purification virus 1998

238 PART VIn - VIRUS ISOLATION AND PURIFICATION

Protocol 28 Virus Purification Protocols

Introduction

Criteria of Purity

There are many criteria of purity, but none is perfect. Ultraviolet light absorption spectra, typical of nucleoproteins, and absorbance ratios at 260 nm and 280 nm (A26o/ A28o) are commonly used to establish the purity of a virus suspension, but some plant nucleoproteins cannot easily be detected in this way (Protocol 29). Serological methods may reveal the presence of plant proteins in the suspension, provided antisera to these contaminants are available (Part XI).

With the electron microscope, impurities can be detected only when their size allows distinction from the virus particles. When small plant proteins are present in rather large amounts, they may be visible as background material on the electron microscope grid (Protocol 31).

The best criterion of purity is equilibrium density-gradient centrifugation, but this method cannot be used for all viruses, as pointed out earlier. In general, it is advised to use more than one criterion to judge the purity of a virus suspension.

In this Protocol, purification procedures are given for 16 viruses from 15 virus groups or genera (Introduction, Table 3). These procedures have given good results in practice. However, the details should not be considered sacrosanct. If need be, suitable modifications may be made, as one's own experience is more important than uncritically following a prescribed route. For propagation hosts of the respective viruses, see also the Appendix to Part III .

• Materials

- Cold-room with bench space - Fume cupboard

Ice flaker Deep-freeze (-20°C)

J. Dijkstra et al., Practical Plant Virology© Springer-Verlag Berlin Heidelberg 1998

Refrigerator (4°C) Oven (120 °C or higher) Water-bath Ice bucket Balance for weighing 0.1-100 g or more Analytical balance pH meter

Protocol 28 - Virus Purification Protocols 239

Hot plate with adjustable temperature range Stirrer with magnetic bars (assorted dimensions) Vortexing machine Peristaltic pump with adjustable speed Gradient-former (Protocol 27, Fig. 1) Waring blender or comparable grinding machine Table-top low-speed centrifuge (up to 6000 rpm), swinging-bucket rotor for 65-400 ml tubes, and tubes Table-top centrifuge for microtubes (microfuge) Cooled medium-speed centrifuge (up to 20,000 rpm), fixed-angle rotors for tubes of approx. 15,50 and 300 ml, and tubes Cooled high-speed centrifuge (up to 50,000 or 60,000 rpm), fixed-angle and swinging-bucket rotors for tubes of 5 to 15 and 30 to 40 ml, and tubes Laboratory tools, such as: thermometer, pincers, glass stirring rods (rounded ends, length 15 cm, diameter 3-5 mm), adjustable-volume micropipettes (100-1000 Ill, 1-5 ml, 2-10 ml), syringes (various capacities and needle lengths), spatulae, microfuge tube racks, micropestles, standard glassware Microfuge tubes, Pasteur pipettes, dialysis tubing, cheesecloth, Parafilm membrane and other disposables

If no rotor type is mentioned, the high-speed centrifugation is in a fixed-angle rotor. All RCF values mentioned are for ray (Introduction, Part VIII). This applies to both high- and medium-speed centrifugations and to fixed-angle as well as swinging-bucket rotors. Different fixed-angle rotors may have different pelleting efficiencies, even when their ray-values are equal. Therefore, check whether RCF values and run times mentioned in the protocols are suitable for the virus at hand. Refer to Introduction Part VIII, specifically the equations for run time (t) and clearing factor (k).

Tubes for medium- and high-speed centrifugation should be balanced pairwise to an accuracy of less than 0.1 g. Tube cavities in fixed-angle rotors which are not used must be fully empty and dry. When using a swinging-bucket rotor, be sure to attach all buckets, including the

240 PART VIII - VIRUS ISOLATION AND PURIFICATION

empty ones, and check whether the latter are really empty and have their caps properly placed.

A supernatant to be discarded should be decanted carefully, preferably in a clean beaker to avoid loss of the pellet (or virus not yet pelleted) along with the supernatant. Resuspending hard pellets may be facilitated by keeping them submerged in buffer overnight and/or rubbing their surface gently with the rounded end of a glass stirring rod.

preparations - Cheesecloth (or muslin): used for straining the plant homogenate. Keep in boiling water for a few minutes to remove any dressing (such as starch).

- Virus-free glassware: The laboratory glassware should be very clean and absolutely virus-free. After thorough washing with detergent, it is rinsed five times in tap water and subsequently three times with distilled or deionised water. Any residual virus and ribonucleases are inactivated by keeping the glassware in an oven at 120 °C for at least 2 h, preferably overnight. To prevent recontamination with ribonuclease from environmental micro-organisms, the glassware should be covered with aluminium foil prior to heating.

- Dialysis tubing: Just before use, the dialysis tubing is boiled for a few minutes in de ionised water to remove the glycerine. Na2-EDTA is added to the water to neutralise the polyvalent cations, which may otherwise generate harmful oxygen radicals.

28.1 Alfalfa Mosaic Alfamovirus

This purification method comprises clarification by organic solvents, concentration of the virus by precipitation with PEG and further fractionation by differential and, if desired, sucrose gradient centrifugation. It is a modification of the method described by Van Vloten-Doting and Jaspars (1972). Yields of up to 150 mg per 100 g ofleaf tissue may be obtained .

• Materials

plant material Alfalfa mosaic virus can be propagated in N. tabacum, N. benthamiana or Phaseolus vulgaris, depending on the virus strain. When N. tabacum "Samsun NN" is used, the risk of contamination with tobacco mosaic Tobamovirus is minimised.


Noninoculated leaves showing symptoms can be harvested 5-10 days after inoculation, depending on the virus strain. If the virus gives systemic necrosis, early harvesting is recommended for maximum virus yield.

High-quality virus can be obtained from leaves inoculated with a high concentration (0.1 mg/ml) of purified virus and harvested 4 or 5 days after inoculation. Freshly harvested leaves are preferred as a source of virus. Freezing and thawing leaves prior to purification may decrease virus yield.

- Chloroform chemicals, - n-Butanol solutions and

Sucrose suspensions - Glycerol - Extraction buffer: 0.1 M KzHP04 ; 0.1 M ascorbic acid; 0.02 M Naz-

EDTA. Adjust to pH 7.1 with KOH. - PEG (Mr 20,000): 30 % (w/v) PEG 20,000 in deionised water - Resuspension buffer: 0.01 M phosphate buffer; 0.001 M ascorbic acid;

0.001 M Naz-EDTA. Adjust to pH 7.0 with NaOH. - Storage (PEN) buffer: 0.01 M phosphate buffer; 0.01 M Naz-EDTA;

0.001 M NaN3 • Adjust to pH 7.0 with NaOH. (Note: Sodium azide (NaN3 ) is highly toxic; it binds to metals forming explosive compounds when kept dry. Wear gloves to dispense.).

Procedure

Purification should preferably be carried out at low temperature, using precooled materials. If no cold-room with bench space is available, vials and containers should be kept in chipped ice and centrifuges set at 4°C.

1. Cut precooled leaves in narrow strips. Homogenise them in a Waring blender in extraction buffer and an ice-cold mixture of equal volumes of chloroform and butanol (1 ml of buffer and 1 ml of the chloroform/ butanol mixture per gram ofleaves).

2. Strain the slurry through preboiled cheesecloth.

3. Break the emulsion by centrifugation in a table-top centrifuge (swinging-bucket rotor) for 5 min at approx. 5000 rpm.

4. Collect the upper water layer and determine its volume.

242 PA RT VIII - VIRUS ISOLATION AND PURI FICATION

5. Add a 30 % solution of PEG 20,000 to a final concentration of5 % (1 vol in 5 vol of water layer).

6. Stir gently for 15 min, maintaining a low temperature.

7. Collect the precipitate by centrifugation, 10 min at 12,000 g.

8. Discard the supernatant and suspend the pellet in resuspension buffer, 1110 of the volume of the water layer.

9. Start two cycles of differential centrifugation, beginning with a lowspeed centrifugation: 10 min at 12,000 g.

10. Discard the pellet. Centrifuge the supernatant at high speed: 3 h at 64,000 g or 1.5 h at 133,000 g.

11. Discard the supernatant and suspend the pellet in resuspension buffer, 1 ml per 20 g of leaves.

12. Centrifuge the virus suspension for 10 min at 12,000 g.

13. Discard the pellet and centrifuge the supernatant at high speed: 3 h at 64,000 g or 1.5 h at 133,000 g.

14. Discard the supernatant and resuspend the pellet in storage buffer to be kept at 4 0c.

15. For long-term storage of the virus at -20°C in storage buffer, the suspension should be mixed with an equal volume of glycerol.

Tips and Troubleshooting

• For extra purity, a gradient centrifugation can be introduced after the differential centrifugation. Instead of step 14, the virus pellet is resuspended in a small volume of phosphate buffer and loaded onto a 6-30 % (w/v) sucrose gradient in phosphate buffer (up to 100 mg of virus per 40 ml gradient). After 3 h centrifugation at 83,000 g in a swingingbucket rotor, the wide virus band (no separation of components) is pipetted off, diluted with an equal volume of phosphate buffer and centrifuged again, as in step 13 but almost twice as long, to collect the virus .

• For obtaining high-quality virus, this gradient centrifugation step can also be introduced immediately after the PEG precipitation (step 8), on the same day as the leaves are harvested. Residual plant components are removed by subsequent differential centrifugation, as described.


28.2 Bean Yellow Mosaic Potyvirus

This procedure (Dijkstra et al. 1996) is suitable for many potyviruses. It comprises clarification by low-speed centrifugation, concentration of the virus by centrifugation through a sucrose cushion and further purification by equilibrium centrifugation. Yields are highly dependent on the virus strain used and may vary from 0.1 to 2 mg per 100 g of tissue .

• Materials

Bean yellow mosaic virus can be propagated in Vicia faba. Systemically infected leaves with clear dark-green and chlorotic mosaic are harvested and used fresh.

- Triton X-I00 - Caesium chloride (CsCI) or caesium sulphate (CS2S04 )

- Sucrose - Extraction buffer: 0.05 M K2HP04; 0.01 M Naz-EDTA; 1 % (w/v)

Na2S03; 5 % (v/v) ethanol. Adjust to pH 7.6. - Resuspension buffer: same as extraction buffer, excluding ethanol - Storage buffer: 0.01 M NaH2P04 adjusted to pH 7.2

Procedure

All steps should be performed in the cold-room or in glassware kept in chipped ice.

1. Grind the leaves in a blender in 3 ml of extraction buffer per gram of leaves.

2. Filter the homogenate through preboiled cheesecloth.

3. Centrifuge the filtrate at 8000 g for 30 min.

4. Discard the pellet. Add Triton X-I 00 to the supernatant to a final concentration of 1 % (v/v) and stir gently for 1 h.

5. Centrifuge the suspension for 3 h at 90,000 g on a sucrose cushion of 20 % (w/v) sucrose in extraction buffer, filling approx. 25 % of each tube.

plant material

chemicals, solutions and suspensions


6. Discard the supernatant and suspend the pellet in resuspension buffer. Let the pellet soak in the buffer overnight. If not yet resuspended completely, rinse the remaining pellet with the virus suspension using a Pasteur pipette. Avoid the formation of foam as this may cause denaturation of the virus coat protein.

7. Centrifuge the suspension at 1000 g for 10 min to remove particulate material.

8. Discard the pellet, the supernatant is .subjected to equilibrium centrifugation with CsCl (Protocol 30).

9. Dissolve CsCl in the virus suspension, adding 3.9 g to each 10 ml. This will give a solution with a density of approx. 1.27 g/cm3 •

10. Divide this solution over 5 ml centrifuge tubes.

11. Centrifuge in a swinging-bucket rotor at 123,000 g for 16 h. Check whether a well-defined virus zone has formed approximately halfway down the tube. If not, continue centrifugation for another 10-16 h.

12. Collect the virus zone with a Pasteur pipette equipped with a bulb. During extraction of the virus fraction, keep the tip of the pipette steady just below the virus zone so as not to disturb the gradient.

13. Dialyse the virus suspension against storage buffer.

• Tips and Troubleshooting

• Purification of the virus in winter in the temperate zone (northern hemisphere: December through April) tends to be less successful than in summer.

• Step 7 may be omitted if this causes unacceptable loss of virus.

• If CsCl is harmful to the virus, CSZS04 may be used in amounts to give a comparable density (3.75 g/10 ml).


28.3 Beet Necrotic Yellow Vein Furovirus

The method comprises clarification with carbon tetrachloride, concentration of the virus with PEG and high-speed centrifugation through sucrose cushions, and further fractionation by equilibrium centrifugation in caesium chloride. Yields of 1 mg per 100 g ofleaf tissue have been obtained .

• Materials

The virus is propagated in Chenopodium quinoa. Systemically infected leaves with symptoms (small yellow spots) are collected. Plants can be kept for additional harvesting later. Virus is purified from fresh leaves.

- Carbon tetrachloride (CCI4 )

- PEG (Mr 6000) Caesium chloride (CsCI)

- Naz-EDTA - Extraction buffer: 0.1 M Tris base; 0.14 M NaCl; 5 % (v/v) ethanol.

Adjust to pH 9.0 with HCl. - Resuspension buffer: 0.1 M Tris base; 5 % (v/v) ethanol. Adjust to pH 9.0

with HCl. - Storage buffer: 0.1 M Tris base. Adjust to pH 9.0 with HCl.

Procedure

1. Homogenise fresh leaves in a blender with 4 ml of extraction buffer per gram of leaves.

2. Add to the homogenate 1.5 ml of CCl4 per gram of leaves and stir for 15 min at 4°C.

3. Centrifuge the mixture for 15 min at 5000 g.

4. Discard the pellet and add to the supernatant PEG 6000 to a final concentration of 5 % (w/v).

5. Stir gently for 30 min at 4 °C and leave the suspension at that temperature for 90 min.

6. Centrifuge the suspension for 20 min at 10,000 g.

plant material



7. Discard the supernatant and resuspend the pellet in 0.8 ml of resuspension buffer per gram of initial leaf weight.

8. Leave the suspension at 4 °C overnight.

9. Centrifuge the suspension for 10 min at 5000 g.

10. Discard the pellet and layer the supernatant onto 20 % (w/v) sucrose cushions in resuspension buffer in 30-40 ml tubes of a swingingbucket rotor. The sucrose solution should fill approximately onethird of each tube.

11. Centrifuge at 48,000 g for 20 min. Particulate plant material will pellet but the sucrose cushion will keep the virus in suspension.

12. Using a Pasteur pipette or a peristaltic pump with narrow tubing, collect all liquid from the tubes, including the sucrose cushion, but leave the pellets.

13. Layer the liquid again onto 20 % sucrose cushions as in step 10.

14. Centrifuge at 104,000 g for 4 h. This time the virus will pellet.

15. Discard the supernatant and suspend each pellet in not more than 1 ml of resuspension buffer.

16. Further purification is by equilibrium centrifugation in CsCl in 5 ml tubes of a swinging-bucket rotor (Protocol 30). Add 0.45 g CsCl for each ml of virus suspension and dissolve. This will give a density of approx. 1.3 g/cm3, increasing the volume by about 12 %. Counter-balancing tubes may be filled with a similar CsCl solution in resuspension buffer without virus.

17. Centrifuge at 110,000 g for 24 h or as much longer as necessary for obtaining sharp virus zone(s) in the gradient.

18. View the tube in the dark under a microscope illuminator and collect the bluish virus zone using a Pasteur pipette or a syringe, with the tip placed at the level of the lower boundary of the zone.

19. Dialyse the virus fraction for 20 h against storage buffer.

20. Collect the virus suspension from the tube and store at 4 0c.


28.4 Carnation Ringspot Dianthovirus

The method (Tremaine et al. 1976) comprises clarification by acidification, concentration of the virus with PEG and further purification by differential centrifugation. Fresh leaves may yield 15 mg virus per 100 g of infected tissue .

• Materials

The virus can be propagated in cowpea (Vigna unguiculata). Leaves are harvested 2 or 3 weeks after inoculation. Freezing of leaves prior to purification results in a loss of yield.

- Acetic acid: 10 % (v/v) - PEG (Mr 6000)

Extraction buffer: 0.2 M sodium acetate buffer; 0.02 M Na-DIECA; 0.1 % (v/v) 2-ME. Adjust to pH 5.0.

- Resuspension buffer: 0.1 M sodium acetate adjusted to pH 5.0

Procedure

1. Homogenise leaves in 2 ml of extraction buffer per gram of leaves.

2. Strain the homogenate through preboiled cheesecloth.

3. Adjust the extract to pH 5.0 with 10 % acetic acid.

4. Leave the extract for 4 h at 5 DC.

5. Centrifuge the extract for 20 min at 8000 g.

6. Discard the pellet and add to the supernatant PEG 6000 to a concentration of 8 % (w/v). Dissolve and leave for 3 h.

7. Centrifuge the suspension at 8000 g for 20 min.

8. Discard the supernatant and suspend the pellet in 10 ml of resuspension buffer per gram of initial leaf weight.

9. Centrifuge the suspension at 10,000 g for 20 min.

plant material



10. Discard the pellet and centrifuge the supernatant for 90 min at 100,000 g.

11. Discard the supernatant and suspend the pellet in a few ml of resuspension buffer.

12. Repeat steps 9-11 until satisfactory purity is reached.

13. The virus can be stored in resuspension buffer at 4 °e.


• In some strains, pH- or temperature-dependent aggregation of particles in vitro has been reported (Tremaine et al. 1976). Aggregates have higher sedimentation coefficients than monomers and are likely to affect results of centrifugation steps. Moreover, spectrophotometric determination of concentration may be hindered by light scattering (Protocol 29).

• Some strains precipitate at pH 5.0. These strains may be purified by extraction in 0.05 M phosphate buffer (pH 7.6), containing 0.1 % (v/v) thioglycollic acid. The extract is clarified with 8.5 % n-butanol overnight in the cold (see procedure for tobacco necrosis Necrovirus). Further purification is by differential centrifugation as described above (steps 9-12).

28.5 Cherry Leaf Roll Nepovirus

The method (Stace-Smith 1966) comprises clarification by freezing and thawing, salting out of plant proteins with ammonium sulphate, and further purification by differential and sucrose-gradient centrifugation. Yields range from 1 to 5 mg per 100 g of leaf tissue.


• Materials

The virus can be propagated in leaves of Chenopodium quinoa, in which it plant material may reach moderately high concentrations

- Ammonium sulphate chemicals and - Sucrose buffer solutions - Extraction buffer: 0.5 M boric acid adjusted to pH 6.7 with NaOH - Resuspension buffer: 0.01 M Na2-EDTA, pH 7.0 - Tris-HCI buffer: 0.01 M Tris-HCI buffer, pH 7.0

• Procedure

1. Homogenise the leaves in 1.5 ml of extraction buffer per gram of leaves.

2. Squeeze the homogenate through preboiled cheesecloth and freeze the extract at -20°C overnight.

3. After thawing, centrifuge the extract for 30 min at 8000 g.

4. Discard the pellet and slowly add to the supernatant 15 g of ammonium sulphate per 100 ml extract.

5. Stir overnight at 4°C. The ammonium sulphate will precipitate a considerable quantity of plant proteins.


7. Discard the pellet and centrifuge the supernatant for 90 min at 78,000 g.

8. Discard the supernatant and suspend the pellet in resuspension buffer.


10. Discard the pellet and centrifuge the supernatant at 133,000 g for 1 h.




13. Discard the pellet and load the supernatant (partially purified virus preparation) onto a 30-40 ml gradient of 10-40 % sucrose in TrisHCI buffer (1-2 ml per gradient) (Protocol 27) and centrifuge in a swinging-bucket rotor for 90 min at 82,500 g.

14. Collect the bands of viral components (see Introduction, Table 3) using a syringe or a fraction collector, and pool them together (no separation of virus components is intended).

15. Dilute the virus preparation with Tris-HCI buffer to an appropriate volume and concentrate the virus by centrifugation in a fixed-angle rotor at 133,000 g for 150 min.

16. Resuspend the pellet in a small volume ofTris-HCI buffer. Store at 4°C.

28.6 Cowpea Mosaic Comovirus

The method is a slight modification of the procedure described by Klootwijk et al. (1977). It comprises clarification by low speed centrifugation, concentration of the virus with PEG and further purification by differential centrifugation. High yields are possible, up to 200 mg per 100 g ofleaf tissue .

• Materials

plant material Cowpea mosaic virus is propagated in mechanically inoculated primary leaves of a susceptible cowpea cultivar. Trifoliolate leaves also contain virus, but yield per gram of these leaves is considerably lower. Plants can be inoculated 9 or 10 days after sowing. Primary leaves are harvested 10-14 days after inoculation. To facilitate removal of plant constituents, the leaves can best be stored frozen and thawed prior to purification.

chemicals and - PEG (Mr 6000) buffer solutions - NaCI

- NaN3 (Note: Sodium azide (NaN3 ) is highly toxic and binds to metals, forming explosive compounds when kept dry. Wear gloves to dispense.)

- Extraction buffer: 0.1 M NaH2P04 adjusted to pH 7.0 with NaOH - Storage buffer: extraction buffer, diluted ten times


Procedure

1. Homogenise leaves in extraction buffer, 2 ml per gram ofleaf material.

2. Squeeze the slurry through preboiled cheesecloth.

3. Centrifuge the extract at 10,000 g for 15 min.

4. Keep the supernatant.

5. Suspend the pellet in extraction buffer, 0.25ml per gram ofleaf material.


7. Discard the pellet and add the supernatant to that of step 4.

8. Add to the combined supernatants, PEG 6000 and NaCI to final concentrations of 4 % (w/v) and 0.2 M, respectively.

9. Stir for 60 min at room temperature.

10. Collect the precipitate by centrifuging at 8000 g for 15 min.

11. Discard the supernatant, suspend the pellet in extraction buffer, 0.5 ml per gram of leaf material.

12. Centrifuge at 10,000 g for 15 min.

13. Keep the supernatant

14. Suspend the pellet in a few ml of extraction buffer.



17. Centrifuge the combined supernatants at 120,000 g for 2 h.

18. Discard the supernatant and suspend the pellet thoroughly in a few ml of storage buffer.


20. Discard the pellet and centrifuge the supernatant at 120,000 g for 2 h.

21. Discard the supernatant and suspend the pellet in a few ml of storage buffer (approx. 1 ml per 20 g of leaf material).


23. Discard the pellet. The supernatant (purified virus suspension) can be stored at 4 DC. Add NaN3 to a final concentration of 0.001 M.



• If a high virus yield is expected, "washings of the pellet" of steps 4-7 and 13-16 may be omitted to shorten the procedure.

• The above procedure may not be suitable for all strains. For example, virus of the K-isolate (found near Kano, Nigeria) is largely lost in the PEG precipitation step. High-speed centrifugation (step 20) may be a suitable alternative for its concentration.

28.7 Cucumber Mosaic Cucumovirus

The method (Mossop et al. 1976) comprises clarification by low-speed centrifugation, concentration of the virus by high-speed centrifugation, and further purification by sucrose gradient centrifugation. Virus yields vary according to virus strain and host plant used, but over 50 mg of virus per 100 g ofleaf tissue has been obtained in some instances.

• Materials

plant material The virus can be propagated in Nicotiana glutinosa, N. clevelandii or susceptible cultivars of N. tabacum. Systemically infected leaves are harvested 12-14 days after inoculation. They are used as fresh leaves.

chemicals and - Triton X-I00 solutions - Sucrose

- Extraction buffer: 0.1 M Na2HP04; 0.1 % (v/v) thioglycollic acid; 0.1 % (w/v) Na-DIECA. Adjust to pH 8.0.

- Resuspension buffer: 0.1 M Na2HP04 - Storage buffer: 10 mM sodium borate buffer adjusted to pH 7.5

Procedure

Purification should preferably be at low temperature, using precooled materials. If the work cannot be done in a cold-room, vials and containers should be kept in chipped ice and centrifuges set at 4 DC.


1. Homogenise the leaves in a blender in extraction buffer, 3 ml per gram of leaves.

2. Strain the extract through preboiled cheesecloth.

3. Centrifuge the extract at 8000 g for 10 min.

4. Discard the pellet and add to the supernatant Triton X-I00 to a final concentration of 2 % (v/v).

5. Stir for 15 min at 4°C.

6. Centrifuge the mixture at 78,000 g for 2 h.

7. Discard the supernatant and resuspend the pellet in extraction buffer, approximately 1/10 of the original volume of extraction buffer (step 1).

8. Centrifuge at 5500 g for 5 min to remove particulate material.

9. Layer the supernatant onto half its volume of 10 % (w/v) sucrose in resuspension buffer and centrifuge at 144,000 g for 45 min.

10. Discard the supernatant and suspend the pellet in resuspension buffer (1 ml per 15 g of original leaf material).

11. Centrifuge at 5500 g for 5 min to remove particulate material.

12. Discard the pellet and layer the supernatant in 2-3 ml portions onto 5-25 % (w/v) sucrose gradients in resuspension buffer (35-40 ml tubes)(see Introduction), centrifuge in a swinging-bucket rotor at 96,000 g for 2.5 h.

13. Collect the virus band using either a syringe or a Pasteur pipette, or a fraction collector.

14. Dilute the virus preparation with an appropriate volume of resuspension buffer and pellet the virus by centrifugation at 78,000 g for 2.5 h.

15. Discard the supernatant and resuspend the pellet in a few ml of storage buffer.


plant material

chemicals, solutions and

suspensions


The above method of purification may be used for strains for which organic solvents are detrimental, such as the M strain. Other strains, for which this method is unsatisfactory, may be purified by a method involving clarification with chloroform and precipitation with PEG (Lot et al. 1972).

28.8 Elderberry Carla virus

The method (Van Lent et al. 1980) comprises clarification by precipitation with salt, concentration of the virus by precipitation with PEG, and further purification by centrifugation through sucrose cushions and a sucrose gradient. Virus yields of 2-3 mg per 100 g of tissue have been obtained.

Materials

The virus can be maintained in the local-lesion host Gomphrena globosa, but it is best purified from infected elderberry leaves (Sambucus nigra or S. racemosa). Prior to purification the leaves can be stored frozen at -20°C.

- PEG Mr 6000 - Sucrose

Extraction buffer: 0.018 M trisodium citrate; 0.165 M Na2HP04; 0.5 % (v/v) 2-ME; 0.1 % (w/v) Na-DIECA. Adjust to pH 9.0.

- Resuspension buffer: 0.018 M trisodium citrate; 0.165 M Na2HP04; 1 % Triton X-100. Adjust to pH 9.0.

- Storage buffer: 0.0018 M trisodium citrate; 0.0165 M Na2HP04' Adjust to pH 9.0.


• Procedure

All manipulations, except centrifugations, can be at room temperature.

1. Homogenise the thawed leaf tissue in a Waring blender in 2 ml extrac-tion buffer per gram of leaves.



4. Discard the pellet and clarify the supernatant with calcium phosphate, formed in situ by slow and simultaneous addition of 1/20 volume of 0.2 M Na2HP04 and 1/100 volume of 1.0 M CaCh with constant stirring for 20 min.


6. Discard the pellet and add to the supernatant PEG 6000 to a final concentration of 6 % (w/v). Keep stirring until all PEG is dissolved.

7. Collect the precipitated virus by centrifugation at 8000 g for 10 min.

8. Discard the supernatant and suspend the pellet in resuspension buffer, 1/10 of the original volume of the leaf extract.


10. Discard the pellet and carefully transfer the supernatant onto a 10 ml cushion of 20 % sucrose in resuspension buffer in a 40 ml centrifuge tube.

11. Centrifuge for 3 h at 70,000 g.

12. Discard the supernatant and suspend the pellet in resuspension buffer as in step 8.


14. Repeat step 10 with a 30 % (w/v) sucrose cushion. (In case of a small volume of virus suspension, as ml sucrose cushion in a 15 ml centrifuge tube may be used, with appropriate change of run time and rotor speed; see Introduction).

15. Centrifuge for 3 h at 78,000 g.

16. Discard the supernatant and resuspend the pellet in a small volume of storage buffer.


plant material

chemicals, buffer solutions and

suspensions


18. Discard the pellet. The supernatant is the partially purified virus. A further purification can be achieved by sucrose-gradient centrifugation, as follows.

19. Prepare 10-40 % (w/v) sucrose gradients in storage buffer in 40 ml tubes of a swinging-bucket rotor (see Introduction). Layer a 2 ml sample of partially purified virus suspension onto each gradient.

20. Centrifuge for 2.5 h at 83,000 g.

21. Collect the virus zone using a syringe or a fraction collector.

22. Collect the virus by centrifugation at 78,000 g for 3 h. If necessary, dilute the virus fraction with storage buffer to the volume required for filling the centrifuge tube(s).

23. Discard the supernatant and resuspend the pellet in a small volume of storage buffer.

28.9 Potato Leafroll Luteovirus

The method (Takanami and Kubo 1979) comprises enzyme-assisted clarification by chloroform and butanol, concentration of the virus by PEG precipitation, and further purification by sucrose gradient centrifugation. Virus yields up to 0.1 mg per 100 g of tissue have been obtained.

Materials

Physalis floridana is a good source for purification of the virus. As the virus is not mechanically transmissible, the plants are inoculated by aphids (Protocol 16). The leaves are frozen prior to purification.

- Chloroform - I-Butanol

Triton X-100 - PEG (Mr 6000) - NaCI - Sucrose


- Extraction buffer: 0.1 M sodium citrate buffer; 0.5 % (w/v) macerozyme R-I0; 0.5 % (w/v) cellulase Onozuka R-I0; 0.1 % (v/v) thioglycollie acid; 5 % (v/v) ethanol. Adjust to pH 6.0.

- Resuspension buffer: 0.1 M sodium citrate; 5 % (v/v) ethanol. Adjust to pH 6.0.

- Storage buffer: 0.1 M sodium citrate adjusted to pH 6.0

Procedure

In this procedure, all centrifugations are at 15°C, except for the density gradient centrifugation, which is at 4°C.

1. Homogenise the leaves in a blender in 2 ml of extraction buffer per gram of leaves.

2. Stir the homogenate at 25°C for 4 h.

3. Emulsify the homogenate in a mixture of equal volumes of chloroform and I-butanol. Use 1 ml of this mixture per gram of initial leaf material.

4. Stir the emulsion vigorously for 5 min.

5. Break the emulsion by centrifugation at 10,000 g for 15 min.

6. Collect the upper aqueous phase, avoiding the interface, and add Triton X-I00 to a final concentration of 1 % (v/v).

7. Stir gently for 30 min and add PEG 6000 to 8 % (w/v), and NaCl to 0.4 M.

8. Stir gently for 1 h at room temperature and allow the virus to precipitate for at least 45 min.

9. Sediment the virus by centrifugation at 10,000 g for 15 min.

10. Discard the supernatant and suspend the pellet in 1 ml of resuspension buffer per gram of initial leaf material.

11. Clarify the suspension by centrifugation at 4000 g for 15 min.

12. Discard the pellet and layer aliquots of the supernatant in 30-40 ml centrifuge tubes onto half their volume of 30 % (w/v) sucrose in storage buffer.

13. Centrifuge at 70,000 g for 4 h.


14. Discard the supernatant and resuspend each pellet in 1 ml of storage buffer.

15. Layer each resuspended pellet onto a 20-50 % sucrose gradient in storage buffer in a 10 to 15-ml swinging-bucket centrifuge tube (Protocol 27).


17. Collect the virus zones using a syringe or Pasteur pipette, or with a fraction collector.

18. Collect the virus by centrifugation at 114,000 g for 4 h.

19. Discard the supernatant and resuspend the pellets in one or a few ml of storage buffer.


• Centrifugations are at 15°C to avoid spontaneous precipitation of the virus.

• Potato leafroll virus multiplication is limited to the phloem tissues. Therefore, after homogenising the leaves in a blender, the pulp is not strained through cheesecloth so as not to remove much of these tissues. Moreover, after centrifugation for breaking the chloroform/butanolbuffer emulsion, the fibrous leaf material will make a solid interface, facilitating decanting of the buffer phase.

• As an alternative to aphid-inoculation of the propagation host P. floridana, infected plants can be multiplied by cuttings.

28.10 Potato X Potexvirus

The method comprises clarification with the aid of carbon tetrachloride, concentration of the virus by PEG precipitation, and further purification by differential, sucrose-gradient and equilibrium centrifugation.


• Materials

The virus is propagated in Nicotiana rustica. Systemically infected leaves are harvested 2-3 weeks after inoculation. The leaves are used fresh.

- Diethyl ether - Carbon tetrachloride (CCl4 )

- PEG (Mr 6000) - Sucrose - Caesium chloride (CsCl) - Extraction buffer: 0.1 M Tris base adjusted to pH 9 with citric acid;

0.01 M sodium thioglycollate; 0.02 M Na-DIECA - Resuspension buffer:

0.1 M Tris base adjusted to pH 9 with citric acid

Procedure

Leaves, buffers and organic solvents are chilled before starting the procedure. During purification, containers are kept in chipped ice.

1. Homogenise the leaves in a blender together with a mixture of S vol of extraction buffer, 1 vol of diethyl ether and 1 vol of CCl4 . Use 2 ml of the mixture per gram of leaves.


3. Centrifuge the extract at SOOO g for 10 min.

4. Discard the pellet and add to the supernatant PEG 6000 to a final con-centration of 5 % (w/v).

5. Stir for 1 h at 4 °C.

6. Collect the precipitate by centrifugation at SOOO g for 15 min.

7. Discard the supernatant and resuspend the pellet in at least 1 ml of extraction buffer per gram of initial leaf weight.

S. Stir overnight at 4 °C.

9. Centrifuge the suspension at SOOO g for 10 min.

10. Discard the pellet and centrifuge the supernatant at 64,000 g for 70 min.

plant material



11. Discard the supernatant and suspend the pellet in 10 ml of resuspen-sian buffer for each 60 g of initial leaf weight.

12. Stir for at least 1 h at 4 0c. l3. Centrifuge the suspension at 8000 g for 10 min.


15. Discard the supernatant and suspend the pellet in 2 ml of resuspension buffer for each 60 g of initial leaf weight.

16. Stir for at least 1 h at 4 0c. 17. Centrifuge the suspension at 8000 g for 10 min.

18. Discard the pellet and layer the supernatant in 1 ml aliquots onto 10-40 % (w/v) sucrose gradients in 35-40 ml tubes for a swinging-bucket rotor (Protocol 27).

19. Centrifuge at 76,000 g for 1.5 h.

20. Collect the virus zone and dilute the suspension 1: 1 with resuspension buffer.

21. Centrifuge the suspension at 54,000 g for 2.5 h.


23. Add to the suspension twice its volume of a solution of 6.23 g of CsCI in 10 ml deionised water and mix well.

24. Centrifuge the mixture in a swinging-bucket rotor at 110,000 g for 24 h or so much longer as necessary to obtain a sharply defined virus zone in the gradient (Protocol 30).

25. View the tube in the dark under a microscope illuminator and collect the bluish virus zone using a Pasteur pipette or a syringe, the tip of which is placed at the level of the lower boundary of the zone.

26. Dialyse for 20 h in the cold against ten times diluted resuspension buffer.

27. Collect the dialysed virus suspension, add an equal volume of glycerol and store at -18°C.


28.11 Sunn-Hemp Mosaic Tobamovirus

The method (Gooding and Hebert 1967) comprises clarification with butanol and concentration of the virus and further purification by repeated PEG precipitations. Virus yields are usually between 100 and 300 mg per 100 g of leaf tissue.

Materials

The virus can be propagated in primary leaves of susceptible cowpeas or in systemically infected Nicotiana benthamiana plants. Cowpea plants are preferably inoculated onto their primary leaves, 9 or 10 days after sowing. The leaves are harvested 10-14 days after inoculation. Storage at -20°C and thawing the leaves prior to purification facilitate removal of plant constituents.

- n-Butanol - PEG (Mr 6000)

NaCI - Extraction buffer: 0.5 M NaH2P04; 1 % (v/v) 2-ME. Adjust to pH 7.2

with NaOH. - Phosphate buffer: 0.01 M NaH2P04 adjusted to pH 7.2 with NaOH - Storage buffer: 0.01 M NaH2P04 ; 0.001 M NaN3 • Adjust to pH 7.2 with

NaOH. (Note: Sodium azide (NaN3 ) is highly toxic; it binds to metals, forming explosive compounds when kept dry. Wear gloves to dispense.)

Procedure

1. Homogenise the leaves in extraction buffer, 1 ml per gram of leaves.

2. Squeeze the slurry through preboiled cheesecloth and determine the volume of the extract.

3. While stirring, add 8 ml n-butanol per 100 ml of extract. After coagulation of the chloroplasts keep stirring for 15 min.

4. Centrifuge the mixture at 10,000 g for 30 min.

5. Collect the supernatant, discard the pellet.

plant material



6. While stirring, add to the supernatant 4 g of PEG 6000 per 100 ml and dissolve.

7. Centrifuge the solution at 10,000 g for 15 min.

8. Discard the supernatant and resuspend the pellet in phosphate buffer, 20 ml per 100 ml of the initial extract (step 2).


10. Discard the pellet, add to the supernatant 0.4 g ofNaCl and 0.4 g of PEG 6000 per 10 ml, while stirring, and dissolve completely.

11. Centrifuge the solution at 10,000 g for 15 min.

12. Discard the supernatant and resuspend the pellet in 2 ml of storage buffer for each 100 ml of initial extract (step 2).


14. Discard the pellet. The supernatant is a milky white suspension of purified virus. Store at 4 DC.


Concentrated purified sunn -hemp mosaic virus may be highly aggregated. Therefore, after dilution, the virus should be allowed to stand for 1 day before determining the concentration or using it in experiments in which the concentration is an important factor, such as infectivity assays.

28.12 Tobacco Mosaic Tobamovirus

Tobacco mosaic virus can be purified by the method described in Subprotoco128.11 for sunn-hemp mosaic virus. Here, a simplified and old procedure is given, comprising clarification by heating, concentration of the virus and further purification by salting out with ammonium sulphate. High virus yields up to 300 mg of virus per 100 g ofleaf tissue can be obtained.


Materials

A suitable host for propagating the virus is Nicotiana tabacum. The cultivar White Burley is commonly used, but any cultivar which can be systemically infected by the virus is suitable. Noninoculated leaves showing the typical symptoms of infection are harvested. They can be stored in the deep-freeze and thawed prior to purification.

- Ammonium sulphate - 0.3 M Na2HP04

• Procedure

1. Homogenise the leaf material and squeeze the homogenate through preboiled cheesecloth.

2. If homogenisation was with pestle and mortar, a higher virus yield may be obtained by adding to the residual pulp 30 ml of 0.3 M Na2HP04 for every 100 ml of extracted sap. After thoroughly grinding this mixture, squeeze again through cheesecloth.

3. Heat the combined extracts at 60 DC in a water-bath for 10 min.

4. Cool the extract with tap water and centrifuge at 2500 g for 20 min.

5. Pour off the supernatant in a measuring cylinder to determine its volume. Discard the pellet.

6. Add 25 g of ammonium sulphate for each 100 ml of extract and dis-solve.

7. Incubate for 30 min at room temperature.

S. Collect the precipitated virus by centrifugation at 12,000 g for 20 min.

9. Discard the supernatant and resuspend the pellet in deionised water to 1/10 to 1/5 of the original volume of the extract (step 5).

10. Dialyse the suspension for 1 day against running tap water.

11. After dialysis, the suspension can be stored as purified virus suspension at 4 DC.

plant material



plant material


suspensions


See Subprotocol 28.11.

28.13 Tobacco Necrosis Necrovirus

The purification method (Temmink 1971) comprises clarification with the aid of butanol, concentration of the virus by high-speed centrifugation, and further purification by differential and sucrose-gradient centrifugation. The method works well with strains of the D-serotype .

• Materials

Tobacco necrosis virus (TNV) has a fairly extensive host range and many plant species are suitable propagation hosts, such as cowpea and Nicotiana tabacum. Systemic infection is, however, rare and infections consist usually of clearly defined local lesions, visible 2 or 3 days after inoculation. Therefore, the virus must be purified from heavily inoculated leaves. Harvested leaves may be stored frozen prior to use as virus source.

- n-Butanol - Sucrose

Extraction buffer: 0.05 M phosphate; 0.1 % (v/v) thioglycollic acid. Adjust to pH 7.6.

- Resuspension buffer: 0.05 M phosphate adjusted to pH 7.6

• Procedure

1. Homogenise (thawed) leaves in a blender in 1 ml of extraction buffer per gram of leaves.

2. Squeeze the homogenate through preboiled cheesecloth.

3. Under constant stirring, add to the extract dropwise 8 ml of n-butanol per 100 ml of extract.

4. Incubate the mixture overnight at 4 0c.


5. Centrifuge the mixture at 8000 g for 15 min.

6. Discard the pellet and centrifuge the supernatant for 2 h at 78,000 g.

7. Discard the supernatant and suspend the pellet in 2 ml of resuspension buffer per 100 g of initial leaf weight.


9. Discard the pellet and centrifuge the supernatant for 2 h at 78,000 g.

10. Discard the supernatant and suspend the pellet in 2 ml of resuspension buffer per 100 g of initial leaf weight.


12. Discard the pellet and layer the supernatant onto 10-40 % (w/v) sucrose gradients in resuspension buffer in 30-40 ml tubes of a swinging-bucket rotor (Introduction).


14. Collect the fraction containing the virus and centrifuge this suspension at 160,000 g for 2 h to pellet the virus.

15. Discard the supernatant and resuspend the pellet in a small volume of deionised water.


• Many isolates of TN V have an associated satellite virus which is dependent upon the TNV for its multiplication. This satellite virus has smaller particles with a lower sedimentation coefficient. In sucrose gradients these particles form a band above the TNV band.

• Elimination of satellite virus from the TNV isolate can be achieved by repeated sucrose gradient centrifugations and, subsequently, inoculation and repeated passages through single local lesions (the bigger local lesions are caused by TNV alone and the smaller ones by TNV in presence of satellite virus).

• An alternative purification procedure for TNV involves salting out with 1.75 M ammonium sulphate (Uyemoto et al. 1968) as the concentration step.


plant material


suspensions

28.14 Tobacco Rattle Tobravirus

The method comprises clarification by low-speed centrifugation, concentration of the virus by PEG precipitation, and further purification by equilibrium centrifugation. Yields are 1-10 mg per 100 g of tissue.

Materials

Tobacco rattle virus is purified from inoculated leaves of Nicotiana clevelandii or N. rustica, harvested 7-10 days after inoculation. The leaves should be used fresh.

- PEG (Mr 6000) - NaCl

Caesium chloride (CsCI) - NarEDTA - Extraction buffer: 0.18 M phosphate-O.Ol M citric acid buffer, pH 7.2

(13.1 volumes of 0.1 M citric acid solution are added to 86.9 volumes of 0.2 M Na2HP04 solution); 0.1 % thioglycollic acid

- Resuspension buffer: same as extraction buffer, but without thioglycollic acid

Procedure

1. Homogenise the leaves in a blender in 1 ml of extraction buffer per gram of leaves.



4. Discard the pellet and determine the volume of the supernatant. Add PEG 6000 to a concentration of 8 % and NaCl to a concentration of 0.2 M.

5. Stir at room temperature for 1 h.

6. Collect the precipitate by centrifugation at 8000 g for 15 min.


7. Discard the supernatant and suspend the pellet in resuspension buffer, 1/10 of the volume of extraction buffer used in step 1.

8. After 30 min, centrifuge the suspension at 8000 g for 10 min.

9. Collect the supernatant.

10. Suspend the pellet in the same volume of resuspension buffer as in step 7.

11. After 30 min, centrifuge this suspension at 8000 g for 10 min.


13. With the combined supernatants, repeat steps 4-6.


15. After 30 min, centrifuge the suspension for 10 min at 8000 g.

16. Discard the pellet and add to the supernatant 4.71 g of CsCI per 10 ml. Dissolve the CsCI thoroughly (the volume will increase by approx. 12 % and the density will be approx. 1.31 g/cm3).

17. Centrifuge the solution in a swinging-bucket rotor at 137,0000 g for 24 h, or longer if necessary (Protocol 30).

18. View the tube in dark under a microscope illuminator and collect the bluish virus zone using a Pasteur pipette or a syringe.

19. Dialyse for 20 h in the cold against ten times diluted resuspension buffer.

28.15 Tobacco Streak lIarvirus

The method (Fulton 1967) comprises clarification with hydrated calcium phosphate, concentration of the virus by high-speed centrifugation, and further purification by acidification and differential centrifugation. Yields up to 40 mg of virus per 100 g of leaf tissue have been reported.

268 PART Vln - VIRUS ISOLATION AND PURIFICATION

• Materials

plant material Datura stramonium is a good propagation host. Heavily inoculated leaves are harvested 10 days after inoculation. The leaves are used fresh. Excessive necrosis of the leaves, occurring at conditions of low-light intensity, can be reduced by additional illumination up to 18 h daily.

chemicals and - Aluminium oxide (AI20 3 )

solutions - Dibasic sodium phosphate (Na2HP04) - CaCl2 - Citric acid - NaOH - Extraction buffer: 0.02 M phosphate buffer; 0.02 M 2-ME. Adjust to

pH 8.0. - Resuspension buffer: 0.01 M Na2-EDTA adjusted to pH 6.0

preparations Hydrated calcium phosphate (HCP) for adsorbing host material is prepared (Fulton 1959) by adding to a 0.1 M solution of Na2HP04 slightly less than an equal volume of 0.1 M CaCI2. When the flocculent white precipitate is settled after 4-6 h, one-third or more of the supernatant is decanted and replaced with distilled water. The precipitate is resuspended. Decantation and resuspension are repeated 15-20 times to remove soluble salts. After these washings the HCP is packed by low-speed centrifugation to the consistency of soft butter and stored at room temperature until use. Drying of the HCP should be prevented .

• Procedure

As an Ilarvirus, tobacco streak virus is unstable in plant extracts. Special measures are necessary to stabilise the virus, such as addition of the antioxidant 2-ME to the extraction buffer.

1. Homogenise the leaves for 5 min in a blender in cold extraction buffer (150 ml for 100 g of tissue), to which Al20 3 has been added to a concentration of 10 % (w/v).

2. Centrifuge the homogenate for 20 min at 1000 g.

3. Discard the pellet and add to the supernatant a volume ofHCP equivalent to 0.8 of the weight of the leaves used. Mix well by stirring.


4. Centrifuge the mixture for 20 min at 1000 g.

5. Discard the pellet and centrifuge the supernatant for 3 h at 78,000 g to sediment the virus.


7. Adjust the pH to 5.0 with citric acid.

8. Collect the precipitate by centrifugation at 8000 g for 15 min.

9. Discard the pellet and readjust the pH of the supernatant to 6.0 with NaOH.




• For isolates of the virus which cannot be purified by the above method, a procedure involving precipitation with 4 % (w/v) PEG 6000 and 0.1 M NaCl may be suitable.

• If the virus source leaves are very necrotic, or the extract is not completely clarified in steps 3 and 4, it may be necessary to increase the volume of HCP used to 0.9 of the weight of the leaves.

28.16 Tomato Spotted Wilt Tospovirus

Tomato spotted wilt virus is the type species of the genus Tospovirus in the family Bunyaviridae. The virions of these viruses consist of nucleocapsids enveloped by a lipid membrane. Both virions and nucleocapsids are infectious when inoculated mechanically. The two infectious entities are purified by different procedures, both of which are given here.

• Materials

The virus is propagated in Nicotiana rustica or N. benthamiana. Leaves plant material showing prominent symptoms are harvested 14 days after inoculation. They are used fresh, as freezing and thawing destroys virus infectivity.



suspensions

- Extraction buffer A: 0.1 M potassium phosphate; 0.01 M Na2S03. Adjust to pH 7.0. Resuspension buffer A: 0.01 M Na2S03

- Sucrose or caesium sulphate (CsS04 )

- Extraction buffer B: 0.1 M Tris base; 0.01 M NarEDTA. Adjust to pH 8.0. Just prior to use add 0.01 M Na2S03.

- Resuspension buffer B: 0.01 M Tris base; 0.01 M NarEDTA. Adjust to pH 7.9. After adjusting the pH, add 1 % Nonidet P-40.

- Storage buffer: 0.01 M Tris base; 0.01 M NarEDTA. Adjust to pH 7.0.

Procedure

Purification of Virions

The method (Gonsalves and Trujillo 1986) comprises clarification by lowspeed centrifugation, concentration of the virus by high-speed centrifugation, and further purification by sucrose-gradient centrifugation. The following procedure should be carried out at 4°C, that is either in a cold-room or in precooled labware held in chipped ice.

1. Grind freshly harvested leaves in a blender in 3 ml of extraction buffer A per gram of leaves.

2. Strain the homogenate through cheesecloth (preboiled for a few min in deionised water containing some NarEDTA).

3. Centrifuge the filtrate at 6500 g for 15 min.

4. Discard the supernatant and suspend the pellet gently but thoroughly in 1 ml of resuspension buffer A per gram ofleaves. Allow the suspension to set for 30 min.



7. Discard the supernatant and suspend the pellet gently in 2.5 ml of resuspension buffer A for maximally 100 g of initial leaf material.



9. Layer the supernatant onto 35 ml gradients of 10-40 % sucrose in resuspension buffer A and centrifuge at 70,000 g for 45 min (Introduction).

10. Collect the virus bands using a syringe.

11. Dilute the virus preparation with an equal volume of resuspension buffer A.

12. Pellet the virus by centrifugation at 70,000 g for 25 min.

13. Suspend the virus in a small volume of resuspension buffer A.

Purification of Nucleocapsids

The method comprises clarification by low-speed centrifugation, concentration of the virus by high-speed centrifugation and further purification by sucrose-gradient or equilibrium centrifugation. The whole procedure should be carried out at 4°C, that is either in a cold-room or in precooled labware, held in chipped ice.

1. Homogenise the leaves in a blender for 30 s in 4 ml of extraction buffer B per gram of leaf material.

2. Strain the homogenate through cheesecloth (preboiled for a few min in deionised water, containing some NarEDTA).

3. Centrifuge the extract at 6500 g for 10 min.

4. Discard the pellet and centrifuge the supernatant for 30 mIll at 49,000 g.

5. Discard the supernatant and suspend the pellet in resuspension buffer Busing 2 ml per gram of leaf material, as follows. Using a spatula, transfer the pellet to a microfuge tube, add a small volume of the resuspension buffer B and gently grind up the pellet with a micropestle. Transfer the contents of the microfuge tube to a small beaker, add the rest of the resuspension buffer B and gently move the beaker until the remaining solid material is resuspended. Leave the beaker for another 30 min.



7. Discard the pellet and layer the supernatant onto a 30 % (w/v) sucrose cushion in resuspension buffer B of almost equal volume. Centrifuge for 1 h in a fixed-angle rotor at 125,000 g.

8. Discard the supernatant and resuspend the pellet in 1 ml of storage buffer.

9. Transfer the suspension to a microfuge tube and centrifuge this tube for a few seconds in a microfuge.

10. Discard any pellet and subject the supernatant to either rate zonal centrifugation (virus from Nicotiana rustica) or equilibrium centrifugation in caesium sulphate (virus from N. benthamiana).

rate zonal lIa.Layer the supernatant onto a 20-40 % sucrose gradient in storage buf-centrifugation fer in 10-15 ml tubes of a swinging-bucket rotor (Introduction).

equilibrium centrifugation

12a.Centrifuge for 2 h at 200,000 g.

13a.Collect the virus bands using a syringe or a fraction collector.

14a.Dialyse overnight against storage buffer to remove the sucrose.

lIb.Prepare 2.2 mlof a 25 % (w/v) solution of CsS04 in storage buffer also containing the supernatant of step 10.

12b.Layer this solution onto 2.2 ml of a 45 % (w/v) solution ofCsS04 in storage buffer in a 5 ml swinging-bucket centrifuge tube.

13b.Centrifuge at 150,000 g for 20 h at 12 DC.

14b.Collect the virus bands by puncturing the tube with a syringe needle from the side at levels of the lower boundaries of the bands.

15b.Dialyse the viral nucleocapsid preparation overnight against storage buffer to remove the caesium sulphate.

References

Dijkstra J, Keesen E, Brink M, Peters D, Lohuis H (1996) Identification and characterisation of a potyvirus of Bambara groundnut. African Crop Sci J 4:97-104

Fulton RW (1959) Purification of sour cherry necrotic ringspot and prune dwarf viruses. Virology 9:522-535

Fulton RW (1967) Purification and some properties of tobacco streak and Tulare apple mosaic viruses. Virology 32:153-162


Gonsalves D, Trujillo EE (1986) Tomato spotted wilt virus in papaya and detection of the virus by ELISA. Plant Dis 70:501-506

Gooding GV, Hebert TT (1967) A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology 57:1285

Klootwijk 1, Klein I, Zabel P, Van Kammen A (1977) Cowpea mosaic virus RNAs have neither m7 GpppN ... nor mono-, di- or triphosphates at their 5' ends. Cell 11:73-82

Lot H, Marrou J, Quiot JB, Esvan C (1972) Contribution a l'etude de virus de la mosalque du concombre (CMV). II Methode de purification rapide du virus. Ann Phytopathol 4:25-38

Mossop DW, Francki RIB, Grivell CJ (1976) Comparative studies on tomato aspermy and cucumber mosaic viruses. V. Purification and properties of a cucumber mosaic virus inducing severe chlorosis. Virology 74:544-546

Stace-Smith R (1966) Purification and properties of tomato ringspot virus and an RNA-deficient component. Virology 29:240-247

Takanami Y, Kubo S (1979) Enzyme-assisted purification of two phloem-limited plant viruses: tobacco necrotic dwarf and potato leafroll. Ann Appl BioI 44:153-159

Temmink JHM (1971) An ultrastructural study of Olpidium brassicae and its transmission of tobacco necrosis virus. Mededelingen Landbouwhogeschool Wageningen 71-6

Tremaine JH, Ronald WP, Valcic A (1976) Aggregation properties of carnation ringspot virus. Phytopathology 66:34-39

Uyemoto JK, Grogan RG, Wakeman JR (1968) Selective activation of satellite virus strains by strains of tobacco necrosis virus. Virology 34:410-418

Van Lent JWM, Wit AJ, Dijkstra J (1980) Characterization of a carlavirus in elderberry (Sambucus spp.). Neth J Plant Pathol 86:117-134

Van Vloten-Doting L, Jaspars EMJ (1973) The uncoating of alfalfa mosaic virus by its own RNA. Virology 48:699-708

purification virus 1998

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