Measuring the amount of virus! - How to calculate the virus concentration? (Mar/22/2006 )

Dear All,
Can anybody tell me how to calculate the 1.5 μmol of viral
membrane phospholipid?

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Reference:
"Virosome-mediated delivery of protein antigens in vivo: efficient
induction of class I MHC-restricted cytotoxic T lymphocyte activity"
Vaccine 23 (2005) 1232–1241

Material and Methods
In short, virus (1.5 μmol of viral
membrane phospholipid) was solubilized in 100mM
octa(ethyleneglycol)-n-dodecyl monoether (C12E8) (Calbiochem,
San Diego, CA) in buffer containing 5mM Hepes,
150mM NaCl and 0.1mM EDTA (HNE buffer).
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Is there any quantifiable marker in your virus, or can you do quantitative (RT)-PCR to determine the amount of viral genomes in 1 ml? From this value you can approximate your number of viral particles in a given volume and (if you know the average size of your virus) estimate how much viral membrane phopholipid you have.

don't know if it would be accurate enough...

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The information I have is that the influenza virus is of 200 nm and each virus has 100,000 viral phospholipid molecules. How about the haemagglutination inhibition or TCID50 for knowing the particles per ml? I am confused about the "quantifiable marker" and we don't have realtime PCR.

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I personally don't like using TCID50 too much, because you have more infectious strains than others (so lower amount of virus for the same TCID50).
With a quantifiable marker, I mean a protein that's used in ELISA, and of which the approximate amount per viral particle is known.

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viral quantification

As per my knowledge you can use RT-PCR based quantification or Filter Hybridization assay to measure the viral titration. I think you can find the protocols.

Brief description of the expt is

Infect the animal cells (time depends upon the viral capablitiy of infection e.g 12 to 24 hrs) with different dilutions of viruses, and transfer the cells to nitrocellulose membrane. use a radio labeled probe to determine the positive signals (by autoradiography) from the viral genome amplifications.

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O’Carroll S. J., Hall A. R., Myers C. J., Braithwaite A. W., Dix B. R. Quantifying adenoviral titers by spectrophotometry. BioTechniques, 28: 408-412, 2000.

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Using those numbers, I calculate 9 x 10^12 virions provide 1.5 umole of lipid. However, I assume the authors of that paper actually measured total lipid phosphorus in their virus stock.

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