1.2 Virus Isolation Lecture Note

1.2 Virus Isolation As mentioned above, there isofnovirus standard waywork for virus purification. A method that is effective for one type may not for another virus that looks similar. Even just different strains require different methods for effective purification. 1.2.1 Selection of Propagation Host Plants Selection ofofpropagation hostVarious plants isimportant an important thing that will determine the success virus isolation. things to pay attention to are as follows: 1.However, The hostit plant for propagation must be easy tofrom grow,seed-borne especially viruses, from seed. must be noted/ensure that it is free for example Soybean mosaic virus(SoMV) which is very effectively transmitted through seedsChenopodium spp. 2. The virus must be present in high concentrations in the host. 3.substances The propagation host mustcompounds, not contain organic (in highacids, concentrations) various such as phenolic gums and blends, proteins and certain enzymes, especially ribonucleases, which can irreversibly inhibit ordifficult coagulateto viruses. For example, various viruses thatmost infectmembers stone fruitof are very isolate from their natural hosts because the Rosaceae contain largehosts amounts of tannin compounds in their leaves. The discovery of alternative in non-Rosaceae such as cucumber (Cucumi sativus L) has made it possible to isolate these viruses. 4.during Material from the host plant should be easily separated from virus particles purification. For example, certain Legium (eg Canavalia ensiformis) contain amounts of Rubisco (protein fraction and oftenlarge purified together with rod-shaped viruses.1) which is often aggregated In practice, various species such as Chenopodium, Cucumis, Nicotiana,ofPetunia, Phaseolusas well asVignais a suitable plant to host the multiplication a large number of viruses. The plantshould speciesbe used, thewhen conditions for growing the plants, and theFortime of harvest chosen the virus concentration is highest. some viruses the concentration increases to a peak after ainfewonedays or weeks and then falls quickly. Sometimes the distribution of viruses plant is uneven, so it is necessary to select parts with high virus content. 1.2.2 Extraction Medium After thethehost plant cell ruptures, andenvironmental the contents ofconditions the cell are released and mixed, virus particles encounter that are foreign tocanthem. Sotheinvirus this case it is necessary tointact use anandartificial extractionwhile mediagoing that keep particles infectious, not aggregated through various stages of extraction and purification. The conditions suitable for virus particles in a pure state may be different from those in crude extracts or semi-pure conditions. And also many different factors can greatly influence the stabilitytheofsuitability virus particles. Amongforthe that determine of the medium virusvarious particlesimportant include thefactors following: pH and buffer system Many viruses are range. stable only overcommonly a narrow pHused range. Thus, the extract must be kept in this pH Buffers for virus extraction include borate, citrate, phosphate and Tris buffers. Reducing agent and protective substance of phenolic components Reducing agentssodium such sulfite, as ascorbic acid, cysteinearehydrochloride, 2mercaptoethanol, or sodium thioglycolate often added to the extraction medium. Additives that can remove plant proteins and ribosomes Many virus particlesmay immediately lose their infectivity under in-vitro conditions. One of the causes be the presence of ribonuclease in the extract or semipurified virus particles. This can be overcome by addition of magnesium bentonite compound.pigments. A type Buffer of charcoal can also bepHadded tocause adsorb host material, especially 0.01 M NaEDTA 7.4 can damage toviruses.Enzymes most ribosomes, thereby avoiding co-sedimentation withpurposes. certain have also been added to extracts for various Pectinase was shoot addedvirus to degrade the sapalso in cocoa leaf extract liquid to results purify Cacao swollen (CSSV). It has been reported that greater have beenresidues obtainedwith in purifying confined of fibrous Driselaseviruses and various otherto phloem enzymes.tissue by incubation Detergents and other additives Non-ionic detergents such as Triton