Cowpea mosaic virus
A. van Kammen
Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands
C. P. de Jager
Department of Virology, Agricultural University, Wageningen, The Netherlands
Chant (1959) and
Van Hoof (1963).
- Cowpea yellow mosaic virus (Rev. appl. Mycol. 39: 204)
- Cowpea mosaic virus, yellow strain (Rev. appl. Mycol. 43: 2773;
An RNA-containing virus with isometric particles about 24 nm in diameter. It
has a limited host range, is transmitted mainly by beetles and readily by sap
inoculation. Infected plants contain two kinds of nucleoprotein particle similar
in size but differing in RNA content. Particles containing no RNA are also
produced by most isolates. The RNA species in different particle types represent
separate parts of the viral genome.
Causes mosaic diseases in Vigna
spp. Leaf area and flower production
are decreased. Yield reductions up to 95% have been reported. Late infections
had less effect on yield than early ones
susceptibility of pigeon pea (Cajanus cajan
) which is widely grown in
the coastal areas of Kenya as a perennial crop and may serve as a reservoir of
Reported from Nigeria, Kenya, Surinam, Cuba and USA.
Host Range and Symptomatology
The host range is rather limited; few hosts are known outside the Leguminosae.
Almost all host species show necrotic or chlorotic lesions in inoculated leaves.
Cowpea varieties differ greatly in type of reaction and severity of symptoms.
Immunity, tolerance and hypersensitivity occur. Symptoms in susceptible varieties
may range from a hardly discernible green mottle to distinct yellow mosaic and
distortion with significantly reduced growth
(Bliss & Robertson, 1971
- Vigna unguiculata (cowpea) cv. Blackeye Early Ramshorn. Chlorotic spots
with diffuse borders (diam. 1-3 mm) are produced in inoculated primary leaves
Trifoliolate leaves develop a bright yellow or light green mosaic of
increasing severity in younger leaves. Distortion and reduction in size are
Plants do not show necrosis.
- Chenopodium amaranticolor. In inoculated leaves, yellow local lesions
(diam. 0.5-1 mm) later becoming necrotic; systemic symptoms are severe mosaic,
chlorotic spots, distortion and puckering
- Vigna unguiculata cv. Blackeye Early Ramshorn is a good source of virus
for purification and for maintaining cultures.
- Phaseolus vulgaris cv. Pinto and Chenopodium amaranticolor are
suitable local lesion hosts. For local lesion transfer Pinto beans should
preferably be used because this host is not systemically infected and the
lesions have a high virus content.
mentioned the possibility that beetle-transmitted cowpea
viruses from Trinidad
classified five isolates into two strains and designated
these as yellow and severe strains. A Nigerian isolate and the isolate SB from
Surinam (collected at Santo Boma;
Van Hoof, 1963
belonged to the yellow strain.
A Trinidad isolate and two other isolates from Surinam represented the severe
strain. Both strains were included in the previous Description of cowpea mosaic
(Van Kammen, 1971
Swaans & Van Kammen (1973)
having made a detailed comparison of the two strains, proposed that they be
regarded as two different viruses of the cowpea mosaic virus group and this
revised Description now deals only with the yellow strain. Because the SB
isolate is designated as the type member of the
the name cowpea mosaic virus is maintained for the yellow strain. The severe
strain will be described separately, as
cowpea severe mosaic virus
Nigerian and SB isolates do not seem to differ in host range and appear to be
serologically identical or very similar
(Agrawal & Maat, 1964).
holds for the isolate from the USA and the SB-isolate
(McLaughlin et al., 1977).
Although there are some differences between host ranges of the Nigerian
and the Kenyan isolates they are closely related serologically
The Cuban isolate
(Kvicala, Smrz & Blanco, 1970)
also has a different host
range but was not tested serologically. However, because it infects
Chenopodium amaranticolor systemically it should probably be grouped
together with the above mentioned isolates of the former yellow strain. In
isolated from the SB-strain two naturally
occurring variants which differed from the parent strain in the relative amount
of RNA-free particles produced.
De Jager (1973)
isolated from the SB-strain two
naturally occurring variants which, unlike the parent strain, infected Early Red
cowpeas systemically. By treatment of virus particles or extracted RNA with nitrous
acid, mutants may readily be obtained. Most of these mutants are characterized
by less conspicuous symptoms and/or decreased virus production
(De Jager & Van Kammen, 1970;
De Jager, 1976;
De Jager et al., 1977).
Siler, Babcock & Bruening (1976)
reported the isolation, after bisulphite treatment, of a
mutant with greater specific infectivity.
Transmission by Vectors
Transmitted by various beetles with biting mouthparts. In Africa the
chrysomelid beetle Ootheca mutabilis
is an efficient vector
but Paraluperodes quaternus
(Curculionidae) were also found to transmit the
(Whitney & Gilmer, 1974
In Surinam and Cuba respectively,
and C. ruciformis
are incriminated as vectors
(Van Hoof, 1963
Kvicala et al., 1970
Jansen & Staples (1971)
listed C. trifurcata, Diabrotica balteata, D. undecimpunctata howardi,
and Acalymma vittatum
(all chrysomelid beetles) as
vectors. Beetle vectors may remain viruliferous for 1-2 to more than 8 days
Jansen & Staples, 1971
Transmission efficiency and retention
of infectivity are correlated with the amount of vector feeding
(Jansen & Staples, 1971
Virus infectivity was found in excrements of beetles
(Kvicala et al., 1970
Whitney & Gilmer (1974)
reported also transmission by
two species of thrips (Sericothrips occipitalis
) and by two species of grasshoppers (Cantotops spissus
and Zonocerus variegatus
Transmission through SeedGilmer, Whitney & Williams (1974)
reported 1-5% seed transmission in
The virus is strongly immunogenic. Standard methods using rabbits give
antisera with titres of 1/1024 in the Ouchterlony double diffusion test, but
higher titres may be obtained
(Agrawal & Maat, 1964
tested by the Ouchterlony method give a single band of precipitate, even
though they contain more than one electrophoretic component.
Little systematic work has been done to compare isolates of the virus from
different parts of the world. However, isolates from Surinam (SB-isolate),
Nigeria, Kenya and USA are closely related serologically. A weak serological
relationship is reported between cowpea mosaic virus as described here and
some other viruses of the comovirus group
cowpea severe mosaic virus
(formerly known as the severe strain of cowpea mosaic virus;
Swaans & Van Kammen, 1973
bean pod mottle virus
(Bancroft, cited by
Agrawal & Maat, 1964
red clover mottle virus
broad bean true mosaic virus
(Jones & Barker, 1976
bean rugose mosaic virus
Stability in Sap
Properties in vitro
reported for the various isolates vary
considerably, probably because of the use of different source plants and/or
assay hosts. Dilution end-points range from 10-4.7
Thermal inactivation points were reported from 55 to 65°C. Longevity
was found to vary from 4 to 10 days.
Yields of virus may reach 2 g/kg leaf tissue from Vigna
at 30°C in a growth chamber. Freshly harvested leaves are homogenized with
twice their weight of 0. 1 M phosphate buffer, pH 7.0. The homogenate is squeezed
through two layers of Miracloth and centrifuged at 15,000 g
min. The supernatant fluid is kept, and the pellet is washed once by suspending
in phosphate buffer (0.25 ml/g leaf tissue) followed by centrifugation at 15,000
. The combined supernatant fluids are stirred for 1 min with 0.7
volume of a 1:1 mixture of chloroform and n
-butanol. After low speed
centrifugation, the clear aqueous layer is removed, and the virus precipitated
by adding polyethylene glycol 6000 (PEG) to 4% and NaCl to 0.2 M, and stirring
for 60 min at room temperature. The precipitate after centrifugation at 15,000
for 15 min is suspended in phosphate buffer (0.5 ml/g leaf tissue).
After centrifugation at 27,000 g
for 15 min, the supernatant fluid
containing the virus is kept and the pellet is extracted once more in a few ml of
phosphate buffer and then centrifuged at 27,000 g
for 15 min. The
combined supernatant fluids are layered on top of 1 ml of 40% (w/v) sucrose in
0.1 M phosphate buffer in a centrifuge tube and then centrifuged at 150,000
for 2.5 h. The clear virus pellet is dissolved in sterile
double-distilled water and centrifuged at 10,000 g
for 15 min to
remove possible contaminants. This procedure
(Klootwijk et al., 1977
provides highly purified virus. It is a combination of the older methods of
purification using chloroform/butanol mixtures
(Bruening & Agrawal, 1967
or PEG and NaCl
(Van Kammen, 1967
Properties of Particles
Purified preparations of virus contain three centrifugal components: empty
protein shells without RNA (T) and two nucleoprotein components (M and B),
containing 25% and 36% RNA respectively. The separated nucleoprotein components
are not infective
(Van Kammen, 1968
but mixtures of the M and B components are.
The infectivity of a mixture depends upon the proportions of the two components
and the concentration of the component present in the lowest amount. Mixtures of
the M or B components with the B or M components of mutants of cowpea mosaic
virus are infective
De Jager & Van Kammen, 1970
De Jager, 1976
but heterologous mixtures of components of cowpea mosaic virus and those
of other comoviruses
Sedimentation coefficients (s20,w) at infinite dilution
(svedbergs): 58 (T), 95 (M), 115 (B). The proportion of M:B is about 1:1.
Molecular weight: 3.80 x 106 (T), 5.15 x 106 (M) and
5.87 x 106 (B).
Isoelectric point: between pH 3.7 and 4.5.
Absorbances at 260 nm (1 mg/ml, 1 cm light path): 6.2 (M), 10.0 (B), at 280
nm (1 mg/ml, 1 cm light path): 1.28 (T).
A260/A280: 1.64 (unfractionated virus),
1.61 (M), 1.74 (B).
Buoyant density in CsCl (g/ml): 1.288 (T), 1.395 (M); component B gives two
bands of density 1.406 and 1.447. The two bands have the same sedimentation
Purified preparations of the virus contain two electrophoretic components,
each of which contains all three centrifugal components
electrophoretic forms can be separated by electrophoresis in a sucrose gradient.
Electrophoretic mobility: -4.0 to -4.25 x 10-5 and -2.6 to -2.8 x
10-5 cm2 sec-1 volt-1 in 0.1 M phosphate
buffer. The slower migrating form predominates in early infection and the faster
one in late infection. The slower migrating form is converted into the faster
migrating form in vivo by loss of a peptide with a M. Wt of approximately
2500 from the smaller capsid protein. The conversion of the slower form into
the faster form can be achieved in vitro by incubation with proteolytic
enzymes. The two electrophoretic forms of the virus have similar infectivity
(Niblett & Semancik, 1969;
Geelen, Rezelman & Van Kammen, 1973).
Particles are icosahedral with 5:3:2 axial symmetry, and a diameter of
By three dimensional image reconstruction of electron
micrographs of particles negatively stained with uranyl acetate a model is
proposed consisting of twelve pentamers of the larger coat protein at the 5-fold
axes and twenty trimers of the smaller coat protein at the 3-fold axes
(Crowther, Geelen & Mellema, 1974
In this model the protein shell consists of 60
subunits each composed of the two structural proteins arranged in interpenetrating
T = 1 lattices.
Particle CompositionNucleic acid:
RNA, single-stranded. Components M and B each have a
single RNA molecule with sedimentation coefficients (s20,w
of 26 S and 34 S and M. Wt of 1.37 x 106
and 2.02 x
(Reijnders et al., 1974
compositions (molar percentages of nucleotides) are, respectively, G 20.7;
A 28.4; C 19.3; U 31.6 and G 22.9; A 28.5; C 17.2; U 31.4
(Van Kammen & Van Griensven, 1970
Both RNA strands contain a sequence of polyadenylic
acid 150-200 residues long at their 3'-end
(El Manna & Bruening, 1973
The two RNAs have no m7
G(5')ppp(5')N...., a structure referred to
as a cap, found with many plant viral RNA species, or (p)(p)pN . . ., at
(Klootwijk et al., 1977
In recombination tests with wild and mutant strains the 26 S RNA
was shown to contain the gene specifying the smaller capsid protein
(Gopo & Frist, 1977)
and to be responsible for the relative amount of T-component
De Jager & Van Kammen, 1970).
The 34 S
RNA seemed to control the rate of conversion of the slower into the faster
electrophoretic form and the specific infectivity
(Siler et al., 1976).
Mutations in both RNA species were found to affect local and systemic
symptoms and systemic spread of the virus in beans and cowpeas
(De Jager & Van Kammen, 1970;
De Jager, 1973;
A mutation causing temperature
sensitivity was located in the 26 S RNA
(De Jager et al., 1977).
Protein: The protein shell of the virus consists of two proteins of
M. Wt c. 37,000 and 22,000 in equimolar amounts
(Wu & Bruening, 1971;
Geelen, Van Kammen & Verduin, 1972).
The coat protein contains about
1.9% carbohydrates covalently linked
(Partridge et al., 1974).
Polyamines: Purified virus particles contain 5.05 µg spermidine
and 0.17 µg spermine per mg virus
(Bruening, El Manna & Wu, 1968;
Nickerson & Lane, 1977).
Relations with Cells and Tissues
The virus particles occur scattered and in clusters throughout the cytoplasm.
They do not form crystalline arrays. Inclusion bodies can be observed in
virus-infected cells by the light microscope, after staining with phloxine,
as a red amorphous mass near or surrounding the nucleus
Swaans & Van Kammen, 1973
Electron micrographs of virus-infected cells show
cytopathic structures, often adjacent to the nucleus
arrays of vesicles forming a kind of reticulum, the spaces between the vesicle
areas being filled with electron-dense material that does not seem to have
obvious structure. Virus particles can be seen embedded in this material. The
vesicles often contain fibrillar material of unknown nature
(De Zoeten, Assink & Van Kammen, 1974
The replication of viral RNA is closely associated
with the membranes of the vesicles of the cytopathic structures as shown by
fractionation of homogenates of virus-infected cells and autoradiography
(Assink, Swaans & Van Kammen, 1973
De Zoeten et al., 1974
leaves contain a RNA-dependent RNA polymerase which is closely
bound to cytoplasmic membranes and is able to synthesize viral RNA in
(Zabel, Weenen-Swaans & Van Kammen, 1974
structures are found in Vigna
mesophyll protoplasts infected with
cowpea mosaic virus, but not in uninfected protoplasts
(Hibi, Rezelman & Van Kammen, 1975
The cowpea mosaic virus described here is the type member of the
Besides the viruses mentioned by Fenner this group includes
pea green mottle virus
(Valenta et al., 1969
pea mild mosaic virus
Andean potato mottle virus
(Fribourg, Jones & Koenig, 1977
and cowpea severe mosaic virus
(formerly called the severe strain of cowpea
mosaic virus). Many viruses from other groups have been isolated from
spp. showing mosaic symptoms. The most notable of these are:
cowpea aphid-borne mosaic virus
(Bock & Conti, 1974
cowpea chlorotic mottle virus
cowpea mild mottle virus
(Brunt & Kenten, 1974
sunnhemp mosaic virus
(Kassanis & Varma, 1975
a strain of
southern bean mosaic virus
a strain of
bean common mosaic virus
(Sachchidananda et al., 1973
Cowpea mosaic virus may be distinguished from these and
other viruses of cowpea by the morphology of its particles and their
behaviour in the ultracentrifuge and by its antigenic properties.
Because of the small plots used for cowpea growing and the extreme prevalence
of the beetle vector, the use of insecticides for vector control is not
practicable. Use of resistant cultivars offers the best means of disease
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Local symptoms in a primary leaf of Vigna unguiculata cv.
Blackeye Early Ramshorn.
Trifoliolate leaves of Vigna unguiculata cv. Blackeye Early
Ramshorn showing systemic mosaic of differing severity.
Symptoms in Chenopodium amaranticolor.
Particles from a purified preparation negatively stained in uranyl
acetate. Bar represents 100 nm.
Electron micrograph of a cytopathic structure in an infected leaf
cell of Vigna unguiculata cv. Blackeye Early Ramshorn. N, nucleus; Ve,
vesicles, some of which contain fibrillar material (arrow); A, amorphous
electron dense material with scattered virus particles. Bar represents 1