Budwo~m - cfs.nrcan.gc.cacfs.nrcan.gc.ca/pubwarehouse/pdfs/8739.pdfRESUME L'auteur inocula des...
Transcript of Budwo~m - cfs.nrcan.gc.cacfs.nrcan.gc.ca/pubwarehouse/pdfs/8739.pdfRESUME L'auteur inocula des...
Effect of a Nuclear Polyhedrosis
Virus of the Spruce Budwo~m on
the Metabolic Processes of Vertebrate Cells
by
Basi l M. Al'if and Peter Dobos
Forest Pest Management Institute
Sault See. Marie, Ontario
and
Department of Microbiology
University of Guelph
Guelph, Ontario
Report FPM-X-lS
Canadian Forestry Service
Environment Canada
August 1979
ISSN O)04-772X
ICi'~PDF
Copie 8 of t his report may be obtained from
DirectorForest Pest Management InstituteCanadian Forestry ServiceEnvironment CanadaBox 490, Sault Ste. Marie, OntarioP6A 5M?
'I'1I1~ l'IW<':ltMI lJF '1'1IE: FOil EST PEST NANACENEN'1' LN~'I'I'I'U'I'E
Active ParticipRegistration Pr,the Developmen t& Guidelines fo
Development &Production ofPest ControlProducts
Developmen t ofTechniques to 0Effectiveness ASpecific. Pests
Determination 0
EnvironmentalSide Effects
Chemical InsecticidesBehavioural & Developmental Uaturally Occurring
Hodifving Chemicals Pathogens - Toxicology
- Pherolnones contact, s tomnch, systemic,- Bacteria residUl1l
- Crowth Regulators - Fungi - Phytotoxicity- Cenetic ~~nipulation - Protozoa - Relative Toxicity to
Virus'esNon-target Organisms-
- Development of Resistance
Application Technology & Field Efficacv- Fotlllula tions - Aircraft & Delivery Systems
\pplicution;>timize - Dosages - Cuidance Systems~ainst - Droplet Spectrum T - Application Paramete~s
- Spray lIardware \leacher, timing
Optimization of Efficacy Against Target
Environmental Accountabilitv
f - Birds - Persistence- MalllD'lals - Degradation Products &- Fish & Other Aqua tic Fauna Pathways- Pollinators & Other Beneficial - Detection & Evaluation
Insects Techniquell
Registration for Forestry Use in Canada- Intilll.3te .interaction with Agriculture Canada, Health
& Welfare, NRC, etc.H ion in the Pest Control Products Act)cess and inof Standards Standards & Guidelines for Forestrv Use
rUse - Federal Interdepartmental Committee on Pesticides,~
Environmental Contaminants Act, Fisheries Act, Higu-tory Birds Convention. etc.
Fundamental&
AppliedResearch
Regulatory&
Advisory
Delivery of Pest HansgcmentSystems & Hethodologi~s toForest Hanagers
v-EFFECTIVE, ECmlOMICAL & EHVIROrrHENTALLY CONSCIOUS
PEST ~\NAGEMENT STRATEGIES
-
Tec.hnologyTransfer
ABSTRACT
Mouse, fish and bird cell cultures were inoculated with a
nuclear polyhedrosis virus of the spruce budworm, Choristoneura
fumiferana. and at one. two or three days after inoculation radio-
active uridine, thymidine or leucine was added. At 2-h intervals
thereafter. the cells were processed and the synthesis of RNA, DNA
or protein was determined. The results, compared to those from
mock infected cells, were presented as qualitative incorporation of
the radioactive precursor into the respective macromolecule. The
data showed that the synthesis of RNA, DNA or protein increases
linearly with time and there was no difference between infected and
mock infected cells observed.
RESUME
L'auteur inocula des cellules de Vertebres (Souris. Poisson.
Oiseau) avec un virus de la polyh~drose nucleaire de la Tordeuse
des Bourgeons de l'Epinette, Choristoneura fumiferana. puis un. deux
ou trois jours apr~s l'inoculation. il ajouta de l'uridine, de la
-thymidine ou de la leucine radioactives. Ensuite. a intervales de
deux heures. les cellules furent travaillees et l'auteur effectua la
synthese de l'ARN. l'ADN ou la proteinp.. II presente les resultats.
compares a ceux de cellules pseudo-infectees. en tant qu'une 1n-
corporation qualitative du precurseur radioactif dans chaque macro-
molecule respective. II observe que la synthese de l'ARN, l'ADN ou la
proteine augmente lineairement avec Ie temps et que nulle difference
observable n'existe entre les cellules vraiment et faussement infectees.
TABLE OF CONTENTS
........ ..............................................
RESULTS AND DISCUSSION ••.
Radioactive Labelling.
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1
2
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2
2
4
4
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12
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................................. .. . . . ... ..................................... ....................................... . .................
Cells ..
Vi rus ..
Protein synthesis ..
RNA synthesis •.
DNA synthesis.
MATERIALS AND METHODS.
INTRODUCTION
REFERENCES... . •••••• ••• .• .•••• • • . • • ••• ••• •••• ••• ••• •• ••• • • • • .••• 16
- 1 -
INTRODUCTION
The spruce budworm. Choristoneura fumiferana, is one of the
most serious forest pest in Eastern Canada and the North Eastern
United States. Every year this insect is responsible for the des
truction of large areas of spruce-fir forest. So far, only chemical
insecticides have been used on a widescale basis to control in
festations. However, the chemical insecticides currently used are
ecologically unacceptable since they affect many non-target organisms.
On the other hand, microbial insecticides generally affect only the
target insect and appear to be safe for vertebrates.
A nuclear polyhedrosis virus (NPV) of the spruce budworm has
been used experimentally as a biological insecticide and to date, over
1600 ha of infested forest have been sprayed with this virus. However,
before widespread use of this NPV as a registered forest control product
its effect on non-target organisms must be assessed.
In collaboration with the Ontario Veterinary College at the
University of Guelph safety tests were undertaken to ascertain the
pathogenicity of spruce budworm NPV to mammals and birds, (1.2). After
extensive testing the results proved conclusively that this insect
virus neither multIplies in vertebrates nor does it seem to alter
their metabolic processes. The effect of NPV on the metabolism of
vertebrates can be elucidated more definitively and accurately by in
oculating cells grown in vitro with virus and studying macromolecular
synthesis in the presence of radioactive precursors. In this report
the effect of NPV on the qualitative biosynthesis of nucleic acids and
proteins is investigated.
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MATERIALS AND METHODS
Virus
Virions were released from purified inclusion bodies by alkali
treatment and were then subjected to rate zonal and equilibrium
centrifugation on sucrose gradients (3). Each preparation was
bioassayed 1n fifth or sixth ius tar larvae and all virion preparations
were infectious.
Cells
The cells to be tested were L cells (mouse epithelial). chick
embryo fibroblasts (CEF) , RTG-2 (trout cells) and fat headed minnow
cells (FHM). They were grown on the bottom of clean. sterile glass
scintillation vials in complete minimum essential medium (HEM) at
the appropriate temperature until confluent monolayers were ob-
tained. One half of the cultures were infected with NPV (6-10 ~g
per culture flask) and the other half were mock infected. The virus
was allowed to adsorb for two hours. At one, two and three days after
infection 2 ml of medium, containing radioactive precursor, was added
to six infected and six uninfected cultures.
Radioactive Labeling
(a) In order to label intracellular proteins, leucine def
3ficient medium was used to which H-leucine was added to contain 5~Ci/ml.
Leucine defficient medium consisted of 5% (v/v) of complete MEM (with-
out calf serum) and 95% leucine free ~ffiM. To this mixture 2% dialyzed
- 3 -
3calf serum was added followed by the addition of H-leucine.
(b) To label intracellula~ RNA, serum free ~ffiM containing
35~Ci/ml of H-uridine, was added to the cell cultures.
(c) For DNA labeling the cells were seeded at lower densities
to give semi-confluent monolayers in 1-2 days. This ensured active
DNA synthesis even after 3 days of incubation. The cells were
3labeled with H-thymidine (7~Ci/ml) in serum free MEM.
Infected and uninfected cultures were analyzed for acid pre-
cipitable radioactivity on days 1. 2 and 3 at 2-h intervals over
a period of 12 h. The radioactive medium was removed and the cells
were rinsed three times with cold phosphate buffered saline (PBS).
three times with ethanol and twice with ether. The precipitated
and fixed monolayers at the bottom of the vials were dried at room
temperature. 3 ml of scintillation cocktail was added and the amount
of incorporated radioactivity was assayed in a liquid scintillation
spectrometer.
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RESULTS AND DISCUSSION
Protein Synthesis
L cells were inoculated with virion and separate cultures were
maintained at 28°C and 37°C. 3H-leucine was added at 1.2 and 3 days
after virus inoculation and the cells were examined for acid pre
cipitable radioactivlty at 2-h intervals thereafter. Mock in
fected cells were processed similarly (Fig. 1). The data presented
in this figure show that virus inoculated cells had the same rate
of protein synthesis as mock infected cells. Even) days after
infection the rate of protein biosynthesis remained the same. These
data show that NPV has no effect on mammalian cellular protein
synthesis.
The cells were maintained at 28°C because it 1s the optimum
temperature for NPV multiplication in the host insect. At 28°C the
level of protein synthesis is lo~er than that at 37°c which is ex
pected since 28°c is considerably below the optimum temperature for
growth of mammalian cells (Fig. 1).
Simdlar data were obtained when using RTG-2 and FHM cells (Fig. 2).
RTG-2 cells cannot grow at temperatures much higher than 22°C and
FHM cells cannot grow at temperatures much lower than 28°c so tests
were conducted only at these temperatures.
Experiments were carried out for only 2 days at 32°C and 37°C
using CEF cells. since these cells begin to deteriorate after this
time (Fig. 3).
2. 4 6 8 10 tz.2. 4 6 8 10 12. 2. 4 6 8 10 12
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- 8 -
It is interesting to note that the rate of protein biosynthesis in
CEF cells was unaltered when the cultures were maintained at 28°C or
37°C. The results summarized in Fig. 2 and 3 show that NPV had no
inhibitory or altering effect on avian or fish cell protein synthesis.
The above data demonstrate that these cells are refractory to
NPV as far as qualitative protein synthesis 1s concerned. Any effect
on the ribosomes. cellular mRNA or tRNA would have been manifested in
an altered rate of protein biosynthesis which was not observed.
RNA Synthesis
The experiments were similar to those used for monitoring the
rate of protein synthesis except that 3H-uridine. a specific RNA
metabolic precursor, was used. L cells grown at 37°C and 28°e
exhibited no difference in the rate of RNA synthesis between infected
and mock infected cultures (Fig. 4). It is clear, however. that the
rate of RNA synthesis at 28°e is much slower than that at 37°e since
the latter is the optimum temperature for vertebrate cells. Similar
data were obtained for CEF cells (Fig. 5) and for the two fish cell
lines (Fig. 6).
It is well documented that a number of viruses inhibit protein
and RNA synthesis in infected cultures (4). It is also clear that
NPV had no effect whatsoever on the quantitative RNA synthesis of
these four cell lines even at a temperature close to the optimum for
NPV multiplication.
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- 12 -
DNA Synthesis
The third parameter studied was the effect of NPV on the
synthesis of DNA in non-target cells. The experiments were carried
out using 3H-Thymidine. a DNA precursor, after inoculation with NPV.
The rate of formation of macromolecular DNA was monitored by the
rate of thymidine uptake. At days I, 2 and 3 after inoculation
3with NPV there was no difference in the rate of H-chymidine 10-
corporation between infected and uninfected cells (Fig. 7. 8 and 9).
It was also observed that at 28°e the rate of DNA synthesis in L
cells (Fig. 7) was much reduced compared to the rate observed at 37°C.
It must be emphasized here that reduced rate of DNA synthesis at 2a D e
is not due to virus infection since mock infected cells behaved
similarly at this temperature.
These cumulative data clearly demonstrated the inability of
spruce budworm NPV to alter or inhibit the rate of protein. RNA and
DNA synthesis in mammalian. avain or fish cell lines. These non-target
cells were maintained at their optimum temperatures as well as the
temperature optimum for NPV replication. NPV had no effect on the
cellular metabolic processes at either temperature. The conclusion
derived from these experiments is that NPV neither replicates nor is
partially expressed in non-target vertebrate cells.
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-14
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- 16 -
REFERENCES
1. Valli, V.E.O .• R. Irving and S. Beck. 1974. Mammalian toxicity
tests of the nuclear polyhedrosis virus of the spruce
budworm Choristoneura fumiferana. A research project
carried out by contract with the Dept. of Pathology,
Ontario Veterinary College, University of Guelph. Guelph,
Ontario.
2. Valli, V.E., C.M. Forsberg, J.e. Claxton and G.E. Fountain. 1975.
Avian toxicity tests of the nuclear polyhedrosis virus
of the spruce budworm Choristoneura fumiferana. A re
search project carried out by contract with th~ Dept. of
Pathology. Ontario Veterinary College, University of
Guelph, Guelph. Ontario.
3. Arif, B.M. and K.W. Brown. 1975. Purification and properties
of a nuclear polyhedrosis virus from Choristoneura
fumiferana. Can. J. Microbial. 21: 1224-1231.
4. Davis, B.D., R. Dulbecco, R.N. Eisen. H.S. Ginsberg and W.B.
Wood. 1973. Multiplication and Genetics of Animal
Viruses. In "Microbiology" pp. 1140-1170. Harper aod
Row Publishers.