Establishment and Quantification of De Novo Lytic Infection by Cell-free Kaposi's Sarcoma-Associated Herpesvirus

Tractable and reproducible systems to model KSHV primary lytic infection are limited. We established a reliable and robust in vitro platform for modeling KSHV de novo lytic infection. It's challenging to model KSHV Lytic infection because of its strong latency bias.

And the few primary cells that support it are delicate and have a limited lifespan, which severely limits reproducibility and scalability. We developed a tractable and reproducible model in HCT116 cells that capture efficient KSHV lytic replication during primary infection, offering a unique platform to study early events in herpes virus replication. To begin, seed five T75 flasks with dox-inducible iSLK cells containing wild-type KSHV BAC16 at approximately 80%confluency.

Incubate the flasks at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 24 hours. Add one microgram per milliliter of doxycycline and one millimolar sodium butyrate to each flask to induce lytic reactivation. Using phase-contrast microscopy, monitor the cells daily.

Collect the supernatant once more than 90%of cells show rounding, detachment from the flask surface, or visible lysis. Then, centrifuge the collected supernatants at 1, 500G for 10 minutes at four degrees Celsius to remove cellular debris. Filter the resulting supernatant through a 0.45 micrometer polyethersulfone membrane filter without disturbing the pellet.

Transfer the filtered supernatants to ultracentrifuge tubes. Centrifuge the tubes at 25, 000G for three hours at four degrees Celsius using an SW 28 rotor to pellet the viral particles. Carefully discard the supernatant into a container with 10%bleach.

Resuspend the viral pellet in one-milliliter of serum-free DMEM. Seed one times 10 to the power of five HCT116 cells per well in a 12-well tissue culture plate. Incubate the plate at 37 degrees Celsius in a humidified incubator with 5%carbon dioxide for 24 hours.

Dilute the concentrated virus stock in prewarmed, serum-free DMEM to a final volume of 250 microliters to achieve a multiplicity of infection, or MOI of 10. Aspirate the culture medium from each well and add 250 microliters of the diluted virus directly onto the cells. Centrifuge the plate at 1, 500G for one hour at 30 degrees Celsius to synchronize infection.

Immediately transfer the plate to a 37 degrees Celsius incubator and continue incubation for one hour. Aspirate the viral inoculum from each well. Wash the cells three times with prewarmed PBS to remove residual virus.

Add one milliliter of complete DMEM to each well. Then, collect one milliliter of culture supernatant from each well. Seed one times 10 to the power of four SLK cells per well in a 96-well plate 24 hours before infection.

Label nine 1.5 milliliter microcentrifuge tubes for a serial two-fold dilution series. Add 200 microliters of virus supernatant to tube one. Add 100 microliters of prewarmed, serum-free DMEM to each of tubes two through nine.

Begin the serial dilution by transferring 100 microliters from tube one to tube two and mixing thoroughly by pipetting. Now, aspirate the culture medium from SLK cells. Add 100 microliters of each virus dilution to the designated wells, using 27 wells in total.

Centrifuge the 96-well plate at 1, 500G for one hour at 30 degrees Celsius to promote viral absorption. Transfer the plate to a 37 degrees Celsius incubator with 5%carbon dioxide and incubate for an additional hour. Remove the inoculum from each well.

Then, gently add 100 microliters of complete DMEM to each well. Incubate the plate for 24 hours at 37 degrees Celsius. Then, analyze the GFP-positive cells.

A two-fold serial dilution of BAC16-derived virus stock showed a progressive decrease in the number of GFP-positive SLK cells, confirming a measurable and dilution-sensitive infectious titer. The calculated infectious titer of the BAC16-derived virus stock was approximately 1.3 million infectious units per milliliter. GFP expression in HCT116 cells increased over time, following de novo infection, with the most intense signal observed at 60 hours post-infection, indicating robust viral replication at that time point.

Flow cytometry analysis confirmed a peak in GFP-positive SLK cells at 60 hours post-infection, reaching 74.4%positivity, followed by a decrease to 62.7%at 96 hours. Quantitative PCR of viral DNA in supernatants showed a sharp increase in viral genome copies, peaking at 60 hours post-infection and declining thereafter.