Protocol for RNA Virus Amplification Using In Vitro Cell Cultures

Given the low titers at which viruses are usually produced, in vitro cell cultures have been utilized for amplifying virions to analytically relevant amounts. The following protocol has been employed for expansion of RNA viruses such as SARS-CoV, FCoV, MHV, and torovirus in cultured cells.

1. Culture Vero cells to ~70-90% confluency.
   The cell culture medium is Dulbecco’s Modified Eagle Medium supplemented with:
• fetal bovine serum (10%)
• penicillin/streptomycin (100 U/mL each)
• HEPES (10 mM)

Adjust the pH to 6.7 with NaOH.

2. Inoculate the cultured cells with the virus of interest (MOI ≥ 3).
3. After 1 hour, replace the inoculum with fresh cell culture medium.
4. At 24 hours post inoculation, replace the cell culture medium with fresh medium.
5. At 48 hours post inoculation, collect the cell culture supernatant.
6. Centrifuge at 10,000 × g for 20 min at 4°C to pellet the cell debris.
7. Harvest the virion-containing supernatant and subsequently process for more stringent purification (e.g., sucrose density gradient ultacentrifugation) or downstream applications.

Reference

For more info, visit our Methods of Virus Purificationpage.

While the above suggested protocol has been tested to be effective, if you are instead looking for a convenient solution for RNA isolation, complete with all buffers and reagents required, we recommend looking at the variety of viral & total RNA extraction kits that we have to offer: