- Factors limiting effective extraction and purification of bean
yellow mosaic virus (BYMV) were investigated. Since the use of published
techniques for purifying BYMV resulted in low yields of partially
purified virus, an improved method for partial purification of this
virus was developed. The following treatments of infectious crude extract
decreased infectivity: (i) mechanical rupture of the chloroplasts,
(ii) homogenization in anionic, nonionic, or cationic surfactants, (iii)
addition of the organic solvents acetone, butanol, ethanol, methanol,
petroleum ether, diethyl ether, toluene, and chloroform, (iv) centrifugation
in high salt concentrations (KBr or CsCl), (v) zone electrophoresis,
(vi) homogenization in ethylenedinitrila-tetra acetic acid
(EDTA) or in thioglycollic acid. The following treatments increased
infectivity of the crude extract when added to the homogenizing
medium: (i) 0.1M KCI, (ii) beta-mercaptoethanol, (iii) urea. Chenopodium amarantciolor, a local lesion host of BYMV,
yielded more virus than the systemically infected hosts evaluated.
Virus los. by sedimentation occurred during initial low-speed centrifugation
of the crude extract from systemically infected hosts. Relatively
little infectivity was lost from C. amaranticolor crude extract
during low-speed centrifugation.
Of four severe BYMV isolates tested, isolate 724 yielded the
highest amount of partially purified virus. Use of isolate 724 cultured
in C. amaranticolor resulted in a high yield of extractable virus.
The best buffer for homogenizing BYMV-infected C. amaranticolor
tissue was 0. 05M diphosphate, 0.1M KC1 within the pH range
8.5-9.0. Re-extraction from pulp increased the total amount of virus
extracted from C. amaranticolor by 30 percent.
Preliminary clarification of crude extract was achieved by a
low-speed centrifugation followed by a moderate-speed centrifugation.
The following treatments decreased yield of partially purified virus:
(i) heating or freezing of the crude extract, (ii) acidification (pH 3,
3.5, 4, 4.5, 5, 5.5, 6) of the crude extract, (iii) addition of 0.25
saturated (NH₄)₂SO₄ to the clarified supernatant, (iv) clarification by
the ether-carbon tetrachloride method, and (v) homogenization of infected
tissue in bentonite. Homogenization in 0.45-0.90M sucrose
yielded an infectious high-speed pellet, and an infectious density-gradient
fraction, but poor clarification. Absorbancy profiles and infectivity of density-gradient (DG) fractions showed that the polyethylene
glycol (PEG) treatment yielded less host contaminants and
better banding of partially purified virus in the DG column than did the
bentonite, sucrose, or ether-carbon tetrachloride treatments.
Sepharose gel filtration or zone electrophoresis of infectious,
concentrated PEG extract did not result in further purification. Zonal
rotor centrifugation of clarified supernatant resulted in a concentration
of BYMV into a narrow band, but the virus sample was still impure.
DG centrifugation of the infectious, concentrated PEG extract
was the best purification method. The most highly purified fraction
occurred 12-16 mm below the meniscus of the DG column.
Electron microscopy showed that the modal length of the virus
particles directly from local lesions was 750 nm, while the modal
length of particles in the most purified DG fraction was 680 nm.
The most highly purified BYMV preparation obtainable had an
ultraviolet absorption spectrum typical of anisometric viruses and a
260/280 ratio of 1.152. The sedimentation coefficient of the most infectious
DG fraction was 2050.5 S. This large value indicated that
particle aggregation occurred.