Preparation of Pseudo-Typed H5 Avian Influenza Viruses with Calcium Phosphate Transfection Method and Measurement of Antibody Neutralizing Activity

This protocol describes the procedures and the critical steps of H5 high pathogen avian influenza pseudo virus preparations and pseudo virus neutralization assays. Also, it discusses the troubleshooting limitation and modifications of these assays. Highly pathogenic pathogens must be carried out in the level three bio safety laboratory, whereas pseudo viruses can be safely handled in a level two bio safety laboratory.

To begin, make a single cell suspension of HEK293 FT cells in a complete DMEM medium. Add 10 milliliters of the single cell suspension to a T75 flask, followed by incubation at 37 degrees Celsius. Two hours before transfection, replace the old medium with 10 milliliters of fresh complete medium with chloroquine.

Mix the reagents and plasmid DNA as described in the manuscript then transfer this mixture into the medium above the cells and rock gently. After incubation, replace the medium with 15 milliliters of fresh complete DMEM medium. After 65 hours, harvest the supernatant by centrifugation.

Collect the supernatant and aliquot it. To detect hemagglutinin protein expression, add 50 microliters of PBS into columns two through 12 of a 96 well round bottom plate. Add 100 microliters of pseudo virus into the wells of column one.

Then transfer 50 microliters of pseudo virus from column one into the wells of column two. Perform this twofold dilution until column 11 followed by gentle mixing. Then discard the extra 50 microliters from column 11.

Add 50 microliters of 0.5%erythrocyte into each well followed by gentle mixing. After 30 to 60 minutes of incubation at room temperature, observe and record hemagglutinin titers of the pseudo virus. For pseudo virus titration, make a single cell suspension of five times 10 to the fourth cells per milliliter of MDCK cells in a complete DMEM medium.

Add 1, 250, 2, 500, 5, 000, 10, 000, 20, 000, and 40, 000 cells to different wells of a flat bottom 96 well plate. After incubation, thaw and vortex the pseudo virus. Add six HAU of pseudo viruses to each well of a 96 well round bottom plate and replenish each well with complete DMEM up to 120 microliters.

Transfer 100 microliters of the mixture to the cells in the 96 well plate. Incubate the culture plate for 48, 60, and 72 hours for viral infection and perform a luciferase assay. To perform the luciferase assay, wash the cells by removing the medium carefully and rinsing it with 200 microliters of PBS.

Remove as much of the PBS as possible, then add 50 milliliters of the lysis buffer to each well. On the next day, equilibrate the plates at room temperature for two hours, then rock the culture plates several times and transfer all the cell lysates to opaque 96 well plates. Add 50 microliters of the substrate to each plate and mix it well.

Record the luciferase titers of the pseudo virus by using the luminometer. Add 100 microliters of the single cell suspension of MDCK cells to each well of a flat bottom 96 well plate and incubate it for 20 hours at 37 degrees Celsius. After the incubation, thaw and vortex the pseudo virus and the sera stored at minus 80 degrees Celsius.

Dilute the sera 20 times in a complete medium. Add 100 microliters of DMEM medium to columns three through 10. Add 200 microliters of the diluted sera into the wells of the second column and perform serial dilution.

Then add 100 microliters of DMEM medium to the 11th column as the negative control. Add 100 microliters of pseudo viruses to each well and mix it. Transfer 100 microliters of the mixture from the 96 well round bottom plate to the corresponding well of the 96 well cell culture plate.

After 60 hours of incubation at 37 degrees Celsius, perform the luciferase assay. The hemagglutinin assay showed that 643 HA units per milliliter of influenza pseudo viruses are present in influenza pseudo virus stocks. The ratios of HA and Gag P24 were within a normal range.

The western blot assay showed the presence of four protein types, including HA0, HA1, HA2, and NA proteins, indicating the similarity of influenza pseudo viruses to that of wild type viruses. Pseudo virus titration results indicate that the suitable conditions for the highest relative luciferase activity include the feeding of 5, 000 cells in a well of a 96 well plate and detecting relative luciferase activity readings after 60 hours of virus infection. The immune sera from the DDV group exhibited high neutralization titers against pseudo virus A Thailand 1 Can 104 strain, whereas sera from the control group did not exhibit any neutralization activity.

Neutralizing activity of the immune sera was potent compared to the control sera, indicating that a lot of antibodies were directed to the HA head in the immune sera. The significant differences between the immune sera and the controls during the assessment of viral entry indicate that antibodies are directed to the HA stem region in the immune sera. When performing this procedure, remember to keep the pH of the transfection solution and the culture medium stable.