Design, construction and evaluation of 1a/JFH1 HCV chimera by replacing the intergenotypic variable region

Faezeh Ghasemi, Majid Ghayour-Mobarhan, Alireza Pasdar, Hamid Pourianfar, Mohammad Reza Aghasadeghi, Hamed Gouklani*, Zahra Meshkat

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)

Abstract

Background: The E2 glycoprotein is an important encoded hepatitis C virus (HCV) protein that contains three different variable regions. Objectives: 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 E2geneof the Japanese Fulminant hepatitis genotype 2a JFH1 virus with a similar region of HCVgenotype 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 by KpnI and BsiWI restriction enzymes, and the fragment was ligated into pFL-J6/JFH. The recombinant vector was transformed into Escherichia coli JM109 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.

Original languageEnglish
Article numbere38261
JournalHepatitis Monthly
Volume16
Issue number10
Early online date7 Aug 2016
DOIs
Publication statusPublished - 1 Oct 2016

Keywords

  • E2 glycoprotein
  • Hepatitis C virus
  • Intergenotypic variable region
  • Real-time PCR

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