Journal of General Virology (2015), 96, 904–914
DOI 10.1099/jgv.0.000023
Isolation of an Adoxophyes orana granulovirus
(AdorGV) occlusion body morphology mutant:
biological activity, genome sequence and
relationship to other isolates of AdorGV
Madoka Nakai,1 Robert L. Harrison,2 Haruaki Uchida,1 Rie Ukuda,1,3
Shohei Hikihara,1 Kazuo Ishii1 and Yasuhisa Kunimi1
Correspondence
1
Madoka Nakai
2
madoka@cc.tuat.ac.jp
Tokyo University of Agriculture and Technology, Saiwai, Fuchu, Tokyo 183-8509, Japan
Invasive Insect Biocontrol and Behavior Laboratory, USDA Agricultural Research Service
(USDA-ARS), Beltsville Agricultural Research Center, 10300 Baltimore Avenue, Beltsville,
MD 20705, USA
3
Yaeyama Branch Office, Okinawa Prefectural Plant Protection Center, 1178-6, Chisokobaru,
Hirae, Ishigaki, Okinawa 907-0003, Japan
Received 12 September 2014
Accepted 30 November 2014
A granulovirus (GV) producing occlusion bodies (OBs) with an unusual appearance was isolated
from Adoxophyes spp. larvae in the field. Ultrastructural observations revealed that its OBs were
significantly larger and cuboidal in shape, rather than the standard ovo-cylindrical shape typical of
GVs. N-terminal amino acid sequence analysis of the OB matrix protein from this virus suggested
that this new isolate was a variant of Adoxophyes orana granulovirus (AdorGV). Bioassays of this
GV (termed AdorGV-M) and an English isolate of AdorGV (termed AdorGV-E) indicated that the
two isolates were equally pathogenic against larvae of Adoxophyes honmai. However, AdorGV-M
retained more infectivity towards larvae after irradiation with UV light than did AdorGV-E.
Sequencing and analysis of the AdorGV-M genome revealed little sequence divergence between
this isolate and AdorGV-E. Comparison of selected genes among the two AdorGV isolates and
other Japanese AdorGV isolates revealed differences that may account for the unusual OB
morphology of AdorGV-M.
INTRODUCTION
Baculoviruses are dsDNA viruses belonging to the family
Baculoviridae, an insect-specific virus family whose members
have been identified in lepidopteran, dipteran and hymenopteran insects. Viruses of this family are distinguished
by rod-shaped virions that are packaged in proteinaceous
occlusion bodies (OBs) (Rohrmann, 2011). Baculoviruses
generally produce two physically and biochemically distinguishable virion phenotypes during infection: occlusionderived viruses (ODVs), which are liberated from the OBs
in the host midgut lumen and establish primary infection
The GenBank/EMBL/DDBJ accession number for the AdorGV-M genome
sequence of the occlusion body morphology mutant is KM226332. The
nucleotide sequences for additional AdorGV sequences from other isolates
are available under accession numbers KM234090–KM234092 (p47),
KM234093–KM234095 (orf112/113), KM234096–KM234098 (orf30),
KM234099–KM234101 (p10 and orf14), KM234102–KM234104 (pep),
KM234105–KM234107 (dbp), KM234108–KM234110 (desmoplakin)
and KM234111–KM234113 (pep-p10).
Four supplementary tables are available with the online Supplementary
Material.
904
of midgut epithelial cells, and budded viruses, which
are released from infected host cells and serve to establish
secondary infection of other host tissues.
The family Baculoviridae is divided into four genera on the
basis of morphology, host taxonomy and phylogenetic
relationships (Herniou et al., 2011). These genera comprise:
Alphabaculovirus, the lepidopteran nucleopolyhedroviruses
(NPVs); Betabaculovirus, the granuloviruses (GVs); and
Gammabaculovirus and Deltabaculovirus, which consist of
NPVs from Hymenoptera and Diptera, respectively. The GVs
have been isolated exclusively from insects of Lepidoptera but
are often distinguished from lepidopteran NPVs by the size
and shape of their OBs and the number of virions occluded
per OB. While alphabaculovirus OBs are usually polyhedral in
shape and 0.4–2.5 mm in diameter, betabaculovirus OBs are
smaller, ovo-cylindrical structures 0.12–0.35 mm in width
by 0.3–0.5 mm in length (Tanada & Hess, 1991). While
alphabaculovirus OBs generally contain multiple ODVs,
betabaculovirus OBs contain one or two ODVs at most.
GVs have been formulated for control of insect pests around
the world. Isolates of Adoxophyes orana granulovirus
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Printed in Great Britain
Genome sequence of granulovirus OB morphology mutant
(AdorGV) are used to control Adoxophyes spp. (Lepidoptera:
Tortricidae) in a variety of crop systems. Initially, AdorGV
was isolated from the apple pest A. orana (Aizawa &
Nakazato, 1963), and the field trials were done in apple
orchards in Japan (Shiga et al., 1973). Since the 1980s,
AdorGV isolates have been developed to control Adoxophyes
honmai on tea (Nakai, 2009). AdorGV was registered in 2003
and commercialized to control A. honmai and A. orana in
tea fields and apple orchards, respectively. There are three
Adoxophyes spp. in Japan, including Adoxophyes dubia,
which also attacks tea in southern Japan. These species are
closely related (Sakamaki & Hayakawa, 2004; Yasuda, 1998).
Similarly, GVs isolated from A. orana and A. honmai yielded
identical restriction fragment patterns by restriction endonuclease analysis, suggesting they are variants of the same
species (Hilton & Winstanley, 2008).
In this study, an OB morphology variant of AdorGV from
Miyazaki, Japan (AdorGV-M) was identified and characterized. In addition to ultrastructural examination of
OBs and evaluation of biological activity, the genome of
this variant was sequenced and compared with that of an
English isolate of AdorGV (AdorGV-E) (Wormleaton et al.,
2003) and with sequences of individual genes from three
other Japanese AdorGV isolates.
(Fig. 1b–d). Rarely, tetrahedral OB sections were observed
(Fig. 1b). The cuboidal OBs ranged from 0.5 to 2 mm in
diameter, but no more than one virion containing a singly
enveloped nucleocapsid was observed in any given OB
section (Fig. 1c).
Purified OBs from larvae infected with this OB morphology variant were loaded and run on an SDS-PAGE gel.
A major band migrating at 25 kDa was observed that
corresponded to the presumptive OB matrix protein
(data not shown). This band was cut out of the gel and
subjected to Edman degradation to determine the amino
acid sequence at the N terminus. This procedure yielded a
36 aa sequence (GYNKSLRYSRHEGTTCVIDNHHLKSLGSVLNDVRRK) that was 100 % identical to residues 2–37 of
the granulins of both AdorGV-E and Epinotia aporema
granulovirus (EpapGV), another GV from a tortricid host
species (Ferrelli et al., 2012). Preliminary amplification and
sequencing with AdorGV-specific granulin, fp25k, p10 and
pep primers yielded nucleotide sequences sharing 99.2–
100 % sequence identity with homologous regions in the
genome of the AdorGV-E (data not shown; Wormleaton
et al., 2003). These results indicate that the Miyazaki A.
honmai GV sample is an isolate of AdorGV.
Characteristics of the relative pathogenicity,
UV inactivation, and viral yield of AdorGV-M and
AdorGV-E
RESULTS AND DISCUSSION
Identification of a GV morphology variant in
Miyazaki Prefecture
In a screen for pathogens of A. honmai, 102 A. honmai larvae
were collected from a tea field in Miyazaki Prefecture, Japan.
Collected larvae were reared on artificial diet and monitored
for pathogenesis and mortality. While approximately onethird of the larvae survived to pupation, 12 exhibited typical
symptoms of GV infection (Table 1). Observation of these
larvae by light microscopy revealed that one of the cadavers
contained cube-shaped OBs that were larger than expected
for a GV (Fig. 1a). The OBs were isolated from the cadaver.
In transmission electron micrographs of larvae infected with
a stock of this virus isolate, the OBs deviated significantly
from the ovo-cylindrical shape normally seen with GV OBs
To determine if the unusual shape and size of the OBs
produced by this virus were associated with altered
pathogenicity and persistence, bioassays were carried out
with the AdorGV-M and AdorGV-E isolates against larvae
(a)
(b)
2.0 mm
(c)
1.0 mm
(d)
Table 1. Mortality of Adoxophyes spp. larvae collected in the
field in Miyazaki Prefecture
Cause of death
GV (large cuboidal OBs)
GV (ovo-cylindrical OBs)
Entomopoxvirus
Fungi
Parasitoid
Unknown
Healthy
Total
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No. larvae collected (%)
1 (1.0)
12 (11.8)
1 (1.0)
12 (11.8)
15 (14.7)
27 (26.5)
34 (33.3)
102 (100)
0.1 mm
0.5 mm
Fig. 1. Morphology of OBs for GVs. (a) Phase-contrast
microscopy of OBs for AdorGV-M. (b, c) Low-magnification (b)
and high-magnification (c) transmission electron micrographs of
OBs for AdorGV-M. (d) Transmission electron micrograph of OBs
for AdorGV-To.
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905
M. Nakai and others
of A. honmai. The two AdorGV isolates killed A. honmai
larvae with similar LC50 (50 % lethal concentration) values
and dose–response curves (Table 2) at both first instar and
fourth instar. The LC50 of AdorGV-M was 2.1- and 2.3-fold
greater than AdorGV-E LC50 against first-instar A. honmai
larvae and fourth-instar larvae, respectively. These differences were significant at the P,0.05 level. No significant
difference was observed for the concentration–response
slope of AdorGV-E and -M with either instar (P.0.05)
(Table 2).
In an experiment examining the loss of pathogenicity
upon irradiation with UV light, the AdorGV-M retained
significantly more pathogenicity towards A. honmai larvae
during prolonged periods of UV irradiation (Fig. 2), as the
relative ratio of inactivation (r) was greater for OBs of
AdorGV-E than of AdorGV-M. The half-life (t1/2) of the
OBs for AdorGV-M was 3.1±0.28 min, which was significantly longer than that for AdorGV-E (0.61±0.04 min;
Table S1, available in the online Supplementary Material).
On the other hand, r and t1/2 values for ODVs from both
strains were not significantly different (P.0.05). This
suggests that the unusual OB morphology of AdorGV-M
was associated with UV resistance.
Yield of infectious progeny virus generated by infection
with AdorGV-E or AdorGV-M was estimated by bioassay
of homogenates prepared from GV-killed larvae (Fig. 3).
The yield of infectious OBs produced per larva inoculated
with AdorGV-E and -M was estimated at 2.0±0.3061011
and 5.5±0.96109 OBs per larva (mean±SE), respectively,
which was significantly different (t55.1, df54, P,0.01).
These results also corresponded to a difference in OBs
produced per unit body mass for AdorGV-E and -M,
which was calculated to be 2.7±0.306109 and 8.9±1.86
107 OBs mg21, respectively (t56.6, df54, P,0.01). The
infectious viral yield of AdorGV-E per larva was 36 times
higher than that of AdorGV-M, and that per unit body
mass was 31 times higher.
Characteristics of the AdorGV-M genome
The AdorGV-M genome was determined initially by Sanger
dideoxy sequencing and subsequently by next-generation
sequencing on an Illumina platform. Both sequencing methods
yielded a consensus genome sequence of 99 507 bp, containing approximately 121 potentially protein-encoding ORFs
(Tables S2 and S3). In the Illumina consensus sequence,
positions 12 506 (within ORF 19/repeat region 2), 12 338
(within ORF 19/repeat region 2) and 77 258 (non-coding
regions between ORFs 95 and 96/repeat region 8) were
found to be highly polymorphic.
The genome sequences of the AdorGV-M and AdorGVE isolates share 99.7 % nucleotide sequence identity by
Martinez/Needleman–Wunsch alignment, with 46 gaps
inserted to optimize the alignment. The AdorGV-M sequence
contains all of the 37 baculovirus core genes and all of the
26 additional genes found in all members of genus
Betabaculovirus (Garavaglia et al., 2012). It also contains
all of the repeat regions and all but one of the ORFs
described for the AdorGV-E isolate, but is 150 bp shorter
than the AdorGV-E isolate due mostly to deletions in the
repeat region sequences of the AdorGV-M genome.
AdorGV-M ORF 52, which is located within repeat region
6 in the AdorGV-E isolate, is truncated at 43 codons in the
AdorGV-M isolate due to a CAA substitution which created
a TAG stop codon. The AdorGV-M sequence has three
ORFs not listed in the sequence of the AdorGV-E isolate,
including ORF 65, ORF 104, and ORF 116. ORF 65 and
ORF 104 are relatively small ORFs of 56 and 75 codons,
respectively, and can also be found in the AdorGV-E isolate
sequence. No homologues for these ORFs are present in
other baculovirus genomes. ORF 116 is a homologue of the
late expression factor-10 (lef-10) gene (Lu & Miller, 1994)
and can also be found in the AdorGV-E isolate. Of the ORFs
shared by AdorGV-M and AdorGV-E, only eight differed in
the occurrence of upstream transcriptional motifs, including
ORFs 22, 28 (odv-e66), 30, 31, 68 (dbp), 85 (vp91), 113 and
120 (egt) (Table S3).
Comparison of selected genes among English and
Japanese AdorGV isolates
Of the 121 ORFs that the AdorGV-M and AdorGV-E
isolates have in common, 66 of them encode amino acid
sequences that share 100 % identity by BLAST. All putative
homologues of AdorGV-M shared with that of AdorGV-E
showed the same gene order (Table S3). Of the remaining
ORFs, ORF 24 encodes the least conserved gene product
with a sequence identity of 96.4 % by BLAST between the
two isolates. This ORF contains all of AdorGV repeat
region 3, with 3 and 24 bp deletions relative to the
sequence of the AdorGV-E. A homologue of ORF 24 can be
Table 2. Dose–response of AdorGV-M and -E on A. honmai larvae
Instar
first
fourth
906
Virus
AdorGV-E
AdorGV-M
AdorGV-E
AdorGV-M
LC50
95 % Confidence limit
(OBs ml”1)
Lower
6.06105
1.36106
1.16107
2.56107
4.66105
9.56105
8.56106
1.96107
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Slope
x2
1.20
1.02
0.96
0.98
0.75
2.58
0.79
0.77
Upper
7.76105
1.76106
1.46107
3.36107
Journal of General Virology 96
Genome sequence of granulovirus OB morphology mutant
100
100
AdorGV-E OB
OAR (%)
OAR (%)
80
60
40
0
1
100
2
t (min)
3
4
0
1
100
AdorGV-M OB
2
3
t (min)
4
5
AdorGV-M ODV
80
OAR (%)
OAR (%)
40
0
5
80
60
40
20
0
60
20
20
0
AdorGV-E ODV
80
60
40
20
0
0
1
2
3
t (min)
4
5
0
1
2
t (min)
3
4
5
Fig. 2. Persistence of AdorGV-E and AdorGV-M exposed to UV-B light. Percentage of original activity remaining (OAR) is
shown for OBs and ODVs from AdorGV-E and -M exposed to UV-B light.
found in the genome of Pieris rapae granulovirus (PiraGV;
Zhang et al., 2012), and other poorly conserved homologues of this ORF appear to be present in other GV
genomes (Wormleaton et al., 2003).
10–10× OBs per larva
(a) 60
50
a
40
30
20
10
b
0
AdorGV–E
AdorGV–M
10–8× OBs (mg body mass)–1
(b)
70
a
60
50
40
30
20
10
0
b
AdorGV-E
AdorGV-M
Fig. 3. Infectious viral yield of A. honmai larvae inoculated with
AdorGV-E and AdorGV-M. Infectious viral yield per larva (a) or
larval body mass (b) at 20 days p.i. Bars indicate SEM. Different
lower-case letters indicate significant difference (P,0.05).
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Among baculovirus genes that are known or suggested to
be involved, directly or indirectly, in baculovirus OB
morphogenesis, the AdorGV granulin (gran, ORF 1), pep-1
(polyhedral envelope protein; ORF 16), and fp25k (ORF
101) genes share 100 % sequence identity at the amino
acid level between the AdorGV-M and AdorGV-E isolates
(Table S3). The p10 gene product (ORF 13) differs in
sequence by one amino acid. Also, genes encoding a second
pep homologue (pep-2; ORF 18) and a protein that appears
to be a fusion of P10 and PEP sequences (ORF 17) also
exhibit differences in amino acid sequence between the
AdorGV-M and AdorGV-E isolates (Table S3).
In this study, AdorGV-M showed higher UV resistance
than AdorGV. Like other baculoviruses, GVs are rapidly
inactivated by exposure to UV light in sunlight (David
et al., 1968). A capacity to resist inactivation by UV light
could conceivably cause an increase in the fitness of a virus
strain that possessed such a trait. GV genes that influence
susceptibility to inactivation by UV light may therefore be
under positive selection pressure (Yang, 2001). To identify
genes in AdorGV-M that may have undergone positive
selection during the divergence of the AdorGV-M and
AdorGV-E isolates, the non-synonymous and synonymous
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907
M. Nakai and others
substitution rates (dN and dS, respectively; Yang, 2001)
between homologous ORF pairs bearing non-synonymous
substitutions were estimated using a maximum-likelihood
method. Of 30 genes examined, two ORFs, ORF 14 and
ORF 68, were found to have dN/dS ratios of 1.3 and 1.1,
respectively, suggesting that these ORFs may have experienced positive selection pressure. Homologues of ORF 14
are present in GVs of Clostera anachoreta (Liang et al., 2011),
Clostera anastomosis (Liang et al., 2013) and PiraGV (Zhang
et al., 2012), but there are no clues about the function of
this gene. ORF 68 encodes a homologue of Autographa
californica multiple nucleopolyhedrovirus (AcMNPV) ac25,
or dbp (DNA-binding protein; Mikhailov et al., 1998).
To identify amino acid differences unique to the AdorGVM that may account for this isolate’s unusual OB morphology, a selection of nine genes were amplified from the
genomes of three additional AdorGV isolates from Tokyo,
Kagoshima and Tokushima, Japan, and sequenced. These
isolates produce OBs with typical GV granule size and
morphology (data not shown). The nine genes selected
included three genes, ORF 13 (p10), ORF 17 (p10-pep) and
ORF 18 (pep-2), putatively involved in OB assembly that
differed in sequence in AdorGV-M, and the two genes with
relatively high dN/dS ratios, ORF 14 and ORF 68 (dbp). In
addition, the sequences for ORF 30 and ORF 112 of the
English isolate also were amplified and sequenced. The
AdorGV-M ORF 30 sequence contains a frameshift at
codon 92 that alters the amino acid sequence relative to
ORF 30 of AdorGV-E. Likewise, the predicted amino acid
sequence of AdorGV-M genome ORF 113 aligns with
positions 35–132 of ORF 112 of the English strain; an
upstream ORF in a different reading frame encodes the
first 17 amino acids of ORF 112 in AdorGV-E, but a
frameshift at this position results in a different sequence in
AdorGV-M. While ORF 30 is unique to the AdorGV
genome, ORF 112/113 has homologues in the A. orana and
A. honmai nucleopolyhedrovirus genome sequences (Hilton
& Winstanley, 2008; Nakai et al., 2003), as well as a less
conserved homologue in the Agrotis segetum granulovirus
sequence (GenBank accession no. YP_006286). Finally,
the p47 (ORF 57) and desmoplakin (ORF 95) genes also
were amplified and sequenced. The p47 gene is a baculovirus
core gene that encodes a subunit of the baculovirus RNA
polymerase (Guarino et al., 1998). The p47 gene of AdorGVE and AdorGV-M genes are distinguished by three nonsynonymous substitutions, with an absence of synonymous
substitutions. The desmoplakin gene, also a core gene, encodes
a nucleocapsid-associated protein that is required for nucleocapsid egress from the nucleus of infected cells (Ke et al.,
2008). Numerous non-synonymous and synonymous substitutions distinguish the AdorGV-M copy of this gene from
that of the AdorGV-E.
Nucleotide sequence alignments and phylogenetic inference with the pep-p10, p47, dbp and desmoplakin coding
sequences indicated that all five AdorGV isolates examined
are likely variants of the same GV species (Fig. 4a). The
sequences of the AdorGV-K, AdorGV-To and AdorGV-E
908
isolates were almost identical, and these taxa were placed
together in a clade with strong bootstrap support (Fig. 4b).
The AdorGV-Tks and AdorGV-M sequences are more
divergent, though AdorGV-Tks was still placed in a larger
group with AdorGV-K, AdorGV-To and AdorGV-E.
Phylogenetic inference with all the nucleotide sequence
data generated for all nine loci produced a tree with very
similar topology, except that AdorGV-Tks was placed into
a group with AdorGV-M with low bootstrap support (data
not shown).
Alignment of the encoded amino acid sequences from the
nine loci revealed that the sequences for the predicted
polypeptides of AdorGV-K, AdorGV-To and AdorGV-E
were identical, while amino acid differences were found in
the AdorGV-M and AdorGV-Tks sequences (Fig. 4). These
two isolates had the same amino acid differences in the p10,
ORF 14 and pep-2 sequences relative to the AdorGV-K/To/
E consensus sequence. The AdorGV-M sequences for pepp10, p47, dbp and desmoplakin contained amino acid
differences that were unique to AdorGV-M. Likewise, the
AdorGV-Tks sequences also contained unique amino acid
differences in its pep-p10, p47 and desmoplakin sequences
(Table 3). While both the AdorGV-M and AdorGV-Tks
isolates bear frameshifts in the same approximate area of
ORF 30 (Fig. 5), only the AdorGV-M contains a frameshift
in ORF 112/113. Since electron microscopy analysis
shows that AdorGV-Tks does not produce OBs that are
cuboidal or unusually large (data not shown), the amino
acid substitutions occurring in pep-p10, p47, dbp, desmoplakin and ORF 112/113 correlate with the distinctive OB
morphology of AdorGV-M and may contribute to the
occurrence of their large size and cuboidal shape.
(a)
pep-p10/p47/dbp/desmoplakin
AdorGV-K
98/99
AdorGV-To
63/– AdorGV-E
AdorGV-Tks
AdorGV-M
PiraGV-E2
0.1
(b)
AdorGV/AdhoGV subtree
54/– AdorGV-K
98/99
AdorGV-To
0.002
63/–
AdorGV-E
AdorGV-Tks
AdorGV-M
Fig. 4. Phylogenetic analysis of nucleotide sequences for ORFs of
the genes pep-p10, p47, dbp and desmoplakin, showing relationships of the AdorGV isolates together with PiraGV (Zhang et al.,
2012) as an outgroup (a) and details of the AdorGV group (b).
Minimum evolution (ME) phylograms inferred from the concatenated alignment are shown with bootstrap values ¢50 % for ME
and maximum-parsimony (MP) trees at each node where available
(ME/MP). Bars, nucleotide substitutions per site.
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Journal of General Virology 96
Genome sequence of granulovirus OB morphology mutant
Table 3. Amino acid substitutions for selected genes of AdorGV-M and AdorGV-Tks isolates relative to the AdorGV-E, AdorGV-K
and AdorGV-To isolates
ORF
Virus isolate*
AdhoGV-M
p10 (ORF 13)
ORF 14
pep-p10 (ORF 17)
pep (ORF 18)
ORF 30
p47 (ORF 57/58)
dbp (ORF 68)
desmoplakin (ORF 95)
ORF 112D
K30R
V5I
Q91R
N102D
S198F
A6T
Indel+frameshift at position 92
A55S
G75E
K221N
E137K
N138D
C57Y
T94A
D104H
N156D
H190Q
E195D
D208E
A265T
S290F
A308V
F393L
Indel+frameshift at position 17
AdhoGV-Tks
K30R
V5I
Q91R
N102D
L236I
A6T
Indel+frameshift at position 94
T12K
Identical to AdorGV-E, -K, -To
C57Y
N156D
H190Q
E195D
D208E
A265T
L279I
S290F
F393L
I421L
Identical to AdorGV-E, -K, -To
*Substitutions listed are relative to orthologues in the English, Kagoshima and Tokyo isolates, which encoded amino acid sequences that were
identical to each other. Substitutions in bold italic indicate differences between the Miyazaki and Tokushima isolates. Substitutions are represented
by the amino acid identity in the AdorGV-E/-K/-To sequence at the indicated position in the sequence, followed by the amino acid identity in the
AdorGV-M or AdorGV-Tks isolate.
DRefers specifically to ORF 112 of the English AdorGV isolate.
Aberrant OB morphology in other baculoviruses
Watanabe & Imanishi (1972) described OBs of an abnormal
shape in GV-infected larvae of A. honmai (previously named
Adoxophyes fasciata), alongside OBs with the standard
GV ovo-cylindrical shape. These GVs were not cubic but
exhibited an elongate or irregular shape, and some appeared
to be ‘compound’ granules consisting of multiple granules
fused together. Cuboidal OBs of an abnormally large size
have been reported for other GVs isolated from Plodia
interpunctella, Choristoneura fumiferana and Cydia pomonella (Bird, 1976; Arnott & Smith, 1968; Stairs, 1964; Stairs
et al., 1966). The cubic granules reported by Arnott & Smith
(1968) occurred alongside elongate and compound granules
in Pl. interpunctella. Both Arnott & Smith (1968) and Stairs
(1964) reported that the cubic granule phenotype persisted
through consecutive passages in healthy insects, suggesting
that this phenotype was genetically encoded by the GVs in
these studies.
Several studies with alphabaculoviruses (NPVs) indicate
that the sequence of the polyhedrin (polh) gene is a primary
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determinant of OB morphology in infected cells (Carstens
et al., 1986; Cheng et al., 1998; Eason et al., 1998; Jarvis
et al., 1991; Katsuma et al., 1999; Lin et al., 2000; López
et al., 2011). In a number of cases, mutation of the
polyhedrin gene resulted in the formation of cuboidal OBs
(Carstens et al., 1986; Jarvis et al., 1991; Lin et al., 2000;
Katsuma et al., 1999). In this study, the AdorGV-M and -E
sequences of the gran genes (the betabaculovirus orthologues of alphabaculovirus polh genes) were identical,
indicating that the mutant morphology of the AdorGV-M
OBs cannot be attributed to mutations in the gran gene.
This observation is consistent with results from other
alphabaculovirus studies showing that the polh sequence is
not the sole determinant of OB morphology (Hu et al.,
1999; Woo et al., 1998). Eason et al. (1998) reported that
recombinant clones of AcMNPV in which the native polh
gene has been replaced by the gran gene of Trichoplusia ni
granulovirus produced large, cuboidal OBs in cell culture,
further indicating that a WT granulin sequence can
generate OBs of the sort observed for AdorGV-M. In
contrast with the OBs formed by AdorGV-M, the granulin
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M. Nakai and others
(a)
23522
AdorGV-E_orf30
401
AdhoGV-K_orf30
AdhoGV-M_orf30 23356
AdhoGV-Tks_orf30
399
AdhoGV-To_orf30
390
Consensus
AdorGV-E_orf30
23557
436
AdhoGV-K_orf30
AdhoGV-M_orf30 23401
449
AdhoGV-Tks_orf30
425
AdhoGV-To_orf30
Consensus
23605
AdorGV-E_orf30
484
AdhoGV-K_orf30
AdhoGV-M_orf30 23451
499
AdhoGV-Tks_orf30
473
AdhoGV-To_orf30
Consensus
(b)
AdorGV-E_orf30
AdhoGV-K_orf30
AdhoGV-M_orf30
AdhoGV-Tks_orf30
AdhoGV-To_orf30
Consensus
81
81
81
81
81
Fig. 5. Alignment of predicted AdorGV ORF 30 nucleotide (a) and amino acid (b) sequences. Nucleotide positions and amino
acid positions from the conceptual translations of the nucleotide sequences are indicated. Nucleotides in the consensus
sequence are denoted by uppercase letters for positions in the alignment with completely identical residues, and lowercase
letters for positions in the alignment with a majority of identical residues.
OBs formed by these recombinant AcMNPV clones
contained few or no virions, as was also the case for the
cuboidal OBs generated by NPV polh mutants. Mutation of
the gene encoding FP25K is also known to reduce virion
occlusion of AcMNPV (Harrison & Summers, 1995);
however, no mutations in the fp25k gene were discovered
in AdorGV-M.
The surface of baculovirus OBs is covered by a layer referred
to as the calyx, or polyhedral envelope, which consists
mostly of carbohydrate (Minion et al., 1979). A phosphorylated protein encoded by the pep (polyhedral envelope
protein) gene is a structural component of this envelope
(Whitt & Manning, 1988). Also, the p10 gene appears to be
involved in the formation of the calyx, though it is not itself
a component of the calyx (Van Oers & Vlak, 1997). The
calyx appears to lend physical stability to the OBs, but it has
also been speculated that the calyx serves to delimit the
boundaries of OBs and define their shape (Eason et al.,
1998). Recombinant clones of the Orgyia pseudotsugata
multiple nucleopolyhedrovirus in which either the pep gene
or both the pep and p10 genes had been disrupted by
insertion of a marker gene produced cuboidal OBs during
infection of cells in culture, similar to the OBs of AdorGVM (Gross et al., 1994). The AdorGV-M ORF 17 (pep-p10)
gene, which encodes a PEP–P10 fusion protein, contains a
serine to phenylalanine mutation (relative to the AdorGV
consensus sequence for this gene; Table 3) that is unique to
910
AdorGV-M. This mutation may account for the large size
and cuboidal morphology of AdorGV-M OBs.
Finally, few differences exist between AdorGV-E and
AdorGV-M in terms of the occurrence and distribution
of transcriptional motifs (Table S3), and no differences
were found in the transcriptional motifs upstream of the
gran, pep-1, pep-2, pep-p10 or p10 genes. Nevertheless,
we cannot formally exclude the possibility that differences
in the expression of one or more of these genes may
contribute to the OB morphology phenotype exhibited by
AdorGV-M.
Increased UV resistance and its possible impact
on fitness
AdorGV-M exhibited an LC50 that was significantly higher
than that of AdorGV-E in bioassays at the P,0.05 level,
but the magnitude of difference was only approximately
twofold. In contrast, AdorGV-M OBs were significantly
more resistant to inactivation by UV irradiation, with a t1/2
that is fivefold longer than that of AdorGV-E OBs. The
larger OBs of AdorGV-M imply that the occluded virions
of this strain are surrounded by a thicker layer of crystalline
granulin matrix than those of AdorGV-E. Studies examining the capacity of the OB crystalline protein matrix to
protect occluded virions against UV inactivation have
provided contradictory results. Ignoffo et al. (1992) found
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Journal of General Virology 96
Genome sequence of granulovirus OB morphology mutant
that OBs of Helicoverpa zea single nucleopolyhedrovirus
actually possessed a slightly shorter t1/2 than alkali-liberated,
non-occluded virion (ODV) when both were exposed to a
simulated sunlight UV source, indicating that the crystalline
protein matrix of OBs provides no protection against UV
inactivation. Behle et al. (2000) found that OBs of an NPV
from Anagrapha falcifera lost a significantly smaller
proportion of activity upon exposure to simulated sunlight
than did non-occluded ODV of the same virus, which
indicated that the OB matrix does provide ODV with some
protection against UV inactivation. If resistance to inactivation by sunlight makes a significant contribution to fitness of
a baculovirus in the field, then one would expect that the
large OB phenotype may provide a fitness advantage
compared with the smaller, typically ovo-cylindrical OB
phenotype. On the other hand, yield of infectious OBs was
approximately 30 times less in AdorGV-M than in AdorGVE. If infectious viral yield reflects number of OBs, it is
consistent with the observation that the numbers of aberrant
cuboidal OBs reported for other baculoviruses tend to be
low when they occur in infected cells. Although the larger
OB phenotype has been reported from time to time among
GVs, the standard ovo-cylindrical shape of GV OBs appears
to be far more prevalent, as was the case with OBs of the GVkilled larvae harvested in Miyazaki (Table 1).
In conclusion, we identified a strain of Adoxophyes orana
granulovirus with an unusual cuboidal morphology that
exhibited an increased degree of resistance to inactivation
by UV irradiation. Genomic sequencing of this virus and
comparison with sequences from other strains of the same
species have revealed the possible genetic basis for the
unusual properties of this virus. Further studies comparing
AdorGV-M and other AdorGV isolates may provide more
insight into the occurrence of aberrant OB morphology
and, more generally, into the molecular basis of baculovirus OB morphogenesis and its evolution.
METHODS
Viruses and insects. The AdorGV-M isolate was obtained from an
infected Adoxophyes spp. larva collected from a tea field in
Kawaminami-cho, Miyazaki Prefecture on 1 June 1999. OBs from
the cadaver were propagated in healthy A. honmai larvae after
removing midguts, because the sample was initially contaminated
with cypovirus. To produce a clonal isolate of AdorGV-M, OBs were
subjected to three passages through A. honmai larvae by low-dose
inoculation. The AdorGV isolate from Tokushima (AdorGV-Tks) was
isolated from Adoxophyes spp. cadavers collected in Yamashiro-cho,
Miyoshi-shi, Tokushima Prefecture; that from Tokyo (AdorGV-To)
was also a field isolate from an A. honmai cadaver collected in
Akiruno-shi, Tokyo; and that from Kagoshima (AdorGV-K) was
provided by the Kagoshima Prefecture Tea Experimental Station.
Insect rearing and inoculation were as described elsewhere (Nakai &
Kunimi, 1997). Larvae were reared on artificial diet (Silkmate 2S;
Nosan) at 25 uC with a 16 h photoperiod (16L : 8D).
Transmission electron microscopy. A. honmai neonate larvae were
infected by allowing them to consume droplets containing 1.06108
OBs ml21 of AdorGV-M or AdorGV-To. Infected fifth-instar larvae
http://vir.sgmjournals.org
showing typical granulosis gross pathology were dissected and the fat
body was fixed, dehydrated, occluded in resin, ultrasectioned and
stained for analysis by electron microscopy (H-7100, Hitachi) at
100 kV as described by Nakai & Kunimi (1997).
N-terminal sequencing of the OB matrix protein. The N-terminal
sequence of the major OB matrix protein from AdorGV-M was
subjected to Edman degradation analysis. AdorGV-M OBs (1.66106)
were boiled in SDS-PAGE sample buffer (50 mM Tris/HCl, 2 % SDS,
6 % 2-meraptoethanol and 10 % glycerol, pH 6.8), and electrophoresed through 15 % SDS-polyacrylamide gels according to Laemmli
(1970). A thick band migrating at 25 kDa was transferred onto PVDF
membrane and sequenced with a pulsed-liquid protein sequencer
(model 494HT, Applied Biosystems) at the National Institute for
Basic Biology, Okazaki-shi, Aichi Prefecture.
Bioassays. Susceptibility of neonate and fourth-instar A. honmai
larvae to OBs of AdhoGV-M and AdhoGV-E was examined by the
food disc and droplet-feeding methods, respectively. For the food disc
method, neonate larvae in 30 mm diameter plastic dishes were
exposed to a disc (6 mm diameter, 6 mm thickness) of Insecta LF diet
(Nihon Nosan-Kogyo) onto which 10 ml OB suspension (104.5, 105,
105.5, 106, 106.5, 107, 107.5 and 108 OBs ml21) had been pipetted. The
control larvae were fed a diet disc treated with 10 ml sterilized distilled
water. After 24 h of feeding at 25 uC, each larva was individually
transferred into a piece of fresh artificial diet in a half-ounce cup
(3 cm diameter62.5 cm height). For the droplet-feeding method,
newly moulted fourth-instar A. honmai larvae were exposed to
droplets containing OB suspension (105.5, 106, 106.5, 107, 107.5, 108,
108.5 and 109 OBs ml21), 1 % red food dye and 10 % sucrose.
Inoculated larvae were reared on artificial diet at 25 uC, with 16L : 8D
photoperiod, until either pupation or larval death. Development and
mortality were observed daily. More than 35 larvae were used for each
treatment with three replicates. The dose–response data were analysed
by probit analysis using POLO-PC (Leora Software).
Resistance of OBs and ODVs to inactivation by UV exposure.
UV tolerance of AdorGV-E and -M OBs was compared by measuring
the mortality of larvae inoculated with OBs exposed to UV light.
Drops of OB suspension (5 ml of each drop containing 8.06107 OBs
ml21 for AdorGV-E and 1.36108 OBs ml21 for AdorGV-M) were
placed on a plastic film (Parafilm) and irradiated with a UV-B lamp
(15 W, G15T8E; Sankyo Denki) in a black box (5061669.51 cm) in
a 4 uC cold room. The distance between the UV light source and the
drops was 9 cm, which corresponded to a light intensity of 390–
410 mW cm22 as measured by a UV meter (UV-340; Custom). After
0, 10, 60, 120, 180 and 300 s of UV exposure, the drops were
transferred to light-shaded plastic tubes and kept at 4 uC until use.
Because of the shorter persistence of AdorGV-E OBs, two additional
exposure times, 30 and 90 s, were used with trials involving AdorGVE. All the drops examined were placed on the plastic film for a total of
300 s, including the time spent exposed to UV light.
Bioassay using the UV-exposed OB was done by the droplet-feeding
method as described above. Newly moulted fourth-instar larvae were
exposed to sucrose/red dye drops containing OB suspensions on
Parafilm that had been exposed to UV light. Larvae showing red
colour in the midgut area were transferred to half-ounce cups
supplied with artificial diet (Insecta LF) and were reared at 25 uC,
16L : 8D. The percentage of larvae exhibiting symptoms of granulosis
was determined by light microscopy. Three biological replicates were
performed.
To measure the resistance of ODVs to UV inactivation, OB
suspensions of both strains were dissolved with DAS buffer (0.1 M
Na2CO3, 0.17 M NaCl, 0.01 M EDTA, pH 10.5) for 30 min, then
neutralized with 0.18 vol 1 M Tris/HCl (pH 7.4) and centrifuged at
5000 g for 10 min at 4 uC to remove undissolved debris. The ODV
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M. Nakai and others
particles were purified by 30–60 % sucrose gradient ultracentrifugation at 120 000 g for 30 min at 4 uC. The visible ODV band was
collected and washed twice with TE solution (10 mM Tris/HCl,
1 mM EDTA, pH 8.0) and suspended in TE solution. The ODV
solution was diluted to an LC70 concentration (as determined by
bioassay), exposed to UV light as described above and kept at 4 uC
until use. Bioassays were set up with 35 newly moulted fourth-instar
larvae per exposure time as described above. Three biological
replicates were performed.
Measuring yield of infectious virus. Neonate larvae were
inoculated with an inoculum corresponding to an LC95 of AdorGVE or AdorGV-M (1.46107 and 5.06107 OBs ml21, respectively) by
the food disc method as described previously (Nakai et al., 2004).
After 20 days, three larvae were collected randomly. Nakai et al.
(2004) observed that the viral yield of AdhoGV (closely related to
AdorGV-E, because same strain as AdorGV-T) reached a maximum
on 14 days post inoculation (p.i.) without significant further increase.
Some larvae infected with AdorGV-M were already dead at 20 days
p.i., and the yield at this harvest time was considered equivalent to the
maximum yield of AdorGV-E and -M. Serial dilutions of each
homogenate (AdorGV-E, 61023, 1023.5, 1024, 1024.5, 1025, 1025.5,
1026, 1026.5, 1027, AdorGV-M; 61022.5, 1023, 1023.5, 1024, 1024.5,
1025, 1025.5, 1026) were administered to 35 neonate larvae by the
food disc method as described above. The dilution points at LC50
were estimated by probit analysis. Infectious viral yield was
determined by comparison with previously obtained linear regressions for known amounts of AdorGV-E and -M OBs. Data were
analysed by t-test with JMP software (SAS).
Viral DNA isolation and sequencing. Viral DNA was isolated from
viral OBs purified as described elsewhere (Nakai & Kunimi, 1997).
The OB suspension was solubilized with 0.1 M Na2CO3 at 37 uC for
30 min and neutralized with 0.04 vol 1 M HCl. Viral DNA was
extracted with phenol/chloroform as described elsewhere (Ishii et al.,
2003). The DNA solution of the AdorGV-M isolate was sequenced
both by primer walking with Sanger dideoxy DNA sequencing and by
next-generation sequencing on an Illumina platform. For primer
walking, the first round of sequence reactions was carried out with a
set of 226 primer pairs designed from the genome sequence of
AdorGV-E to amplify regions of approximately 1200 bp. Fragments
amplified by PCR were sequenced from both ends. The amplicons
generated with these primers overlapped each other by 200 bp.
Fragments were assembled into contigs, and a second round of
amplicons overlapping the first set was generated and sequenced
using an additional set of 246 primer pairs. The contigs from this
second round were assembled with those of the first round to produce
a draft of the genome sequence.
To determine the AdorGV-M genome sequence with next-generation
sequencing technology, a library was prepared from fragmented
AdorGV-M DNA and used to generate 7 710 053 sequencing reads
(76 cycles, single-end) with the Genome Analyser IIx (Illumina).
Sequencing reads were mapped on the genome DNA sequence of
AdorGV-E with Bowtie2 v. 2.2.3 (Langmead & Salzberg, 2012), and
automated correction of genome sequence errors was performed by
SAMtools (Li et al., 2009) and in-house Perl (v. 5.10.1) scripts. The
final sequence draft was confirmed with the IGV (v. 2.3) genome
browser (Robinson et al., 2011).
In addition to sequencing of the AdorGV-M genome, individual loci
from AdorGV isolates collected in Tokushima, Kagoshima and Tokyo
were PCR amplified and sequenced. To obtain template for PCR,
granules were solubilized as previously described (Rowley et al.,
2010), and viral DNA was extracted from occluded virus by phenol/
chloroform extraction and ethanol precipitation. Two microlitres of a
100-fold dilution of the DNA templates was used in 50 ml PCRs as
described by Rowley et al. (2010) using primers designed to amplify
912
the AdorGV ORFs p10, ORF 14, pep-p10, pep-2, ORF 30, p47, dbp,
desmoplakin and AdorGV-E ORF 112 (Table S4). Amplimers were
precipitated away from excess primers and nucleotides using 20 %
PEG/2.5 M NaCl and subsequently sequenced with gene-specific
primers as described by Harrison & Lynn (2007).
Genome annotation, selection pressure analysis, and phylogenetic inference. The GeneQuest program of the Lasergene
suite (version 10) was used to identify ORFs of at least 50
codons in the AdorGV-M sequence. Those ORFs that did not overlap
adjacent ORFs by more than 75 bp were selected for annotation,
BLASTP queries and further characterization. If two ORFs overlapped
by more than 75 bp, the larger ORF was selected for annotation, as
were ORFs for which annotated homologues in other baculovirus
genomes were identified.
DNASTAR
The ORFs in AdorGV-M were compared with homologous ORFs in
the genome of the AdorGV-E isolate. For those ORFs distinguished by
non-synonymous substitutions, the rates of non-synonymous substitution per non-synonymous site (dN) and of synonymous substitution per synonymous site (dS) between the AdorGV-M and -E
sequences were calculated using the CODEML program of PAML v. 4.4c
(Yang, 2007) with the individual frequency of each codon as a free
parameter.
Alignments of AdorGV nucleotide and conceptual amino acid
sequences were performed by CLUSTAL W using the MEGALIGN program
of Lasergene v. 10 set at default values for gap and gap-length
penalties. Pairwise alignments of nucleotide sequences were carried
out using the Martinez/Wunsch–Needleman algorithm of the same
program. For phylogenetic inference, alignments of individual genes
were concatenated with BioEdit (Hall, 1999). Phylogenetic relationships were inferred by minimum evolution and maximum-parsimony
using MEGA4 (Tamura et al., 2007) with parameters as described
previously (Harrison et al., 2008; Rowley et al., 2010).
ACKNOWLEDGEMENTS
The authors sincerely acknowledge the contribution of Miku Suzuki
and Kento Abe for electron microscopy and DNA isolation. This work
was supported by the JSPS KAKENHI grant nos 18380038 and
21580064 and the US Department of Agriculture. Mention of
trade names or commercial products in this publication is solely
for the purpose of providing specific information and does not
imply recommendation or endorsement by the US Department of
Agriculture.
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