Journal of Invertebrate Pathology 75, 279 –287 (2000)
doi:10.1006/jipa.2000.4933, available online at http://www.idealibrary.com on
Characterization of a Single-Nucleocapsid Nucleopolyhedrovirus of
Thysanoplusia orichalcea L. (Lepidoptera: Noctuidae) from Indonesia
Xiao-Wen Cheng 1 and Gerald R. Carner
Department of Entomology, 113 Long Hall, Clemson University, Clemson, South Carolina 29634-0365
Received August 16, 1999; accepted March 8, 2000
A single-nucleocapsid nucleopolyhedrovirus (NPV)
isolated from Thysanoplusia orichalcea L. (Lepidoptera:
Noctuidae) (ThorNPV) in Indonesia has tetrahedral occlusion bodies (OBs) with a width of 1.22 mm (range 5
0.803–1.931 mm). The length of the virion with an envelope averaged 0.29 and 0.23 mm without an envelope.
ThorNPV was propagated in Pseudoplusia includens
(Walker) and its authenticity was confirmed by sequence analysis of the polyhedrin gene of the ThorNPV
produced in T. orichalcea and P. includens. Polyhedrin
amino acid sequence analysis revealed that ThorNPV
belongs to Group II of baculoviruses and is closely related to Trichoplusia ni single nucleocapsid NPV, sharing 97.6% sequence identity. Infectivity of ThorNPV
against third instar P. includens was low, with a LD 50
value of 65,636 OBs/larva. Electron microscopy of infected tissues showed many polyhedra without virions
embedded, which might explain the low virulence
against P. includens. Differences in virion occlusion
rates between individual cells in the same tissue suggested that the inoculum consisted of at least two variants that differed in the gene(s) controlling virion occlusion. In a host range test using the LD 50 value to P.
includens against Spodoptera exigua, S. frugiperda, S.
eridania, Anticarsia gemmatalis, Helicoverpa zea,
Trichoplusia ni, and P. includens, P. includens was the
only species infected. The virus infected primarily the
fat body, tracheal epithelium, and hypodermis. The
genomic size of the ThorNPV is 135 kb. © 2000 Academic Press
Key Words: Thysanoplusia orichalcea; Pseudoplusia
includens; NPV; bioassay; host range; histopathology;
morphology; DNA restriction profile; genomic size; Indonesia.
MATERIALS AND METHODS
1. Insect Rearing
INTRODUCTION
Nucleopolyhedroviruses (NPVs) in the family Baculoviridae are common natural microbial control agents
1
that have been used in a number of integrated pest
management programs (Podgwaite, 1985; Shepard and
Shepard, 1997). NPVs have been the virus of choice for
insect control because of their stability and ease of
handling. Although 488 isolates of NPVs have been
reported (Murphy et al., 1995), relatively few have
been developed as microbial insecticides, and efforts
are still underway to locate improved strains and species.
An NPV was isolated from loopers, Thysanoplusia
orichalcea (Lepidoptera:Noctuidae), collected from carrots in West Java, Indonesia, in 1992. Initial observations by light microscopy revealed that the occlusion
bodies (OBs) of the virus were quite small and had an
unusual tetrahedral shape. The virus was brought to
Clemson University for further characterization and
for testing against local species of loopers. In initial
tests, the crude preparation of the virus showed activity against soybean looper, Pseudoplusia includens
(Walker), and cabbage looper, Trichoplusia ni (Hubner). However, this 1992 shipment was subsequently
found to contain a mixture of viruses including two
NPVs (one is tetrahedral and one is polyhedral in
shape) and a cytoplasmic polyhedrosis virus (CPV). A
second shipment of NPV-infected T. orichalcea in 1996
contained only the tetrahedral form. When the tetrahedral virus was separated and purified, it showed no
activity against T. ni.
In this paper, the tetrahedral ThorNPV is characterized as to its morphology, biological activity, histopathology, and molecular biology.
Present address: Laboratory for Molecular Virology, Great Lakes
Forestry Center, 1219 Queen St. E., Sault Ste. Marie, ON P6A 5M7
Canada.
P. includens were from a laboratory colony established using eggs obtained from the USDA-ARS,
Southern Insect Management Laboratory, Stoneville,
Mississippi. All other insects used in the host range
test were colonies maintained in our insect rearing
facility. Larvae of all insects species were reared individually on pinto bean-based artificial diet (Burton,
279
0022-2011/00 $35.00
Copyright © 2000 by Academic Press
All rights of reproduction in any form reserved.
280
CHENG AND CARNER
1969) at 27 6 1°C, 75% relative humidity, and 16:8 h
(L:D) photoperiod. Fifty pupa were randomly selected
and transferred into a 4-liter glass jar. Emerged adults
were fed with 10% honey water. A 10-cm-wide and
25-cm-long paper towel strip was hung in the jar for
egg deposition. Eggs on the paper towel strip were
transferred into another clean glass container for
hatching. Neonates were transferred individually onto
the surface of the diet in 31-ml (one ounce) artificial
diet cups for development to the pupal stage.
2. Propagation and Purification of ThorNPV
Infected T. orichalcea larvae were collected in 1996
from carrots in Indonesia and stored at 220°C until
they were transported to the United States. The larvae
were macerated in a glass homogenizer with distilled
water, and the homogenate was filtered through four
layers of cheesecloth. This crude NPV suspension was
used to prepare an inoculum (1 3 10 7 OBs/ml) that was
spread on the surface of the diet (0.07 ml/cup) on which
third instar soybean loopers were feeding. Infected larvae showing virus infection symptoms were collected
and stored at 220°C.
OBs from infected larvae were purified by differential and sucrose gradient centrifugation using the
method of Cheng et al., (1990). The purified OB pellet
was suspended in 0.5 ml of distilled water and stored at
220°C.
Purification of virions followed the methods of
Harrap and Longworth (1974) and Harrap et al.,
(1977). Briefly, virions were released from OBs by
treatment with 0.1 M sodium carbonate (pH 10.5) and
gentle agitation at 37°C until the suspension was relatively transparent and colored light brown. These
virions were purified by centrifugation on a linear sucrose gradient (25–50%) at 80,000g for 1 h at 4°C. This
gradient was made by freezing 30 ml of 37.5% sucrose
(w/v) in the centrifuge tube and thawing at 4°C for
15 h. The virion band was collected with a pasteur
pipet, diluted with distilled water, and pelleted at
46,000g for 1 h at 4°C. The purified virion pellet was
stored at 220°C.
3. DNA Analysis
The DNA from purified virions of ThorNPV was isolated using the resin-based Qiagen Genomic DNA Isolation Kit 100/G (Qiagen). Isolation of DNA followed
the cell culture protocol of the kit except that a higher
DNA yield was obtained by not vortexing to avoid
shearing and performing proteinase K digestion overnight at 37°C. The concentration of isolated DNA was
determined by measuring the absorbance at 260 nm
and the purity was estimated by calculating the 260:
280 nm absorbance ratio. Autographa californica NPV,
AcMNPV (AcUW1-LacZ, a clone from Pharmigen),
which was used in the following comparative restric-
tion endonuclease (REN) analysis, was propagated in
Sf21 cells and virions were harvested by ultracentrifugation (O’Reilly et al., 1992). DNA from AcMNPV
virions was purified as above.
REN analysis consisted of digesting 0.5 mg of purified DNA from ThorNPV and AcMNPV with HindIII,
EcoRI, PstI, and XbaI (Promega or BioLab) in 20-ml
reaction volumes under the conditions recommended
by the manufacturer. Digested fragments were separated in a 0.7% agarose gel using l-HindIII DNA and 1
kb DNA ladder (GIBCO/BRL) as molecular size markers. DNA from ThorNPV and AcMNPV digested with
the same REN was loaded in the gel side by side. The
gel was stained with ethidium bromide and photographed under an UV transilluminator with an IS1000 Digital Imaging System (Alpha Innotech Corporation, San Leandro, CA). The accompanying software
(V. 2.02) was used to estimate the molecular weight of
DNA fragments.
Since ThorNPV was propagated in an alternate host
other than the original one, confirmation of authenticity
was needed. OBs and virions were extracted from a cadaver of T. orichalcea infected by ThorNPV as above.
Because the original sample was small in virion yield,
phenol– chloroform–isoamylalcohol (25:24:1) DNA extraction was performed following proteinase K digestion
as above. To clone the open reading frame (ORF) of the
polyhedrin gene for sequencing, the polymerase chain
reaction (PCR) method was employed. Basically, a pair of
20-mer primers was chosen from the flanking sequence of
the polyhedrin gene ORF of ThorNPV propagated in P.
includens (Forward, 59-GCG TCC GTG TAG ATG TAA
AG-39; Reverse, 59-CTA TAG CAG CTT CGC GTC TA-39).
The forward and reverse primers were located at 2208
and 1852 nt positions where the A in the translation
initiation codon ATG of the polyhedrin gene was set as
position 1 (Cheng et al., 1998). Viral DNA from ThorNPV
propagated in P. includens was used as a control template in the PCR reaction. Standard PCR protocol was
followed. The PCR product was gel purified and ligated
into pGEM-T vector (Promega) before transforming
DH5a competent cells by the electroporation method
(Sambrook et al., 1989). Positive clones were selected on
LB plates with ampicilin/X-gal/IPTG. Plasmid DNA was
purified from three clones carrying the ThorNPV polyhedrin ORF insert. Sequencing of the insert was performed using dye-labeled ALF M13 universal and reverse
primers in a Cy5 AutoRead Sequencing Kit with an ALFexpress DNA Analysis System (Pharmacia Biotech).
The sequence was processed by a biocomputing software,
LASERGENE (DNASTAR, Inc.). This sequence was
searched in GenBank (NCBI) using the BLAST search
engine.
To establish the phylogenetic relationship of this
virus to other members in the baculovirus family, the
ThorNPV polyhedrin amino acid sequence was aligned
with all the homologous polyhedrin/granulin protein
CHARACTERIZATION OF ThorNPV
281
FIG. 1. Comparative restriction enzyme digestion profile of ThorNPV and AcMNPV DNA using the restriction endonucleases HindIII,
EcoRI, PstI, and XbaI. One-kilobase DNA ladder and l-HindIII marker were used as molecular size markers. Each visible band was assigned
one or more letters depending on the number of fragments in each band.
hits from the BLAST searches in GenBank using the
CLUSTAL method with LASERGENE. The closely related NPVs plus AcMNPV were used for phylogenetic
analysis by LASERGENE and PAUP 3.0r (Written by
D. L. Swofford).
4. Bioassay
Second instar P. includens usually climbed to the
paper lids of the diet cups and were transferred individually using a camel hair brush to 1.5-ml Eppendorf
tubes with a 1-mm hole punched on the lid of each tube.
These larvae were starved overnight, and by the next
day they were in early third instar. Fresh tomato leaf
disks (4 mm in diameter) were soaked in 0.02% Triton
X-100 for 5 min and were washed three times in distilled water. Excess water was removed by blotting
with a paper towel. Each disk was then treated with 1
ml of ThorNPV suspension at one of five concentrations:
4 3 10 5, 4 3 10 6, 4 3 10 7, 4 3 10 8, and 4 3 10 9 OBs/ml.
For the control, leaf disks were treated with 1 ml of
distilled water. After the leaf disks had air-dried, they
were placed individually into the Eppendorf tubes containing the starved soybean looper larvae. One hundred loopers were used to test each concentration, with
4 replications of 25 larvae each. The Eppendorf tubes
containing loopers and leaf disks were put into sealed
plastic bags with a piece of damp towel in each bag.
Two to three holes (2 mm) were punched in the plastic
bags to prevent condensation. After approximately
24 h, most loopers had consumed the leaf disks; they
were then transferred individually into 31-ml (one
ounce) artificial diet cups. Loopers that had not completely consumed the leaf disks were discarded.
The bioassay was conducted in an incubation chamber where environmental conditions were the same as
in the rearing room. Mortality of loopers was checked
and recorded daily until adults emerged from pupae
that had not died.
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CHENG AND CARNER
The dose–mortality data were analyzed with a computer program, POLO-PC (Le Ora Software, Inc.,
Berkeley, CA), based on the probit analysis method
described by Finney (1971).
TABLE 1
Size of ThorNPV DNA Fragments Generated by Digestion
with the Restriction Enzymes HindIII, EcoRI, PstI, and XbaI
Size (kb)
Fragment
HindIII
EcoRI
PstI
XbaI
17.3
14.2
14.2
12.2
8.7
7.1
7.1
6.0
5.0
4.7
4.2
4.2
3.9
3.7
3.4
3.2
3.0
2.9
1.9
1.6
1.6
1.1
1.1
0.8
0.6
23.1
13.8
9.9
9.3
7.5
7.5
6.9
6.9
6.2
4.7
4.7
4.5
4.0
3.8
3.2
3.2
2.8
2.7
2.5
2.3
2.1
1.8
0.9
0.8
0.4
18.1
18.1
11.2
7.1
7.1
6.2
5.9
5.5
5.3
5.0
4.3
4.3
4.3
3.9
3.8
3.7
3.5
2.6
2.4
2.4
1.8
1.8
1.8
1.6
1.3
17.3
12.6
10.7
8.1
7.8
7.5
6.6
6.6
6.3
5.0
5.0
5.0
4.8
4.0
4.0
3.9
3.8
3.7
2.8
2.3
1.8
1.6
1.4
1.3
0.9
Total
134.3
Average 6 SEM 134.8 6 0.4
135.5
5. Host Range Test
Purified ThorNPV OB suspension was used to prepare an inoculum (1 3 10 7 OBs/ml) that was spread on
the surface of the diet in 31-ml diet cups (730 OBs/
mm 2). Thirty to eighty third instar larvae of Spodoptera exigua, S. frugiperda, S. eridania, Anticarsia gemmatalis, Helicoverpa zea, Trichoplusia ni, and P. includens were transferred individually to the viruscontaminated diet. Development of larvae was checked
daily.
6. Electron Microscopy
For scanning electron microscopy (SEM), the purified NPV pellet was suspended in 5 ml of distilled
water. One microliter of the viral suspension was
spread onto a 1 3 1-cm glass slide, dehydrated in an
oven at 37°C for 30 min, and coated with gold. The
coated sample was observed under the SEM (JEOL
848) and pictures were taken and saved on an optical
disk for measurement of virus structures using the
PGT IMIX software in a Sun workstation.
For transmission electron microscopy (TEM), 30
third instar soybean loopers were starved for 6 h. Loopers were held individually in 1.5-ml Eppendorf tubes
with a hole in the lid and allowed to feed on a 4-mmdiameter tomato leaf disc treated with 1 ml of virus
suspension. When the loopers consumed the leaf disc
(about 5 h), they were transferred into artificial diet
cups. Every 12 h postinoculation (p.i.), a looper was
taken out and dissected to collect the fat body. At 7
days p.i., the midgut, tracheae, hypodermis, silk
glands, Malpighian tubules, and testes were also collected. Tissue samples were immersed in 2% phosphate-buffered glutaraldehyde for 2 h. Processing of
samples for TEM followed the methods of Hamm and
Styer (1985). The polymerized samples were sectioned
with a diamond knife on a microtome (Reichert-Jung
Ultracut-E), poststained with uranyl acetate and lead
citrate (Venabel and Coggleshall, 1965), and examined
under TEM (Hitachi H600 AB).
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Y
134
135.6
RESULTS
A single band formed in the sucrose gradient centrifugation of dissolved OBs. This indicated that the virions were all of the same size. The virions were contained at about the 35% (w/v) region of the sucrose
gradient. The freeze-and-thaw method for formation of
the linear sucrose gradient provided a very efficient
and simple way to purify the virus particles.
Comparative REN analysis of the ThorNPV genomic
DNA with AcMNPV is shown in Fig. 1. There were 26
HindIII, 25 EcoRI, 26 PstI, and 26 XbaI REN recognition sites in the ThorNPV genome. The genome size of
the ThorNPV was estimated to be 134.8 6 0.4 kb
(Table 1). REN profiles also showed that the ThorNPV
FIG. 2. Alignment of polyhedrin amino acid sequences from NPVs that are closely related to ThorNPV. Residues in boxes represent those
that differ from the consensus. Dots in the consensus indicate that not all residues match. Autographa californica NPV, AcMNPV (Hooft van
Iddekinge et al., 1983); Leucania seperata NPV, LeseNPV (GenBank Access No. U30302); Mamestra brassicae NPV, MbMNPV (Cameron and
Possee, 1989); Mamestra configurata NPV, MacoNPV (Li et al., 1997); Panolis flammea NPV, PaflNPV (Oakey et al., 1989); Buzura
suppressaria single-nucleocapsid NPV, BusuNPV (Hu et al., 1993); Orgyia pseudotsugata single-nucleocapsid NPV, OpSNPV (Leisy et al.,
1986); Ectropis obliqua single-nucleocapsid NPV, EcobNPV (U95014); Trichoplusia ni single-nucleocapsid NPV, TrniNPV (Fielding and
Davison, 1999); Thysanoplusia orichalcea single-nucleocapsid NPV, ThorNPV (Cheng et al., 1998); Spodoptera exigua NPV SeMNPV (Van
Strien et al., 1992); Spodoptera frugipeda NPV SfMNPV (Gonzalez et al., 1989); Spodoptera litura NPV, SpliNPV (X94437).
CHARACTERIZATION OF ThorNPV
283
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CHENG AND CARNER
FIG. 4. Accumulated mortality of third instar soybean loopers
treated with different dosages of ThorNPV using a leaf disk bioassay.
Control, 0 OBs/larva. The vertical line for each dose represents the
standard error for the mean, n 5 74.
FIG. 3. The most parsimony tree (unrooted) of some polyhedrin
genes from NPVs closely related to ThorNPV after initial alignment
of all NPV polyhedrin genes available in GenBank as of January
2000 with Clustal method. The tree was constructed after 100 replicates of bootstrap branch-and-bound search using PAUP 3.0r. I and
II are two groups of baculoviruses (Zanotto et al., 1993). Percentages
in the parentheses beside the virus name are polyhedrin sequence
identities with ThorNPV. Numbers above the lines are the number of
changes between the node and the species. Numbers in italics below
the line indicate the frequency of that cluster after bootstrap analysis. (CI 5 0.78; RI 5 0.72).
was readily distinguishable from the type species of
baculovirus, AcMNPV (Fig. 1).
When ThorNPV DNAs from OBs propagated in both
T. orichalcea and P. includens were used as templates
in the PCR amplification, both PCR reactions produced
an expected 1-kb fragment (photo not shown). After the
1-kb polyhedrin PCR product from ThorNPV originating in T. orichalcea was cloned and sequenced, a search
of GenBank using BLAST revealed that this ThorNPV
polyhedrin gene from virus produced in T. orichalcea
was identical to that of ThorNPV recovered from P.
includens (Cheng et al., 1998) (Access Number
AF019882).
Polyhedrin gene sequence alignment showed that
ThorNPV was a new member of Group II of the Baculoviridae (Zanotto et al., 1993). Based on polyhedrin
protein sequence within Group II, ThorNPV formed a
subcluster with other NPVs, with its closest relative
being T. ni NPV (TrniNPV) (Fielding and Davison,
1999) (Figs. 2 and 3). The two viruses share 97.6%
polyhedrin amino acid sequence identity. There is only
a five amino acid difference between the ThorNPV and
TrniNPV polyhedrin protein sequence (Fig. 2).
About 4000 OBs of ThorNPV per larva were required
to produce noticeable mortality in third instar soybean
loopers. Concentrations lower than 4000 OBs per larva
did not produce mortality (Fig. 4). The LD 10, LD 50, and
LD 90 values for the ThorNPV against third instar soy-
bean loopers were 5194, 65,636, and 829,394 OBs/
larva, respectively (Table 2). The equation of the log
dose–probit regression line was Y 5 1.1633x 2 5.6040
(x 2 5 5.2759; df 5 3), the standard error (SE) of the
slope was found to be 0.0880, and the t value was
13.2206. The estimated t value (13.2206) greatly exceeded 2.576 (1% significance point for the t distribution with ` df), which implied the probit response is a
linear function of log dose in this bioassay.
Seven species of Noctuidae, including P. includens,
were tested for infection by ThorNPV. Only P. includens was infected by this virus (54.7%), which was
determined by examining the dead larvae for large
numbers of tetrahedral NPV OBs. However, this NPV
was found to be infective against another looper, Chrysodeixis chalcites, in Indonesia (unpublished data). The
amount of inoculum used in the host range test was
approximately the LD 50 value (65,636 OBs/larva) to P.
includens.
As seen by SEM, the occlusion shape of the ThorNPV
is roughly tetrahedral. The triangular faces were usually not uniform and bulbous protrusions were observed on most OBs. Some virions were not completely
embedded and “pits” were left on the OBs when virions
were lost during the preparation (Fig. 5). The average
TABLE 2
Dosage Mortality Response of Third Instar Soybean
Looper, P. includens, to ThorNPV a
95% Confidence limits
Entry
Value
Upper
Lower
LD 10 (OBs/larva)
LD 50 (OBs/larva)
LD 90 (OBs/larva)
Slope
5,194
6,5636
829,394
1.163
12,443
135,908
3,510,570
1,216
31,615
347,528
a
LD values in the table were calculated with a computer program,
POLO-PC.
CHARACTERIZATION OF ThorNPV
FIG. 5. Scanning electron micrograph (15 kV, 38000) showing
the ThorNPV occlusion bodies. Solid arrow indicates “pits” in the
OBs where virions were embedded but were lost in the preparation.
BS, bulbous surface.
285
size of the OBs was 1.37 6 0.43 mm with a range of
0.803 to 1.931 mm.
Ultrathin sections of the ThorNPV revealed that this
was a single-nucleocapsid NPV (Fig. 6A). Among the
tissues examined, it was found that ThorNPV replicated mainly in the nuclei of fat body and tracheal
epithelium cells. Although hypodermis was not observed under TEM, it was probably also infected because the integument became very fragile in late
stages of infection. Virus nucleocapsid assembly occurred in the virogenic stroma in the nuclei of infected
cells in tracheal epithelium and fat body (Fig. 6B). The
size of the virion with envelope was 0.290 6 0.06 3
0.068 6 0.004 mm, and the size without the envelope
was 0.230 6 .06 3 0.035 6 .004 mm. Abnormal OBs
were found with few or no virions and many virions
were not occluded in the later stages of infection (Fig.
FIG. 6. Transmission electron micrographs of soybean looper fat body cells infected with ThorNPV. (A) One hundred eighty hours
postinfection (p.i.). Arrow indicates a normal occlusion body with virions embedded. (B) Seventy-two hours p.i. showing the virogenic stroma
(VS), nucleocapsids (NC) without envelopes, virions (V) and polyhedrin membrane (PM) scattered in the nucleus of the cell. (C) One hundred
eight hours p.i. Arrowhead indicates occlusion body without virions embedded. Solid arrow points to virions not occluded. (D) OBs in two
adjacent cells in the tracheal hypodermis. One cell has normal OBs with embedded virions (a) and the other has abnormal OBs with few or
no virions embedded (b).
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CHENG AND CARNER
6C). In some cells, clumps of polyhedrin could be seen
scattered through the nucleus (Fig. 6B). This was observed in both fat body and tracheal epithelium tissues. In examining clusters of infected cells, we would
often observe one cell with fully formed normal OBs
and an adjacent cell would contain abnormal OBs with
no embedded virions (Fig. 6D).
DISCUSSION
The genomic size is an important characteristic of a
baculovirus at the molecular level. It has been reported
that the genome of baculoviruses is highly complex and
occurs as circular, supercoiled, dsDNA of 88 to 160 kb
(Blissard and Rohrmann, 1990). The genome size of
ThorNPV (134.8 kb) falls within this reported range.
Use of REN digestion for the estimation of viral genome size in this research resulted in a very small
variation in size estimates (Table 1).
There were concerns that the ThorNPV produced in
P. includens was not authentic. However, a comparison
of polyhedrin sequences revealed that virus recovered
from P. includens was the same as that recovered from
the original host. This was further confirmed by a side
by side comparison of HindIII DNA profile of ThorNPV
produced in the original host, T. orichalcea, and the
alternate host, P. includens, in which no noticeable
difference was observed (photo not shown). The
ThorNPV was found to be closely related to TrniNPV,
based on high polyhedrin sequence identity (97.6%).
Both are single-nucleocapsid NPVs, but they are not
identical (Figs. 2 and 3). They differ not only in polyhedrin sequence but also in the shape of the OB, with
TrniNPV forming a polyhedron and ThorNPV a tetrahedron (Fielding and Davison, 1999; Cheng et al.,
1998). Furthermore, ThorNPV is not able to infect T. ni
larvae. The tetrahedral shape of ThorNPV did not
change when it was produced in the alternate host, P.
includens. Similar results were also reported by Young
and Yearian (1983) who observed that the tetrahedral
shape of Rachiplusia nu NPV (RanuNPV) did not
change when it was passed through an alternate host,
Rachiplusia ou.
The ThorNPV showed very low virulence to third
instar P. includens (Table 2), which may be explained
by the fact that P. includens is not the native host for
ThorNPV. Livingston et al. (1980) reported an NPV
from P. includens and found this P. includens singlenucleocapsid NPV was very virulent to third instar
soybean loopers (LC 50 5 5.7 PIB/mm 2).
Tetrahedral-shaped NPVs are rarely isolated, and
this is one of the reasons for our interest in studying
ThorNPV. There are a few reports of other NPVs with
a similar shape. Young and Yearian (1983) reported an
NPV from Rachiplusia nu with a tetrahedral shape
and Lymantria monacha (Linn.) and Hyphantria cunea
(Drury) also have NPVs with tetrahedral OBs (Smith,
1976). However, no detailed molecular studies have
been conducted with these other NPVs.
The size of the ThorNPV polyhedra is at the lower
end of the size range (0.5–15 mm) reported for NPVs
(Tanada and Kaya, 1993). The tetrahedral-shaped
RanuNPV reported by Young and Yearian (1983)
showed a similar size of 1.16 mm with a range of 0.72 to
1.66 mm.
Abnormal OBs were found with few or no virions or
with the virions not completely occluded (Figs. 5 and
6A). Similar abnormalities have also been found in S.
exigua infected with the S. frugiperda MNPV (Hamm
and Styer, 1985) and Lambdina fiscellaria somniaria
NPV in a cell line of Malacosoma distria hemocytes
(Sohi and Cunningham, 1972). These reports suggest
that malformation of OBs may be due to replication in
an alternate host rather than in the native host. However, an examination of the inoculum of ThorNPV from
its original host also showed some OBs with few or no
virions (photo not shown). A difference in virion occlusion was observed in adjacent cells in the same tissue
(Fig. 6D). If we assume that the two neighboring cells
present the same conditions for virus replication, then
the virion occlusion difference may reside in the virus
itself. Most NPVs that are isolated from the field and
propagated in laboratory hosts are a mixture of different variants of the same virus. Some of the cells may
have been infected by variants with a malfunctioned
gene(s) that controls virion occlusion. Evidence of this
was presented by Slavicek et al. (1998) who identified a
novel Lymantria dispar NPV mutant (PFM-1) that had
very few viral nucleocapsids in the OBs. By using
marker rescue studies, they gave proof of the existence
of genes other than polyhedrin and 25 K FP that directly impact virion occlusion. This occurrence of OBs
with few or no virions may contribute in part to the low
virulence of this virus to the soybean looper, since OB
counts are used in dosage determination. Thus, it
would be important for commercial producers to check
abnormality rates when mass-producing baculoviruses
as viral insecticides.
In conclusion, based on the polyhedrin gene sequence searches in GenBank, ThorNPV is a new member of the family of Baculoviridae. Its potential as a
candidate for microbial control of local looper species is
very questionable. However, the embedding abnormality of ThorNPV in vivo may be useful in studying the
factor(s) that govern this abnormality.
ACKNOWLEDGMENTS
The authors thank Drs. Eleanor and Merle Shepard for collection
of NPV sample in Indonesia. We also thank JoAn S. Hudson and
Ai-Teh Zhang of the Clemson Electron Microscope facility for assistance in the operation of both TEM and SEM and Dr. Basil Arif for
help in completing the manuscript. Financial support was provided
by HATCH Project 1614 and by a USAID Project, Integrated Pest
Management Research, Development and Training Activities for
CHARACTERIZATION OF ThorNPV
Palawija Crops in Indonesia. This paper is Technical Contribution
4398 of the South Carolina Agricultural Experiment Station, Clemson University.
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