title: Design, Construction and Evaluation of 1a/JFH1 HCV Chimera by Replacing the Intergenotypic Variable Region.
abstract: BACKGROUND
  The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions.
  
  
  OBJECTIVE
  The aim of the present study was to construct an HCV 1a/JFH1 chimeric virus by replacing the intergenotypic variable region (igVR) fragment of the highly variable region of the E2 gene of the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCV genotype 1a. This chimera was produced as a model virus with the ability to be cultured. We analyzed the adapted virus and the variations of nucleic acids within it.
  
  
  METHODS
  Specific primers were designed for the igVR of HCV genotype 1a followed by the overlap-PCR method for the synthesis of the desired DNA fragment. The amplified igVR-1a chimera gene and pFL-J6/JFH were digested byKpnI andBsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed intoEscherichia coliJM109 strain competent cells. All clones were confirmed by colony PCR using specific primers, and the confirmed recombinant vector was sequenced. The recombinant vector was targeted for RNA synthesis by T7 RNA polymerase enzyme. RNA transfection was performed in the Huh7.5 cell line. Virus production in several passages and the evaluated viral load were studied using quantitative real-time PCR and ELISA methods. After 30 passages, the RNA virus was extracted and cloned in PCDNA3.1 vector, and was then sequenced.
  
  
  RESULTS
  Quantitative real-time PCR results showed 11,292,514 copies/mL of chimeric virus production in cell culture. The virus production was confirmed using ELISA, which showed a virus core production of 808.2 pg/mL. The results of cloning and sequencing showed that some of the nucleic acids in the chimera virus were changed, affecting the viral behavior in the cell culture.
  
  
  CONCLUSIONS
  Real-time PCR and ELISA showed high levels of production of 1a/JFH1 chimeric HCV in the Huh7.5 cell culture. The constructed virus can be used for future studies, including the development of new HCV drugs and vaccines.
authors.affiliation: Department of Medical Biotechnology, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran.
authors.forename: Faezeh
authors.initials: F
authors.lastname: Ghasemi
authors.affiliation: Biochemistry of Nutrition Research Center, School of Medicine, Mashhad University of Medical Sciences, Mashhad, IR Iran.
authors.forename: Majid
authors.initials: M
authors.lastname: Ghayour-Mobarhan
authors.affiliation: Faculty of Medicine, Department of New Sciences and Technology, Mashhad University of Medical Sciences, Mashhad, IR Iran; Division of Applied Medicine, Medical School, University of Aberdeen, Foresterhill, Aberdeen, UK.
authors.forename: Alireza
authors.initials: A
authors.lastname: Pasdar
authors.affiliation: Industrial Biotechnology Research Institute, Iranian Academic Centre for Education, Culture and Research Mashhad Branch, Mashhad, IR Iran.
authors.forename: Hamid
authors.initials: H
authors.lastname: Pourianfar
authors.affiliation: Hepatitis and AIDS Department, Pasteur Institute of Iran, Tehran, IR Iran.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Reza Aghasadeghi
authors.affiliation: Molecular Medicine Research Center, Hormozgan University of Medical Sciences, Bandar Abbas, IR Iran.
authors.forename: Hamed
authors.initials: H
authors.lastname: Gouklani
authors.affiliation: Antimicrobial Resistance Research Center, Mashhad University of Medical Sciences, Mashhad, IR Iran.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Meshkat
keywords: E2 Glycoprotein; Hepatitis C Virus; Intergenotypic Variable Region; Real-time PCR