Virus Purification Techniques: From Extraction to Purification

1.4 Virus Purification Virus purification is amacro seriesmolecules, of tasks towhich obtainarepure virus particles. Virus particles are basically composed of nucleic acid molecules as their genome which are wrapped in protein molecules as their shell. Certain virus particles will have a certain shape, size will and composition of nucleic acids and proteins. Thus, certain virus particles have a certain specific gravity. That is why virus particles can be purified by centrifugation. 1.4.1 Virus Purification Procedure In general, purification of plant ofvirus particles canthebenext divided into two stages, namely clarification/purification the extract and stage of purification of virus particles. Clarification/Clearification of extracts This crude extract consists of target virus particles mixed with material resulting from the breakdown of host cells. This crude extract is then treated with phenol/chloroform compounds to coagulate/precipitate the crude material thatto makes up the host cells. The preparation is then centrifuged at low speed precipitate theFrom coarse components of plant tissue that have coagulated or precipitated. here, a preparation is obtained containing target virus particles with host cellandcomponents such(micromolecules). as various organelles, large moleculesmixed (macromolecules) small molecules Next stage of purification of virus particles This stage aims toand concentrate particles and remove low molecular weight host components also largervirus components. Among those frequently used are: High Speed Deposition oftheVirus Particles . Thisandmethod is a any very remaining useful step.plant This stage will concentrate virus particles remove material in the supernatant fluid. Density . Thea cesium gradientchloride that is often usedThis is using a seacanof sucrose.gradient A bettercentrifugation gradient can use solution. process be used to concentratevirus virus particles particles and remove host components without pelleting/precipitating which could potentially damage the particles in high-speed virus particle deposition. 1.4.2 Example of Virus Particle Purification Steps The technicalities described here are based on the author's experience during further studies at Okayama University, Japan. Meanwhile, the purified virus particle is MyRV-1 (Mycoreovirus-1) which infects the fungus Cryponectria parasitica. comparison.For readers who have other experiences, please use them as a The steps are as follows: 1.namely Cultivate thedextrose C. parasitica fungus infected with MyRV-1 in one liquidweek. medium, Potato broth (PDB) as much as 2 liters for Also culture healthy/virus-free C. parasitica fungi as a comparison. 2.theHarvest the mushroom mycelium bymedium filteringbyitwrapping using Miracloth. Then roll upin miracloth. Absorb the extracted the miracloth roll tissue paper. To speed upthedrying, you can replace theabout tissue9 grams paper oftwomycelium or three times. After that, weigh mycelium. You will get each. 3.mortar Transfer the mycelium into the cooled mortar.for(Cooling is donePour by placing the or pestle in the refrigerator at -80oC one night). the liquid nitrogen into the fine mortar/mycelium, then grindliquid it usingnitrogen a pestlecanuntil itdone becomes fine flour. To get mycelium flour, pouring be more than once. 4.until Pourit melts. 36 ml ofThen 0.1 Mtransfer phosphate buffer (NaPO4) into the musselium flour.using Waita the preparation into a beaker. Homogenize stirrer in ice for 30 aminutes. ThenThis transfer theyield preparation into a falcon tube byof filtering it using chessloth. will (approximately) 22.5 ml preparation fluid. 5.spinTousing the preparation add 15% CCL4 (3.5After ml) then shake vigorously. Afterfalcon that, a stirrer for 10 minutes in ice. that, insert it into two new tubes. 6.Then Centrifuge at 3,500into rpmtwofornew 20 minutes. You will get a clearusing whitea chessloth. supernatant. each transfer crystal tubes by filtering Toa prepare for ultracentrifugation, equalize the weight of each pair of tubes using balance. 7.Once Thenfinished, centrifuge at a the speedsupernatant. of 25,000 rpm for 3 hours using anclear anglewhite rotor.in discard The pellets are round, color, slightly yellowish in the middle. 8.thenThecombined, pellets in each tube 3were added with 1.5 mlThis of 0.1preparation M phosphate buffer, so that ml was obtained. was then homogenized by rotating using a stirrer for 1 hour on ice. Once finished, then put it into a microcentrifuge tube. 9.TheThesupernatant preparationwas is then centrifuged atintoa speed of microcentrifuge 10,000 rpm for 10tube. minutes. taken and put a new The preparations were stored on ice at 4°C overnight. 10. The next stepgradient is to prepare the sucrose gradient for use theof next day. The finished sucrose was stored at 4oC. The preparation these sucrose gradients is described elsewhere in this chapter. 11. On the next day, the preparation (supernatant) istube. added/dropped over theof sucrose gradient until it is almost at the mouth of the So there are a total 8fungi tubes, which 4 tubes are fungi asinfected with viruses and 4 tubes are for thatofare not infected withforviruses a comparison. 12. The weights each rotor pair atof 35,000 tubes rpm are for equalized using a balance. Then centrifuged using ofa swing 25 minutes. 13. 0.5From ml ofeachThe virus-infected fungithen is taken from toeach tube2 ml, on athen banddiluted that looks dark. tubes were combined obtain with phosphate buffer 2 times to obtain 4 ml. Equalize the weight of the pair of tubes using a balance and prepare for ultra centrifugation. 14. Centrifuge at 45,000 rpm for 2 hours using a fixed/angle rotor. 15. Discardbuffer. the supernatant. phosphate Then storeSuspend at -80oC.the pellet from each tube in 50 µl of 0.1 M 16. Preparation is ready for the next stage.