You could check the medium that the cells are growing in by coimmunoprecipitation using an antibody against the EBV envelope (an anti-gp350 antibody may work). The antibody will pulldown envelope protein on its own and also virions. Then perform a western blot of the beads and probe for the capsid - the capsid should only be present if virions are present (ie, envelope p[rotein should not pull down capsid too).

Hi Lindsay. Do you know good working antibodies against gp350 and the EBV capsid? Another problem is to get an adequate positive control. At the momemt I have no cell line which is definitely able to produce EBV particles.

Daniel, I only used EBV to immortalise B cells so I cannot suggest which antibodies you could use, but I would expect there to be plenty of commercially available ones that would do and I seem to recall the 72A1 antibody clone being used to immunoprecipiate EBV virions. For your positive control I would suggest the B95-8 marmoset cell line which releases EBV. Eric's suggestion of PCR amplifying DNAse-treated medium is good as a cheaper alternative, but that would also require a positive control with B95-8 cells. I guess the B95-8 cell line is pretty expensive if you only wish to determine whether or not your two cell lines are producing EBV. Unfortunately I no longer work with EBV so I cannot send you any B95-8 cells or virions.

the best way to detect the virus in the supernatant of culture is to detect the viral genome by PCR

To check the presence of ebb lytic cycle. I would suggest to do a DNA zol extraction so that the viral DNA is also spooled out then proceed wit a qpcr for genes gp350, bzlf1, borf1/2. Bzlf would suggest the activation of virus lytic cycle and additionally you can complement your results by doing an RT pcr as suggested above. One other method maybe a bit tedious is to naive infect B cells or blasts and use immunofluroscence for gp350/220 in a time kinetic (choose points as mentioned in literature ) and check on the microscope. Or if you have acces to electron microscopy just use the supernatant of ur current culture and look for the virions.

We currently performed genome detection by Real time PCR but this does not give information of infectiousness of particles...

Do PCR using primers directed to a conserved region of the virus

The suggestion by Eric Pringle and Sri Harsha Meghadri to PCR for one or several late genes of EBV such as the glycoproteins makes sense. Late genes are not expressed in an infection unless early genes are expressed and turn the late genes on. You can use an Invitrogen viral DNA extraction kit - PureLink if that is in your budget - it works well on cell free supernatants.

Hi, first you need to establish if your cells contain the lytic phase virus as

you will find virions in your supernatant if EBV is in the lytic cycle. PCRing for Bzlf genes should confirm EBV lytic cycle. As already suggested by someone else an IP for capsid protein would also help you identify the virions. However, detection of virions may not necessarily means infectivity too.

If you decide to go for the PCR I would use material from both cells and cell supernatant as this will be more informative for the effect you want to see. good luck

Personally, I would not look for BZLF1 RNA as there is always a little bit of early lytic gene expression even in cell lines that never release infectious virus (eg LCLs made from cord blood/some tumour cell lines). I would do QPCR on DNA from the supernatant. Simply lyse the s/n in 1x PCR buffer + proteinase K, heat inactivate the PK, then run QPCR for either a late lytic gene (like gp350) or the viral polymerase; BALF5. If you used a plasmid standard as already suggested you could then quantitate the amount of virus, although it is very easy to contaminate pipettes etc when using DNA standards in QPCR so adequate care and negative controls should always be used. If you wanted to check that you really had infectious virions use a transformation assay using your s/n to transform resting B cells. Again, as already suggested you could also look for polymerase RNA in the cells but be aware that with very sensitive QPCR you find low levels of lytic transcripts even in cells that are not producing virus. As for positive control - any LCL made with either B95.8 or any wild type virus on resting B cells from a normal healthy donor should contain at least 1-2% lytic cells at any given time, if left untreated. One final thing to consider is what strain of virus was initially used to immortalise your lines - some modified lab strains contain GFP and that can be used to titre the virus - search for 'green Raji units' in the literature and you should find a method - Raji is a Burkitt lymphoma cell line and you will need to get hold of this if you want to use this method. gp350 Western blot will also work but is nowhere near as sensitive as QPCR and I'm pretty sure that there are good commercial Abs. As suggested the widely available B95.8 cell line would work as a good positive control for this (B95.8 is used interchangably to refer to the strain of virus released from this cell line and the cell line itself) but be careful with the loading control as it is from a monkey! (Standard GAPDH PCR primers bind poorly to B95.8 but I'm not sure about the antibody - just don't be surprised if human gene controls don't work). Hope that helps.

It would be helpful to know what you need the information for? Do you need a cell line which is completely incapable of producing infectious virus? Does it have to be a lymphoblastoid cell line or can it be a lymphoma cell line? If you can use an EBV-infected Burkitt lymphoma line that is incapable of producing virus, Raji cells are a prime example, carrying a deletion of an essential lytic gene.

All LCLs are likely to have some level of lytic gene expression although the level of actual virion production is slight and can vary considerably among cell lines. A simple and highly sensitive way of comparing the level of lytic gene expression in addition to the RT-PCR suggested above is to stain the cells for surface gp350 and just examine them under fluorescent microscopy. This usually correlates well with the ability to produce virus. If you need to establish whether infectious virus is being made, and the virus does not express GFP, you can infect an EBV-negative cell line or primary B cells with filtered supernatant and stain for EBNA expression in the infected cells.

I would like to thank all of you very much for your helpful answers.

Indeed we need cell lines which are completely incapable of producing infectious virus. We would like to work with the two cell lines MEC-1 and JVM-3. Both cell lines were EBV-transformed with supernatants of B95.8 cells and are described as 'true' B-cell chronic lymphocytic leukemia (B-CLL) cell lines (that's what we want). Until now it is not clearly shown that these cell lines are completely incapable of producing infectious EBV particles. It would be very helpful for us if we could provide evidence of this incapability.

We do this for EBV and HHV-8 perfectly well using our established qPCR assays. If you need more advice you can contact me.

Not sure if IHC is something you want to do but you could always make a smear and use EBER.

http://www.leicabiosystems.com/ihc-ish/novocastra-reagents/ish-probes-reagents/details/product/eber-probe/

Dear all,

I would like to know if there is a method to understand if B95-8 cell line produces virus. I tried with PBMC infection in titration but it is not clear at all. Is it possible doing the PCR of cell line supernatant?

thanks in advance