I have done plaque assays of herpesviruses in animal tissues and have seen the following over the last couple of decades:

1) At high concentrations of tissue homogenate, I have observed either a lack of plaques or a low number of plaques showing up on an intact cell monolayer and that the number of plaques increased in the next serial dilution and then decreased in dilutions thereafter. We attributed that phenomenon to components in the homogenates (IFNs or ILs) that at high concentrations could inhibit the virus growth without visible effects on the cell monolayer. The inhibition required tissue from an infected animal, since mixing known PFU of virus with homogenates from uninfected animals did not show the inhibitory effect. My workaround that I used (and was detailed by others) was to increase the sensitivity in the lowest dilution of clarified homogenate by using it to infect a much larger monolayer of cells in a bigger dish/flask/plate. That way, the inhibitors would dilute out and allow for a quantitative plaque formation. Sometimes, the number of plaques in this larger format assay would be significantly higher than that predicted by the smaller assay with multiple dilutions.

2) Also, just by chance, sometimes the few PFU in the sample would not end up in the lowest dilution but end up in latter dilutions. This will happen sporadically, but if you see the number of plaques increasing with increasing homogenate concentration and then stay the same or decrease in the highest dilution, then you might want to check for inhibition as above. Note that even in replicates using inbred mice, some mice had more inhibition of plaque formation than others.

Hope that helps.

Dear Li-Ming

I suppose that a high amount of virus would cause all cells in your dish to lyze, thus giving a max response. So no plaques at high conc but at dilutions probably points more towrd a handling/sterility problem. Do you routinely run controls with no virus but with dilutions?

I have done plaque assays of herpesviruses in animal tissues and have seen the following over the last couple of decades:

1) At high concentrations of tissue homogenate, I have observed either a lack of plaques or a low number of plaques showing up on an intact cell monolayer and that the number of plaques increased in the next serial dilution and then decreased in dilutions thereafter. We attributed that phenomenon to components in the homogenates (IFNs or ILs) that at high concentrations could inhibit the virus growth without visible effects on the cell monolayer. The inhibition required tissue from an infected animal, since mixing known PFU of virus with homogenates from uninfected animals did not show the inhibitory effect. My workaround that I used (and was detailed by others) was to increase the sensitivity in the lowest dilution of clarified homogenate by using it to infect a much larger monolayer of cells in a bigger dish/flask/plate. That way, the inhibitors would dilute out and allow for a quantitative plaque formation. Sometimes, the number of plaques in this larger format assay would be significantly higher than that predicted by the smaller assay with multiple dilutions.

2) Also, just by chance, sometimes the few PFU in the sample would not end up in the lowest dilution but end up in latter dilutions. This will happen sporadically, but if you see the number of plaques increasing with increasing homogenate concentration and then stay the same or decrease in the highest dilution, then you might want to check for inhibition as above. Note that even in replicates using inbred mice, some mice had more inhibition of plaque formation than others.

Hope that helps.

 Dear Dr. Brodin and Dr. Morello:

Thank you so much for your suggestions and experiences sharing.

I guess I will evaluate whether the same phenomenon only existed in influenza A lung infected mouse tissue, but not in uninfected mouse'. Special thanks to Dr. Morello for pointing out IFN and IL as key players in the immune inhibitory effect on virus growth. It provides a lot of directions for troubleshooting and is really helpful.

Best Wishes to you both.

Li-Ming

What you are describing happens all the time for many bunya and arena-viruses.  It is also known as the "pro-zone effect", and certainly for arenas, it occurs because these viruses make defective interfering (DI) particles that prevent a cell from becoming productively infected (ie- a cell infected with a 'normal' virus and a DI will not go on to produce more virions, because the DI blocks the ability of the 'normal' virion to establish an infection).  At the most concentrated dilutions, you have enough DI particles in the stock to prevent all the cells in the well from becoming infected (because it only takes one DI particle to stop even a large number of normal virions from establishing infection in a given cell).  As you dilute out the viral stock, you dilute out the DI particles as well, and the likelihood of a given cell being infected by both kinds of virus drops, so you begin to see single plaques again.  Influenza also makes DI particles, and they are known to vary by cell type (they are affected by lots of other things too, but this is not well understood).  So- what you may be seeing is an effect on DI production- perhaps specific to your drug treatment, perhaps specific to the way you are growing your virus.  If you see changes in this prozone effect with drug treatment I'd say its worth paying attention to, as altering the DI levels can also (in theory) affect virulence/persistence given the DI particles reduction of cyopathic effect.

As mentioned by Emily above Arena viruses are notorious for this . . . on of our models is LCMV and if you don't know what you are looking at a 10 fold dilution from viral supernatant looks like there is no virus - no plaques - the monolayer has however a "slightly" different color after either Neutral Red or Crystal Violet stain and if you know what to look for and have a clear picture in your mind what an uninfected monolayer looks like you CAN "see" IT.

As for Influenza . . . I'm assuming you are using MDCK cells for your plaque assay? and add Trypsin? In the lower wells both the "non-infectious particles" and the (limiting) amount of Trypsin may play a role in the lack of apparent plaques.

 Dear Dr. Bruce and Dr. Raue:

Thank you so much for providing different points of view and the detailed explanation.

Yes, I used MDCK cells for my assay and use trypsin as well.

From my data, if I withdrawal the virus after a hour incubation, the "pro-zone effect" was not shown, but if I let virus particle from lung tissue stayed on MDCK cells after a hour incubation up to 72 hrs, then staining with crystal violate. The "pro-zone effect" was found. Therefore, I guess so far it might be the effect of IL and IFN that Dr. Morello  mentioned earlier. And the effect of these cytokines decreased with the dilution, so I can only see the "pro-zone effect" in the first two dilution.

Best Regards,

Li-Ming