Download Complete comparative genomic analysis of two field isolates of

Journal of General Virology (2005), 86, 91–105
DOI 10.1099/vir.0.80488-0
Complete comparative genomic analysis of
two field isolates of Mamestra configurata
nucleopolyhedrovirus-A
Lulin Li,13 Qianjun Li,24 Leslie G. Willis,1 Martin Erlandson,2
David A. Theilmann1 and Cam Donly3
Correspondence
1
Pacific Agri-Food Research Centre, AAFC, Summerland, BC, Canada
Cam Donly
2
[email protected]
3
Saskatoon Research Centre, AAFC-Saskatoon, SK, Canada
Received 30 July 2004
Accepted 20 September 2004
Southern Crop Protection and Food Research Centre, AAFC, London, ON, Canada
A second genotype of Mamestra configurata nucleopolyhedrovirus-A (MacoNPV-A), variant
90/4 (v90/4), was identified due to its altered restriction endonuclease profile and reduced
virulence for the host insect, M. configurata, relative to the archetypal genotype, MacoNPV-A
variant 90/2 (v90/2). To investigate the genetic differences between these two variants, the
genome of v90/4 was sequenced completely. The MacoNPV-A v90/4 genome is 153 656 bp in
size, 1404 bp smaller than the v90/2 genome. Sequence alignment showed that there was
99?5 % nucleotide sequence identity between the genomes of v90/4 and v90/2. However, the
v90/4 genome has 521 point mutations and numerous deletions and insertions when compared
to the genome of v90/2. Gene content and organization in the genome of v90/4 is identical to
that in v90/2, except for an additional bro gene that is found in the v90/2 genome. The region
between hr1 and orf31 shows the greatest divergence between the two genomes. This region
contains three bro genes, which are among the most variable baculovirus genes. These results,
together with other published data, suggest that bro genes may influence baculovirus genome
diversity and may be involved in recombination between baculovirus genomes. Many ambiguous
residues found in the v90/4 sequence also reveal the presence of 214 sequence polymorphisms.
Sequence analysis of cloned HindIII fragments of the original MacoNPV field isolate that the
90/4 variant was derived from indicates that v90/4 is an authentic variant and may represent
approximately 25 % of the genotypes in the field isolate. These results provide evidence of
extensive sequence variation among the individual genomes comprising a natural baculovirus
outbreak in a continuous host population.
INTRODUCTION
Baculoviruses are pathogenic for arthropods, mainly insects
of the Lepidoptera, Hymenoptera and Diptera. These
viruses have been investigated because of their potential as
3Present address: Animal Disease Research Institute, 3851 Fallowfield
Rd, Ottawa, ON, Canada, K2H 8P9.
4Present address: Department of Medicine/Division of Geographic
Medicine, University of Alabama at Birmingham, BBRB 203, 845 South
19th Street, Birmingham, AL 35294-2170, USA.
The GenBank/EMBL/DDBJ accession number for the sequence
reported in this paper is AF539999.
Figures showing mutations in the promoter regions of lef-7 (orf16) and
orf25, an alignment of the LEF-9 C-terminal amino acid sequences of
v90/4 and v90/2 with those of 13 lepidopteran baculoviruses and an
alignment of the 59-end sequences of bro-b between v90/4 and v90/2
are available as supplementary material in JGV Online.
0008-0488
Printed in Great Britain
biological control agents of agricultural and forest pests.
Baculoviruses contain circular, double-stranded DNA
genomes of 80–180 kb. To date, the genomes of 26
nucleopolyhedroviruses (NPVs) have been sequenced
completely.
The bertha armyworm, Mamestra configurata, is an important pest of cruciferous oilseed crops in western Canada,
from which a number of NPVs have been isolated from
field populations. In exploring the potential of these viruses
for control of M. configurata and other pest insects, the
viral isolates have been characterized with respect to their
virulence in M. configurata, as well as their genomic restriction endonuclease (REN) profiles (Erlandson, 1990).
Analysis of M. configurata NPV (MacoNPV) isolates has
revealed significant diversity in their biological properties
and genetics. Recently, we reported the complete genome
analysis of two MacoNPV species (Li et al., 2002a, b). These
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L. Li and others
two viruses are closely related, but have evolved divergently into two separate baculovirus species, designated
MacoNPV-A and MacoNPV-B.
Very little is known about the genetic diversity of baculoviruses in field populations. Restriction fragment length
polymorphisms have been reported for many species,
suggesting that some level of natural genome variation is
common among baculoviruses (Croizier & Ribeiro, 1992;
Garcia-Maruniak et al., 1996; Hodgson et al., 2001;
McIntosh et al., 1987; Muñoz et al., 1999). In addition,
it has recently been reported that field isolates of the
archetypal baculovirus Autographa californica multiple
NPV (AcMNPV) contain additional genes to those in the
previously reported genome sequence (Lu et al., 1996;
Schetter et al., 1990; Yanase et al., 2000). These observations
suggest that baculovirus genomes are quite dynamic and
that this variability may provide selective or evolutionary
advantages to the virus population. In this study, we
describe the sequence of a second MacoNPV-A genome,
variant 90/4 (v90/4), which was initially identified due to
REN profile and biological differences in comparison to
the archetypal MacoNPV-A variant, 90/2 (v90/2). This is
the first study to perform a complete comparative analysis
of two genomes of the same species from the same virus
outbreak in a wild insect population. The genomes of
v90/4 and v90/2 reveal that significant sequence variation
exists between genotypes within the same virus species.
METHODS
Insects and viruses. Larvae from a laboratory culture of M. config-
urata were maintained on a semi-synthetic diet (Bucher & Bracken,
1976) at 21 uC, 60 % relative humidity and an 18 : 6 light : dark
photoperiod.
The v90/4 isolate was derived from a single larval cadaver that was
collected near Lamont, Alberta, Canada (53u 509 N 112u 389 W), in
1990. The Lamont virus isolate was amplified by infection in vivo.
Initial REN analysis indicated that this isolate contained a heterogeneous mixture of several genotypes. Haemolymph was collected
from fourth-instar bertha armyworm larvae 4 days after being
infected with the Lamont isolate and processed for infection of
insect cell culture. Briefly, haemolymph samples were collected into
1?5 ml centrifuge tubes containing 0?5 ml Grace’s tissue-culture
medium (Gibco-BRL) on ice. Haemocytes were pelleted by lowspeed centrifugation (1400 g for 5 min) and the supernatant was
transferred to 0?45 mm SPIN-X centrifuge tube filters (COSTAR) and
centrifuged for 1 min in a benchtop centrifuge (Eppendorf 5415
C). The filtrate was then used to infect Mamestra brassicae cells
(IZD-MB-0503, ATCC CRL 8003) in plaque assays. A series of 10
plaques was selected and replaqued a second time. Because the
production of virus progeny was not very efficient in this cell line
(approx. 105 TCID50 units ml21), plaque isolates were amplified in
M. configurata larvae for further study. A single plaque isolate, v90/4,
was chosen for sequencing.
The v90/2 isolate was derived from a single larval cadaver that was
collected near Wilkie, Saskatchewan, Canada (52u 309 N 108u 419 W),
in 1990. It was amplified by infection in vivo and cloned by using an
in vivo isolation technique, as described by Smith & Crook (1988).
REN analysis of the virus isolate did not reveal the presence of
92
submolar fragments (Li et al., 1997) and, in subsequent analysis of the
genome sequence data, very few (<50) nucleotide polymorphisms
were detected, indicating that the isolate was genetically homogeneous.
Stocks of MacoNPV were produced by infection of fourth-instar
bertha armyworm larvae by contamination of the diet with 1?46104
PIB per cm2 of diet surface. Virus production and polyhedral inclusion
body (PIB) isolation, virion purification and viral DNA extraction
essentially followed previously described methods (Erlandson, 1990;
Li et al., 1997).
REN analysis of viral DNA. REN analysis of viral DNA was per-
formed as described previously (Li et al., 1997). Briefly, purified
DNA of MacoNPV-A v90/4 and v90/2 was digested with HindIII at
37 uC for 3 h, then separated on 0?7 % agarose gels in 0?56 TBE
(45 mM Tris/borate, 1 mM EDTA) at 20–40 V for 15–22 h. Gels
were stained with ethidium bromide and photographed.
Bioassays. Bioassays were carried out with neonate bertha armyworm larvae by using a droplet-feeding bioassay with five virus
doses and 100 larvae per dose, as described previously (Erlandson,
1990). Those larvae consuming virus inoculum during a 30 min
exposure period were included in the assay and were transferred to
an artificial diet and incubated at 21 uC, with fresh diet added as
needed for the duration of the bioassay. Mortality was tabulated
daily and mortality response data were analysed on the basis of
mortality on day 14 post-infection. LD50 estimates were determined
by using SAS-PROBIT (version 8, SAS Institute).
DNA sequencing and sequence analysis. The MacoNPV-A
(v90/4) genome was sequenced by using a shotgun approach, as
described previously (Li et al., 2002a). In total, 1929 sequencing
runs of 500–600 readable bases were assembled into 15 contigs by
using Sequencher 4.0 software (Gene Codes Corporation). PCR was
performed to synthesize DNA fragments bridging the gaps between
contigs by using MacoNPV-A (v90/4) genomic DNA as template.
PCR products were sequenced from both ends. The sequences were
assembled with the initial contigs into a single, circular contig.
Sequences were analysed with Wisconsin Genetics Computer Group
programs (Devereux et al., 1984), GeneWorks 2.3 (IntelliGenetics)
and MacVector 7.1 (Accelrys). Homology searches were carried out
with GenBank/EMBL, SWISSPROT and PIR databases by using the
BLAST algorithm (Altschul & Lipman, 1990). Multiple sequence
alignments were performed by using CLUSTAL W (Thompson et al.,
1994). MacoNPV genome sequence accession numbers are AF539999
for MacoNPV-A (v90/4), AF467808 for MacoNPV-A (v90/2) and
AY126275 for MacoNPV-B.
Sequence analysis of HindIII fragments cloned from fieldisolated virus. Occluded virus of the non-plaque-purified
MacoNPV Lamont field isolate, from which MacoNPV-A v90/4 was
derived, was purified from infected M. configurata larvae (as
described above). Occluded virus DNA was digested with HindIII,
separated on 0?7 % agarose gels and selected HindIII fragments were
purified from gel slices (QIAquick Gel Extraction kit; Qiagen) and
cloned in vector pUC18. Eight clones for each HindIII fragment,
3051–4547 and 67365–70629, were sequenced. The DNA sequences
were aligned with respect to MacoNPV-A v90/2 and v90/4 genome
sequences (LaserGene, Seqman) and compared.
RESULTS
LD50 and REN profile of MacoNPV-A (v90/4)
The v90/4 virus, along with a number of other MacoNPV
isolates, including v90/2, was originally collected from NPV
epizootics in an outbreak population of M. configurata
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Journal of General Virology 86
Comparative genomic analysis of MacoNPV-A
M
(bp)
v90/4 v90/2
(bp)
12 000
7 000
5 000
4 000
3 000
6 602
5 309
4 442
2 923
2 000
1 650
1 527
1 000
850
650
500
Fig. 1. REN profiles of MacoNPV-A v90/4 and v90/2 isolates.
DNA was isolated as described in Methods and 1 mg DNA
was digested with HindIII and separated through a 0?7 % agarose gel in 0?56 TBE buffer. Names of isolates are given
above each lane. M, Marker lane containing 1 kb Plus DNA
ladder (Invitrogen), with the sizes of DNA fragments indicated
to the left of the panel. The major differences between the
two MacoNPV-A lanes, including two fragments (6602 and
4442 bp) unique to v90/2 and three fragments (5309, 2923
and 1527 bp) unique to v90/4, are marked by arrows.
throughout western Canada in 1990. Preliminary screening
of these isolates by using REN profile analysis showed that
v90/4 was very similar to v90/2, but contained some
restriction fragment length polymorphisms, as shown in
Fig. 1. Bioassays with v90/4 and v90/2 in neonate M.
configurata demonstrated that v90/4 was less virulent, as its
LD50 value was 128?4 (95 % confidence interval, 96–171)
PIB per larva, tenfold higher than that of v90/2 at 11?9
(95 % confidence interval, 8?6–15?6) PIB per larva.
Genome sequence comparison of v90/4 and
v90/2
The genome of v90/4 is 153 656 bp, 1404 bp smaller than
that of v90/2. A complete sequence alignment showed that
99?5 % of the v90/4 genome sequence is identical to that of
v90/2. As shown in Fig. 2, there are 521 nucleotide changes
in aligned regions when the genomes of v90/4 and v90/2
are compared. In addition, relative to v90/2, the v90/4
genome has 31 deletions and 14 insertions, comprising 1527
and 123 bp, respectively.
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Analysis of all variations showed that 398 point mutations,
six insertions totalling 30 bp and 20 deletions totalling
1326 bp occur in predicted ORFs; 65 point mutations, three
insertions (13 bp) and five deletions (102 bp) occur in
intergenic regions; and 58 point mutations, two insertions
(79 bp) and six deletions (98 bp) occur in homologous
repeated (hr) regions. Only 27 % of the mutations cause
amino acid sequence substitutions.
Although point mutations, insertions and deletions are
dispersed throughout the genome, specific regions have a
significantly higher density of changes (Fig. 2). The most
variable region is located between hr1 and orf31 (bro-c)
(v90/4, 15?0–27?1 kb; v90/2, 15?0–28?3 kb). In this 12?1 kb
region (7?7 % of the genome), there are 261 of the 521
point mutations, accounting for 50 % of the total nucleotide changes. Of the 261 point mutations, 82 cause nonsynonymous changes and only the chitinase and orf27
genes do not have any amino acid changes. This region
also contains multiple deletions in the v90/4 genome. The
largest deletion is a 1165 bp fragment that contains orf21
(bro-a) and a portion of orf20. An alignment of the v90/4
and v90/2 sequences around the junction regions of the
1165 bp deletion is shown in Fig. 3. The 1165 bp fragment
contains a palindromic sequence (TCTAATTAGA) at its
59 end and another palindromic sequence (AAATATTT) at
the 39 end.
In total, 417 ORFs of 150 bp or longer, starting with an
ATG, were detected in the v90/4 genome. Of these, 168
have minimal overlap with adjacent ORFs or hr regions,
or showed significant homology to genes in GenBank. Gene
content and arrangement are almost identical between
v90/4 and v90/2 (Fig. 2). However, there is a single gene
difference between the two viruses. As indicated above,
bro-a is absent in v90/4. Of the 168 common ORFs, 49
ORFs show 1–12 % amino acid sequence variation and 12
ORFs vary in size (see Table 1). Among the 63 ORFs that
are common to all lepidopteran baculoviruses (Chen et al.,
2002; Li et al., 2002a), eight ORFs have amino acid substitutions in their encoded products. These include me53,
lef-1, tlp-20, lef-8, lef-9, orf80, odv-e66 (orf144) and ie-1. Five
of 12 ORFs that are unique to v90/2 and v90/4, orf5, orf10,
orf18, orf23 and orf64 (Li et al., 2002b), have amino acid
sequence substitutions in their putative gene products.
Alterations in the regulation of gene expression can have
significant effects on gene function; therefore, the promoter
regions of all genes were analysed for variations in known
regulatory motifs. Table 2 lists the nucleotide variations
between v90/2 and v90/4 that occur in promoter regions
located within 150 bp of an ORF. The promoter motifs in
these regions are also presented. For example, the T to
A substitution at 245 upstream of orf16 (lef-7) forms a
TATA box in this region in v90/4 that is not found in v90/2
(see Supplementary Fig. S1, available in JGV Online). orf25
in v90/2 contains an early gene motif (249-TATAAA, 221CAGT); in v90/4, there is a C to T substitution at 222 that
mutates the potential transcriptional start site, CAGT, into
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L. Li and others
Fig. 2. Comparison of genome structure between MacoNPV-A v90/4 and v90/2. The figure depicts a schematic
representation of the MacoNPV-A genome, with map positions of the 169 ORFs of MacoNPV-A v90/2 (Li et al., 2002b)
represented by arrows indicating transcriptional direction and relative size. Numbers above arrows represent the number of
each ORF (Li et al., 2002b). Red vertical lines represent the location of point mutations in the v90/4 genome; dark blue bars
(lowered) represent deletions and light blue bars (raised) represent insertions in v90/4, compared to v90/2. Yellow arrows
represent ORFs with changes in amino acid sequences. Green arrows represent ORFs with identical amino acid sequences.
hr sequences and their positions on the genome are indicated by empty boxes. Numbers below arrows represent the genome
position relative to base 1.
TAGT (see Supplementary Fig. S1, available in JGV Online).
Pullen & Friesen (1995) showed that mutation of this base
in the AcMNPV ie-1 promoter reduced gene expression
dramatically.
The hr sequences have been shown in numerous studies
to be important as origins of DNA replication and as
transcriptional enhancers (reviewed by Friesen, 1997; Lu
et al., 1997). Comparing v90/2 and v90/4, hr1, hr3 and hr4
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all show sequence variation but, interestingly, no changes
were observed in hr2 (Fig. 2). Relative to v90/2, the v90/4
hr3 has a deletion of 78 bp and hr4 has an insertion of
76 bp, both of which represent a single hr repeat unit. This
is similar to what was observed in hr elements of fieldisolated variants of Spodoptera exigua multiple NPV
(SeMNPV) (Muñoz et al., 1999). An alignment of hr4
sequences of v90/4 and v90/2 shows the significant changes
that can occur in the hr elements (Fig. 4). The v90/4 hr4,
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Journal of General Virology 86
Comparative genomic analysis of MacoNPV-A
Fig. 3. Comparison of the sequences in the 1165 bp deletion/insertion region between MacoNPV-A v90/4 and v90/2. The
v90/2 1165 bp fragment, missing in v90/4, contains a single bro-a gene (shown as the grey arrow) and a portion of orf20. At
the ends of the 1165 bp fragment, there are two different palindromic sequences, TCTAATTAGA and AAATATTT
(underlined). The identical sequences between v90/4 and v90/2 are linked by vertical lines, whilst nucleotides missing in
either v90/4 or v90/2 are represented by dashes.
in addition to the 76 bp insertion, also contains five deletions (totalling 20 bp) and 33 point mutations. The hr1 and
hr3 elements have eight and 17 point mutations, respectively, relative to v90/2.
mixture of genotypes in the original MacoNPV field isolate. MacoNPV-A v90/4 appears to represent approximately 25 % of the mixed genotype population, as measured
by cloned fragment pools.
Sequence polymorphism in the MacoNPV-A
genome
Variations in structural protein genes
In the process of assembling the v90/4 sequence from
shotgun clones, clear alternative base readings or polymorphisms were occasionally observed for specific positions.
After ruling out sequencing errors by close examination of
all related electropherograms, 214 sequence polymorphisms
were detected. As shown in Table 3, the majority (186) of
the polymorphisms occurred in ORF regions, but they
only caused 47 amino acid polymorphisms in 26 ORFs.
Among the ORFs that contain polymorphisms, 14 are
homologous to known genes. These include the potential
structural protein genes gp37, gp41, 91k, vef and odv-e66 and
the viral DNA replication- and transcription-associated
genes lef-8 and ae, as well as homologues of fgf, cg30, hoar,
bjdp, bro-d and bro-e.
The MacoNPV field isolate from which v90/4 is derived
was determined to be heterogeneous, based on REN analysis showing submolar ratios of some REN fragments. In
an attempt to determine whether v90/4 is representative
of the genotypes within the heterogeneous field isolate,
selected HindIII fragments were cloned and sequenced. The
sequences of HindIII fragment 3051–4547 clones that were
taken directly from the field isolate fell into two groups.
Two clones had sequences identical to that of MacoNPV-A
v90/4 and the remaining six clones were of a genotype
that differed from both v90/4 and v90/2 (Fig. 5). Similarly,
two of eight HindIII fragment 67365–70629 clones were
identical to MacoNPV-A v90/4 sequence, with the remaining six clones representing two additional genotypes (5 : 1).
The sequence data indicate that MacoNPV-A v90/4 is an
authentic genotype that was found in the heterogeneous
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Seven genes encoding known structural proteins contain
variations in amino acid sequences between v90/4 and v90/2
or have amino acid sequence polymorphisms in v90/4.
Among these genes is the viral enhancing factor gene (vef).
VEF is a metalloprotease that is known to enhance viral
infectivity and is present in the viral occlusion bodies of
granuloviruses and a few NPVs. MacoNPV-A VEF is 847 aa
in size and has been shown to enhance infection of AcMNPV
(Li et al., 2003). There are two amino acid substitutions
in the putative VEF protein of v90/4 relative to v90/2, both
occurring in the C-terminal region. At aa 758 and 779,
v90/2 has an asparagine and a threonine, whereas v90/4
has an aspartic acid and an alanine, respectively (Table 1).
In addition, v90/4 VEF has three amino acid polymorphisms, an asparagine to aspartic acid and two leucine to
phenylalanine polymorphisms, at aa 536, 800 and 804,
respectively (Table 3). As these variations occur outside the
known functional domain region of VEF, it is unknown
whether these amino acid residues are important for
activity.
Two additional genes encoding structural proteins, p87/
vp80 (orf82) and odv-e66 (orf144), have amino acid sequence
substitutions in v90/4 relative to v90/2 (Table 1). P87/VP80
has a single substitution at aa 327, an alanine in v90/2 and
serine in v90/4. In v90/4, ORF144 (ODV-E66) has a single
substitution at aa 36, where a lysine is replaced by an
asparagine, and a leucine to isoleucine polymorphism at aa
405. ORF78 is a second ODV-E66 homologue. It contains a
threonine to isoleucine polymorphism and an alanine to
serine polymorphism at aa 407 and 451, respectively. It is
notable that all of the substitutions and polymorphisms
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Table 1. Amino acid sequence variations between v90/2 and v90/4
-, Deletion of amino acid residues; (+), conserved mutations as defined by
ORF Name
5
7
8
10
15
16
18
me53
xe
lef-7
19
20
21
23
bro-a
24
bro-b
26
28
29
30
96
Sequence variation
Position
v90/2
v90/4
49
83
119
288
6–8
251
11
45
154
183
10–12
111
135
151
L
E
D
A
DRR
G
A
D
K
E
IIT
E
M
D
S
K(+)
Y
T
AVG
E
P
N
E(+)
D(+)
V(+)FI
V
I(+)
Y
49
51
74–82
31
34
39, 40
45–47
51, 52
54–57
59
61–63
64–93
96–106
109–111
113–116
169
174, 175
177, 178
186
207
246
249
140
151, 152
268
10
140
155
220
22
80–83
86
28
50
R
H
P
S
CFYFELYL FI(+)QI(+)
LF(+)KY
S
P
R
K(+)
K, A
V, L
VAT
I(+)WS(+)
FQ
EP
QRFW K(+)K(+)NL
E
K(+)
KSY
Q(+)PF(+)
----------TSL
VT(+)S
VQAK L(+)HPQ(+)
T
A
VI
AV(+)
SV
A(+)A
P
L
M
I(+)
D
E(+)
E
Q(+)
Y
C
SK
PE(+)
Q
K(+)
Q
E(+)
R
K(+)
T
I
L
P
E
K(+)
KDET
----D
A
M
I
N
D(+)
BLAST
ORF size Identity ORF
(90/2, 90/4) (%)
terms.
Name
Position v90/2
98
319, 330
99
94
99
99
99
98
97
175, 65
95
85, 82
88
372, 331
90
31
bro-c
32
34
35
40
45
55
56
59
he65
61
98
98
110, 106
95
214, 213
90
63
64
66
70
72
80
82
89
Sequence variation
lef-1
pkip
52
54
106
164–166
168–170
172
175, 176
179
181
184
186
193
205
210
142
148
207
211
213
219
230
235–237
239
247, 248
91
87
77
99
165, 166
83
283
88/89
112
142
147
109
114
154
159, 160
192
Q
L
G
VSN
FAC
N
YA
P
E
S
T
N
V
E
K
K
K
Q
L
M
N
ALT
E
KI
S
K
R
F
KQ
S
T
----E
I
D
T
D
R
HY
R
v90/4
-F
D
DIQ
VVR
I
LP
E
D(+)
A(+)
I
S(+)
I(+)
D(+)
E(+)
R(+)
Q(+)
K(+)
M(+)
T
G
EM(+)K
Q(+)
NV
N(+)
R(+)
K(+)
L
NK(+)
F
A
EEEE
-V(+)
N(+)
I
N
K(+)
----G
9
T
-sod
120
E
K(+)
105, 106 SM N(+)I(+)
146
Q
K(+)
185
I
V(+)
280
Q
R(+)
207
E
D(+)
p87/vp80
327
A
S(+)
vef
758
N
D(+)
779
T
A
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ORF size Identity
(90/2, 90/4) (%)
97
338, 341
99
99
99
99
98
99
99
98
225, 223
98
120, 145
100
99
99
99
168, 169
99
99
99
99
Journal of General Virology 86
Comparative genomic analysis of MacoNPV-A
Table 1. cont.
ORF
Name
Sequence variation
Position
90
bro-e
96
102
108
118
123
124
bro-g
lef-9
126
p94
tlp20
350
355
172
138
156
20
146
301
341
467
161
202
v90/2
v90/4
T
K
V
S
E
L
S
Y
I
G
Y
E
LI
Q(+)
A
F
G
V(+)
F
H(+)
M(+)
V
H(+)
D(+)
ORF size
(90/2, 90/4)
Identity
(%)
360, 361
99
ORF
99
99
99
99
99
99
127
130
141
144
147
99
154
162
appear at the C termini of the ODV-E66 proteins and do not
occur in any of the predicted transmembrane domains or
the nuclear-targeting signal (Hong et al., 1994).
In addition to VEF and ODV-E66, amino acid sequence
polymorphisms also occur in the putative products of gp37,
gp41 and 91k in v90/4 (Table 3), but none of the amino
acid changes occur in regions that have known or predicted function. The 91k gene contains a polymorphism
that causes an insertion of a single serine residue at aa
660/661 in one of the two putative translation products.
The gp41 gene, which encodes an ODV-specific protein,
contains a C to T polymorphism, changing a glutamine
codon to a stop codon. This will result in two different
proteins potentially being produced, one that is 333 aa in
size and is identical to that of v90/2 and a second that is
146 aa, with the C-terminal sequence truncated. If translated, the smaller protein may have altered or antagonistic
functions relative to the full-length GP41. Olszewski &
Miller (1997) showed that a single base mutation in the
C terminus of gp41 was responsible for a temperaturesensitive mutation that inactivated GP41 in AcMNPVinfected cells. Loss of functional GP41 resulted in the
inhibition of virus production.
Name
bro-h
lef-8
odv-e66
ie-1
Sequence variation
Position
v90/2
v90/4
425
492
585
20
65
545
36
42
84
62
26/27
K
I
Q
G
E
S
K
T
S
I
--
R(+)
M(+)
K(+)
A
K(+)
N(+)
N
I
N(+)
V(+)
D
ORF size
(90/2, 90/4)
Identity
(%)
99
99
99
99
98
606, 607
99
99
amino acid substitutions relative to v90/2 LEF-9: tyrosine
to histidine, isoleucine to methionine and glycine to valine
at aa 301, 341 and 467, respectively (Table 1). LEF-8 has a
single serine to asparagine substitution between v90/2 and
v90/4 at aa 545, which is in a less conserved region of this
protein. In addition, v90/4 LEF-8 has an arginine to cysteine
polymorphism and an asparagine to threonine polymorphism at aa 364 and 632, respectively. LEF-1 and LEF-7 have
single amino acid sequence substitutions, from arginine to
lysine and aspartic acid to asparagine, at aa 77 and 45,
respectively, between v90/2 and v90/4 (Table 1). In view
of the importance of these genes in viral DNA replication
and/or transcription, these changes could potentially affect
virus replication. Kamita & Maeda (1997) reported that
mutation of two adjacent nucleotides in the AcMNPV
helicase gene, which causes a single amino acid change,
resulted in host-range expansion (Argaud et al., 1998;
Kamita & Maeda, 1997).
Variations in DNA replication and transcription
regulatory genes
In v90/4 IE-1, a single aspartic acid residue is inserted at aa
26 compared to v90/2, which is within the acidic activation domain. The insertion increases the acidic nature of
this domain, possibly increasing the transactivation potential of this region. In addition, AE, an exo-alkaline nuclease
that is hypothesized to be involved in the processing of
DNA replication intermediates (Li & Rohrmann, 2000), has
a glutamine to histidine polymorphism at aa 273 in v90/4.
Among the v90/4 homologues of viral genes that are
involved in DNA replication and transcription, ie-1, lef-1,
lef-7, lef-8 and lef-9 contain sequence variations relative to
v90/2 [Table 1, Supplementary Fig. S2 (available in JGV
Online)]. In transient assays, AcMNPV IE-1 and LEF-1 are
required for viral DNA replication (Kool et al., 1994),
whereas LEF-7, LEF-8 and LEF-9 are required for late gene
expression (Lu & Miller, 1995). LEF-1 has been characterized as a primase (Mikhailov & Rohrmann, 2002). AcMNPV
LEF-8 and LEF-9 are subunits of the viral RNA polymerase
II complex (Guarino et al., 1998). v90/4 LEF-9 has three
The me53 and cg30 genes are putative transcription regulatory genes, but their actual function during the baculovirus life cycle has yet to be determined. Homologues of
me53 are conserved in all lepidopteran baculoviruses that
have been sequenced to date. Both ME53 and CG30 contain
RING finger and leucine zipper domains that are found in
other polypeptides known to be involved in gene regulation
(Knebel-Mörsdorf et al., 1993; Thiem & Miller, 1989). The
ME53 homologue in v90/4 has a single alanine to threonine
substitution at aa 288 relative to v90/2, which is within the
conserved RING finger domain region. However, the amino
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Table 2. Changes in promoter regions between v90/4 and v90/2
ORF
4
5
6
8
16
18
22
23
24
Name
hoar
odvp-6e
lef-7
chit
bro-b
25
27
28
29
30
31
50
52
54
57
59
61
62
63
68
77
90
91
97
103
116
117
127
128
129
130
139
141
142
143
147
149
150
157
158
159
bro-c
ae
xe
bro-e
bro-h
iap3
lef-8
lef-11
39k
p10
Change*
PromoterD
ATTTTAAAAGT>278
A>271
A277G
T237A
T245A
G2141T
GA279,78AC, T274C
A249G
G2114A, CT2110,109TG, G239T, T237A, TACTT234–230-,
G228A, G226A, A223T, T215A, A210T, A28T
T274C, 260-, A233G, C222T, A29T
G2135C, A2131G, A2100G, G255A, G253T, A251G,
CC248,247TT, GTA229–227CAC
TAC281–279GTG, T257C, C255A, C253T, T28C
T2125C, A269T, T261C
AG2128,2127GA, A2125G, G2123A, T2121C, CC222,221TT
A2109G, C2107T, T2105C, CT2103,2102TC, G278A, A263G
C229T, C227G, G224A
C2129T
G294A
A2110G
C2146T, T265C
T218C, A210G
A276G
A2128G, T224A
C2122T
A211C
C231T
T2129G, C25T
T273C
C26T
G2145A, T2130A, A2109C
G2109A
G2105A
C285T
C285T
A294G
C2137T, A23C297G
AA223,222TT
T280C
A2113G, C257T, G225A
T257C
A223G
T231C
A285G
T2132C
Promoter motifsd
E
L
274-TATAA, 245-CAGT
263-GTAAG
E
L
260-TATAAA, 227-CAGT
218-ATAAG
E
249-TATAAA
E
277-TATAAA, 251-CAGT
E
273-TATAAA, 251-CAGT
E
243-TATAA, 237-CATT
E
262-TATATA, 226-CAGT
L
L
L
242-ATAAG
236-ATAAG
226-ATAAG
L
2128-ATAAG, 280-GTAAG
L
L
L
L
2148-ATAAG, 2102-ATAAG
244-TTAAG
219-ATAAG
239-ATAAG
*Numbers represent the location of the nucleotide changes in each promoter region; letters in front of the numbers are sequences present in
v90/4; letters behind the numbers are sequences present in v90/2; sequences in front of ‘>’ are insertions and ‘-’ behind the numbers indicates
deletions in v90/4 relative to v90/2.
DE, Early; L, late.
dNumbers indicate the locations of motifs in each promoter region.
98
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Journal of General Virology 86
Comparative genomic analysis of MacoNPV-A
Fig. 4. Nucleotide alignment of the hr4 sequences of v90/4 (nt 134258–135356) and v90/2 (nt 135725–136764), showing
multiple point mutations and insertion/deletions. The large 76 bp insertion in v90/4 hr4 represents a single repeat unit.
Individual repeat units are separated by slashes. Identical sequences between v90/4 and v90/2 are linked by vertical lines,
whilst nucleotides missing in either v90/4 or v90/2 are represented by dashes.
acid at this site is divergent among baculoviruses, so it is
unlikely that this will alter the function of the RING finger.
The v90/4 CG30 has three amino acid polymorphisms:
aspartic acid to asparagine, glutamic acid to aspartic acid
and serine to glycine at aa 147, 154 and 158, respectively (see
Table 3).
bro gene variations
There are seven and eight bro genes in the v90/4 and v90/2
genomes, respectively. As described above, bro-a is the sole
ORF that exists in v90/2 but is missing in v90/4. v90/2 BROA shows low sequence identity to other BROs in the v90/2
and v90/4 genomes, with 27 % identity to v90/2 BRO-C
being the highest. Other proteins related to v90/2 BRO-A
are Spodoptera litura NPV (SpltNPV) BRO-B (27 %),
Xestia c-nigrum granulovirus (XecnGV) BRO-A (25 %),
XecnGV BRO-F (24 %), Helicoverpa armigera single NPV
(HearSNPV) BRO-C (25 %), Bombyx mori NPV (BmNPV)
BRO-C (23 %) and BmNPV BRO-B (23 %). As shown in
Fig. 2, bro-a, bro-b and bro-c are located in the most highly
variable region of the MacoNPV-A genome. In addition to
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missing bro-a, v90/4 bro-b and bro-c have 73 and 55 point
mutations, respectively (see Supplementary Fig. S3, available in JGV Online). The nucleotide point mutations in
these two bro genes account for 25 % of the point mutations
in the whole genome.
All of the bro genes except for bro-d and bro-f contain
mutations that cause amino acid substitutions (Table 1).
BRO-B has 41 aa deleted between aa 65 and 105, as well as
32 amino acid substitutions; BRO-C has 13 amino acid
substitutions; and BRO-E, -G and -H each have a single
amino acid substitution. Polymorphisms also cause amino
acid variation in BRO-E and -F (Table 3). These variation
levels suggest strongly that bro genes, especially bro-a, -b
and -c, are hot spots for MacoNPV-A mutations and
genome variation.
Additional ORFs with amino acid sequence
mutations or polymorphisms
The other known genes whose encoded protein sequences
vary between v90/4 and v90/2 or have polymorphisms in
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Table 3. Sequence polymorphisms in the MacoNPV-A (v90/4) genome
int, Intergenic (not including hr elements); :, nucleotide deletion; -, deletion of an amino acid residue; *, stop codon. Numbers between
two letters in the amino acid variation columns indicate the positions where the amino acid polymorphisms occurred in the predicted gene
products.
ORF
(name)
Position
4 (hoar)
4471
4549
4605
4705
8522
8974
9026+1
9030
9034
9073
11732
18272
18489
22787
22999
23002
23012
23327
23369
23390
32936
33064
33068
33107
33178
33269
33272
37183
37267
37268
38864
43371
43389
43450
43466
43593
44951
44953
44956
45133
45229
45242
45248+1
45248+2
45339
45479
45481
45557
7
int
8
10
19
26
27
37 (gp37)
42
48
int
49
50
int
int
int
int
51 (fgf)
100
Variation
nt
A/G
T/A
T/C
C/G
T/C
A/T
:/A
T/A
T/:
T/C
T/C
A/G
C/A
G/A
C/T
G/A
G/A
G/A
G/A
T/C
C/T
C/T
C/T
C/T
C/A
C/T
G/A
G/A
G/A
G/A
T/A
C/G
C/A
T/C
T/A
C/T
C/T
C/G
G/A
T/C
T/:
T/C
:/T
:/T
G/A
A/C
C/G
T/G
aa
ORF
(name)
M217V
51
E251G
Y129C
C140F
P211S
E212K
R215H
52
53
54 (ae)
S143L
56
Q181K
int
59
60
T750I
E218V
Q418E
75
S25T
P67L
78 (odv-e66)
79
80
L298V
R297P
N275T
Position
45579
45606
45703
45871
45997
46180
46236
46243
46297
46360
46544
46771
47077
47499
47509
47689
47734
47817
47941
47977
48025
48112
49809
49902
50040
50280
52492
52812
52893
53172
53180
53295
65102
65147
67187
67212
67262
67358
68203
68334
68666
68930
69098
69104
69943
69944
70019
70334
Variation
nt
aa
A/G
G/A
C/T
G/T
A/G
G/A
A/G
A/T
G/A
G/A
C/T
T/C
A/G
C/T
A/G
A/T
C/G
A/G
T/C
T/C
T/C
C/T
C/T
C/T
G/A
A/G
C/T
G/A
A/G
T/G
G/A
G/A
T/C
T/A
G/A
A/C
A/G
C/T
C/T
G/T
G/A
G/T
T/C
T/A
G/A
G/A
G/A
A/G
L268V
P259S
ORF
(name)
82
83
89 (vef)
89
A352S
90 (bro-e)
H273Q
int
91
int
92
95
int
100 (cg30)
T407I
A451S
R69S
H151Y
101 (91k)
102
104 (gp41)
106
111
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Position
Variation
nt
71336
72022
72128
72583
72631
72754
77387
77549
77723
77717
78182
78327
78626
79121
79131
79279
79290
79304
79308
79309
79310
79981
79984
80001
80332
80386
80548
80567
80746
80870
81277
86167
90037
90499
90522
90532
90654
90723
91407
91410
91415
91416
91419
92273
93776
94905
96263
100124
C/T
G/A
C/T
C/T
T/G
G/A
C/T
G/A
T/C
C/A
A/G
A/G
C/T
C/G
T/C
C/T
T/G
A/G
G/:
T/:
A/:
C/G
A/G
T/C
A/G
A/G
A/G
C/T
A/G
A/G
C/T
C/T
G/A
G/A
C/A
A/G
A/G
G/A
T/C
T/C
A/:
T/:
G/:
G/A
T/C
C/T
A/G
A/G
aa
C136W
D536N
F800L
F804L
Q356K
I351T
L350-
S119G
R100Q
D147N
E154D
S158G
S661-
Q147*
V194A
Journal of General Virology 86
Comparative genomic analysis of MacoNPV-A
Table 3. cont.
ORF
(name)
115
int
int
int
116
117
118
119
122 (bro-f)
hr3
int
140 (bjdp)
141 (lef-8)
Position
Variation
nt
100125
107315
107441
107480
107481
107482
107676
107880
108001
108022
108037
108042
108055
108952
109289
110959
110961
123281
123304
123976
125057
125247
126182
127368
A/G
A/G
C/T
C/:
A/:
A/:
G/A
A/G
G/T
T/A
T/C
G/A
A/T
T/C
T/C
G/A
T/C
C/T
G/A
C/G
A/T
A/T
G/C
C/T
ORF
(name)
aa
I297V
L338M
T26S
R364C
int
int
int
int
int
142
int
int
144 (odv-e66)
148
v90/4 include homologues of fgf, p94, lsxe, pkip, tlp-20, sod,
hoar and bjdp (Tables 1 and 2). Homologues exist in all
of the sequenced lepidopteran baculovirus genomes for
the predicted product of AcMNPV fgf, which is similar
to fibroblast growth factors (Ayres et al., 1994). It was
proposed that the expression of baculovirus FGF might
facilitate the infection of tracheal cells, which serve as
conduits for establishment of systemic AcMNPV infection
(reviewed by Hayakawa et al., 2000). The v90/4 FGF
contains five amino acid polymorphisms (Table 3).
AcMNPV p94 was hypothesized to be associated with the
triggering of apoptosis induced by viral infection (Clem
et al., 1994). The v90/4 P94 homologue has five amino acid
substitutions relative to v90/2; of note is a methionine
residue substituted for a conserved isoleucine residue at aa
492 (Table 1).
In addition to the above ORFs, 28 other ORFs that have
not been characterized or have no predicted function have
mutations resulting in amino acid substitutions in v90/4
relative to v90/2 (Tables 1 and 2). The most variable protein
observed was ORF30, which shows 12 % sequence variation.
This ORF, previously described as a unique MacoNPV ORF
(Li et al., 2002b), is homologous to SpltNPV ORF106 (31 %
amino acid sequence identity).
http://vir.sgmjournals.org
Position
Variation
nt
128123
128173
128192
128459
128597
128630
128915
128916
128917
128918
128919
128920
128921
128927
128937
129238
129422
129423
129724
130138
130491
136072
136237
136393
G/T
A/C
T/C
T/C
G/A
G/A
A/:
T/:
A/:
G/:
A/:
C/:
A/:
C/T
T/C
C/T
A/T
A/T
G/A
T/C
G/T
T/C
C/T
C/A
aa
N632T
ORF
(name)
156
int
158
int
160
162
I54M
int
164
165
L405I
167
168
Position
Variation
nt
136625
140772
140790
140826
140883
141135
141176
141942
142281
142747
143045
145423
145509
145635
145726
145738
147561
147752
147999
148011
150381
150455
aa
C/T
T/A
T/C
T/C
C/T
C/T
A/G
C/T
C/T
A/G
T/C
G/A
G/A
A/G
A/G
G/A
T/:
G/A
G/A
A/G
G/A
C/T
DISCUSSION
The MacoNPV-A viruses v90/4 and v90/2 were originally
isolated from M. configurata larvae in relatively close
geographical proximity during the same outbreak of this
pest. The complete genome sequence of v90/4, which was
found to be at least ten times less virulent for this host, has
been determined and compared to that of the previously
sequenced v90/2 genome. The results showed that the
genome of v90/4 had 99?5 % sequence identity to that of
v90/2 and nearly identical gene content and arrangement.
Surprisingly, however, 49 ORFs were identified that contained nucleotide point mutations, insertions or deletions
resulting in amino acid substitutions, as well as one ORF
that was present in v90/2, but not in v90/4. These genetic
variations underlie the biological differences between these
isolates. This is the first study to determine the extent of
sequence variability between baculovirus genomes isolated
from the same naturally occurring field population.
Variability was not distributed evenly throughout the viral
genomes, as the sequence data show that the region with
the most mutations in v90/4 relative to v90/2 is located
between hr1 and orf31 (bro-c) (Fig. 2). This region contains
three genes, orf18, orf23 and orf30, that are unique to
MacoNPV-A and it does not contain any genes that are
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L. Li and others
102
Journal of General Virology 86
Fig. 5. Sequence comparison of clones from the MacoNPV Lamont field isolate. The HindIII fragment representing nt 3051–4547 was isolated and
cloned from genomic DNA extracted directly from the Lamont field isolate. Eight separate clones were sequenced and compared to the sequence of the
plaque-purified MacoNPV v90/4 and the archetypal sequence of MacoNPV v90/2. The eight Lamont clone sequences were of two types, two clones
having sequences identical to that of MacoNPV-A v90/4 (Lamont type 2 in the comparison) and six clones were of a genotype differing from both v90/4
and v90/2 (Lamont type 1 in the comparison). Green shading, no identity to either v90/4 or v90/2; red shading, identity only to v90/4; blue shading,
identity only to v90/2.
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Comparative genomic analysis of MacoNPV-A
conserved in all baculoviruses sequenced to date. Interestingly, we have shown previously that this region is also the
most variable region between MacoNPV-A and the closely
related species MacoNPV-B (Li et al., 2002a). Even when
compared with more distantly related baculoviruses, this
region appears to be more variable. For example, the gene
arrangement of SeMNPV is highly collinear to that of v90/2;
however, comparison of the two genomes shows that the
relocation and inversion of a cluster of ORFs, as well as
various insertions and deletions, have occurred in this highly
variable region (Li et al., 2002b). In addition, an SeMNPV
mutant with a single deletion of 25 kb encompassing orf15–
orf41, a region homologous to MacoNPV-A orf16–orf54, can
be isolated routinely in cell culture (Heldens et al., 1996).
The SeMNPV deletion mutant did not cause host larval
mortality or morbidity, suggesting that the 25 kb deletion
contains information that is critical for virus virulence
in vivo. MacoNPV homologues of SeMNPV genes in this
region that vary between v90/4 and v90/2 include lef-7,
he65, orf34 and orf40. In addition, lef-7, chitinase, orf25,
orf50 and orf54 contain variations in their upstream promoter regions that could potentially affect gene expression.
The largest single difference between v90/4 and v90/2 is the
deletion of the bro-a gene. The bro genes are a family of
ORFs with sequence homology to AcMNPV orf2 that have
been identified in a number of baculoviruses. Lymantria
dispar multiple NPV (LdMNPV) has 16 bro-related genes
(Kuzio et al., 1999). AcMNPV, Epiphyas postvittana NPV,
Orgyia pseudotsugata NPV, BmNPV, HearSNPV, Helicoverpa
zea single NPV (HzSNPV), SpltNPV, Culex nigripalpus NPV,
Cydia pomonella granulovirus and XecnGV have between one
and seven bro genes (Afonso et al., 2001; Ahrens et al., 1997;
Ayres et al., 1994; Chen et al., 2001, 2002; Gomi et al., 1999;
Hayakawa et al., 1999; Hyink et al., 2002; Luque et al., 2001;
Pang et al., 2001). SeMNPV was previously reported to lack
a bro gene, but the SeMNPV ORF13 was recently reported
to be a BRO homologue of MacoNPV-A bro-g (IJkel et al.,
1999; Li et al., 2002b). Only Plutella xylostella granulovirus
has been found to lack a bro gene homologue (Hashimoto
et al., 2000).
The BmNPV bro genes are transcribed as delayed-early
genes. Functional studies suggested that BmNPV bro-a, -c
and -d are essential for viral infection, but bro-a and bro-c
could complement each other functionally (Kang et al.,
1999). BmNPV BRO-A, -C and -D have nucleic acidbinding activities and are located in nucleoprotein complexes in the nuclei of infected cells. It has been proposed
that BRO-A and -C may influence host DNA replication
and/or transcription (Zemskov et al., 2000). The BRO N
domain, proposed to be a DNA-binding domain, has
recently been shown to be homologous to a family of
proteins from other viruses, bacterial phages and bacteria
(Iyer et al., 2002). Based on the apparently essential nature
of some of the BmNPV bro genes, it is possible that the
virulence difference that we observed between v90/4 and
v90/2 may be due to the presence or absence of bro-a.
http://vir.sgmjournals.org
AcMNPV (C6) was originally reported as having only a
single bro gene (Ayres et al., 1994). However, another bro
gene was later identified in four AcMNPV variants isolated
from Galleria mellonella, S. exigua, S. litura and X. c-nigrum
(Yanase et al., 2000). In the variant isolated from S. litura,
the second bro gene is contained within a 1?1 kb insert
between AcMNPV orf30 and orf31. We have also previously
reported that bro genes were found to be associated with a
region of the MacoNPV-B genome (orf 53–orf 58) that was
possibly derived by recombination with a distantly related
virus, XecnGV (Li et al., 2002a). In this study with
MacoNPV-A, three bro genes (-a, -b and -c) were found
to be located in the most highly variable region of the
genome, with bro-b and bro-c containing 25 % of all point
mutations in the v90/4 genome relative to that of v90/2.
Alignments of the homologous bro genes of MacoNPV-A
v90/2 and MacoNPV-B showed an average of 81 % sequence
identity, which is much lower than the overall 87?6 %
sequence identity between the two genomes (Li et al.,
2002a). A high degree of sequence variability has also been
observed among the bro genes of other viruses. The
sequences of HearSNPV and HzSNPV have recently been
reported and they have been shown to be variants of the
same virus species. Interestingly, the most divergent ORFs
between these two viruses are two bro genes (Chen et al.,
2002). In BmNPV, among the five bro genes, bro-d is
related most closely to AcMNPV orf2, with 80 % amino acid
sequence identity. This is much lower than the average
identity level of predicted proteins from these two viruses,
which is 93 % (Gomi et al., 1999). These data indicate that
bro genes are highly variable relative to other baculovirus
genes. Furthermore, where multiple bro genes exist within
a baculovirus, they are usually highly divergent amongst
themselves, as has been shown in MacoNPV-A v90/2 and
LdMNPV (Kuzio et al., 1999; Li et al., 2002b). It is likely
that the highly variable bro genes are functionally different
genes. Overall, the results of this study and those described
above suggest strongly that bro genes are associated with
baculovirus genome variation, but the reasons for this
association are yet to be determined.
Analysis of the v90/4 genome identified 214 nucleotide
polymorphisms. Sequence polymorphisms are common
in genomes of humans, viruses and other organisms and
contribute to important phenotypic diversities. This is the
first paper to report all polymorphisms for an entire baculovirus genome. The v90/4 viral DNA used for sequencing was
from a plaque-purified clone that was amplified in larvae
of M. configurata. The presence of a pool of polymorphisms
may be a more efficient mechanism than direct substitution,
insertion or deletion in adapting to a changeable environment and may provide an evolutionary advantage by having
a population of variable genomes.
The results of this study describe the extensive sequence
variability that exists in natural baculovirus populations.
The sequence does not provide direct answers as to why
v90/4 is less virulent than v90/2, but suggests that the bro
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L. Li and others
genes are potentially involved and may play a significant
role in generating baculovirus diversity. In addition, the
data indicate that natural baculovirus populations represent a spectrum of genotypes that may have significant
ecological implications for the survival and adaptation of
these viruses in the face of potentially dynamic changes
in host species subpopulations, changing climatic conditions and/or availability of alternative host species.
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ACKNOWLEDGEMENTS
Guarino, L. A., Xu, B., Jin, J. & Dong, W. (1998). A virus-encoded
The technical assistance of Alison Paton and Keith Moore (AAFC,
Saskatoon Research Centre) for insect rearing and MacoNPV
bioassays, respectively, is greatly acknowledged. The support of
Dr Jim Brandle (DNA Sequencing Laboratory), AAFC, Southern
Crop Protection and Food Research Centre (London, ON, Canada)
and the excellent technical contributions of LouAnn Verellen are
gratefully acknowledged.
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