engleski

Genetic Variability of Hepatitis C Virus in Patients Infected from a Common Source

INTRODUCTION. Infection with hepatitis C virus (HCV) is the leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, affecting over 170 million people worldwide. Despite the absence of a satisfactory tissue culture system or a small animal model to study HCV replication, detailed analysis of HCV genomic variability early after infection has also been hampered by the fact that HCV infecting strains are usually heterogeneous. Furthermore, samples and clinical data from acute phase and follow up after infection are generally not available since patients are frequently asymptomatic. In this study we have had the unique opportunity for analyzing the HCV population in patients infected from a common source at early times after infection and follow up those with chronic infection. METHODS. Two out of seven patients spontaneously clarified the virus, while the other five were persistently infected. HCV-RNA was extracted from serum by trizol method, and RT and nested-PCR reactions were performed by standard protocols. All reactions were carried out using the Expand High Fidelity PCR System (Roche) for diminishing genetic background during DNA amplifications. We amplified a fragment of 396 nt of the E2 gene encompassing the hipervariable region 1 (HVR-1). At least two different PCR amplifications were done from each cDNA in order to avoid molecular bottleneck in ulterior clonal analyses. RESULTS. Two different groups of patients could be defined by HVC viral load values, one including the two spontaneous patients and one chronic (average 6.9 x103 IU/ml, ranged 5 to 7.4 x103), and other for the rest of chronic patients (average 346.5 x103 IU/ml, range 76 to 857 x103). Amino acid consensus sequences were almost identical, indicating the oligoclonal nature of this infection. Quasispecies analyses of samples taken at early times revealed a great heterogeneity for the lowest viral load group in contrast to the highest one, with average Shannon entropy values of 0.839&plusmn ; 0.049 vs. 0.427&plusmn ; 0.084, respectively. CONCLUSION. HCV infection establishment after transmission is a bottleneck event. Viruses must find genomes with higher replication rates (fitness) to counteract the immune defense of the new host. Once a master sequence is established, infection goes on to chronicity. However, at early times after infection, host response could force viruses to replicate with lower efficiencies, leading to a quasispecies structure without a predominant master sequence and with genomes at the edge of fitness threshold.

HCV; quasispecies; hepatitis; genetic variability; viral fitness; bottleneck effect; HVR-1

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