The following points highlight the nine main steps involved in the purification of virus in plants. The steps are: 1. Virus Propagation in a Suitable Host Plant 2. Selection of Infected Part of the Plant 3. Factors 4. Extraction of Virus Using a Suitable Buffer 5. Infectivity Test 6. Criteria for Purity of Virus 7. Virus Yield 8. Storage of Purified Virus 9. Uses of Purified Virus.
Step # 1. Virus Propagation in a Suitable Host Plant:
A large amount of infected plant material is needed to purify the virus. It can be achieved by inoculation of a water or buffer extract of the infected plant to a number of suitable hosts or to the same healthy host. If a virus cannot be transmitted by mechanical means, it can also be transmitted by grafting or by suitable vector to a healthy host for multiplication only those suitable plants as hosts are selected which are free from the chemicals which inhibit infectivity of virus (Table 1) for e.g., cow pea, Petunia hybrida, Chenopodium and certain tobacco varieties.
Step # 2. Selection of Infected Part of the Plant:
It is also essential because mid rib and petioles contain low concentration of the virus and should be separated before use for virus extraction. Young leaves should be preferred to older leaves because inhibitory materials that adsorb or adhere to the virus are frequently low in concentration in young leaves. Plant parts to be used for virus isolation should not the contaminated by another virus or strain of the same virus. Roots of pea plant are best to extract clover yellow mosaic virus (C1YMV).
Step # 3. Factors:
Certain climatic factors like temperature, light intensity and its duration, host nutrition and time after inoculation affect the virus concentration in the host plant. Some virus attain peak concentration in about 12 days after inoculation, rapidly decreased in the next four days and reaches at very low concentration by 48 days.
So, the conditions for growth of the infected plants and time of harvest should be standardized for each virus to maximize the virus yield.
Step # 4. Extraction of Virus Using a Suitable Buffer:
An extraction medium must enable extraction of maximum amount of virus from the infected plant material, keep the virus in stable, infective, un-aggregation condition and minimize host contaminants. A buffer of suitable pH and molarity with additives to prevent oxidation and avoid co-precipitation other materials with the virus, yet retain virus infectivity is chosen by trial and error based on the knowledge of the methods used for other viruses.
Phosphate acetate and borate buffers are commonly used at different pH and molarity. Some virus need a cation like Ca2+or Mg2+ to preserve their infectivity, besides the ionic strength of the buffer. Mortar pestle, food blenders, meat mincer and the electrically operated glass mortar and pestle are useful to homogenize the virus infected plant material in a suitable buffer.
All extractions of virus are done in cold conditions at 4°C or using salt-ice bath around mortar to prevent oxidation reactions. Cell components (Ribosomes, RuBP carboxylase protein from chloroplast, fragments of lower molecular weight compounds should be removed in the extraction process, leaving the infective virus in the solution.
It can be achieved by the following methods:
Heating:
Heating the extract for a few minutes at 50°-60° helps to coagulate the unwanted materials. It is very good method to extract stable virus TMV.
Centrifugation:
Centrifugation at different speeds separates virus particles and host components of different densities. Low speed 500-10,000 rpm centrifugation, is used in the initial stages of clarifying the crude infective plant extract to sediment the gross host material. High speed centrifugation at speeds of 30,000 rpm is used at later stages to get relatively pure virus, devoid at most of the host components.
Crystallization or Salt precipitation:
Ammonium sulphate to one third saturation of the crude extract is used to precipitate TMV, which can be suspended in suitable buffer. (Stanley, 1935).
Precipitation at Isoelectric Point:
Virus which are nucleoproteins are precipitated at specific pH. The precipitate is collected by low speed centrifugation and re-suspended in suitable buffer.
Coacervation:
It involves separation of macromolecules in two liquid layers, one of which is rich in colloid. A virus separates into one of the two layers.
Gel Filtration:
Sephadex, agar and agarose remove smaller host components by adsorption.
Density Gradient Electrophoresis:
Virus components can be separated by layering the preparation on a suitable buffered density gradient formed in a U-tube and applying electrical charge.
Step # 5. Infectivity Test:
Infectivity of the purified mechanically transmissible virus can be checked by inoculating to a suitable host.
Step # 6. Criteria for Purity of Virus:
Criteria for purity of a virus as a single entity may be by specific characteristic of that virus in a specific test plant, serological tests, UV spectrophotometric absorption data, gel electrophoresis, PCR analysis and molecular characterization.
Step # 7. Virus Yield:
The total weight of purified virus is called virus yield. It varies with the virus and plant used. It ranges from 0.05 ug/g leaf with Barley yellow Dwarf-virus (BYDV) to 2000 µg TMV/g tobacco.
Step # 8. Storage of Purified Virus:
Extracted or purified virus is stored in small quantities in vials with equal volume of glycerol and 3 or 4 crystals of thymol or sodium azide (to prevent microbial growth) at 4°C or frozen.
Step # 9. Uses of Purified Virus:
Purified virus free from contaminants is required to determine its structure, biochemical and molecular composition in relation to function and development of probable practicable control or management measures for the disease by various methods, including manipulation of parts of its genome.