Production and Titration of Chimeric HCV Viruses

The sequence of the genotype 2a/2a chimera J6/JFH1 has been deposited in a gene bank (accession number JF343782). Construction of Jc1 (54 (link)), and mCD81/Jc1 (24 (link)) (mtHCV) was described elsewhere. BiCre-Jc1/mCD81 was generated by introducing the L216F, V388G, and M405T point mutations into BiCre-Jc1 (16 (link)) via site-directed mutagenesis. Jc1(p7nsGluc2a) and Jc1/mCD81(p7nsGluc2a) were generated by ligating an EcoRI/BsaBI fragment containing core, E1, and parts of E2 into the EcoRI/BsaBI backbone of Jc1/Flag2(p7nsGluc2a) (29 (link)). To produce infectious virus, Huh-7.5.1 (52 (link)) or Huh-7.5 (55 (link)) cells were electroporated with in vitro-transcribed full-length HCV RNA (T7 RiboMAX express large-scale RNA production system; Promega, Madison, WI). Seventy-two hours postelectroporation, the medium was replaced with DMEM without FBS, and supernatants were harvested every 6 h starting from 72 h. Pooled supernatants were filtered through a 0.45-µm bottle top filter (Millipore, Billerica, MA) and concentrated using a stirred cell (Millipore). Viral titers (TCID₅₀) were determined using Huh-7.5 cells as previously described (56 (link)).

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von Schaewen M., Dorner M., Hueging K., Foquet L., Gerges S., Hrebikova G., Heller B., Bitzegeio J., Doerrbecker J., Horwitz J.A., Gerold G., Suerbaum S., Rice C.M., Meuleman P., Pietschmann T, & Ploss A. (2016). Expanding the Host Range of Hepatitis C Virus through Viral Adaptation. mBio, 7(6), e01915-16.

Publication 2016

T7 RiboMAX express large-scale RNA production system 0.45-µm bottle top filter stirred cell

Backbone Cells Chimera Ecori Gene Genotype Infectious Point mutations Promega Site directed mutagenesis Virus

Corresponding Organization :

Other organizations : Princeton University, Rockefeller University, Center for Experimental and Clinical Infection Research, Ghent University Hospital, Medizinische Hochschule Hannover

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Variable analysis

independent variables

Introduction of the L216F, V388G, and M405T point mutations into BiCre-Jc1 via site-directed mutagenesis

dependent variables

Production of infectious virus

control variables

Huh-7.5.1 or Huh-7.5 cells used for electroporation and viral titer determination
DMEM without FBS used for harvesting supernatants

controls

Positive control: Jc1(p7nsGluc2a) and Jc1/mCD81(p7nsGluc2a)
Negative control: Not explicitly mentioned

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