Isolation and Purification of Viruses from Infected Insect Bodies

Abstract

Source: Biesbrock, A. M. et al. Propagation of Homalodisca coagulata virus-01 via Homalodisca vitripennis Cell Culture. J. Vis. Exp. (2014)

This video demonstrates the in vitro propagation of Homalodisca coagulata Virus-01 using insect-derived host cells. It outlines the steps for virus purification, inoculation, and replication within cultured insect cells.

Protocol

1. Whole Virus Extraction

1. Homogenize whole bodies of virus-positive H. vitripennis in phosphate buffer, pH ~7.2, with 0.02% sodium diethyldithiocarbamate trihydrate (DETCA) by vortexing for ~10 sec intervals until there are no more large clumps of tissue present. Extract virus via superspeed centrifugation at 124,500 x g for 4 hr at 4 °C or 22,000 x g for 16 hr at 4 °C. If a precipitate forms at the top, remove it with a sterile cotton swab.
2. Discard the supernatant.
3. Collect the pellet and dissolve with 5 ml of 10 mM phosphate buffer (no DETCA), pH ~7.2, containing 0.4% Na-deoxycholic acid and 4% polyethylene glycol hexadecyl ether (Brij 52).
   1. To mix the pellet well, remove the pellet from the side of the tube and crush it up until it is in solution, if necessary.
   2. Add an additional 10 mM phosphate buffer in ~5 ml increments to help the pellet go into solution if needed, and combine into 2 tubes.
4. Centrifuge the solution at 300 x g for 15 min.
5. Remove the supernatant and pass the solution through a 0.45 μm filter, and collect the filtrate in a large collection tube.
6. Transfer filtrate to a dialysis membrane with a molecular weight cut off (MWCO) of 3.5 kD, using small amounts of 10 mM phosphate buffer (pH 7) containing no DETCA if needed.
7. Place the dialysis membrane in a large beaker filled with ddH₂O at 4 °C. Change the ddH₂O every hour for 5 - 6 hr, until a white precipitate forms in the membrane.
8. Collect the purified virus from within the membrane and subject the 100% virus solution to a 10-fold dilution series by adding 10 μl of solution to 90 μl of ddH₂O. Subsequently, add 10 μl of the dilution to another 90 μl of ddH₂O until reaching a dilution of 1:100,000.
9. Store virus solution at -80 °C.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Corning cell culture flasks	Sigma Aldrich	CLS430168	Surface area 25 cm², canted neck, cap (plug seal)
Olympus DP30BW, IX2-SP, IX71	Olympus		Inverted microscope and camera
Greiner CELLSTAR multiwell culture plates	Sigma Aldrich	M8937	48 wells (TC treated with lid)
DETCA	Sigma Aldrich	228680	Sodium diethyldithiocarbamate trihydrate
Corning bottle-top vacuum filter system	Sigma Aldrich	CLS431206	Cellulose acetate membrane, pore size 0.45 μm, membrane area 54.5 cm², filter capacity 500 ml
Brij 52	Sigma Aldrich	388831	Polyethylene glycol hexadecyl ether
Phosphate buffer solution	Sigma Aldrich	P5244	Received as 100 mM diluted to 10 mM with sterile water
PBS	Sigma Aldrich	P5368	Phosphate buffered saline
Bovine serum albumin (BSA)	Sigma Aldrich	A2153
Grace’s Insect medium (supplemented, 1x)	Sigma Aldrich	G8142	H2G+ leafhopper medium component
L-histidine monohydrate	Sigma Aldrich	H8125	H2G+ leafhopper medium component
Medium 199 (10x)	Sigma Aldrich	M4530	H2G+ leafhopper medium component
Medium 1066 (1x)	Sigma Aldrich	C0422	H2G+ leafhopper medium component
Hank’s Balanced Salts (1x)	Sigma Aldrich	51322C	H2G+ leafhopper medium component
L-Glutamine (100x)	Sigma Aldrich	G3126	H2G+ leafhopper medium component
MEM, amino acid mix (50x)	Sigma Aldrich	56419C	H2G+ leafhopper medium component
1 M MgCl solution	Sigma Aldrich	M8266	H2G+ leafhopper medium component
Pen-Strep	Sigma Aldrich	G6784	H2G+ leafhopper medium component
Nystatin	Sigma Aldrich	N6261	H2G+ leafhopper medium component
Gentamycin	Sigma Aldrich	46305	H2G+ leafhopper medium component
Dextrose	Sigma Aldrich	D9434	H2G+ leafhopper medium component
Fetal Bovine Serum	Sigma Aldrich	F2442	H2G+ leafhopper medium component