Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

The overall goal of this procedure is to determine the antiviral effect of test compounds against specific steps of early viral entry. This is accomplished by first seeding the host cells for the viral infection. The second step is to perform the viral infection and the test compound treatments at different times and temperatures.

Next, the drug treated inoculums are washed away and the cells are overlaid with basal medium. The final step is to incubate the cells. Ultimately, viral infectivity is analyzed to determine the influence of the test compounds on the early viral entry steps of the viral infection.

For example, a luminometer is used to assess the luciferase activity in the cell snat from infection by a reporter virus. The main advantage of this technique is that it can allow a more mechanism based approach in identifying antiviral agents, particularly those targeting viral entry. To begin, first, prepare stock solutions of the test compounds, trilogy acid or CHLA and PUNIC collagen or PUG in DMSO dissolve heparin for a positive control in sterile double distilled water to prepare host cells for the assay culture.

Human hepato carcinoma, HUH 7.5 cells overnight in fresh DMAM supplemented with FBS and antibiotics. Remember to follow biosafety level two procedures throughout the experiment to test the cytotoxicity of the test compounds. Treat the cells with increasing concentrations of CHLA or PUG from zero to 500 micromolar diluted in basal medium, or 1%DMSO as a negative control, and then incubate the cells for 72 hours at 37 degrees Celsius in a cell culture incubator.

Remove the medium and wash the cells with 200 microliters of PBS twice. Next, add 100 microliters of XT T assay reagent, and incubate at 37 degrees Celsius for three hours. Then use a microplate reader to determine the absorbance at 492 nanometers with a reference wavelength at 690 nanometers.

Calculate the percentage of viability with a given formula from these values. Determine the 50%cellular cytotoxicity concentrations for each compound. To begin the viral inactivation assay.

First plate, one times 10 to the fourth cells per well. In a 96 well plate incubate the cells at 37 degrees Celsius with 5%carbon dioxide overnight for attachment for infection. Prepare Gaia luciferase reporter tagged hepatitis C virus or HCV particles as referenced in the text protocol.

In a sterile tube mix 100 microliters of 100 micromolar CHLA or PUG with 100 microliters of 10 to the fourth. Focus forming units or FFU of HCV for a positive control mix. HCV with heparin at a final concentration of 1000 micrograms per milliliter.

Incubate at 37 degrees Celsius for three hours. After three hours, dilute the virus compound mixture 50 fold with 9.8 milliliters of basal medium at room temperature. Then prepare a new virus compound mixture, and immediately dilute this mixture in basal medium for the zero hour incubation sample.

After removing the incubation medium from the cells, add 100 microliters of the diluted virus drug solution per well in triplicate. The solution now contains 10 to the second FFU per Well incubate a 37 degree Celsius and 5%carbon dioxide for three hours to allow viral infection of the cells. After incubation, remove the viral suspension from the wells and gently wash the cells with 200 microliters of PBS.Twice.

Incubate the cells in 100 microliters of basal medium for 72 hours for viral replication and release of luciferase reporter into the supernatant. Then collect the supernat from the cultures and micro tubes and centrifuge at 17, 000 times G for five minutes at four degrees Celsius. To remove cellular debris, mix 20 microliters of each supernat with 50 microliters of gaussi luciferase assay reagent, and measure the luminescence from the luciferase reporter activity in a luminometer.

According to the manufacturer's instructions for the viral attachment assay plate, one times 10 to the fourth cells per well. In a 96 well plate and incubate overnight for cell attachment the next morning. First chill, the 96 well plate containing the cell monolayer at four degree Celsius for one hour.

Prepare the test compounds and virus mixture on ice. For example, H-C-V-C-H-L-A-H-C-V 1%DMSO or positive control HCV Heparin solutions with 10 to the second FFU virus. For each treatment, well aspirate the medium from the cell culture wells gently.

Then immediately add 100 microliters of virus test compound mixture in triplicate wells, taking care not to raise the temperature. Incubate the plate in a refrigerator maintained at four degrees Celsius for three hours. Virus particles will attach on the cell service at four degrees Celsius, but will not be internalized.

Next, aspirate the supernatants. Gently wash the cells twice with 200 microliters of ice cold. PBS add 100 microliters of basal medium to each well, and then incubate at 37 degrees Celsius for 72 hours.

Finally, for quantitating the release luciferase reporter from the infected cells. Collect the supernat from each sample well and perform the Gaussian Luciferase assay for assaying viral fusion and entry. Pre chill the cell culture plate at four degree Celsius for one hour.

After the overnight incubation for cell attachment, remove the existing medium from the wells and infect the cells with 10 to the second F-F-U-H-C-V on ice. Incubate the plate at four degrees Celsius for three hours. Gently wash the cells twice with 200 microliters of ice cold PBS to remove the non-ad adhered virus.

Then on ice, treat the cells with the test compounds dissolved in basal medium or basal medium with 1%DMSO as a control and incubate the plate at 37 degrees Celsius for three hours. This allows assessment of the test compounds on viral entry, which occurs at 37 degrees Celsius. Aspirate the medium and wash the non internalized viruses twice with 200 microliters of citrate buffer or PBS.

Add 100 microliters of basal medium per well and incubate at 37 degrees Celsius for another 72 hours. Collect the supernat and perform the Gaussian Luciferase assay as demonstrated before for detecting the released luciferase reporter shown Here are the effects of the test compounds on HCV. In activation, the virus was incubated with the test compounds for zero or three hours prior to infection.

The percentage of inhibition of viral infection is plotted for each treatment. Negative controlled DMSO shows no inhibition. CHLA exhibits high inhibition upon contact, following zero hour incubation, as well as after the longer incubation for three hours.

Additional results with another test. Compound PUG are shown here, adapting this general experimental design, CHLA and PUG are shown to additionally inhibit the infectivity of human cytomegalovirus or HCMV in embryonic lung fibroblasts. The effect of CHLA and PUG on HCV attachment is shown here.

Recall that the cells were incubated with the virus in the presence or absence of the test compounds at four degrees Celsius, and then removing the unbound virus cells were incubated at 37 degrees Celsius for infection by the virus. The shaded bars demonstrate the effect of C-H-L-A-P-U-G or heparin on HCV entry into the cells. Note that in this case, the viral attachment was first carried out at four degree Celsius.

Then after removing unbound virus, the cells were incubated with test compounds at 37 degrees Celsius for viral entry. Similarly, in the case of HCMV, infection of embryonic lung fibroblasts treatment with CHLA or PUG shows strong inhibition of viral attachment and viral entry. While attempting this procedure, it's important to remember to perform the steps at the indicated temperature, which will influence the accuracy of the results.