We would like to test antiviral activity of compounds against Herpes simplex. What could be the most simple way to do it, maybe in cell cultures (usually we are doing anticancer experiments)?
Dear Dr. Petrikaite,
To check the antiviral effect of the compound, you should first confirm the infectivity of the virus. For that Culture the cells with the virus and confirm the infectivity by either PFU, CPE, TCID50, PRNT, imaging or by mRNA. Find the optimum concentration needed to infect the N number of cells.
Once the infective dose of virus is optimized, the antiviral activity can be confirmed by two ways depending upon the blocking mechanism of the compound.
First is culture the cells with the compound before infecting the virus.
Second is incubate the compound with the virus before infection.
Hope this helps
All the best
This depends on the mechanism of the compound. But as Prashant said the first thing you need is a good system to grow and detect the virus. With Herpes virus this should not be too difficult. Ensure you have a well characterized and productive virus stock. Then titrate your compound on the virus and/or cells (depends on the expected mechanism). Look for a change in titer from the untreated culture. You should be able to come up with an EC-50 for your compound, if it has a significant effect.
Just some details regarding the previous answers. For the Herpes simplex virus we used A-549, L-41 or MA-104 cell lines. All were sensitive and showed a good CPE. The best (and simpliest) way to quantify the CPE is the MTT-method. We always used two variants: 1) preincubation of a cell culture with the tested compound 1 h, then inoculation of a virus (an in vitro analogue of the "prophylactic" activity); 2) infection of a cell culture with a virus, 30 min incubation, washing out of the non-fixed virus, introduction of a compound under investigation. Good CPE became evident in 3-4 days after infection. After OD mesurement of MTT-reaction you calucate the reduction of -lg TCID50 comparing virus control for several concentrations of a compound. The best way to calculate is the non-linear regression model (the GhraphPad Prism software). Then you calculate EC50 and SI = IC50 (average toxiciy of a compound)/EC50.
Yes, we use the tissue culture for testing the antiviral compounds of avian influenza. But if you have an access to the embryonated chicken eggs, it works better than the TC.
Inoculate the eggs at age of 9-11 days through allantoic sac route with different dilutions of the material.
Thanks for all valuable suggestions! Is it OK to do antiviral experiments in incubators with other cells, or the risk of contamination is too high?
It is necessary to use separate CO2-incubator and strongly recommended to carry out all procedures in the separate laminar box because the HSV is very contagious!
What Mikhail Yurievich Eropkin suggested is to test antiviral activity by pretreating the cells prior to virus infection. There is several ways to test antiviral function of your compund, which has been mentioned in the paper of Article In vitro anti-Herpes simplex virus activity of crude extract...
Yes, there is more risk of contamination with the tissue culture than with the embryos. The egg is a sealed sterile room full of antibodies (igA, IgY, IgM) against most contaminant, it is a living creature with all his systems, as immune system (although not fully functional) which is not the case with the tissue culture.
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