Mar 19, 2025

Public workspaceSeV cbVG high (HD) stock preparation from recombinant fluorescent reporter virus 

Journal of Virology
DOI
https://dx.doi.org/10.17504/protocols.io.j8nlk9xqwv5r/v1
  • Carolina Lopez1
  • 1Washington University
Carolina Lopez
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlk9xqwv5r/v1
External link: https://doi.org/10.1128/jvi.00894-25
Protocol Citation: Carolina Lopez 2025. SeV cbVG high (HD) stock preparation from recombinant fluorescent reporter virus . protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk9xqwv5r/v1
Manuscript citation:
Pye SE, Achouri E, Yang Y, Musa A, López CB Validation of diverse and previously untraceable Sendai virus copyback viral genomes by direct RNA sequencing. Journal of Virology 99(8). doi: 10.1128/jvi.00894-25
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 20, 2025
Last Modified: March 19, 2025
Protocol Integer ID: 123173
Keywords: generating cbvg high virus stock, cbvg high virus stock, stock preparation from recombinant fluorescent reporter virus, recombinant fluorescent reporter virus, rescued recombinant virus, recombinant virus, cbvg, stock preparation, virus
Abstract
Protocol for generating cbVG high virus stocks from newly rescued recombinant viruses
Materials
  1. Cultured LLCMK2 cells (ATCC, #CCL-7)
  2. Tissue culture media and Infection media (See media prepare protocol)
  3. 0.05% Trypsin (Thermo, #25300054)
  4. PBS (Phosphate-buffered saline)
  5. Cell counter and Trypan blue
  6. T75 flask, 96 well plate
  7. Pipettes
  8. 2.0ml Cryogenic vials (Corning, #430488)
  9. TPCK-Trypsin (for Sendai virus infection, see SeV infection protocol)
Troubleshooting
Seed LLCMK2 cells to T75 flask
  1. Check LLCMK2 cells to ensure they are in good condition, approximately 90% confluent.
  2. Wash cells twice with PBS.
  3. Add Amount3 mL 0.05% trypsin-EDTA to the T75 flask, incubate at room temparture for 3-4 minutes.
  4. Add Amount7 mL TCM to the flask to stop trypsinization and collect cells.
  5. Combine all cells together.
  6. Mix Amount10 µL cells and Amount10 µL Trypan blue, then add Amount10 µL mixture to a cell counter slide and count the cells.
  7. Plate 2,000,000 cells in Amount10 mL of TCM media to T75 flask. One more flask will be needed for cell counting.
  8. Incubate cells at 37 °C overnight.

Note: The size of the flask depends on the final volume you need. You can scale down to use a T25 flask or a 6-well plate. However, since high cbVG (HD) virus stocks usually have a lower titer, and the process to create a HD stock is time-consuming, a T75 flask is recommended.

Infect LLCMK2 cells with new rescued virus with a MOI of 10
  1. Prepare infection media containing TPCK-trypsin: Add TPCK-trypsin to the infection media at a final concentration of 2 µg/mL. Prepare Amount15 mL of the media for each flask.
  2. Collect the cells from T75 flask seeded for counting cells and collect cells number as above.
  3. Using the number of cells in T75 flask and the MOI of you virus stock to calculate the virus volume needed for infection: Virus Volume (µL)= MOI×Number of Cells / Virus Titer (TCID50/mL).
  4. Wash cells twice with PBS.
  5. Add the virus and prepared infection media containing TPCK-trypsin at final volume of Amount5 mL to cells.
  6. Incubate the flask at 37 °C for 1 hour and gently shake it every 15 minutes.
  7. Remove the infection media from flask.
  8. Wash the cells twice with PBS.
  9. Incubate the cells at 37 °C for 5 days.

Note: In this protocol, we are using rSeVCeGFP as an example.

Virus stock titration
Follow the protocol "SeV fluorescent reporter virus titration by TCID50" to do the titration
Protocol
NAME

SeV fluorescent reporter virus titration by TCID50

CREATED BY
Carolina Lopez


Harvest the passage 1 HD virus stock
  1. Check the infected cells, you should be able see eGFP signal in all cells at the Passage 1. During later Passage HD stocks infection, eGFP signal may only be seen in some of the cells not all cells.
  2. Collect the supernatant, and span it at Centrifigation2000 rpm, 4°C for 5 minutes to remove cells debris.
  3. Aliquot the virus, the volume of each tube is depended on how you will use it. Generally, Amount500 µL per tube is recommended.
  4. Quickly freeze the virus using a dry ice-ethanol mixture.
  5. Label them and store them at Temperature-80 °C

Make P2, P3... HD stocks
Repeat the whole process to make P2, P3, ... HD stocks