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TECHNIQUES IN PLANT VIROLOGYCIP Training Manual5.0 VIRUS PURIFICATION
Section 5.2Protocols for thePurification of Plant Viruses
Purified virus preparations are needed to study the physical andbiochemical properties, to produce specific antisera, to prepare nucleicacid probes complementary to the viral sequence, and to determinetranslation products. Generally, purification consists of the followingstages:
P.V. • Sec 5.2 –99 • Page 2 - INTERNATIONAL POTATO CENTER
Once the virus is purified, its concentration must be determined. For thispurpose the virus is diluted (usually 1/20) and read at absorbency OD =
Extraction
Clarification(low speed)
Precipitation(low speed)
Clarification(low speed)
Sedimentation(high speed)
Clarification(low speed)
Density gradients centrifugation(high speed)
Fractionating
Sedimentation(high speed)
PURIFIED VIRUS
P.V. • Sec 5.2 –99 • Page 3 - INTERNATIONAL POTATO CENTER
260. The reading is then adjusted to the dilution level used and divided bythe virus extinction coefficient. This coefficient is characteristic for eachvirus. It represents the absorbency of a virus concentration of 0.1%(1mg/ml) with an optical path length of radiation of 1cm and a wavelengthof 260 nm. The extinction coefficient is symbolized as follows:
For instance, an APLV (bottom component) preparation diluted at 1/20has an OD260 value of 0.37 and an extinction coefficient of 9.6. The virusconcentration is therefore:
0.37 x 20 = 7.4
7.4/9.6 = 0.77 mg/ml
A simple calculation will show the total mg of virus obtained in thepurification and the virus yield per kg in the host plant.
Small samples should be taken at each step of the purification for viewingin the electron microscope. This determines the concentration of the virusand whether or not it is free of host plant components. Below are themost common virus purification protocols.
0.1% E
1 cm, 260 nm
P.V. • Sec 5.2 –99 • Page 4 - INTERNATIONAL POTATO CENTER
POTATO VIRUS M (PVM)
Host plant Datura metel or Lycopersicon pimpinelifoliumHarvest 18–20 days after inoculationWorking temperature 4°C on ice
ExtractionHomogenize plant material with 2 volumes (w/v) of 0.1 M phosphate
buffer pH 8.0 and 0.5% 2-mercaptoethanol
Filter through 3 layers of guazeAdd 10% (v/v) carbon tetrachloride and 8% (v/v) 1-butanol
Emulsify for one minute
Clarify7800 g for 10 minutes
Remove pellet
Remove supernatant
Process supernatant
Process pelletResuspend overnight in 0.01M
phosphate buffer pH 7.5 (1/10 of theoriginal volume)
Sediment72,500 g for 90 minutes
Clarify7800 g for 10 minutes
Remove pellet
Remove supernatant
Remove pellet
Remove supernatant
Process supernatant
Process pelletResuspend overnight in0.01M phosphate bufferpH 7.5 (1/50 orig. vol.)
Process supernatant
Process pelletResuspend overnight in0.01M phosphate buffer
pH 7.5 (1/100 of theoriginal volume)
SedimentOn sucrose cushions 20% (w/v). 1 ml
per tube, 160,400 g for 90 minutes
Clarify7800 g for 10 minutes
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in a swinging bucket rotor
96,500 g for 120 minutes
FractionateCollect the virus band in the gradient
Process fractionsDilute 1:3 in 0.01M phosphate buffer pH 7.5
Sediment102,600 g for 60 minutes
P.V. • Sec 5.2 –99 • Page 6 - INTERNATIONAL POTATO CENTER
Remove pellet Process supernatant
Clarify7900 g for 10 minutes
Centrifuge in performed density gradientsof cesium chloride (30, 60, and 90%) 128,000 g for 5 hours
FractionateCollect the virus-containing band in the gradient
Dialyze Overnighttwice with 2 liters 0.01 M phosphate buffer pH 7.5
PURIFIED VIRUS
Read absorbencyin a spectrophotometer
P.V. • Sec 5.2 –99 • Page 7 - INTERNATIONAL POTATO CENTER
POTATO VIRUS X (PVX)
Host plant Nicotiana glutinosaHarvest 20 days after inoculation
ExtractionHomogenize plant material (50 to 60 g) with 2 volumes (w/v) in 0.1M
phosphate buffer pH 8.0 with 0.2% of 2-mercaptoethanol and 10%ethanol
Filterthrough 3 layers of gauze
Clarify7800 g for 20 minutes at 4°C
Remove pellet
Remove pellet
Process supernatantAdd 1% Triton X-100. Stir 1 hour at
4°C
Process supernatantAdd 0.2 M NaCl and 4% PEG (MW6000 to 8000). Stir for 1 hour at 4°C.
Incubate for 1 hour at roomtemperature
Clarify5500 g for 20 minutes
Precipitate7800 g for 30 minutes
P.V. • Sec 5.2 –99 • Page 8 - INTERNATIONAL POTATO CENTER
Remove supernatant
Remove pellet
Remove supernatant
Remove pellet
Process pellet (at 4°°°°C)Resuspend in 8 ml 0.05 M phosphate
buffer pH 8.0 with 1% of Triton X-100and rinse the tubes immediately with 2
ml of the same buffer
Process supernatant
Process pelletResuspend in 0.5 ml 0.05 M
phosphate pH 7.2 using a rotatingstirrer at 200 rpm overnight
Process supernatant
Clarify7800 g for 10 minutes
CentrifugeOn sucrose cushion 30% (w/v)
72,500 g for 150 minutes
Clarify2000 g for 5 minutes at 4°C
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.01 M phosphate buffer pH 7.2
with 0.01 M EDTA in a swinging bucket rotor96,500 g for 75 minutes
FractionateCollect the virus band in the gradient
P.V. • Sec 5.2 –99 • Page 9 - INTERNATIONAL POTATO CENTER
Remove supernatant
Remove pellet
Process pelletResuspend in 0.4 ml 0.01 M
phosphate buffer pH 7.2Stir 4ºC overnight
Supernatant
Process fractionsDilute in the same volume of 0.1M phosphate buffer pH
7.2
Sediment102,600 g for 60 minutes
Clarify5000 g for 10 minutes
PURIFIED VIRUS
Read absorbency in a spectrophotometer
P.V. • Sec 5.2 –99 • Page 10 - INTERNATIONAL POTATO CENTER
POTATO VIRUS A (PVA)
Host plant Nicotiana occidentalisHarvest 18–20 days after inoculation. Use leaves with central vein
removedWorking temperature 4°C on ice
ExtractionHomogenize plant material with 2 volumes (w/v) of 0.2 M phosphate
buffer pH 8.0 and 0.5% 2-mercaptoethanol, 1M of urea and 0.01MEDTA
Clarify7800 g for 30 minutes
CentrifugeOn sucrose cushion of 30% (w/v) in 0.05M citrate bufferpH 6.2 with 0.01M EDTA at 72,500 g for 180 minutes
Remove pellet
Remove pellet
Remove supernatant
Process supernatantAdd 1% Triton X-100. Stir at
4°C for 3 hours
Process supernatant
Process pelletResuspend in 0.05M citrate buffer pH6.2 with 0.01M EDTA and 1% Triton
X-100. Stir at 4°C overnight
Clarify7800 g for 10 minutes
P.V. • Sec 5.2 –99 • Page 11 - INTERNATIONAL POTATO CENTER
CentrifugeOn sucrose cushion of 30% (w/v) in 0.01M citrate buffer pH 6.2
with 0.01M EDTA 160,400 g for 150 minutes
Remove pellet
Remove supernatant
Process supernatant
Process pelletResuspend in 0.05M citrate buffer pH 6.2 with
0.01M EDTA at 4°C overnight
Clarify5500 g for 15 minutes
Clarify5500 g for 15 minutes
Remove pellet Process supernatant
Centrifuge in density gradientsof sucrose (10 to 40%, v/v) in 0.01M citrate buffer pH 6.2 with
0.01M EDTA in a swinging bucket rotor. 128,000 g for 40minutes.
FractionateCollect the virus band in the gradient
P.V. • Sec 5.2 –99 • Page 12 - INTERNATIONAL POTATO CENTER
Sediment102,600 g for 60 minutes
Remove supernatantProcess pellet
Resuspend in 0.01M phosphate buffer pH7.2, stirring gently for 6 hours at 4°C.
Clarify5500 g for 10 minutes
Remove pellet Supernatant
PURIFIED VIRUS
Read absorbency in a spectrophotometer
P.V. • Sec 5.2 –99 • Page 13 - INTERNATIONAL POTATO CENTER
POTATO VIRUS S (PVS)
Host plant: Chenopodium quinoa (Andean strain) or Nicotiana debneyii (other strain).Harvest: 18 to 20 days after inoculation.
ExtractionHomogenize the plant material with 2 volumes (w/v) in 0.1M phosphate
buffer pH 8.0 with 0.15% of 2-mercaptoethanol and 10% of ethanol
FilterThrough 3 layers of gauze
Clarify5000 g for 20 minutes
Remove pelletProcess supernatant
Add 4% of PEG (MW 8000) and 1% of Triton X-100.Stir at 4°C for 60 minutes
Precipitate5000 g for 10 minutes
Remove supernatantProcess pellet
Resuspend in 0.05M phosphate buffer pH 8.0 (1/10 ofthe original volume) with 1 mM of EDTA
Clarify10,000 g for 20 minutes
Remove pellet Process supernatant
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Clarify5100 g for 15 minutes
Remove supernatant Resuspend in 0.05M phosphate buffer pH 8.0 with1 mM of EDTA
Clarify5100 g for 15 minutes
Remove pellet Process supernatant
Centrifuge in density gradientsof sucrose (10 to 40% w/v) in 0.05M phosphate buffer pH 8.0 with
1mM EDTA in swinging bucket rotor.100,000 g for 60 minutes
FractionateCollect the virus band in the gradient
Sediment102,700 g for 60 minutes
Remove supernatantProcess pellet
Resuspend in 0.05M phosphate buffer pH 8.0 with1 mM of EDTA at 4°C overnight
CentrifugeOn sucrose cushion 20% (w/v) 160,000 g for 3 hours
P.V. • Sec 5.2 –99 • Page 15 - INTERNATIONAL POTATO CENTER
Remove pellet Process supernatant
Centrifuge in density gradientsof sucrose (10 to 40% w/v) in 0.01M phosphate buffer, pH 8.0 with
0.01M EDTA in swinging bucket rotor.128,000 g for 40 minutes
FractionateCollect the virus band in the gradient
Sediment102,700 g for 60 minutes
Remove supernatantProcess pellet
Resuspend with 0.01M phosphate buffer pH 8.0(final volume: 1 or 2 ml)
Clarify5100 g for 10 minutes
Remove pellet Supernatant
PURIFIED VIRUS
Read absorbency in a spectrophotometer
P.V. • Sec 5.2 –99 • Page 16 - INTERNATIONAL POTATO CENTER
POTATO VIRUS Y (PVY)
Host plant: Nicotiana occidentalisHarvest: 15 to 20 days after inoculation. Use leaves with central vein removed.
ExtractionHomogenize the plant material with 2 volumes (w/v) of 0.2M
sodium and potassium buffer pH 8.0 with 0.2% of2-mercaptoethanol and 0.01M of EDTA
Remove supernatant
Filterthrough 3 layers of gauze
Process pelletResuspend in 0.02M Na and K phosphate buffer pH 7.2with 1% of Triton X-100 (1/5 of the original volume) at
4°C overnight
Clarify7800 g for 20 minutes
Remove pelletProcess supernatant
Add 1% of Triton X-100Stir at 4°C for 120 minutes
Clarify7800 g for 20 minutes
Remove pelletProcess supernatant
Add 4% of PEG (PM 6000 to 8000) and 0.2M of NaClStir at 4°C for 60 minutes and incubate at room
temperature for 60 minutes
Precipitate7800 g for 20 minutes
P.V. • Sec 5.2 –99 • Page 17 - INTERNATIONAL POTATO CENTER
FractionateCollect the band, which contain the virus in the gradient
Process fractionsAdd an equal volume of 0.01M Na and K phosphate buffer pH 7.2
with 0.01M of EDTA
Clarify5100 g for 10 minutes
Remove pellet Process supernatant
Centrifugeon sucrose cushion 30% (w/v) in 0.05M Na and K phosphate buffer
pH 7.2 (15 ml of sample for 5 ml 30% sucrose)72,500 g for 150 minutes
Remove supernatantProcess pellet
Resuspend in 0.01M Na and K phosphate buffer pH 7.2with 0.01M EDTA (1/30 of the original volume) at 4°C
for 4 hours
Clarify5100 g for 10 minutes
Remove pelletProcess supernatant
Add 0.5 g of CsCl for each 1.5 ml of sample
Centrifugeon cesium chloride gradients (30, 60, and 90%, w/v) in 0.01M Naand K phosphate buffer pH 7.2 with 0.01M of EDTA (1.5 ml of
sample for each 3 ml gradient) in swinging bucket rotor.128,000 g for 3 hours
P.V. • Sec 5.2 –99 • Page 18 - INTERNATIONAL POTATO CENTER
Sediment102,700 g for 60 minutes
Remove supernatantProcess pellet
Resuspend in 0.01M Na and K phosphate buffer pH 7.2(1/50 of the original volume) overnight at 4°C
Clarify5100 g for 60 minutes
Remove pellet Supernatant
PURIFIED VIRUS
Read absorbency in a spectrophotometer
P.V. • Sec 5.2 –99 • Page 19 - INTERNATIONAL POTATO CENTER
ANDEAN POTATO MOTTLE VIRUS (APMV) ANDANDEAN POTATO LATENT VIRUS (APLV)
Host plant: Hybrid Nicotiana clevelandii x Nicotiana bigelovii.Harvest: 18 to 20 days after inoculation.
ExtractionHomogenize the plant material with two volumes (w/v) of 0.2Msodium and potassium phosphate buffer pH 8.0 with 0.2% of 2-
mercaptoethanol and 5mM of MgCl2
Filterthrough 3 layers of gauze
Emulsify with 20% chloroform (v/v)
Clarify5500 g for 20 minutes
Remove pelletProcess supernatant
Add 0.2M of NaCl and 6% (w/v) of PEG (MW 6000–8000), mix and incubate at 4°C for 120 minutes
Precipitate5500 g for 30 minutes
Process pelletResuspend with 0.05M sodium and potassium phosphate
buffer pH 8.0 with 5mM of MgCl2 (40% of originalvolume). Stir for 120 minutes
Remove supernatant
Clarify2500 g for 10 minutes
P.V. • Sec 5.2 –99 • Page 20 - INTERNATIONAL POTATO CENTER
Remove pellet
Sediment170,000 g for 120 minutes
Process supernatant
Centrifugeon a 20% sucrose cushion (w/v) 1 ml per tube
160,500 g for 60 minutes
Process pelletResuspend in 0.05M Na and K phosphate pH 8.0
with 5mM of MgCl2 overnight at 4°CRemove supernatant
Clarify2500 g for 10 minutes
Remove pellet Process supernatant
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.05M Na and K phosphate buffer
pH 8.0 with 5 mM of MgCl2 in swinging bucket rotor.100,000 g for 3 hours
FractionateCollect the band that contains the virus in the gradient
Process the fractionsDilute in 0.05M Na and K phosphate
pH 8.0 and 5 mM of MgCl2
P.V. • Sec 5.2 –99 • Page 21 - INTERNATIONAL POTATO CENTER
Remove supernatant
Process pelletResuspend in 0.05M Na and K phosphate
pH 8.0 with 5 mM of MgCl2
Process pelletResuspend in 0.001 M Na and K phosphate
pH 8.0
Remove supernatant
PURIFIED VIRUS
Clarify2500 g for 10 minutes
Read absorbency in a spectrophotometer
Remove pellet Process supernatant
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.05M Na and K phosphate pH 8.0
with 5mM of MgCl2in swinging bucket rotor.
100,000 g for 3 hours
FractionateCollect the band that contains the virus in the gradient
Process the fractionsDilute 1:1 (v/v) in 0.05 M Na and K phosphate
pH 8.0 with 5 mM of MgCl2
Sediment170,000 g for 120 minutes
P.V. • Sec 5.2 –99 • Page 22 - INTERNATIONAL POTATO CENTER
TOBACCO RINGSPOT VIRUS (TRSV)
Host plant: Hybrid Nicotiana clevelandii x Nicotiana bigeloviiHarvest: 28 days after inoculation
ExtractionHomogenize the plant material with 1 volume (w/v) of 0.07M
phosphate buffer pH 7.0 with 0.01M of EDTA and 0.1% ofthyoglicolic acid
Clarify5500 g for 10 minutes
Filterthrough 3 layers of gauze.
Emulsify with 1 volume chloroform
Remove pellet
ProcessStir well and incubate for 1 hour
Collect the aqueous phase
Process supernatant
Clarify8000 g for 20 minutes
Process supernatantAdd 6% PEG (MW 8000) and 0.3 M
of NaClStir for 1 hour at 4°C
Remove pellet
Precipitate9600 g for 20 minutes
Process pelletResuspend in 10% of original volume 0.07 M phosphate
pH 7.0 with 0.01M of EDTA for 3 hoursRemove supernatant
P.V. • Sec 5.2 –99 • Page 23 - INTERNATIONAL POTATO CENTER
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.07M phosphate pH 7.0 in
swinging bucket rotor96500 g for 45 minutes
FractionateCollect the band that contains the virus in the gradient
Sediment160400 g for 60 minutes
Remove supernatant Resuspend the pelletin 0.07 M phosphate pH 7.0
PURIFIED VIRUS
Read absorbency in a spectrophotometer
P.V. • Sec 5.2 –99 • Page 24 - INTERNATIONAL POTATO CENTER
POTATO LEAFROLL VIRUS (PLRV)
Host plant: Physalis floridana or Solanum tuberosumHarvest: 35 days after inoculation. Storage the leaves at –70°C, one day before purification
ExtractionGrind plant material to a powder with liquid nitrogen. Add 2 volumes
(w/v) of 0.1 M trisodic citrate pH 6.2 with 10mM of EDTA,1% Na2 SO3, 1.5% celluclast, and 0.1% of thioglycolic acid and
stir overnight at room temperature.
HomogenizePlant material in a blender
Filterthrough 3 layers of gauze. Emulsify with 25% ofthe initial volume, of chloroform-1-butanol (1:1)
for 5 minutes
Remove pellet
Remove supernatant
Process supernatantAdd 0.4 M of NaCl and 8% of PEG(MW 6000–8000) and stir 2 hours at
room temperature
Process pelletResuspend in 0.01 M sodium
phosphate buffer pH 7.6.Overnight at 4°C.
Precipitate9600 g for 45 minutes
Clarify9600 g for 25 minutes
P.V. • Sec 5.2 –99 • Page 25 - INTERNATIONAL POTATO CENTER
Remove pellet
Remove supernatant
Remove supernatant
Process supernatant
Process pelletResuspend in 10mM sodium
phosphate buffer pH 7.6.Overnight at 4°C
Process pelletResuspend in 10 mM sodium
phosphate buffer pH 7.6.Overnight at 4°C
Clarify11290 g for 20 minutes
Centrifugeon sucrose cushion (20% w/v) in 10 mM sodium
phosphate pH 7.6.72530 g for 120 minutes
Centrifuge on sucrose cushion (20% w/v) in 10 mM sodium phosphate
buffer pH 7.672530 g for 120 minutes
Clarify7840 g for 10 minutes
Remove pellet Process supernatant
Clarify6200 g for 15 minutes
Remove pellet Process supernatant
P.V. • Sec 5.2 –99 • Page 26 - INTERNATIONAL POTATO CENTER
Remove pellet
Remove pellet
Process supernatant
Process supernatant
Clarify5000 g for 10 minutes
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 10 mM sodium phosphate buffer,
pH 7.6 in a swinging bucket rotor114500 g for 45 minutes
FractionCollect the band that contains virus in the gradient. Dilute
in 10mM sodium phosphate buffer pH 7.6
Sediment164,400 g for 60 minutes
Clarify5500 g for 10 minutes
Remove supernatant
Process pelletResuspend in 10 mM sodium
phosphate buffer pH 7.6Shake overnight at 4°C
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 10 mM sodium phosphate buffer
pH 7.6 in a swinging bucket rotor114500 g for 45 minutes
P.V. • Sec 5.2 –99 • Page 27 - INTERNATIONAL POTATO CENTER
PURIFIED VIRUS
Read absorbency in a spectrophotometer
FractionateCollect the band that contains virus in the gradient. Dilute
in 10mM sodium phosphate buffer,pH 7.6
Sediment160400 g for 60 minutes
Remove supernatant
Process pelletResuspend in 10 mM sodium
phosphate buffer pH 7.6Overnight at 4°C
P.V. • Sec 5.2 –99 • Page 28 - INTERNATIONAL POTATO CENTER
POTATO VIRUS T (PVT)
Host plant: Chenopodium quinoaHarvest: 15 to 20 days after inoculation
ExtractionHomogenize plant material with 2 volumes (w/v) in 0.06 M phosphate
buffer, pH 7.0.
FilterThrough 3 layers of gauze.
Remove pellet Process supernatantFilter through glass wool.
Add 5% PEG (MW 8000). Stir for 1 hourat 4°C. Remove from shaker and let sit at
room temperature for 10 minutes.
Precipitate10000 g for 20 minutes
Clarify8000 g for 20 minutes
Remove supernatant
Process pelletResuspend in 0.06 M
phosphate buffer, pH 7.0Overnight at 4°C
Remove pellet Process supernatant
Clarify2000 g for 8 minutes
P.V. • Sec 5.2 –99 • Page 29 - INTERNATIONAL POTATO CENTER
Sediment130000 g for 60 minutes
Remove supernatantProcess pellet
Resuspend in 0.06 Mphosphate buffer, pH 7.0
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.06 M phosphate buffer, pH 7.0 in
swinging bucket rotor84200 g for 45 minutes
FractionateCollect the virus band in the gradient
Sediment160500 g for 60 minutes
Remove supernatantResuspend pellet
in 0.06 M phosphate bufferpH 7.0. Overnight at 4°C
Centrifuge in density gradientsof sucrose (10 to 40%, w/v) in 0.06 M phosphate buffer, pH 7.0 in
swinging bucket rotor84200 g for 45 minutes
FractionateCollect the virus band in the gradient
Sediment160500 g for 60 minutes
P.V. • Sec 5.2 –99 • Page 30 - INTERNATIONAL POTATO CENTER
Remove supernatantResuspend pellet
in 0.06 M phosphatebuffer, pH 7.0. Overnight
at 4°C
PURIFIED VIRUS
Read absorbency in a spectrophotometer