医療微生物学と診断

Virus-Free Synthesis of a Hepatitis C Virus P7 cDNA through a Three- Steps Polymerase Chain Reaction

Abstract

Emilia Zicca, Nadia Marascio, Grazia Pavia, Francesco Bombardiere, Sabrina D’Agostino, Fernanda Fabiani, Anna Bilotta, Nicola Perrotti, Francesco Trapasso, Maria Carla Liberto and Alfredo Focà

Hepatitis C virus (HCV) infection represents a great public healthcare challenge as it affects nearly 170 million individuals worldwide. Therefore, the deep investigation of the mechanisms involved in the pathogenesis of chronic hepatitis induced by HCV is a crucial step in the design of novel targeted therapies for the treatment of this condition. However, techniques of molecular biology to characterize HCV proteins can suffer of intrinsic limitations due to high mutation rates of the virus genome.

In this study, we propose a novel strategy to synthesize a viral cDNA sequence corresponding to the p7 gene in HCV genome-free conditions. Our approach consists of a three-steps polymerase chain reactions (PCRs) by using a set of four large overlapping synthetic oligonucleotides aimed to separately amplify both 5’ and 3’ ends of the p7 gene; 5’ and 3’ products, overlapping themselves, were then used as a template in a third PCR amplification in order to get a full-length p7 cDNA.

Our methodology represents an interesting proof-of-principle as it allows for the safe manipulation of short viral genes. Moreover, this new technique overcomes the elevated genetic variability of HCV genomes without affecting the antigenic characteristics of the putative viral protein.