Morphological, phylogenetic and biological characteristics of Ectropis obliqua single-nucleocapsid nucleopolyhedrovirus.

Abstract

The tea looper caterpillar, Ectropis obliqua, is one of the major pests of tea bushes. E. obliqua single-nucleocapsid nucleopolyhedrovirus (EcobSNPV) has been used as a commercial pesticide for biocontrol of this insect. However only limited genetic analysis for this important virus has been done up to now. EcobSNPV was characterized in this study. Electron microscopy analysis of the occlusion body showed polyhedra of 0.7 to 1.7 mum in diameter containing a single nucleocapsid per envelope of the virion. A 15.5 kb genomic fragment containing EcoRI-L, EcoRI-N and HindIII-F fragments, was sequenced. Analysis of the sequence revealed that the fragment contained eleven potential open reading frames (ORFs): lef-1, egt, 38.7k, rr1, polyhedrin, orf1629, pk-1, hoar and homologues to Spodoptera exigua multicapsid NPV (SeMNPV) ORFs 15, 28, and 29. Gene arrangement and phylogeny analysis suggest that EcobSNPV is closely related to the previously described Group II NPV. Bioassays on lethal concentration (LC(50) and LC(90)) and lethal time (LT(50) and LT(90)) were conducted to test the susceptibility of E. obliqua larvae to the virus.

Full text

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✽ To whom correspondence should be addressed.
(Tel) 86-571-86971697; (Fax) 86-571-86971697
(E-mail) chxzhang@zju.edu.cn
The Journal of Microbiology, February 2006, p.77-82
Copyright ⓒ 2006, The Microbiological Society of Korea
Vol. 44, No. 1
Morphological, Phylogenetic and Biological Characteristics of Ectropis
obliqua Single-Nucleocapsid Nucleopolyhedrovirus
Xiu-cui Ma1, Hai-Jun Xu1, Mei-Jun Tang2, Qiang Xiao2, Jian Hong1 and Chuan-Xi Zhang1,*
1Institute of Insect Sciences, Zhejiang University, 268 Kaixuan Road, Hangzhou, Zhejiang 310029, China
2Tea Research Institute, Chinese Academy of Agricultural Sciences, Hangzhou, Zhejiang 310000, China
(Received November 2, 2005 / Accepted January 18, 2006)
The tea looper caterpillar, Ectropis obliqua, is one of the major pests of tea bushes. E. obliqua
single-nucleocapsid nucleopolyhedrovirus (EcobSNPV) has been used as a commercial pesticide
for biocontrol of this insect. However only limited genetic analysis for this important virus has
been done up to now. EcobSNPV was characterized in this study. Electron microscopy analysis
of the occlusion body showed polyhedra of 0.7 to 1.7 μm in diameter containing a single nucleo-
capsid per envelope of the virion. A 15.5 kb genomic fragment containing EcoRI-L, EcoRI-N and
HindIII-F fragments, was sequenced. Analysis of the sequence revealed that the fragment con-
tained eleven potential open reading frames (ORFs): lef-1, egt, 38.7k, rr1, polyhedrin, orf1629,
pk-1, hoar and homologues to Spodoptera exigua multicapsid NPV (SeMNPV) ORFs 15, 28, and
29. Gene arrangement and phylogeny analysis suggest that EcobSNPV is closely related to the
previously described Group II NPV. Bioassays on lethal concentration (LC50 and LC90) and lethal
time (LT50 and LT90) were conducted to test the susceptibility of E. obliqua larvae to the virus.
Keywords: Ectropis obliqua, single-nucleocapsid nucleopolyhedrovirus, polyhedrin, phylogeny, bio-
assay
The Baculoviridae is a family of rod-shaped viruses
with large, circular, covalently closed, double-stranded
DNA genomes. Their DNA range in size from 81.7
kb for Neodiprion lecontei nucleopolyhedrovirus
(NeleNPV) to 178.7 kb for Xestia c-nigrum GV
(XecnGV). Two genera, Nucleopolyhedrovirus (NPV)
and Granulovirus (GV), have been recognized and
distinguished by the morphology of their occlusion
bodies. NPVs are designated single (S) or multiple
(M) based on the number of nucleocapsids contained
within their virions (Blissard et al., 2000). NPVs have
been subdivided into groups I and II based on their
molecular phylogenies (Zanotto
Baculoviruses are specific pathogens for invertebrates,
especially insects of the order Lepidoptera. They are
being extensively studied for their usage in the ex-
pression of recombinant proteins and biological con-
trol of insect pests. The improvement of both applica-
tions requires a detailed knowledge of distinct baculo-
virus features and the extent of their diversity. In or-
der to better understand the evolution of baculoviruses
and the molecular mechanism behind baculovirus in-
et al., 1993).
fection and replication, the sequencing of baculovirus
genomes has been undertaken by a number of re-
search groups (Zhang et al., 2005).
Ectropis obliqua SNPV (EcobSNPV) is a singly
embedded NPV pathogenic to the tea looper, E. obli-
qua Prout (Lepideptera: Geometride), one of the ma-
jor pests of tea bushes in East Asia (Chen and Huang,
2001). The economic importance for the host of
EcobSNPV makes it an important virus to study. It
has been demonstrated that EcobSNPV is an effective
and environmentally sound alternative to chemical in-
secticides (Hu et al., 1994). The virus has been used
to control the tea looper (Yin et al., 2003) and was
recently developed as a commercially available bio-pesti-
cide agent with a registration number of LS20052031.
About one thousand kilograms of EcobSNPV suspen-
sion at a concentration of 1×1010 PIB (polyhedral in-
clusion body)/kg is produced every year for control-
ling the tea looper in East China. Although the re-
striction maps of EcobSNPV have been assembled (Li
et al., 1983), little is known about its genetic analysis.
In this study, we present the sequence analysis of a
15.5 kb region from the genome of EcobSNPV and
compare it to the corresponding sequence and genetic
organization from other baculoviruses. The larvae of E.
obliqua were tested for their susceptibility to this vi-

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78 Ma et al.J. Microbiol.
rus employing the lethal concentration (LC50) and le-
thal time (LT50) bioassay.
Materials and Methods
Virus and DNA
The virus used in this experiment was originally isolated
from the tea looper, E. obliqua in Anhui Province in the
People’s Republic of China. The virus was propagated in
4th- instar larvae of E. obliqua and occlusion bodies
were purified by sucrose-gradient centrifugation
(O’Reilly et al, 1992). Viral genomic DNA was iso-
lated from purified occlusion bodies by dissolution in
0.1 M Na2CO3 and 0.01 M NaCl (pH 10.5), followed
by proteinase K and SDS treatment, phenol-chloro-
form extraction, and precipitation in ethyl alcohol.
The DNA was then dissolved in 0.1 × TE buffer.
Electronic microscopy
Midgut tissue of infected larvae was dissected and
fixed in 2.5% glutaraldehyde in 0.05 M cacodylate
buffer, and post-fixed in 1% osmium tetroxide in the
same buffer. Fixed samples were dehydrated through
a graded series of ethanol solutions and embedded in
Spurr’s resin. Sections were cut, stained with uranyl
acetate and lead citrate, and examined under a trans-
mission electron microscope.
Restriction endonuclease (REN) analysis, PCR, and
cloning
EcobSNPV genomic DNA was digested with various
restriction endonucleases (EcoRI, EcoRV, HindIII, PstI
and XhoI) and separated by 0.7% agarose gel electro-
phoresis (25 V, overnight) using standard techniques
(Sambrook et al., 1989). The size of restriction endo-
nuclease DNA fragments was estimated from com-
parative mobility using a graphical method and
HindIII λDNA markers. Most of the EcoRI-fragments
smaller than 10 kb were cloned and sequenced (Table
1). Some fragments with sizes larger than 10 kb were
analyzed with double enzyme digestions for con-
firmation of their sizes. The polymerase chain re-
action (PCR) method was employed to check the rela-
tionship between EcoRI-L and EcoRI-N fragments.
The following primers were used: 5’-CCG CTG TGG
ACA AAC AC-3’ (forward) and 5’-TCA AGT GTA
GGC GAA GG-3’ (reverse). PCR reaction was per-
formed by standard protocols with annealing at 52°C.
The PCR product was then gel-purified.
EcoRI-L (5.2 kb), EcoRI-N (3.8 kb), and HindIII-F
(8.5 kb) fragments were cloned into plasmid pUC19,
and the purified PCR product was cloned into T-easy
vector. The recombinant plasmids were then trans-
formed into E. coli TG1 using standard techniques
(Sambrook et al., 1989).
Nucleotide sequence analysis
Sequencing was carried out with the dideoxynucleo-
tide chain terminating method using Sequenase™
Version 2.0 DNA sequencing kit (USB). The deduced
amino acid sequences were compared with the up-
dated GenBank/EMBL, SWISSPROT, and PIR data-
bases using BLAST and FASTA programs (Pearson et
al., 1990; Altschul et al., 1997). The data on other
baculovirus genes compared in this paper were cited
from GenBank or from published papers. The amino
acid sequences were aligned based on a distance ap-
proach using CLUSTAL X version 1.81 with different
gap opening and gap extension values. Following
alignment, a phylogenetic tree was constructed for the
combination of polyhedrin, lef-1, and pk-1 genes by
the N-J method (PAUP* 4.0, beta 10 version) with
the default settings of random break tie and the dis-
tance option of mean character difference. Statistical
support for each node was evaluated by bootstrap
analysis with 1,000 replicates. The tree was reformed
by using TREEVIEW (V. 1.6.6), and the Plutella xy-
lostella granulovirus (PxGV) sequence was used as an
outgroup to estimate the position of the tree root.
The nucleotide sequence reported here was submitted
to GenBank under the access number AF107100.
Bioassays
The occlusion bodies were suspended in sterile water
at a concentration of 2 × 107 PIB/ml. A series of 10-
fold dilutions was prepared from the OB stock
solution. Six concentrations (20,000 PIB/ml, 2,000
PIB/ml, 200 PIB/ml, 20 PIB/ml, 2 PIB/ml, 0.2
PIB/ml) were used for the bioassay. Twigged tea
leaves were dipped in their respective concentrations
of PIB suspensions, allowed to air dry at room tem-
perature, and were then fed to larvae. The leaves
treated with sterile water were used as a control.
Bioassays were performed by continuous feeding of
EcobSNPV OB to second- instar larvae of E. obliqua
on fresh tea leaf surfaces. Larvae were fed on normal
fresh diets at 3 days post-inoculation. These larvae
were observed daily until they died or pupated.
Experiments were performed with 51-72 larvae per
dose in triplicate. All analyses, including evaluation
of virulence indices (LC50, LC90, LT50 and LT90),
were performed using DPS software (Feng, 1998).
Results and Discussion
Transmission electron microscopy
Occlusion bodies (OBs) of EcobSNPV were observed
in the infected midgut tissues of E. obliqua under a
transmission electron microscope. The micrograph
showed that EcobSNPV OBs were of irregular shape
and ranged in size from 0.7 to 1.7 μm (1.15 ± 0.27

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Vol. 44, No. 1Characterization of EcobSNPV79
A B
Fig. 1. Electron micrographs of polyhedra from E. obliqua sin-
gle-nucleocapsid nucleopolyhedrovirus.
Fig. 2. Restriction endonuclease (REN) digestion fragments of
EcobSNPV for EcoRI, EcoRV, HindIII, PstI and XhoI. λ DNA di-
gested with HindIII is included as a molecular size marker. The
fragments were designated alphabetically starting with A for the
largest fragment for each REN digest. Each visible band was as-
signed one or more letters depending on the number of fragments
in each band. Lanes: 1, λDNA / HindIII; 2, EcoRI; 3, EcoRV; 4,
HindIII; 5, PstI; 6, XhoI.
Table 1. Estimated sizes of EcobSNPV DNA restriction fragments
(kb)
Fragment EcoRI
EcoRV HindIII PstI XhoI
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
22
20
11
10
9.163*
8.097*
7.976*
6.4
6.201*
6.1
5.209*
5.152*
4.720*
3.831*
3.057*
3.002*
2.094*
1.45
1.20
25
19
12
12
8.3
7.8
7.0
6.8
4.45
3.70
3.60
2.85
2.54
2.36
1.48
1.33
1.17
1.17
23
20
18
13.3
10.5
9.911*
8.6
7.8
5.152*
4.20
2.70
2.45
2.08
1.95
1.72
1.16
27
25
19.5
19.5
14.0
8.5
8.3
3.07
1.75
1.75
22
20
18
9.9
9.8
9.6
7.0
7.0
5.0
4.80
3.83
1.75
1.65
1.22
Total 134.5121.4132.5 128.4121.5
* These fragments were cloned and sequenced.
μm: mean ± SD) in diameter (data not shown).
Multiple rod-shaped virions, measuring about 250 nm
in length and 40 nm in width, were embedded in each
OB with a single nucleocapsid packaged within the
envelope of the virion (Fig. 1).
Restriction enzyme profile
Digests of the EcobSNPV genome with the restriction
enzymes EcoRI, EcoRV, HindIII, PstI, and XhoI re-
sulted in a total of 77 fragments larger than 1.0 kb
(Fig. 2 and Table. 1). Based on estimated REN frag-
ment sizes, the EcobSNPV genome was predicted to
be about 127.7 kb. This is similar to the genomic size
of 67.55-85.14×106 Da (102-129kb) reported by Li et
al (1983). Fragments smaller than 1.0 kb were not
figured into calculations. Southern blot analysis using
Bombyx mori NPV (BmNPV)
Autographa californica MNPV (AcMNPV) egt probes
indicated that overlapping fragments HindIII-F and
EcoRI-N contained the EcobSNPV polyhedrin gene.
HindIII-I and EcoRI-L fragments contained the egt
gene (data not shown). Moreover, a PCR product was
further sequenced to confirm the junction of EcoRI-L
and EcoRI-N, which were separated from each other
by a 465 bp EcoRI fragment. Therefore, the REN
map of the whole fragment containing EcoRI-L,
EcoRI-N, and HindIII-F was constructed by cloning
the three fragments. It was sequenced to confirm the
accurate localization and orientation of polyhedrin,
egt, and other genes (Fig. 3).
polyhedrin and
Sequence determination and gene organization
The 15,528 bp EcobSNPV fragment was sequenced.
Eleven ORFs homologous to baculovirus proteins
were identified within the sequenced region: a late ex-
pression factor 1 gene (lef-1), a homologue to the
Spodoptera exigua multicapsid NPV (SeMNPV) ORF15
(Eo-se15), an ecdysteroid UDP-glucosyltransferase gene
(egt), a homologue to the SeMNPV ORF28 (Eo-se28), a
homologue to the SeMNPV ORF29 (Eo-se29), a 38.7
kD protein (38.7k), a ribonucleotide reductase (rr1), a

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80 Ma et al.J. Microbiol.
Fig. 3. Gene organization of the egt-polh region of EcobSNPV and its comparison with the corresponding regions of SeMNPV. The arrows
represent the ORFs and point in the directions of their transcription.
Fig. 4. Phylogenetic analysis using the predicted amino acid resi-
dues from polyhedrin, lef-1 and pk-1. The tree was constructed us-
ing N-J method (PAUP* 4.0, beta 10 version) and branch numbers
represent bootstrap probabilities (%) of 1,000 replicates. The tree
was reformed by using TREEVIEW (V. 1.6.6), and the Plutella xy-
lostella granulovirus (PxGV) was used as an outgroup.
major occlusion protein (polyhedrin), a virus repli-
cation-essential protein (orf1629), a protein kinase
(pk-1) and a homologue of the HzSNPV ORF4 (hoar)
(Fig. 3).
To investigate the relatedness of EcobSNPV to oth-
er baculoviruses, we compared the gene order in the
egt-polh region of EcobSNPV with fully-sequenced
lepidopteran NPVs. Fig. 3 shows a representative com-
parison of the gene arrangements between EcobSNPV
and SeMNPV. The genetic organization and the puta-
tive map of transcripts of this region indicated that a
core gene cluster of three genes, polyhedrin, orf1629,
and pk-1, was present in both aforementioned
baculoviruses. Further study indicated that the set of
these three genes has remained in the same relative
position (orientation may be different) in all of the se-
quenced genomes from lepidopteran NPVs with the
exception of Adoxophyes honmai NPV (AdhoNPV).
In this virus, alk-exo and another ORF are inserted
between polyhedrin and orf1629. The fact that these
genes are found in the same relative position in most
lepidopteran NPVs supports the use of the polyhedrin
gene as point of reference to orient baculovirus phys-
ical maps. This also indicates that there may be some
physical constraint preventing them, or at least these
DNA sequences, from being separated. The gene map
also suggested that three gene clusters are conserved
between EcobSNPV and SeMNPV. The first cluster
includes rr1, polyhedrin, orf1629, pk-1, and hoar, in
turn, with same gene orientation and genomic position
in these two viruses. The second gene cluster includes
38.7k, lef-1, and Se15 homologues. The third cluster
includes egt, Se28, and Se29 homologues, with differ-
ent orientation and position in the genomes between
these two viruses, due to rearrangements and inversions
of the gene cluster. Further investigation showed that all
of the aforementioned three clusters were also found
in Mamestra configurata NPV-A (MacoNPV-A) and
MacoNPV-B genomes. In addition, the following clus-
ters were found to be present in other group II NPV
genomes: clusters 1 and 2 within Chrysodeixis chal-
cites NPV (ChchNPV) and Trichoplusia ni SNPV
(TrniSNPV), clusters 2 and 3 within AdhoNPV, clus-
ter 2 within Spodoptera litura MNPV (SpltMNPV)
and cluster 3 within Lymantria dispar MNPV
(LdMNPV). Surprisingly, none of the above three
clusters could be found in Group I NPVs like
AcMNPV, BmNPV, Orgyia pseudotsugata MNPV
(OpMNPV), Epiphyas postvittana NPV (EppoNPV),
and others. The conserved clusters gave evidence that
EcobSNPV is more closely related to group II NPVs.
Phylogenetic position within NPVs
Polyhedrin is the most extensively studied baculovirus
gene and usually used to understand the phylogenetic
relationships of baculoviruses. Sequence alignment for
amino acids encoded by 20 NPV polyhedrin genes in-
dicates that EcobSNPV polyhedrin shares a high de-
gree of homology with 17 other lepidopteran NPVs, with
identities of 82-94.3%. As an outgroup in Fig. 4, PxGV
granulin shares 54.1% identity with EcobSNPV
polyhedrin. In contrast, polyhedrin genes from two
fully sequenced hymenopteran NPVs, Neodiprion le-

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Vol. 44, No. 1Characterization of EcobSNPV81
Table 2. LC50 and LC90 values for second instar tea looper ex-
posed to EcobSNPV at different time post-inoculation
DaysLC50 ± S.E.LC90 ± S.E.
37.33 ± 0.49 8.70 ± 0.53
46.11 ± 0.307.48 ± 0.35
55.55 ± 0.246.92 ± 0.29
64.68 ± 0.166.04 ± 0.21
74.09 ± 0.13 5.45 ± 0.17
83.54 ± 0.114.91 ± 0.14
93.10 ± 0.104.47 ± 0.12
10 2.15 ± 0.103.52 ± 0.10
111.80 ± 0.113.17 ± 0.10
12 1.39 ± 0.122.77 ± 0.10
131.08 ± 0.12 2.45 ± 0.10
140.80 ± 0.122.17 ± 0.10
Table 3. LT50 and LT90 values for EcobSNPV against second instar
tea looper at different concentrations
Concentration
(PIB/ml)
LT50
LT90
20,000 6.6 9.3
2,000 8.510.7
200 9.811.6
20 12.3-
contei NPV (NeleNPV) and Neodiprion sertifer NPV
(NeseNPV) share very low identities (44.7% and
47.2%, respectively) with that of EcobSNPV (data not
shown), indicating that hymenopteran NPV (NeleNPV
and NeseNPV) may have existed before the di-
vergence of lepidopteran NPV and GV (Lauzon et al.,
2004; Garcia-Maruniak et al., 2005).
The phylogenetic tree (Fig. 4) of NPVs based on
the combined sequences of polyhedrin, lef-1, and pk-1
indicated that EcobSNPV was most closely related to
Helicoverpa armigera
Helicoverpa zea SNPV (HzSNPV). It also appeared
that the relationships between SeMNPV, MacoNPV,
AdhoNPV, ChchNPV, TrniSNPV, LdMNPV, and
EcobSNPV were closer. It is more distantly related to
other lepidopteran NPVs, such as AcMNPV, BmNPV,
OpMNPV, Choristoneura fumiferana MNPV (CfMNPV)
and EppoNPV. This evidence strongly suggested that
EcobSNPV is a member of the Group II NPVs.
Based on DNA polymerase gene sequence alignments,
Bulach et al. (1999) described LdMNPV as a Group
II NPV. Three Group II NPV subclades were classi-
fied as A, B, and C. Further phylogenetic analysis of
polyhedrin gene suggested that EcobSNPV belonged
to subgroup II-C.
Although polyhedrin is still considered a reasonable
marker for identification of its neighbors, Herniou et
al. (2003) and Lange et al. (2004) argued that it might
not be the best baculovirus gene for phylogenetic
studies because polyhedrin phylogenies often disagree
with other gene phylogenies. While other phylogenetic
analyses consistently group AcMNPV and BmNPV
together, phylogenies based on polyhedrin have
AcMNPV as a sister group to the rest of the group I
NPVs (Herniou et al., 2003). Phylogenies based on
combined sequences of shared genes have been found
to be more robust than those based on the sequences
of individual genes (Herniou et al., 2001; 2003). Thus
in this study, we selected polyhedrin, lef-1, and pk-1
gene sequences to construct a baculovirus phyloge-
netic tree based on their presence in all sequenced
NPV and GV genomes. Of course, the concatenation
of more genes in common between the genomes of
interest may provide more reliable information for the
phylogenesis after the genome of EcobSNPV is fully
sequenced.
SNPV (HaSNPV) and
Biological activity
EcobSNPV was evaluated for its infectivity in sec-
ond-instar larvae of E. obliqua. A bioassay was de-
signed to determine both lethal concentration of virus
and lethal time of incubation. The results showed that
mortality of E. obliqua larvae increased and the lethal
time was shortened with increasing concentration of
EcobSNPV. When the second instar larvae were treat-
ed with high concentrations of EcobSNPV between
20,000 and 2,000 PIB/ml, all died within 10 to 14
days. However, only 30.6% of the larvae died at 18
days post-inoculation when they were treated with a
concentration of 0.2 PIB/ml. The time-dose-mortality
analysis showed that the value of LC50 was 104.09
PIB/ml at 7 days post-inoculation and 100.8 PIB/ml at
14 days post-inoculation (Table 2). At a concentration
of 2,000 PIB/ml, LT50 was 8.5 days and LT90 was
10.7 days (Table 3).
In conclusion, we report here the initial character-
ization of EcobSNPV, a baculovirus that infects the
tea looper, E. obliqua Prout, an insect that poses im-
portant economic concerns. A 15.5 kb genomic DNA
sequence was analyzed and 11 genes were identified.
The genetic organization and transcription profile of
the EcobSNPV egt-polh region, like the sequence
alignment of other baculoviruses, showed a consid-
erable degree of similarity to SeMNPV, MacoNPV-A,
MacoNPV-B, and other Group II NPVs. Baculovirus