eoe sub articles

EoE Sub Articles

1. Production of Recombinant HBV Encoding the Reporter Protein
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	<li>Preparation of HepG2 cells
	<ol>
		<li>Prepare cell culture medium (Dulbecco&#39;s modified Eagle&#39;s medium or DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 &#181;g/ml streptomycin, and 100 U/ml nonessential amino acids).</li>
		<li>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...

an in vitro recombinant virus reporter system for the detection of viral infection

Source: Nishitsuji, H., et al. <a href="https://app.jove.com/v/54849">Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle.</a> 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 &#8212; helping detect viral infection.

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

1. Production of Recombinant HBV Encoding the Reporter Protein

	Preparation of HepG2 cells
	
		Prepare cell culture medium (Dulbecco&#39;s modified ...

Take a human liver cancer cell culture, genetically modified to express sodium taurocholate co-transporting polypeptide or NTCP &#8212; a transmembrane receptor.
Add recombinant hepatitis B virus, or HBV. The virus comprises an outer envelope containing surface proteins and an inner nucleocapsid enclosing the genome. Insertion of a luciferase reporter gene inhibits the virus&#39;s reproductive ability.
Viral envelope protein binds to the host cell&#39;s NTCP receptor.
NTCP interacts with the epidermal growth factor receptor, EGFR, and oligomerizes, inducing EGFR-mediated endocytosis of the virus.
The viral envelope fuses with endosome membrane to release the nucleocapsid, which disassembles at the nuclear pore complex.
The viral genome enters the nucleus and converts to covalently closed circular DNA or cccDNA &#8212; a stable template &#8212; encoding viral proteins, including luciferase.
At predefined intervals, harvest the cells and lyse to release intracellular luciferase.
Introduce a reporter substrate. Luciferase oxidizes the substrate, producing a signal.
A post-infection increase in luciferase activity indicates continuous expression of the viral genome.