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0022-538X/84/121024-04$02.00/0
Copyright C) 1984, American Society for Microbiology
Laboratoire Associe 203, Equipe de Virologie du Centre National de la Recherche Scientifique, Universite de Rouen,
Rouen, France,' and Centre de Recherche en Virologie, Institut Armand-Frappier, C.P. 100, Laval, Quebec,
Canada H7N 4Z32
Received 2 December 1983/Accepted 27 August 1984
The efficiency of replication of a cytoplasmic polyhedrosis virus isolated from a member of the order
Cytoplasmic polyhedrosis viruses (CPVs) are one of the larvae (24), and the procedure proposed by Payne and
six genuses in the Reoviridae family and are found mainly in Tinsley (22) was adopted for purification. Established cell
insects (14). Thus, they possess double-stranded RNA lines from Bombyx mori (strain SPC Bm 36) (9), Choriston-
genome in the form of 10 segments that are not linked eura fumiferana (IPRICF 124) (27), Choristoneura fumifer-
covalently, and their coding assignment for one type of CPV ana Ti (IPRICF-124) (2), Lymantria dispar (SCLd 135) (23),
has been established (15; P. P. C. Mertens, Ph.D. thesis, Malacosoma disstria (IPRIMd 66) (9), Papilio xuthius
Oxford University, England, 1979). Differences in the elec- (strains Px 58 and Px 64) (16); and Trichoplusia ni (8) were
trophoretic mobilities of genome segments between different propagated in plastic flasks (25 cm2) (Bioblock, Strasbourg,
isolates of CPV provides a basis for a provisional classifica- France) at 28°C using the insect medium of Grace (4)
tion of 13 distincts virus types of CPV (20, 21). supplemented with 10% fetal calf serum and antibiotics (100
Apart from host range and morphology, CPVs are distin- U of penicillin and 100 pLg of streptomycin per ml of culture
guished among the Reoviridae by the production of large medium). Before infection, exponential growth cultures of
polyhedral inclusion bodies (polyhedra), containing four to cells were seeded in multiwell plates (1.5 cm in diameter), set
six polypeptides (21), in the cytoplasm of virus-infected cells at 4 x 105 cells per ml, and incubated at 28°C. After 24 h, the
(for a review, see references 18 and 20). medium was discarded, and 0.2 ml of viral suspension was
Although several reports have described the polypeptide added to each well for an adsorption period of 1 h. The cell
composition of CPV purified polyhedra and viral particles (7, sheet was then washed three times with culture medium, and
12, 19, 21, 22), there are no descriptions of intracellular 0.5 ml of medium was added to each well. The viral
polypeptide synthesis of viral origin after CPV infection in a suspension used was titrated at 106.23 50% tissue culture
tissue culture system. Recently it was reported by McCrae infective dose per ml with a technique described by Bellon-
(15) that the absence of a suitable tissue culture system for cik and Chagnon (1). For radiolabeling of cells, at the
CPV growth in an impediment to the study of the molecular indicated times postinfection the insect medium of Grace
details of viral replication and gene expression. However, without methionine and supplemented with 2% dialyzed fetal
this assertion must be denied since successful infection of calf serum was added to the cultures for 1 h. Then it was
invertebrate cell lines with different types of CPV has been replaced by the same medium containing 20 pCi of L-
reported (4, 6, 7, 13, 24). In our efforts to understand the [35S] methionine (specific activity, 1,465 Ci/mmol; Radio-
replication strategies of CPVs in cell culture we report here chemical Centre, Amersham, England) per ml for 2 h. Cell
the infection of eight lepidopterean cell lines and the poly- lysates for SDS-PAGE analysis were prepared by discarding
peptide patterns of one of these cell lines (Lymantria dispar) the supernatant of cultures and resuspending the cells in the
after infection with a CPV isolation from the lepidopterean following buffer: 50 mM Tris-hydrochloride pH (6.8) con-
Euxoa scandens (EsCPV) (24). Finally, we adapted an taining 2% SDS, 2% 3-mercaptoethanol, and 15% glycerol.
indirect immunoperoxidase technique to identify EsCPV After the trichloracetic acid-precipitable material was boiled
proteins after sodium dodecyl sulfate-polyacrylamide gel for 2 min, the radioactivity incorporated in it was evaluated
electrophoresis (SDS-PAGE) was blotted to a nitrocellulose in duplicate 5-,u portions from all samples dried onto What-
membrane. man 3 filter paper disks (2 by 2 cm). The filters were
The seed virus (EsCPV) was obtained from diseased immersed into ice-cold 5% trichloroacetic acid, washed
twice with absolute ethanol, and dried in benzidine and air.
The radioactivity was then counted in 5 ml of a toluene-
* Corresponding author. based scintillation fluid with a liquid spectrometer (Inter-
1024
;~ ~ .
VOL. 52, 1984 NOTES 1025
TABLE 1. Yield of CPV polyhedra in various lepidoptera cell (heavy plus light chain), and peroxidase labeled (Institut
linesa Pasteur Production, Paris, France). After extensive wash-
No. of cells No. of ings, the peroxidase activity was revealed with the medium
Cell line containing Nol of polyhe- polyhe- of Graham and Karnovsky (5).
polyhedra dra (x 104) dra per In Table 1 we show that the L. dispar cell line is the more
(%) cell
efficient to support EsCPV replication as compared with
Lymantria dispar 57.7 ± 5.6
714 ± 25.8 12.21 seven other tested, established lepidopterean cell lines.
Bombyx mori 32.8 ± 6.8 411.3 ± 59.5 6.98 Results presented in this table were obtained, after an
Choriston eura fumiferana 6.8 ± 2.9 65.6 ± 5.2 0.46 incubation time of 96 h at an optimum temperature of 28°C;
Choristoneura fumiferana ti 5.1 + 2.5 37.0 ± 18.7 0.40 increasing incubation time to 168 h did not significantly alter
Trichoplusia ni 6.1 ± 1.2 21.6 ± 6.6 0.26
Malacosoma disstria 3.3 ± 2.8 5.2 ± 0.9 0.14 the yield of infection. At that time efforts to clone L. dispar
Papilio xuthus strain 58 1.2 ± 0.3 4.1 ± 1.5 0.05 cells with improved susceptibility to EsCPV infection were
Papilio xuthus strain 64 2.2 ± 0.3 6.5 + 1.8 0.07 unsuccessful. It is noteworthy to mention that the EsCPV
a Results of this table are the average of six different experiments. Incuba-
polyhedra obtained in all of these cell cultures are of cuboid
shape, compared with the spherical shape obtained with the
X .112V
92
66- -66 p
15- - 4 p
t
I t 1 0 .1
I
# 0, "o $.
.1
4, '. ."i
I
ilim"
t .
O.-
10
..,*
49 39
- v
31-
28 p
T 1 2 4 8 12 18 24 36 48 72 g9
FIG. 1. Appearance of [35S]methionine-labeled EsCPV polypeptides in L. dispar-infected cells. Uninfected cellular polypeptides (T) were
pulse-labeled for 2 h, and infected cell polypeptides were pulse-labeled for 2 h at the indicated times after infection. The migration of the
standard polypeptides used is indicated in the left margin in kilodaltons: carbonic anhydrase (31), ovalbumin (45), bovine serum albumin (66),
and phosphorylase B (92). p, Polyhedral polypeptide; v, viral-particle polypeptide.
1026 NOTES J. VIROL.
- 120 p
'- 112 V
- 110 p
92 -
66 - - 66 p
- 46 p
45-
31 - _ms w m 6i.u~hEMS- 28 P
.~.4*
FIG. 3. Electrophoretic blotting of SDS-PAGE of the polypeptide profiles of infected cells to a nitrocellulose membrane. Staining of the
blot by the indirect immunoperoxidase technique is as described in the text. The far right lane contains purified CPV polyhedra and viral
particles. p, Polyhedral polypeptides; v, viral-particle polypeptide.
VOL. 52, 1984 NOTES 1027
The fact that the rate of synthesis of the major polyhedral mic polyhedrosis virus, wound tumor virus, and reovirus. J.
polypeptide (28K) increases during pulse periods at 36, 48, Virol. 10:1053-1070.
72, and 96 h after infection suggests the regulation of 13. Longworth, J. F. 1981. The replication of a cytoplasmic polyhe-
translation or transcription of viral mRNA for the polyhedrin drosis virus from Chrysodeixis eriosoma (Lepidoptera: Noctui-
gene. Further studies on CPV replication in L. dispar cells tae) in Spodopterafrugiperda cells. J. Invertebr. Pathol. 37:54-
should serve as a model to help understand the regulation of 61.
14. Matthews, R. E. F. 1982. Classification and nomenclature of
CPV transcription in vivo either by host cellular or nascent viruses. Intervirology 17:1-199.
viral proteins, as previously suggested by Smith and Furui- 15. McCrae, M. A. 1982. Coding assignments for the genus of a
chi (25). cytoplasmic polyhedrosis virus, p. 20-24. Proceedings of the
We express our appreciation to S. Barray for helpful discussions, Third International Colloquium on Invertebrate Pathology. Uni-
J. Lecomte and M. Henrichon for the densitometric analysis of the versity of Sussex, Brighton, England.
autoradiograms, and D. Rouleau for her excellent technical assist- 16. Mitsuhashi, J. 1973. Establishment of cell lines from the pupal
ance. ovaries of the swallow tail, Papilio xuthus Linne (Lepidoptera:
We acknowledge the financial assistance of the Centre National Papillonidae). Appl. Entom. Zool. 8:64-72.
17. Payment, P., D. J. S. Arora, and S. Belloncik. 1982. An enzyme-