I have done 3 blind passages to concentrate the viral stock in the supernatant without adding TPCK Trypsin. After passage 1 my Ct value for INF A was 36.96. After P2 the value decreased to 33.36. However, I was expecting a much lower value in the mid-20s. I am freeze-thawing the flasks at -20 degrees to lyse the cells and release the virus into the medium. Is it the correct approach? and can I get a much higher titre without adding TPCK Trypsin?
Basically, it depends on the virus subtype, i.e., HPAIVs do not require TPCK for propagation, while LPAIVs do require the addition of TPCK.
You can try to dilute the virus stock 1000-fold dilution, then infect the cells with this dilution and incubate at 37C for 1 h. Then, wash out and replace with infection media, and incubate for 3 days. This approach will prevent or avoid the DI phenomenon for influenza viruses.
Furthermore, I think that freezing and thawing the flask before harvesting would inactivate the virus. Instead, you can just harvest the supernatant at the end of incubation time, centrifuge to get rid of lysed cells, and aliquot the virus.
Good luck!
thanks for the suggestion. I too thought that DI particles are interfering with the formation of whole virions but i am skeptical that will diluting actually work.
- Badges
- Science method