Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

This protocol allows researchers to generate pseudotype viruses that can be safely used to study viral entry events of highly pathogenic viruses, such as sal coronavirus and most coronavirus. This versatile technique is based on the easy setup transient transfection. This system can also be applied to produce particles pseudotyped with fusion proteins of other viruses, not just coronavirus S.For best results it is important to monitor cell health and density for transfection as well as for pseudotype virus infection.

Demonstrating this procedure will be Tiffany Tang, and Lakshmi Nathan. Graduate students from my laboratory. To begin, carefully wash HEK293T cells with 10 milliliters of 37 degrees Celsius, pre-warmed DPBS twice.

Next, aspirate the supernatant and attach cells with one milliliter of 25%trypsin solution that has been pre-warmed at 37 degrees Celsius. Than, incubate the flask of cells at 37 degrees Celsius in 5%carbon dioxide environment for three to five minutes until cells start detaching. Add four milliliters of complete DMEM to deactivate the trypsin solution.

And then count cells using a cell counting slide and a light microscope. Dilute cells to 500, 000 cells per milliliter with complete DMEM. Next, seed two milliliter per well of the cell solution in a 6-well tissue culture plate.

And gently move the plate back and forth and side to side to evenly distribute cells. After making sure that cells are evenly distributed incubate the plate at 37 degrees Celsius and 5%carbon dioxide environment overnight or 16 to 18 hours. To preform three plasmid cotransfection, first mix calculated volumes of plasmids and the reduced serum cell culture medium in a micro centrifuge tube.

Incubate the mixture at room temperature for five minutes. Then add three microliters per well of the lipid based transfection reagent to 47 microliters per well of the reduced serum cell culture medium. Incubate the mixture at room temperature for five minutes.

Next, in a micro centrifuge tube, mix equal amounts of the transfection reagent and the plasmid solutions by pipe heading up and down several times. Incubate the mixture at room temperature for 20 minutes. After overnight incubation of the cell plate, use an inverted light microscope to examine cells for their morphology and density.

Then, aspirate the spent medium of cells and gently add one milliliter per well of the pre-warmed reduced serum cell culture medium to each well. Next, add 100 microliters of the transfection solution to each well drop wise. And incubate the cells at 37 degrees Celsius in 5%carbon dioxide for four to six hours.

At the end of the incubation, add one milliliter of pre-warmed antibiotic free transfection DMEM, at 37 degrees Celsius to each well. And incubate the cells at 37 degrees Celsius in 5%carbon dioxide for 48 hours. To begin pseudotype particles collection use an inverted light microscope to exam the cells for their morphology and general condition.

The color of the medium should be light pink or slightly orange. Then, transfer the supernatants of the transfected cells to 50 milliliters conical centrifuge tubes. Centrifuge the tubes at 290 times gravity for seven minutes to remove cell debris.

Next, filter clarified supernatant through a sterile 0.45 micron pore size filter. Make small volume aliquots of pseudotype virus solution in cryo vials. And store them at negative 80 degrees Celsius.

To perform pseudotyped particle infection, first exam cells under light microscope to confirm that there is a confluent carpet of cells. Then, wash the cells three times with 0.5 milliliters of the pre-warmed DPBS at 37 degrees Celsius. Next, aspirate the supernatants of cells and inoculate the cells with 200 microliters of the thawed pseudotyped particle solution.

Incubate the cells at 37 degrees Celsius in 5%carbon dioxide for one to two hours. After the incubation period, add 300 microliters of the pre-warmed DMEMc at 37 degrees Celsius. And incubate the infected cells at 37 degrees Celsius in 5%carbon dioxide for 72 hours.

Finally aspirate the supernatants of the infected cells. And precede to luciferase reporter assay. To quantify the infectivity of pseudotyped particles first thaw luciferin substrate, stored at minus 80 degrees Celsius, at five time luciferase assay lysis buffer stored at minus 20 degrees Celsius to room temperature.

Then, dilute the luciferase assay lysis buffer to one times concentration with sterile water. Next, add 100 microliters of the diluted buffer to each well. And incubate at room temperature on a rocker for 15 minutes.

To perform luciferase activity measurement, one well at a time, first add 20 microliters of luciferin substrate to a micro centrifuge tube. Then, add 10 microliters of the lysate to the tube. Mix the contents by gently flicking the tubes.

And placed the tube in a luminometer device. Close the lid and measure the luminescence value of the tube. Record the relative light units measurement, and precede to data analysis.

Infectivity assays of SARS-Spp and MERS-Spp in susceptible host cells, showed a strong average infectivity, compared to the expected positive control particles. Non infected controls and the negative control particles lacking viral envelope glycoproteins. Also luciferase activity assays showed concentration dependence of SARS-Spp and MERS-Spp infectivity.

An increase of SARS-Spp or MERS-Spp infectivity in poorly permissive target cells, transfected to express ACE2 receptor of SARS-Spp, or DPP4 receptor of MERS-Spp, confirms that receptor usage of pseudovirions is similar to that of nato viruses for mediating attachment and entry of pseudovirions. Double check calculations prior to performing this step. And make sure all solutions are mixed well during the step.

A western blot assay can be performed on concentrated pseudotyped particles to assess S glycoprotein incorporation into the pseudovirions. While the pseudotyped particles described here are safer surrogates than nato viruses, they still require biosafety level two precautions.