Journal of General Virology(1992), 73, 1621 1626. Printedin Great Britain
1621
Physical map of the Cryptophlebia leucotreta granulosis virus genome and
its relationship to the genome of Cydia pomoneila granulosis virus
Johannes Alois Jehle, 1. Horst Backhaus, 1 Eva Fritsch 2 and Jiirg Huber 2
Federal Biological Research Centre for Agriculture and Forestry, l Institute of Biochemistry and Plant Virology,
Messeweg 11-12, 3300 Braunschweig and 2Institute for Biological Control, Heinrichstrasse 243, 6100 Darmstadt,
Germany
A physical map o f t h e genome o f Cryptophlebia
leucotreta granulosis virus (CIGV) was constructed for
the restriction enzymes BamHI, EcoRI, Kpnl, NdeI,
NruI, SacI and XhoI using hybridization techniques.
The size o f the viral genome was determined to be
112.4 kbp. A restriction fragment library covering
almost the entire genome o f CIGV was constructed,
and the position o f the granulin gene was identified
by cross-hybridization with granulin coding fragments
o f Cydia pomonella granulosis virus (CpGV). Two
further regions o f intergenomic similarity between
CIGV and C p G V were mapped. These regions were
aligned and show a collinear arrangement.
Introduction
However, the susceptibility of C. leucotreta to C p G V is
about 1000 times lower than that to C1GV (Fritsch et al.,
1990).
In contrast to nuclear polyhedrosis viruses, little is
known about the molecular biology of GVs (for review
see Crook, 1991). The granulin gene is the only one of any
GV genome which has been sequenced (Akiyoshi et al.,
1985; Chakerian et al., 1985) and restriction maps have
been constructed for only a few GVs, e.g. C p G V (Crook
et al., 1985) and Pieris (= Artogeia) rapae GV (Dwyer &
Granados, 1987; Smith & Crook, 1988a). As a first step
towards a more detailed characterization of the genome
of CIGV and its relationship to other GVs a physical
map of the genome of the Cape Verde Island CIGV
isolate was constructed and the genomic similarity to
C p G V was examined.
Cryptophlebia leucotreta granulosis virus (C1GV) is
considered to be a highly specific and effective control
agent of the false codling moth, C. leucotreta, which is a
serious pest of cotton, citrus, maize and many other crop
plants in equatorial and tropical Africa (Fritsch, 1988).
CIGV belongs to the family Baculoviridae [genus
granulosis virus (GV)] and has 50 to 80 nm × 200 to 400
nm sized virions which are occluded by a matrix protein,
called granulin, which has an Mr of about 29K. The
genome consists of double-stranded, supercoiled, circular DNA. Since the first description of CIGV, which had
been isolated from infected larvae from the Ivory Coast
(Angelini et al., 1965), further isolates have been found.
One was isolated from laboratory colony larvae from the
Hoechst Corporation which originated in South Africa.
Another one was obtained from diseased larvae which
were collected on the Cape Verde Islands (Miick, 1985).
All these isolates can be clearly distinguished by
restriction analysis.
A biochemical comparison between CIGV and Cydia
pomonella granulosis virus (CpGV), the G V of the closely
related codling moth, showed several similarities in the
protein patterns after SDS gel electrophoresis but no
similarity in the restriction patterns of the D N A s
(Fritsch et al., 1990). Moreover, an asymmetric crossinfectivity of the two viruses has been observed. In
bioassays with neonatal larvae C1GV infects larvae of
C. leucotreta, but not C. pomonella, whereas C p G V is
virulent for both C. leucotreta and C. pomonella.
0001-0616 © 1992 SGM
Methods
CIGV and CpGV propagation. CIGV-CV3 was isolated by in vivo
cloning (Smith & Crook, 1988b) of the CIGV wild-type which was
found on the Cape Verde Islands and was propagated in C. leucotreta
larvae (Fritsch et al., 1990).CpGV derives from the Mexican isolateof
CpGV (Tanada, 1964)and was propagated in larvae of C. pomonellaat
the institute in Darmstadt, Germany.
Infected larvae were homogenizedin 50 mM-Tris-HC1pH 8-0 and
centrifuged in 50% sucrose (w/w) at 40000 g for 60 min. The pellet
was resuspended and the granules were purified by centrifugation
(225000g, 90 min) through a linear 50% to 60% (w/w)sucrosegradient,
generating a virus band which was then repeatedly washed in Tris
buffer and pelleted at 40000 g for 20 min to remove residual sucrose,
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1622
J. A. Jehle and others
DNA purification and restr&tion endonuclease analysis. Virus granules
were dissolved in 0-05 bl-Na.,CO3, 1 ~ SDS, followed by double phenol
extraction and dialysis against 10 mM-Tris-HCl, 1 mM-EDTA pH 8-0.
Viral DNA was digested with restriction enzymes and electrophoresed
using standard methods (Sambrook et al., 1989). Single restriction
fragments were purified from agarose gels by precipitation with glass
milk (Geneclean, Biol01).
Cloning of restriction fragments. EcoRl or NdeI restriction fragments
of CIGV and CpGV DNA were cloned into pGem-3Zf(+) and pGem5Zf(+) phagemid vectors (Promega) using standard methods (Sambrook et al., 1989). The plasmids were propagated in Escheriehia coli
Sure cells (Stratagene) and screened for lacZ complementation.
DNA hybridization and detection. Purified fragments or plasmids
containing unique restriction fragments were nick-translated (Rigby et
al., 1977) using biotin-14-dATP (BRL). After gel electrophoresis of
viral DNA digests the DNA fragments were transferred to Hybond-N
(Amersham) (Southern, 1975). Hybridization was carried out at 42 °C
in 50% formamide according to standard methods (Sambrook et al.,
1989), followed by chemiluminescent dephosphorylation of AMPPD
(PhotoGene, BRL). For low stringency hybridization the formamide
concentration was reduced to 37.5~ or 20~, and the hybridization
temperature was lowered to 35°C. The temperature and salt
concentration of the post-hybridization washes were also adjusted to
stringency levels (Howley et al., 1979). Semi-quantitative dot blot
hybridization of viral DNA was carried out according to Kafatos et al.
(1979) and Possee & Kelly (1988) and quantified by scanning exposed
X-ray films (Anderson & Young, 1985).
Table 1. Size* of CIG V restr&tionfragments
Fragment
A
B
C
D
E
F
G
H
I
EcoR1
17-00
11.60t
8"50t
8-30t
8-30t
7'60t
7.30t
5.35t
5"00t
J
5.00t
K
4.50t
NdeI
27.00
20.20
14-60
9.40
6-80t
5"40t
4.75
4.25t
3.75t
L
4"00t
Y50t
3.05t
2'75t
M
N
4"00t
3"70t
1"95t
1"95t
0
P
2.60t
2.255
1.45t
0.90
Q
R
1.75t
1.65t
0.65t
s
T
1.00t
0.85t
U
V
W
X
Total
0"75
0'53
0"46
0.44
112.43 112-35
BamHl
KpnI
NruI
Xhol
32.00
16.20
9.85
9-75
7-70
7.50
6.85
6.50
6.20
27.40
27.00
16.20
i5.00
10-00
6.00
3.30
2.30
1-90
44.50
16-80
15.80
12.50
8-20
7.10
4-45
1-85
0.90
3 6 . 9 0 56.60
24.00 30.50
22-60 25.20
I2.i0
12-10
4.65
0.30
5.00
1.65
2-60
1-10
2'10
0"55
SacI
112.25 112.40 112.40 112.35 112.30
* Expressed in kbp relative to DNA molecular size standards.
t Cloned into phagemids.
Results and Discussion
Physical map of CIG V genome
For the construction of restriction maps for BamHI,
EcoRI, KpnI, NdeI, NruI, SacI and XhoI, an in vivo
cloned and purified isolate of C1GV obtained from the
Cape Verde Islands, designated CIGV-CV3, was used.
The resulting restriction patterns are illustrated in Fig. 1.
Fragment sizes were estimated by comparison with size
markers in adjacent lanes. For correct size estimation,
the fragments were separated in agarose gels of different
concentrations. The size of large fragments was
estimated by double digestion. By adding the sizes
of fragments generated by the restriction enzymes,
the size of the entire genome was calculated to be
112.35 kbp (Table 1), about 14 kbp smaller than the
genome of CpGV, which is about 126 kbp (Crook et aL,
1985).
DNA fragments of EcoRI and NdeI digests were
cloned into pGem-3Zf(+) and pGem-5Zf(+) phagemids. All EcoRI fragments between 850 bp (fragment U)
and 11.5 bp (fragment B) and all NdeI fragments
spanning the missing EcoRI fragment A (EcoRI-A) were
cloned in this way to establish a phagemid library of
nearly the entire genome (Table 1).
To construct a physical map of the CIGV genome the
restriction sites on the isolated or cloned fragment were
examined. Viral DNA was also digested with each of the
seven restriction enzymes, electrophoresed, Southerntransferred and hybridized to the biotinylated fragments
or plasmid clones of C1GV. Since the vector did not show
any cross-hybridization with virus DNA the undigested
plasmids were biotinylated and used as probes.
By analysing the data of the digestions and
hybridizations it was possible to construct a physical
map of C1GV DNA containing 84 restriction sites (Fig.
2). The hybridization data did not show any repeated
sequences, but some very weak bands which were not
visible in ethidium bromide-stained gels could be
detected. These were identified as submolar bands
indicating some genetic heterogeneity of the in vivo
cloned virus isolate.
Orientation of the restriction map
Following the proposal of Vlak & Smith (1982), who
defined the restriction site adjacent to the smallest
restriction fragment containing the polyhedrin gene as
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Physical map of CIGV genome
1
2
3
4
5
6
7
1623
8
jA
,B
D
F
,N
Fig. 1. Electrophoresisthrougha 0.7%agarosegel of purifiedCIGV-CV3DNA digestedwithEcoRI (lane 1), NdeI (lane 2), BamHI,
(lane 3), KpnI (lane4), NruI (lane5), XhoI (lane6) and SacI (lane7). Restrictionfragmentsare letteredin sequentialorderof theirsize;
the HindlII digest of lambda DNA (lane 8) showsthe size standard.
the zero point of the physical maps of nuclear polyhedrosis viruses, the position of the granulin gene is used
as the zero point for mapping the genomes. For the
identification of the granulin gene of C1GV the cloned
EcoRI-I and -L fragments of CpGV, which contain the
granulin gene of CpGV (Crook et al., 1985), were used as
probes. Both biotinylated fragments hybridized within
the NdeI-F of CIGV (compare Fig. 3c, lanes 3 and 4, in
which the NdeI-F of C1GV DNA hybridized to the
granulin gene-containing fragments EcoRI-I, -L and
HindlII-I, -K of CpGV). With an EcoRI and a NruI
digest of CIGV, the labelled EcoRI-I of CpGV hybridized to EcoRI-L and NruI-E of C1GV, whereas the
EcoRI-L of CpGV hybridized to EcoRI-A and NruI-I
(data not shown).
From these results the orientation of the map of CIGV
was established corresponding to the order of the EcoRII and -L of CpGV. The restriction site between the NruIE and NruI-I was chosen as the zero point (Fig. 2 and Fig.
4). As granulin gene-containing fragments of CpGV
hybridize to C1GV DNA, we conclude that the corresponding homologous C1GV fragments constitute the
granulin gene.
Comparative hybrid&ation of the genomes of CIG V and
CpG V
To gain a more complete impression of the genomic
relationship between C1GV and CpGV Southern blot
studies were carried out using EcoRI and BamHI digests
of CpGV and EcoRI and NdeI digests of CIGV. When
the entire genomes of the viruses were used as probes
only a few fragments gave strong signals. In addition
several fragments with very weak hybrid formation were
found (Fig. 3). The CpGV probe hybridized strongly to
the EcoRI-A, -G and -L and the corresponding NdeI-B
and -F of CIGV, while weaker signals were obtained
with EcoRI-B and NdeI-E. Labelled C1GV DNA
hybridized to EcoRI-B, -I and -L and to BamHI-B and -I
of CpGV and more weakly to EcoRI-A and BamHI-J.
The same hybridization patterns were obtained when
only NdeI-F, EcoRI-G and EcoRI-E of CIGV were used
as probes. Matching these homologies in the restriction
maps revealed two regions with high similarity and a
third with weaker hybridization (Fig. 3 and 4). As
mentioned, region I comprises the granulin-coding
region. The second homologous region was mapped
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1624
J. A. Jehle and others
BamHI
I
J
I
H
F
A
EcoRI
KpnI
I
I
I H
=
Jill
C
I
~
IKI
E
I
=
F
Nru[
IIIHI
E
C
i
0
m.u.
I
0
D
I
U
II
-=
D
I K
'
H I I
I
i
10
I
10
V,W
N IIQI
A
l
30
F
I
I
I
D
B
D
A
I
T
M IIPI
lOt
I
E
1
C
J
IRI
I
I
I
S
II
C
L I
B
I
wJ
o
I I J I
B
I Q'J I
ed
C
D
INI
~
G
I
D
B
~
I
E
I
r
I
A
i
50
I
40
=
~
C
i
40
I
30
G
I
I
I
wi
F
B
•
I
B
I
20
X
I
]
I
20
I'•'I
~:~iK
B
!
G
C
A
1
Kbp
I
IG IHI K
.¢
ILIKI
SacI
XhoI
F
A
co
NdeI
I
I
i
60
I
50
i
70
t
60
i
80
I
70
F I
E
i
90
I
80
I
i
100
l
112
|
90
I
100
Fig. 2. Physical map of CIGV-CV3 DNA for EcoRI, NdeI, BamHI, KpnI, NruI, XhoI and Sacl. The circular genome was linearized
between Nrul fragments E and J; m.u., map units•
within EcoRI-G of CIGV and the overlapping part of
BamHI-B and EcoRI-B of CpGV. The third region lay
between NdeI-E of C1GV and CpGV EcoRI-A and
BamHI-J.
Southern blot hybridization at lower stringency conditions (37-5~ formamide, 37 °C) showed intergenomic
relationships between C1GV and CpGV which were
distributed over almost the entire genomes (data not
shown). All CIGV and CpGV fragments greater than 2
kbp gave signals. An area which did not show any
hybridization under less stringent conditions (neither in
37-5~ nor in 2 0 ~ formamide) could be mapped within
EcoRI-H and NdeI-H, -M and -Q of CIGV.
The signal strength of all the intergenomic hybridizations was much lower than the controls obtained by selfhybridization of CIGV or CpGV DNA. Hence, only
parts, not the whole, of the signal-giving fragments
hybridize in reality. The extent of intergenomic identity
was estimated by semi-quantitative dot blot analysis
which was carried out according to Kafatos et al. (1979)
and Possee & Kelly (1988). Whereas with stringent
hybridization conditions (50~ formamide, 42°C)
sequence matching of less than 1% was detected, the
intergenomic hybridization between CIGV and C9GV
increased at lower levels of stringency (37"5~o fcrmamide, 37 °C and 20% formamide, 37 °C) to 4 ~ and 9~o,
respectively.
Southern blot analysis showed some common sequences between the DNAs of CpGV and C1GV, with a
few genomic regions considerably more conserved than
others. One of the mapped related regions overlaps with
the position of the granulin gene, but nothing is known
about the genes in the other regions. However, their
collinear arrangement on both genomes implies that the
structural organization of the two viral genomes has been
conserved to a fair degree during evolution.
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Physical map of CIG V genome
(a)
(b)
1
2
I¢-L--
(c)
3
4
(d)
3
4
1625
(e)
3
4
3
Fig. 3. (a) Southern blot hybridization (50% formamide, 42 °C) of biotinylated CpGV D N A with CIGV D N A digested with EcoRI
(lane 1) and NdeI (lane 2). (b to e) Southern blot hybridization of different biotinylated CIGV D N A probes with CpGV D N A digested
with EcoRI (lanes 3) and BamHI (lanes 4); (b) hybridized with entire CIGV DNA, (c) hybridized with CIGV D N A NdeI fragment F, (d)
hybridized with CIGV D N A EcoRI fragment G, (e) hybridized with CIGV D N A EcoRI fragment E.
A
E¢oRI
~\~,,,~,\\\~.\\\.~ H I
NdeI
•
F
L = ~ P E M, QH
'" ~~'~\~\~'~ I -
E
I
=
t
O
F
i K X N~IWQ~\x\\\\\\G\\~\,~
.V,
(3
A
I
B
B
,O, MTpt J IRI I I
C
S L~','*
~.x~.'
O
ClGV
I
i
~Ia
iIII
i
II
Mapped regions
of similarity
Ib
I
EcoRI
LM
A
~,~x~I K ~ x \ ~ ' ~ , ~ , ~ . , . \ , , \ ~
BamHI
~'-'-\\\\~-\'~ F
Kbp
I
~
~
~
~
~
~
~
~
~
~
0
10
20
30
40
50
60
70
80
90
100
i
j
~\,.x-.-.\~
B
J IG
~
C
H
z
g~'~'~ CpGV
D
B
~\\.<\~~.~.\~
E
ill
G
I
A
I M~K\~
I
I
110
120
Fig. 4. Alignment of the linearized restriction mal~s of CpGV (Crook et al. 1985) and C1GV. All restriction fragments giving strong
hybridization signals are hatched. Black bars and dashed lines indicate related regions (I to III) and their position in each genome.
Regions Ia and Ib encompass the granulin gene.
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1626
J. A. Jehle and others
This work was supported in part by a grant of the German Ministry
of Science and Technology (no. 0319306A).
References
AKIYOSHI, D., CHAKERIAN, R., ROHRMANN, G. F., NESSON, M. H. &
BEAUDREAU, G. S. (1985). Cloning and sequencing of the granulin
gene from Trichoplusia ni granulosis virus. Virology 141, 328-332.
ANDERSON, M. L. M. & YOUNG, B. D. (1985). Quantitative filter
hybridization. In Nucleic Acid Hybridization: A Practical Approach,
pp. 73-111. Edited by B. D. Hames & S. J. Higgins. Oxford: IRL
Press.
ANGELINI, A., AMARGIER,A., VANDAMME,P. & DUTHOIT, J. L. (1965).
Une virose /t granules chez le 16pidopt~re Argyroploce leucotreta.
Coton et Fibres Tropicales 20, 277-282.
CHAKERIAN, R., ROHRMANN, G. F., NESSON, M. H., LEISY, D. J. &
BEAUDREAU, G. S. (1985). The nucleotide sequence of the Pieris
brassicae granulosis virus granulin gene. Journal of General Virology
66, 1263-1269.
CROOK, N. E. (1991). Baculoviridae: subgroup B. Comparative aspects
of granulosis viruses. In Virusesoflnvertebrates, pp. 73-110. Edited
by E. Kurstak. New York: Marcel Dekker.
CROOK, N. E., SPENCER, R. A., PAYNE, C. C. & LElSY, D. J. (1985).
Variation in Cydia pomonella granulosis virus isolates and physical
maps of the DNA from three variants. Journalof General Virology66,
2423-2430.
DWYER, K. G. & GRANADOS, R. R. (1987). A physical map of the Pieris
rapae granulosis virus genome. Journal of General Virology68, 14711476.
FRITSCH, E. (1988). Biologische Bek~mpfung des Falschen Apfelwicklers, Cryptophlebia leucotreta (Meyrick) (Lep. Tortricidae) mit
Granuloseviren. Mitteilungen Deutsche Gesellschaft far Allgemeine
und Angewandte Entomologie 6, 280-283.
FRITSC~t, E., HUBER, J. & BACm-t~US,H. (1990). CpGV as a tool in the
risk assessment of genetically engineered baculoviruses. In Proceed-
ings and Abstracts, Vth International Colloquium on Invertebrate
Pathology and Microbial Control, Adelaide, pp. 439-443.
HOWLEY, P. M., ISRAEL, M. A., LAW, M. & MARTIN, M. (1979). A rapid
method for detecting and mapping homology between heterologous
DNA. Journal of Biological Chemistry 254, 4876-4883.
KAFATOS, F. C., JONES, C. W. & EFSTRATIADlS,A. (1979). Determination of nucleic acid sequence homologies and relative concentrations
by a dot hybridization procedure. Nucleic Acid Research 7, 15411552.
M~2CK, O. (1985). Biologie, Verhalten und wirtschaftliche Bedeutung
von Parasiten sch~idlicher Lepidopteren auf den Kapverden. Neue
Entomologische Nachrichten 18, 1 168.
POSSEE, R. D. & KELLY, D. C. (1988). Physical map and comparative
DNA hybridization of Mamestra brassicae and Panolis flammea
nuclear polyhedrosis virus genomes. Journal of General Virology 69,
1285-1298.
RIGBY, P. W. J., DIECKMANN, M., RHODES, C. & BERG, P. (1977).
Labelling deoxyribonucleic acid to high specific activity in vitro by
nick translation with DNA poly~nerase I. Journal of Molecular
Biology 113, 237-251.
SAMBROOK, J., FRITSCH, E. F. & MANIATIS, T. (1989). Molecular
Cloning: A Laboratory Manual, 2nd edn. New York: Cold Spring
Harbor Laboratory.
SMITH, I. R. L. & CROOK, N. E. (1988a). Physical maps of the genomes
of four variants of Artogeia rapae granulosis virus. Journal of General
Virology 69, 1741 1747.
SMITrl, I. R. L. & CROOK, N. E. (1988b). In vivo isolation of baculovirus
genotypes. Virology 166, 240-244.
SOUTHERN, E. M. (1975). Detection of specific sequences among DNA
fragments separated by gel electrophoresis. Journal of Molecular
Biology 98, 503-517.
TANADA, Y. (1964). A granulosis virus of the codling moth, Carpocapsa
pomonella L. Journal of Insect Pathology 6, 378-380.
VLAK, J. M. & SMITH, G. E. (1982). Orientation of the genome of
Autographa californicanuclear polyhedrosis virus: a proposal. Journal
of Virology 41, 1118-1121.
(Received 12 September 1991; Accepted 28 February 1992)
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