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Chronic bee paralysis virus in the nerve ganglia of the adult honey bee
JOURNAL
OF
INVERTEBRATE
Chronic
PATHOLOGY
Bee
7,
Paralysis
170-174
(1965)
Virus in the Nerve Adult Honey Bee1
Ganglia
of
the
PETER E. LEE AND BASIL FURCALA Plant
Research
Institute
and Entomology Agriculture, Accepted
Research Institute, Ottazua, Canada December
Canada
Department
of
15, 1963
A virus of irregular shape was isolated from paralyzed honey bees (Apis melliferaLinnaeus), This virus is considered to be chronic bee paralysis virus (CBPV) Thin sections revealed that CBPV is a nonoccluded virus located in the thoracic and abdominal ganglia of afflicted honey bees. Examination of fat and muscle tissue from paralytic bees, and comparative tissue from healthy bees, failed to disclose virus particles,
A paralysis of adult honey bees (Apis me& Linnaeus) in Britain is caused by a virus called chronic bee paralysis virus (CBPV) (Bailey et al., 1963). Although a virus is suspectedto cause paralysis of adult bees in Canada, the infectious principle has not been isolated. This paper presentsevidence that CBPV is present in Canada. Emphasisis placed on the location of the virus in paralytic bees.
lijera
EXPERIMENTAL Material
nates 4,900 g/l 5 minutes; supernatants 78,OOOg/60 minutes) the pellets from highspeedcentrifugation were dispersedin distilled water, and portions prepared for electron microscopy by staining with phosphotungstic acid (Brenner and Horne, 1959). Particles with an average size of 40 X 27 rnkl were observed in the extract from paralytic bees (Fig. 1) but not in the extract from healthy bees. These particles are similar to those reported by Bailey et al. (1963). The extract from paralytic bees in 50 percent sucrose caused symptoms typical of bee paralysis within 7 days when test bees were administered inoculum by either spraying or feeding.
Two samplesof bees were obtained from a commercial apiary near Ottawa, Canada. One Location of CBPV in Paralyzed Bees sample was removed from a colony showing Paralyzed and healthy bees were dissected paralytic symptoms, the other from a colony in saline and pieces of fat, thoracic muscle, which did not show symptoms of paralysis. and nerve ganglia from the thorax and abIsolation of CBPV and Infectivity Tests domen removed and fixed in Palade’s fixative Beesfrom each samplewere homogenizedin (Palade, 1952). The tissueswere dehydrated a water/carbon tetrachloride mixture (Bailey in alcohol and embeddedin Epon 812 (Luft, 1961) . Following polymerization at 6O”C, et al., 1963). After one cycle of differential sections were cut with a diamond knife on a centrifugation of the homogenates (homogePorter Blum microtome, collected on Form1 Joint contribution from the Plant Research Invar-carbon grids, stained with uranyl acetate stitute (Contribution No. 421) and the Entomology and examined in a SiemensElmiskop I. Research Institute, Research Branch, Canada DeVirus particles, round to oval in shape,were partment of Agriculture, Ottawa. 170
PARALYSIS
F ‘IG. rep ularly
1.
VIRUS
IN
NERVE
171
GANGLIA
Suspension from diseased honey bees negatively stained with phosphotungstic shaped particles of chronic bee paralysis virus (CBPV). 160,000 X.
acid
showing
ir-
172
LEE
FIG.
2.
CBPV
particles
in thin
AND
section
FURGALA
of thoracic
nerve
ganglion.
80,000
X
PARALYSIS
FIG. 3. Thin 40,000 X. Fl G. 4.
section Abdominal
of
abdominal nerve
ganglion.
VIRUS
nerve Note
IN
ganglion particles
NERVE
173
GANGLIA
showing
CBPV
filling
inclusion
particles
free
of the ganglion.
in
the
cj
40.000
x
~plasm
174
LEE AND FURGALA
observed in the thoracic and abdominal ganglia (Figs. 2-4). Particles appeared either in inclusions (Fig. 4)) or free in the cytoplasm of the nerve cells (Fig. 3). The CBPV particles are not enclosed in a protein matrix, unlike the polyhedrosis and granulosis viruses. No particles were observed in fat or muscle tissue of afflicted bees. These observations, however, do not preclude the presence of the virus in fat or muscle. Virus particles were not observed in comparable tissue from healthy bees. Although insect viruses have been reported to infect nervous tissue (Lotmar, 1941: Martignoni. 1954; Smith and Xeros, 1954; Benz, 1963), to our knowledge, this is the first report of unenclosed insect virus in nerve cells. Also noteworthy is the fact that the morphology of CBPV resembles a new type of insect virus recently described by Vago (1963). ACXNOWLEDGI\IENTS
We thank Dr. Eric Smith, Mines Branch, Mines and Technical Surveys, Ottawa, for use of the Siemens Elmiskop I electron microscope, and also Miss J, Ng. Y&m for assistance in the preparation of Formvar-carbon substrates.
REFERENCES BAILEY,
Two
L., GIBBS, A. J., AND WOODS, R. D. viruses from adult honey bees (Apis
1963. mel-
lifera L.), Virology, 21, 390-395. BENZ, GEORC 1963. A nuclear polyhedrosis of Malacosoma alpicola (Staudinger). J. Insect Pathol., 5, 215-241. BRENNER,
S., AND
HORNE,
staining method for microscopy of viruses. 34, 103-110.
R. W. 1959. A negative high resolution electron Biochim. Biophys. Acta,
LOTMAR,
R. 1941. Die Polyederkrankheit Kleidermotte (Tineola biselliella. Mitt. Entomol. Ges., 18, 372-373.
LUFT,
J. H. embedding 9, 409-414.
1961. Improvements methods. J. Biophys.
der Schweis.
in epoxy resin Biochem. Cytol.,
M. E. 1954. Uber zwei Viruskrankheiten von Forstinsekten im Engadin. Mitt. Schweis. Enlom. Ges., 27, 147-152.
MIIRTIGNONI,
P.~L.~DE,
tron Shfrm,
G. E. 1952. A study of fixation for elecmicroscopy. J. Exptl. Med., 95, 285-298.
K. M., ASD XEROS, r\:. 1954. Electron and light microscope studies of the development of the virus rods of insect polyhedroses. Pavasitology, 44, 71-80.
VACO, C. Insect
1963. Pathol.,
A
new type 5, 275-276.
of
insect
virus.
J.
OF
INVERTEBRATE
Chronic
PATHOLOGY
Bee
7,
Paralysis
170-174
(1965)
Virus in the Nerve Adult Honey Bee1
Ganglia
of
the
PETER E. LEE AND BASIL FURCALA Plant
Research
Institute
and Entomology Agriculture, Accepted
Research Institute, Ottazua, Canada December
Canada
Department
of
15, 1963
A virus of irregular shape was isolated from paralyzed honey bees (Apis melliferaLinnaeus), This virus is considered to be chronic bee paralysis virus (CBPV) Thin sections revealed that CBPV is a nonoccluded virus located in the thoracic and abdominal ganglia of afflicted honey bees. Examination of fat and muscle tissue from paralytic bees, and comparative tissue from healthy bees, failed to disclose virus particles,
A paralysis of adult honey bees (Apis me& Linnaeus) in Britain is caused by a virus called chronic bee paralysis virus (CBPV) (Bailey et al., 1963). Although a virus is suspectedto cause paralysis of adult bees in Canada, the infectious principle has not been isolated. This paper presentsevidence that CBPV is present in Canada. Emphasisis placed on the location of the virus in paralytic bees.
lijera
EXPERIMENTAL Material
nates 4,900 g/l 5 minutes; supernatants 78,OOOg/60 minutes) the pellets from highspeedcentrifugation were dispersedin distilled water, and portions prepared for electron microscopy by staining with phosphotungstic acid (Brenner and Horne, 1959). Particles with an average size of 40 X 27 rnkl were observed in the extract from paralytic bees (Fig. 1) but not in the extract from healthy bees. These particles are similar to those reported by Bailey et al. (1963). The extract from paralytic bees in 50 percent sucrose caused symptoms typical of bee paralysis within 7 days when test bees were administered inoculum by either spraying or feeding.
Two samplesof bees were obtained from a commercial apiary near Ottawa, Canada. One Location of CBPV in Paralyzed Bees sample was removed from a colony showing Paralyzed and healthy bees were dissected paralytic symptoms, the other from a colony in saline and pieces of fat, thoracic muscle, which did not show symptoms of paralysis. and nerve ganglia from the thorax and abIsolation of CBPV and Infectivity Tests domen removed and fixed in Palade’s fixative Beesfrom each samplewere homogenizedin (Palade, 1952). The tissueswere dehydrated a water/carbon tetrachloride mixture (Bailey in alcohol and embeddedin Epon 812 (Luft, 1961) . Following polymerization at 6O”C, et al., 1963). After one cycle of differential sections were cut with a diamond knife on a centrifugation of the homogenates (homogePorter Blum microtome, collected on Form1 Joint contribution from the Plant Research Invar-carbon grids, stained with uranyl acetate stitute (Contribution No. 421) and the Entomology and examined in a SiemensElmiskop I. Research Institute, Research Branch, Canada DeVirus particles, round to oval in shape,were partment of Agriculture, Ottawa. 170
PARALYSIS
F ‘IG. rep ularly
1.
VIRUS
IN
NERVE
171
GANGLIA
Suspension from diseased honey bees negatively stained with phosphotungstic shaped particles of chronic bee paralysis virus (CBPV). 160,000 X.
acid
showing
ir-
172
LEE
FIG.
2.
CBPV
particles
in thin
AND
section
FURGALA
of thoracic
nerve
ganglion.
80,000
X
PARALYSIS
FIG. 3. Thin 40,000 X. Fl G. 4.
section Abdominal
of
abdominal nerve
ganglion.
VIRUS
nerve Note
IN
ganglion particles
NERVE
173
GANGLIA
showing
CBPV
filling
inclusion
particles
free
of the ganglion.
in
the
cj
40.000
x
~plasm
174
LEE AND FURGALA
observed in the thoracic and abdominal ganglia (Figs. 2-4). Particles appeared either in inclusions (Fig. 4)) or free in the cytoplasm of the nerve cells (Fig. 3). The CBPV particles are not enclosed in a protein matrix, unlike the polyhedrosis and granulosis viruses. No particles were observed in fat or muscle tissue of afflicted bees. These observations, however, do not preclude the presence of the virus in fat or muscle. Virus particles were not observed in comparable tissue from healthy bees. Although insect viruses have been reported to infect nervous tissue (Lotmar, 1941: Martignoni. 1954; Smith and Xeros, 1954; Benz, 1963), to our knowledge, this is the first report of unenclosed insect virus in nerve cells. Also noteworthy is the fact that the morphology of CBPV resembles a new type of insect virus recently described by Vago (1963). ACXNOWLEDGI\IENTS
We thank Dr. Eric Smith, Mines Branch, Mines and Technical Surveys, Ottawa, for use of the Siemens Elmiskop I electron microscope, and also Miss J, Ng. Y&m for assistance in the preparation of Formvar-carbon substrates.
REFERENCES BAILEY,
Two
L., GIBBS, A. J., AND WOODS, R. D. viruses from adult honey bees (Apis
1963. mel-
lifera L.), Virology, 21, 390-395. BENZ, GEORC 1963. A nuclear polyhedrosis of Malacosoma alpicola (Staudinger). J. Insect Pathol., 5, 215-241. BRENNER,
S., AND
HORNE,
staining method for microscopy of viruses. 34, 103-110.
R. W. 1959. A negative high resolution electron Biochim. Biophys. Acta,
LOTMAR,
R. 1941. Die Polyederkrankheit Kleidermotte (Tineola biselliella. Mitt. Entomol. Ges., 18, 372-373.
LUFT,
J. H. embedding 9, 409-414.
1961. Improvements methods. J. Biophys.
der Schweis.
in epoxy resin Biochem. Cytol.,
M. E. 1954. Uber zwei Viruskrankheiten von Forstinsekten im Engadin. Mitt. Schweis. Enlom. Ges., 27, 147-152.
MIIRTIGNONI,
P.~L.~DE,
tron Shfrm,
G. E. 1952. A study of fixation for elecmicroscopy. J. Exptl. Med., 95, 285-298.
K. M., ASD XEROS, r\:. 1954. Electron and light microscope studies of the development of the virus rods of insect polyhedroses. Pavasitology, 44, 71-80.
VACO, C. Insect
1963. Pathol.,
A
new type 5, 275-276.
of
insect
virus.
J.