An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection

Published: October 31, 2023

Abstract

Source: Nishitsuji, H., et al. Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle. J. Vis. Exp. (2017).

This video demonstrates an in vitro technique to detect hepatitis B virus (HBV) infection via a recombinant HBV reporter system. Upon infecting host cells, the recombinant viral genome expresses the luciferase reporter enzyme, which produces bioluminescence upon oxidizing its substrate — helping detect viral infection.

Protocol

1. Production of Recombinant HBV Encoding the Reporter Protein Preparation of HepG2 cells Prepare cell culture medium (Dulbecco's modified Eagle's medium or DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 100 U/ml nonessential amino acids). Plate 4 x 106 HepG2 cells in a 10-cm collagen-coated dish in 10 ml of culture medium the day before transfection. Incubate HepG2 cells at…

Disclosures

The authors have nothing to disclose.

Materials

Nano-Glo Luciferase Assay Regent Promega N1110
Penicillin-Streptomycin Mixed Solution Nacalai tesque 09367-34
MEM Non-Essential Amino Acids Solution Thermo Fisher Scientific 11140050
DMEM Thermo Fisher Scientific 11995065
Opti-MEM I Reduced Serum Medium Thermo Fisher Scientific 31985070
100 mm/collagen-coated dish Iwaki 4020-010
Lipofectamine 3000 Transfection Reagent Thermo Fisher Scientific L3000001
Polyethylene glycol (PEG) 6000 Sigma-Aldrich 81255
Polyethylene glycol (PEG) 8000 Sigma-Aldrich 89510
NaCl Nacalai tesque 31319-45
0.5 mol/l-EDTA Solution Nacalai tesque 06894-14
Tris-HCl Nacalai tesque 35434-21
Millex-HP, 0.45 μm, polyethethersulfone, filter Merck Millipore SLHP033RS
Dimethyl sulfoxide (DMSO) Sigma-Aldrich D2650
Collagen coated 96-well plate Corning NO3585
Passive Lysis 5xBuffer Promega E1941
GloMax 96 Microplate Luminometer Promega E6501
Sucrose Nacalai tesque 30403-55
Luminometer plate Greiner bio-one 655075
HepG2-NTCP1-myc-clone22 Nishitsuji, H., et al. Cancer. Sci. (2015)
pUC1.2HBV delta epsilon Nishitsuji, H., et al. Cancer. Sci. (2015)
pUC1.2HBV/NL Nishitsuji, H., et al. Cancer. Sci. (2015)
50 ml tube Violamo 1-3500-02
Anti-Myc antibody Sigma-Aldrich C3956
HBIG Japan Blood Products Organization
IFN-β Mochida Pharmaceutical 1.49872E+13
Heparin Sigma-Aldrich H3393

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Cite This Article
An In Vitro Recombinant Virus Reporter System for the Detection of Viral Infection. J. Vis. Exp. (Pending Publication), e21756, doi: (2023).

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