24 mai 2006

Is there some silent HCV in my cell culture ?

The question is the following:

Is there some "silent" hepatitis C virus in my cell culture in vitro ?

Indeed, the virus might not be detected under normal, conventional cell cultures conditions, but might be "awaked" under some peculiar cell culture conditions.

What are these peculiar cell culture conditions? Simply:

Incubating the cells without cell culture medium replacement, for a whole week.

The following cell lines can be tested:

    - HepG2

    - Huh-7

    - HepaRG

After a week of cell culture without medium change, the following analyses can be performed:

   - RT-PCR of the HCV RNA in the cell culture supernatant ;

   - RT-PCR of the HCV RNA in the cells.

A parallel control involving the same cells undergoing regular cell culture medium change (i.e.: every 2 days) must be performed, as a negative control.

It will appear that the result of the RT-PCR will be sometimes positive, when no cell culture medium change has been performed. This might be due to the fact that the cap-dependent translation is no longer efficient or extremely low under these cell culture conditions, and that the cap-independent translation on the HCV IRES mRNA has been activated to allow virus synthesis.

It could also be, as a working hypothesis, that the HCV RNA is kept as a silent,  negative strand in the cells that undergo regular cell culture medium change.

Hepatocyte cell lines must always be considered as potentially silently infected with hepatitis C virus. A regular analysis of the HCV RNA in these cells by RT-PCR might be performed, with the RT involving generation of cDNAs on BOTH the positive-stranded and the negative-stranded HCV RNA templates.

keywords: hepatitis C virus, HCV, hepatocyte cell line, hepatocyte, cell culture, in vitro culture, RNA, positive strand, negative strand, medium change, hepatocellular carcinoma, silent virus, HepG2, Hep-G2, Hep G2, HuH-7, HuH7, HuH 7, HepaRG, Hepa-RG, Hepa RG, RT-PCR, reverse transcription, polymerase chain reaction, supernatant, translation, protein synthesis, IRES, internal ribosome entry site, initiation factors, reactivation, activation, virus synthesis.

Posté par hepatocyte à 10:45 - Commentaires [0] - Permalien [#]

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