This technique of producing influenza viral PP and determining its infectivity can simplify the research of influenza virus in a BSL-2 city. We normalized the PPS for the infections based on qRT-PCR data of PPS. Two glycoprotein expression plasmid are encoded, which can simplify the research on virus reassortment.
One day after cell seeding, check the cell morphology and density under an inverted light microscope. Ideally the cell should be approximately 85%confluent at transfection. Replace the medium with one milliliter of serum-free DMEM per well, and put the plate back in the incubator.
For each well of cells to be transfected, dilute eight microliters of the transfection reagent to a volume of 150 microliters with reduced serum medium. Mix the solution gently and let it sit at room temperature for five minutes. Meanwhile, dilute 2.5 micrograms of plasmid DNA into 158 microliters of reduced serum medium.
After the five minute incubation, combine the diluted DNA with diluted transfection reagent, gently mix the solution and leave it at room temperature for 15 minutes. Add the DNA lipid complex to the corresponding wells with the cells in serum-free medium. Then mix the plate gently by rocking back and forth.
Incubate the plate at 37 degree Celsius and 5%carbon dioxide for four to six hours. After the incubation, remove the medium and replace it with two milliliters of DMEM per well. Then incubate it for another 36 to 48 hours.
Seed each type of susceptible cell at 10, 000 cells per well and a 96 well plate. Then leave the plate over night in a 37 degree Celsius and five percent carbon dioxide incubator. On the third day after transfection, check the color of the medium which should light pink or slightly orange and examine the cells with an inverted florescent biological microscope under 440 to 460 nanometer wavelength.
At 38 to 48 hours after post-transfection, harvest the pseuotyped particles or PPS by passaging them through a 0.45 micrometer polyvinylidene fluoride membrane filter to eliminate cell debris, then divide them into small volume aliquots and store them at minus 80 degree Celsius. To quantify the PPS, transfer 30 microliters of the purified viral particles to a 1.5 microliter RNase-free tube and add one microliter of Benzonase Nuclease. Incubate the tube at 37 degrees Celsius for one hour to eliminate any DNA and RNA contamination.
After the incubation, freeze the sample to minus 70 degrees Celsius to active the nuclease and add two microliters of Proteinase K.Incubate the mixture for 30 minutes to digest the envelope proteins and release the CMV-GFP RNA. Then inactive the Proteinase K at 100 degrees Celsius for three minutes. Quantify the PPS with quantitative reverse transcription RTPCR using the Universal Probe One-Step RT-qPCR Kit in the primers and probes specified in the text manuscript.
Normalize the PPS to four times 10 to fifth RNA copies per milliliter. Then add TPCK-trypsin to a final concentration of 10 micrograms per milliliter into the PPS that harbors H7N9 hemagglutinin. Incubate the PPS at 37 degrees Celsius for one hour to form the functional subunits HA1 and HA2.
Mix the normalized PPS with DMEM medium at a one to one ratio, then bring the plate containing susceptible cells to the biosafety cabinet. Aspirate the supernatant and wash the cells once with 100 microliters of pre-warmed PBS. Add 100 microliters of PPS-DMEM mixture into each well, triplicating the infectivity tests of each type of PPS to one susceptible cell line.
Incubate the plate for four to six hours at 37 degrees Celsius and 5%carbon dioxide. Then aspirate the supernatant and replace it with 100 microliters of DCM Incubate the plate for another 24 to 36 hours before infectivity detection. Our results are PPS are considered now infectious to people.
So necessary safe guard procedures are recommended to take such as wearing a mask and performing the infectivity assays in a biosafety cabinet. This protocol was generate to generate 10 types of pseudotyped particles or PPS with two grouped hemaglutinin and neuraminidase vesicular stomatitis virus G glycoprotein or no envelope glycoproteins. The PPS were imaged with transmission electron microscopy and their infectivity were evaluated in two cell lines, A549 and MDCK.
In the PPS group harboring H5, the infectivity of H5N1, H5 plus N9 and H5 was approximately 90%18%and 10%for cell line A549 and 40%5%and 5%for MDCK, respectively. In the PPS group harboring H7, the infectivity of H7N9, H7 plus N1 and H7 was approximately 10%7%and 1%for cell line A549 and 8%4%and 1%for MDCK, respectively. The infectivity of vesicular stomatitis virus G glycoprotein PP was about 21%for A549 and 16%for MDCK cells.
While the delta envelope glycoproteins PP showed no infectivity. It's critical to remember that the PP producer HEK-293T/17 cells constitute to the foundation of this procedure. So the optimal growth teach us HEK-293T/17 cells is most important.
Following this procedure or some other test such as L2 recept the bounding assay can be performed. This assay can almost give rid of the biological peculiarities of the receptor preference and the pattern will reveal the tropism of the PPS. Since they took the glycoproteins from influenza virus are cloned into two plus max.
HA and the NA proteins can be studied separately so we reassortment between them. The mutants and the mature effects can be explored.
