a E1-E2 was fully cloned. As diversity index mean Hamming distance within sample clones was calculated. This mean ranged from 0,0056 in 2006 post treatment sample, to 0,0165 in 1999 sample, where all 3 genotypes were detected. ML tree topography of cloned and plasma sequences was compatible with quasispecies selection trough the time. Conclusions Genotype 1a, 2a and 3a coevolved through all the infection period analyzed Genotype 1a and 3a showed an steady state probably ruled by normalizing selection. However genotype 2a showed higher substitution rate and a clone subpopulations dynamic compatible with a continues selection over quasispecies populations. Interplay of several factors as natural history of infection, host´s immune responses and viral genetic itself may drive the intra-host HCV evolution.