Virus Genes (2013) 47:133–151 DOI 10.1007/s11262-013-0922-2
Complete genomic sequences and comparative analysis of Mamestra brassicae nucleopolyhedrovirus isolated in Korea Jae Bang Choi • Won Il Heo • Tae Young Shin • Sung Min Bae Woo Jin Kim • Ju Il Kim • Min Kwon • Jae Young Choi • Yeon Ho Je • Byung Rae Jin • Soo Dong Woo
•
Received: 7 February 2013 / Accepted: 15 May 2013 / Published online: 28 May 2013 Ó Springer Science+Business Media New York 2013
Abstract Mamestra brassicae nucleopolyhedrovirus-K1 (MabrNPV-K1) was isolated from naturally infected M. brassicae (Lepidoptera: Noctuidae) larvae in Korea. The full genome sequences of MabrNPV-K1 were determined, analysed and compared to those of other baculoviruses. The MabrNPV-K1 genome consisted of 152,710 bp and had an overall G ? C content of 39.9 %. Computer-assisted analysis predicted 158 open reading frames (ORFs) of 150 nucleotides or greater that showed minimal overlap. Two inhibitor of apoptosis (iap) and six baculovirus repeated ORFs were interspersed in the MabrNPV-K1 genome. The unique MabrNPV-K1 ORF133 was identified in the MabrNPV-K1 genome that was not previously reported in baculoviruses. The gene content and arrangement in MabrNPV-K1 had the highest similarity with those of Helicoverpa armigera MNPV
(HearMNPV) and Mamestra configurata NPV-B (MacoNPVB), and their shared homologous genes were 99 % collinear. The MabrNPV-K1 genome contained four homologous repeat regions (hr1, hr2, hr3 and hr4) that accounted for 3.3 % of the genome. The genomic positions of the four MabrNPV-K1 hr regions were conserved among those of HearMNPV and MacoNPV-B. The gene parity plot, percent identity of the gene homologues and a phylogenetic analysis suggested that these three viruses are closely related not only to each other but also to the same virus strains rather than different virus species. Keywords Baculovirus Nucleopolyhedrovirus Mamestra brassicae NPV-K1 Genomic sequence Comparative analysis
Introduction J. B. Choi W. I. Heo T. Y. Shin S. M. Bae S. D. Woo (&) Department of Agricultural Biology, College of Agriculture, Life and Environment Sciences, Chungbuk National University, Cheongju, Republic of Korea e-mail:
[email protected] W. J. Kim Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea J. I. Kim M. Kwon National Institute of Highland Agriculture, RDA, Pyeongchang, Republic of Korea J. Y. Choi Y. H. Je Department of Agricultural Biotechnology, College of Agriculture and Life Sciences, Seoul National University, Seoul, Republic of Korea B. R. Jin College of Natural Resources and Life Science, Dong-A University, Busan, Republic of Korea
Baculoviruses belong to the Baculoviridae family, which are rod-shaped, arthropod-specific viruses with covalently closed, double-stranded and large circular DNA genomes of 80–180 kb [1]. The family Baculoviridae recently had a proposed revision into four genera, i.e. Alphabaculovirus (lepidopteran-specific nucleopolyhedroviruses (NPVs)), Betabaculovirus (lepidopteran-specific granuloviruses (GVs)), Gammabaculovirus (hymenopteran-specific NPVs) and Deltabaculovirus (dipteran-specific NPVs), based on molecular phylogeny and host insects [2]. Lepidopteran NPVs can be further classified into two sub-groups, I and II, based on their envelope fusion proteins and polyhedrin gene, which are essential for the spread of infection in the insect and are required for efficient virus budding [3, 4]. GP64 exists only in Group I NPVs and is utilised as an envelope fusion protein, whereas Group II NPVs lack GP64 and has the F (fusion) protein [5]. NPVs form a distinguishable occlusion body
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called a polyhedron, containing virions with nucleocapsids, and the single-nucleocapsid (S) or multiple-nucleocapsid (M) NPVs are designated according to the number of nucleocapsids packaged in the virion. Presently, full genome sequences of more than fifty baculovirus isolates have been determined [6]. Baculoviruses have mostly been investigated among insect viruses because of their potential for use as biological control agents of agricultural and forest pests, protein expression vectors and biotechnological tools [7]. The cabbage armyworm, Mamestra brassicae, is an important insect pest of numerous vegetables and ornamental plants worldwide [8]. Although there have been many efforts, controlling them is difficult because they rapidly develop resistance to chemical insecticides. To overcome this limitation, the isolation and characterisation of several NPVs from M. brassicae have been reported [9]. Until now, studies on M. brassicae NPVs (MabrNPVs) have focused on investigating the pathogenicity and characteristics of some major viral genes. The variations of susceptibility and virulence between MabrNPV, Helicoverpa armigera MNPV (HearMNPV) and M. configurata NPV (MacoNPV) were evaluated for several related insect hosts, and these studies showed that these viruses are closely related in terms of biological and biochemical characteristics [9–12]. The gene and genomic hybridisation analyses showed also the close relationship between these viruses [12]. In addition, a comparison of the genomic analyses revealed the full viral DNA sequences of HearMNPV and MacoNPV showed the high degree of similarity between these two viruses by high overall gene content and arrangement [13]. From these results, it was carefully suggested that the HearMNPV, MacoNPV and MabrNPV are derived from same NPV species. However, further comparison of these NPVs at a genomic level could not be performed because the genome sequence of MabrNPV was unknown. Recently, we reported the isolation and various characteristics, including pathogenicity, of the MabrNPV that was isolated in Korea and given the name MabrNPV-K1 [14, 15]. In this study, the complete genomic sequence of MabrNPV-K1 was determined and analysed. A comparison of MabrNPV-K1 with the closely related viruses HearMNPV and MacoNPV-B at the genome level suggested that these viruses are closely related and that these virus strains are derived from the same ancestor.
Materials and methods Insect, virus and viral DNA isolation Mamestra brassicae larvae were maintained on a Chinese cabbage diet at 25 °C, 60 % relative humidity and an
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18L:6D photoperiod at the laboratory. The MabrNPV-K1 was propagated in third instar larvae of M. brassicae by oral infection of polyhedra. The MabrNPV-K1 was isolated in Korea and derived from a single larval cadaver from a single collection site in 2008 [14]. The viral isolate was amplified and cloned by the previously reported in vivo isolation method [16]. To purify viral polyhedra, the virusinfected cadavers were homogenised in 0.5 % sodium dodecyl sulphate (SDS) using a Bullet Blender (Nest advance Co, USA), and the homogenate was filtered through four sterilised cheesecloths, which were subsequently washed with washing buffer (50 mM Tris–HCl, pH 8.0, 10 mM EDTA, 5 % b-mercaptoethanol, 4 % SDS). To extract genomic DNA, the purified polyhedra were resuspended in 0.1 M sodium carbonate and incubated at 37 °C overnight with 0.5 % SDS and proteinase K (0.2 mg/ml). A further extraction was performed with phenol/chloroform and isoamyl-alcohol (24:1) and precipitated in 70 % ethanol. The DNA was resuspended in TE buffer [10 mM Tris–HCl, pH 8, 1 mM EDTA]. The quantity and quality of extracted DNA were determined by UV-spectrophotometer and electrophoresis in a 0.7 % agarose gel. Nucleotide sequence determination The complete nucleotide sequence of MabrNPV-K1 genomic DNA was determined using the 454 pyrosequencing method [17] from a sequencing company (Macrogen, Korea). A MabrNPV-K1 DNA fragment was used to prepare the 454 sequencing library using the GS DNA Library Preparation Kit (Roche, Switzerland). The MabrNPV-K1 genome was sequenced to 200-fold coverage using a 454 approach. The determined nucleotide sequences were assembled using the GS De Novo Assembler version 2.6 (http://www.454.com/products-solutions/analysis-tools/gsde-novo-assembler.asp). The sequence gaps of the resulting contigs were filled by PCR and Sanger sequencing. Sequence data analysis of the MabrNPV-K1 genome Sequence data were assembled and analysed using Lasergene7 software (DNASTAR). Putative open reading frames (ORFs) were analysed using the FGENESV0 (http://linux1. softberry.com/berry.phtml) and the NCBI ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). ORFs encoding more than 50 amino acids were designated as putative genes with minimal overlap. The NCBI web blast script was used to submit relevant ORFs to the GenBank. Relevant ORFs were checked for alignment with other known baculovirus gene homologues and compared using the standard protein–protein BLAST algorithm (http://blast.ncbi.nlm.nih. gov/Blast.cgi). Gene parity plot analysis was performed to compare the gene organisation in the MabrNPV-K1 genome
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Total RNA was isolated from infected M. brassicae larvae using RiboEx_column Kit (GeneAll, Korea). Reverse transcription polymerase chain reaction (RT-PCR) was performed using the following primers for Mb133-F; 50 -GTAAACTTCTCGGTAGCACGTG-30 , Mb133-R; 50 -T CAATGACAAACCGCTACGG-30 , MbPol-F; 50 -ATGTA TACCCGTTACAGTTAC-30 and MbPol-R; 50 -TTAA TAGGCGGGTCCGTTG-30 with a Maxime RT-PCR PreMix Kit (Intron biotechnology, Korea). The PCR reaction was carried out with an initialisation step (45 °C/30 min, 94 °C/5 min) and 30 amplification cycles (94 °C/30 s, 52 °C/30 s, 72 °C/1 min) in a TaKaRa PCR Thermal Cycler Dice Gradient (TaKaRa, Japan). The 50 -end was confirmed by rapid amplification of cDNA ends (RACE) using SMARTerTM RACE cDNA Amplification Kit (Clontech, United States). Following cDNA synthesis, PCR amplification was carried out using gene specific primer, 50 -AACCGCTACGGCAGCTGTTTGTTGTGCG-30 , and 50 -RACE anchor primer according to the manufacturer’s instruction.
genome sequence of MabrNPV-K1 is highly AT-rich, its G ? C content is 39.9 %, and it is 99.0 and 98.8 % identical to that of HearMNPV and MacoNPV-B, respectively. Conversely, Autographa californica MNPV (AcMNPV), Orgyia pseudotsugata MNPV (OpMNPV), Spodoptera exigua MNPV (SeMNPV) and Plutella xylostella GV (PlxyGV) had low similarity with MabrNPV-K1. The genomic DNA sequence of MabrNPV-K1 was deposited in GenBank under the accession number JQ798165.1. The genome size and nucleotide sequence identities of MabrNPV-K1 were most similar to those of HearMNPV and MacoNPV-B, which also have a similar host range with MabrNPV. The genome sequence of HearMNPV was reported recently [13] and shares the most homology with MacoNPV-B, but it was different from the previously reported HearSNPV sequence, which was isolated from the same host but has different host ranges with HearMNPV. MabrNPV-K1 has also similar host ranges as HearMNPV, and has little genomic similarity to HearSNPV. These results support the existence of a close relationship among MabrNPV-K1, HearMNPV and MacoNPV-B. A total of 158 putative ORFs of at least 50 codons in length with minimal overlap of larger ORFs or shared significant sequence identities with previously characterised baculovirus ORFs were identified (Fig. 1; Table 2). The adenine residue at the translation initiation codon of the polh gene (ORF1) represented the zero point on the MabrNPV-K1 genome map (Fig. 1). Table 2 shows the characteristics of the 158 MabrNPV-K1 ORFs, including the order, size, orientation and homology level to the genes of the previously reported eight NPV genomes. Fifty-four percent (85 ORFs) of the ORFs are oriented clockwise, and 46 % (73 ORFs) are oriented counterclockwise with respect to the orientation of the polh gene [20]. The directions of the transcripts are indicated by arrows (Fig. 1; Table 2). The MabrNPV-K1 genome had 4 hrs and 6 baculovirus repeated ORFs (bro), which was similar to the HearMNPV and MacoNPV-A and -B genomes. Almost 157 MabrNPV-K1 ORFs except ORF133 have an assigned function or homologues in other baculoviruses. The unique MabrNPV-K1 ORF133 had no similarity to previously reported Baculovirus ORFs. Thirty-five conserved genes in all Baculovirus genomes were also found [21].
Results and discussion
Comparison of the MabrNPV-K1 ORFs to other baculoviruses
with that of other baculoviruses using the chart program packaged in Microsoft Excel software. The Tandem Repeats Finder (http://tandem.bu.edu/trf/trf.html) and JDotter (http:// pgrc.ipk-gatersleben.de/jdotter) programs were used to locate homologous repeat regions (hrs).
Phylogenetic analysis A phylogenetic tree was generated using combined amino acid sequences derived from the pif-2, lef-8 and lef-9 genes of the 49 baculoviruses that had completely sequenced genomes at the time of analysis. Multiple amino acid sequence alignments were performed using Cygwin software (http://www.cygwin.com). Phylogenetic analysis was inferred using the neighbour-joining method with MEGA version 5.0 [18]. Culex nigripalpus (Cuni) NPV (GenBank accession no. NC_003084.1; [19]) was selected as the outlying group. Statistical support for each node was evaluated by bootstrap analysis with 2,000 replications.
RT-PCR and 50 RACE
Genome sequence analysis of the MabrNPV-K1 The circular MabrNPV-K1 genome consisted of 152,710 nucleotides, a size closest to HearMNPV when compared with other related and important NPVs (Table 1). The
The gene content and organisation of MabrNPV-K1 were compared with other baculoviruses using BLAST (Table 2). MabrNPV-K1 shared 157 ORFs with HearMNPV, 156 ORFs with MacoNPV-B, 155 ORFs with MacoNPV-A, 121 ORFs with SeMNPV, 110 ORFs with HearSNPV-G4,
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Table 1 Characteristics of baculovirus genomes Characteristic
MabrNPVK1
HearMNPV
HearSNPVG4
MacoNPVB
MacoNPVA
SeMNPV
AcMNPV
OpMNPV
PlxyGV
Size (bp)
152,710
154,169
131,405
158,482
155,060
135,611
133,894
131,995
100,999
G ? C content (%)
40
40
39
40
42
44
41
55
41
Coding sequence (%)
90
90
86
89
90
88
91
89
88
Total ORFs
158
162
135
168
169
139
154
152
120
Number of hrs
4
4
5
4
4
6
9
5
4
Number of bro
6
6
3
7
8
0
1
2
0
Identity of MabrNPVK1 (%)a
100
99.0
40.4
98.8
87.8
59.5
39.0
37.9
35.3
GenBank assession no.
JQ798165
EU730893
NC_002654
AY126275
U59461
NC_002169
NC_001623
NC_001875
NC_002593
a
Nucleotide sequence identity between two complete genomes
Fig. 1 Circular map of the 158 predicted ORFs of the complete MabrNPV-K1 genome. ORFs are represented by arrows with position and direction. Homologous repeat regions are represented by square boxes
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Virus Genes (2013) 47:133–151
137
Table 2 Features of the MbNPV-K1 predicted ORFs ORF no.
Name
Position
Length (aa)
Promoter
Homologous (ORF number, size, aa identity %) HearMNPV
HearSNPV-G4
MacoNPV-B
MacoNPV-A
1
1
246
100
1
2
512
99
2
1
polh
1 ? 741
246
L
1
246
100
2
orf1629
790 / 2328
512
L
2
522
96
3
pk1
2327 ? 3145
272
3
272
99
3
267
49
3
272
100
3
4
hoar
4
756
26
4
736
97
4
5
190
98
5
5
3216 / 5441
741
E
4
742
99
5956 ? 6522
188
E
5
188
99
246
87
6
odv-e56
6604 ? 7725
373
L
6
373
100
15
354
52
6
373
100
6
7 8
me53 Fusion protein
7868 / 8932 9537 ? 11573
354 678
L
7 8
354 678
99 99
16 133
359 677
30 39
7 8
354 678
100 99
7 9
9 10
gp16
11
p24
14
319
E
9
319
100
9
319
99
10
12696 / 12983
95
L
10
95
100
119
94
32
10
95
100
11
118
248
49
11
229
100
12
12
103
99
13
13
215
99
14
12996 / 13685
229
L
11
229
100
13752 ? 14066
104
L
12
103
99
14020 ? 14667
215
13
215
99
xe
14763 ? 15146
127
E
14
127
98
14
127
98
15
hr1
15222–16382 L
17
12 13
11692 / 12651
lef-2
117
241
48
16388 / 16684
98
15
98
99
15
98
100
16
16750 ? 17349
199
16
199
99
16
199
99
17
17502 ? 18212
236
17
236
99
18275 / 19963
562
L
18
562
99
41
570
63
19
562
99
22
60
527
51
15
Endonuclease
18
Chitinase
19
ro-a
18 19
20167 ? 21633
488
19
332
78
20
353
75
24
20
21679 ? 22104
141
E
20
141
99
21
141
99
25
21 22
22205 ? 23014 23125 / 23760
269 211
E
21 22
269 213
99 97
22 23
269 211
99 99
26 27
23
23954 ? 24268
104
L
23
104
100
24
104
99
29 30
57
195
30
24398 / 25033
211
E
24
211
98
25
211
99
Helicase2
25258 / 26625
455
E
25
455
99
26
455
99
26
he65
26760 / 28514
584
26
584
99
61
236
29
27
584
99
32
27
Cathepsin
28579 ? 29604
341
27
341
100
56
365
47
28
341
100
33
29601 / 29948
115
L
28
115
100
29
115
100
34
24 25
28 29
lef-1
29976 ? 30623
215
29
215
99
124
245
48
30
215
99
35
30
38.7k
30623 ? 31672
349
L
30
349
99
123
385
35
31
349
100
36
58
279
62
32
262
99
37
33
179
99
38
31
gp37
31724 ? 32512
262
L
31
262
99
32
ptp2
32469 / 33008
179
L
32
179
99
33
egt
33076 ? 34662
528
E
33
528
99
126
515
52
34
516
99
39
E
34
34831 ? 35367
178
34
178
99
127
192
23
35
178
99
40
35
35367 ? 36017
216
35
215
99
128
266
28
36
213
98
41
36
36053 / 38608
851
36
851
99
129
947
26
37
851
99
42
37 38
chtB2
38666 ? 39106 39137 ? 39664
146 175
L L
37 38
146 175
99 99
83
165
33
38 39
146 175
99 99
43 44
39
pkip
39685 ? 40194
169
L
39
169
100
130
169
35
40
169
100
45
40216 / 40557
113
40
113
100
41
113
100
46
40563 / 41435
290
41
290
99
131
265
30
42
290
99
47
40 41
arif
42
pif2
41191 ? 42450
419
42
419
99
132
383
69
43
419
99
48
43
pif1
42465 ? 44054
529
43
529
99
111
528
43
44
529
99
49
44051 ? 44296
81
44
81
100
45
81
100
50
44331 / 45425
364
45
364
99
46
364
99
51
44 45
fgf
113
301
34
123
138
Virus Genes (2013) 47:133–151
Table 2 coninued ORF no.
Name
Position
Length (aa)
Promoter
Homologous (ORF number, size, aa identity %) HearMNPV
46 47
alk-exo
48
HearSNPV-G4
MacoNPV-B
MacoNPV-A
45461 ? 46366
301
46
301
99
47
301
99
53
46407 / 47594
395
L
47
395
99
114
428
41
48
395
99
54
47824 / 48162
112
L
48
112
100
115
129
27
49
112
100
55
L
49
48161 ? 49324
387
49
387
99
50
387
99
56
50
49363 / 49764
133
50
133
99
51
133
100
57
51 52 53
rr2b
49836 ? 50777
313
51
313
100
52
313
99
58
calyx
50785 / 51816 51843 / 52820
343 325
52 53
349 325
95 100
120
340
43
53 61
209 325
100 100
59 60 62
L
54
53090 / 53431
113
54
112
100
110
88
40
62
113
100
55
53383 / 53745
120
E
55
120
100
109
118
35
63
120
100
63
56
53922 / 54536
204
E, L
56
204
99
64
204
100
65
54601 / 55056
151
57
151
100
55113 ? 55478
121
58
121
100
57
sod
58 59
55504 ? 56115
203
56081 ? 56551
156
56616 ? 58070
484
58093 ? 58734
213
nrk1
58769 / 59878
hr2
60156 - 61490
pif3
60 61
parg
62 63
L
106 98
159
59
203
99
60
156
99
199
L
61
484
99
100
510
L
62
213
100
101
253
369
E
63
369
75 52
65
151
100
66
66
121
100
67
67
203
99
68
68
156
99
69
24
69
484
99
70
57
70
213
100
71
99
71
359
100
72
61602 ? 62078
158
L
64
158
99
72
158
93
73
65
Dutpase
62138 ? 62566
142
E
65
114
99
73
142
93
74
66 67
bro-b p13
62669 ? 63676 63726 ? 64562
335 278
L
67 68
335 278
98 100
74 75
326 278
78 99
75 76
68
xe
64617 ? 65141
174
69
174
99
76
174
99
77
69
odv-e66a
64
70
60 97
527 276
52 58
65253 ? 67271
672
E, L
70
672
99
96
672
59
77
672
99
78
67268 / 67579
103
L
71
103
100
95
94
45
78
103
99
79
L
71
odv-ec43
67621 / 68691
356
72
vp80
68851 / 70497
548
73
p45
70525 ? 71658
377
72
356
100
94
361
50
79
356
100
80
74
548
99
92
605
27
81
548
99
82
E, L
75
377
100
91
377
58
82
377
99
83 84
74
p12
71645 ? 71953
102
L
76
102
100
90
122
42
83
102
100
75
p40
71979 ? 73073
364
L
77
364
99
89
369
53
84
364
99
85
76
p6.9
73132 ? 73365
77
L
78
77
100
85
77
100
86
77
lef-5
73362 / 74183
273
79
273
99
87
315
48
86
273
99
87
78
38 k
74082 ? 74984
300
L
80
300
99
86
321
53
87
300
100
88
L
79
vef
75023 ? 77569
848
81
848
99
88
848
99
89
80
bro-d
77574 / 78644
356
82
356
99
89
356
99
90
78738 / 79166
142
83
142
99
90
142
100
91
82 83
odv-e28 helicase1
79200 / 79718 79675 ? 83304
172 1209
L
84 85
172 1209
99 99
85 84
173 1253
63 47
91 92
172 1209
99 99
92 93
84
odv-e25
83404 / 84054
216
L
86
216
100
82
230
65
93
216
100
94
L
81
85
p18
84051 / 84536
161
86
p33
84535 ? 85293
252
85403 ? 85918
171
88
lef-4
85950 / 87314
454
89
vp39
87313 ? 88299
328
90
cg30
88382 ? 89206
274
87
123
E, L
87
161
100
81
162
69
94
161
100
95
88
252
99
80
254
57
95
252
99
96
89
171
99
96
174
98
97
90
454
99
79
461
48
97
454
99
98
L
91
329
99
78
293
51
98
328
99
99
E
92
274
99
77
283
22
99
274
99
100
Virus Genes (2013) 47:133–151
139
Table 2 coninued ORF no.
Name
91
vp91
89262 / 91700
812
L
93
812
92
tlp-20
91669 ? 92256
195
L
94
195
Length (aa)
Promoter
Homologous (ORF number, size, aa identity %)
92081 ? 92803
240
L
95
L
HearMNPV
93 94
Position
gp41
95
92772 ? 93773
333
93653 ? 94108
151
HearSNPV-G4
MacoNPV-B
MacoNPV-A
99
76
816
44
100
812
99
101
99
75
225
54
101
195
99
102
240
99
74
241
65
102
240
99
96
333
100
73
322
58
103
333
100
97
151
100
72
110
41
104
151
100
105
104
96
vlf-1
94110 ? 95252
380
L
98
380
100
71
412
67
105
380
100
106
97 98
ctl
95249 / 95401 95473 / 96567
50 364
L E
99 100
50 364
98 99
34
359
24
106 107
50 364
96 99
107 108
E
99
p26
96689 / 97423
244
101
244
99
22
267
28
108
244
99
109
100
iap-2
97472 / 98218
248
102
248
99
62
250
43
109
248
99
110
101
mt
98163 / 98990
275
103
271
99
63
274
50
110
275
99
111
98974 / 99339
121
104
121
100
64
133
55
111
121
100
112
99338 ? 100519
393
105
393
99
65
379
31
112
393
99
113
99
102 103
lef-3
104
Desmoplakin
100579 / 102834
751
106
753
99
66
785
60
113
752
105
dnapol
102833 ? 105835
1000
107
1000
99
67
1020
59
114
1000 100
114 115
106
105869 / 106258
129
L
108
129
100
69
127
40
115
129
100
116
107
106269 / 106526
85
L
109
85
100
70
85
71
116
85
100
117
117
246
98
118
57
195
32
118
181
99
119
108
106618 ? 107358
246
110
246
98
109
107350 / 107895
181
111
181
99
110
107930 ? 108391
153
111
108446 ? 109093
215
L
112
153
99
119
153
99
120
113
215
98
120
215
98
121
114 115
352 229
91 98
59
244
26
121 122
349 229
94 99
122 123
123
497
100
124
112 113
bro-e bro-f
109134 / 110171 110225 / 110914
345 229
114
lef-9
110995 / 112488
497
L
116
497
99
55
519
71
L
117
195
100
53
217
70
118
834
99
115
fp25k
112566 ? 113153
195
116
p94
113230 ? 115734
834
124
195
100
125
125
834
99
126
117
bro-g
115807 ? 116295
162
L
119
179
99
60
527
50
126
162
99
127
118
chab2
116327 ? 116602
91
L
120
91
100
52
88
57
127
91
100
128
119
chab1
116616 ? 117104
162
121
169
97
51
160
59
128
179
98
129
120
117097 / 117576
159
E
122
159
99
50
171
40
129
159
100
130
121
117826 / 118095
89
L
123
89
100
49
64
51
130
89
100
131
118037 / 118246
69
124
69
100
48
68
49
131
69
100
132
123
vp1054
118372 / 119382
336
E, L
125
336
99
47
351
53
132
336
99
133
124
lef-10
119243 / 119470
75
L
126
75
98
46
71
50
133
75
99
134
122
125
119430 ? 119657
75
L
127
75
99
45
75
36
134
75
99
135
126
119671 ? 120678
335
L
128
328
98
44
378
30
135
321
96
136
127
120683 / 121156
157
L
129
157
100
43
136
57
136
157
100
137
128
167
130
167
100
42
180
27
137
167
100
138
hr3
121155 ? 121658 121692 - 122922
iap-3
123181 ? 124038
285
131
285
99
103
268
33
138
285
99
139
130
bjdp
124077 / 125231
384
132
384
99
39
194
33
139
384
99
140
131
lef-8
125252 ? 127888
878
133
878
99
38
901
68
140
878
99
141
127917 / 128381
154
134
154
99
141
154
99
142
136
663
99
96
672
35
143
663
99
144
137
397
99
35
333
58
144
397
99
145
129
132
128410 ? 128628
72
134
odv-e66b
128673 / 130664
663
135
p47
130712 ? 131905
397
133
L
L
123
140
Virus Genes (2013) 47:133–151
Table 2 cotinued ORF no.
Name
Position
Length (aa)
Promoter
Homologous (ORF number, size, aa identity %) HearMNPV
131916 / 132965
136 hr4 137
349
HearSNPV-G4
MacoNPV-B
MacoNPV-A
138
349
99
145
349
99
146
146
190
99
147 148
133044 - 134395 134457 ? 135029
190
E
140
190
99
138
bv-e31
135091 ? 135795
234
E, L
141
234
100
33
238
67
147
234
100
139
lef-11
135720 ? 136094
124
L
142
124
99
32
127
50
148
124
99
149
140
39k
136063 ? 136917
284
L
143
284
99
31
311
33
149
284
99
150
141
Ubiquitin
137109 / 137411
100
L
145
100
100
151
100
100
152
137467 ? 138012
181
L
146
181
99
27
255
50
152
181
99
153
142
138363 / 138719
118
L
147
118
100
26
133
37
153
118
99
154
144
dbp
138808 ? 139788
326
E
148
326
99
25
323
42
154
326
99
155
145 146
lef-6
139794 ? 140219 140260 / 140505
141 81
L
149 150
141 81
99 100
24 23
187 67
45 56
155 156
141 81
99 100
156 157
147
p26
140621 ? 141421
266
L
151
266
99
22
267
46
157
266
99
158
148
p10
141460 ? 141711
83
L
152
83
100
21
87
47
158
83
100
159
149
p74
141798 / 143771
657
L
153
657
99
20
688
56
159
657
99
160
143852 ? 144103
83
E, L
154
83
99
160
83
100
161 162
143
150 151
ie-1
144139 / 145953
604
155
601
99
14
655
42
161
603
99
152
ep23
145995 ? 146570
191
L
156
191
99
13
203
33
162
191
99
163
153
chtb1
146629 / 146907
92
L
157
92
100
12
92
55
163
92
100
164
154
odv-ec27
146910 / 147746
278
L
158
278
100
11
284
60
164
278
100
165
155
odv-e18
147785 / 148042
85
L
159
85
100
10
81
54
165
85
100
166 167
156
p49
148044 / 149429
461
L
160
461
99
9
468
57
166
461
99
157
ie-0
149447 / 150151
234
L
161
234
99
8
285
43
167
234
100
168
158
rr1
150316 / 152601
761
E
162
761
99
168
761
99
169
ORF no.
Homologous (ORF number, size, aa identity %) MacoNPV-A
SeMNPV
1
246
98
1
246
92
8
245
90
2
466
83
2
462
43
9
543
23
3
272
88
3
295
55
10
272
4
722
74
4
724
34
5 6
202 373
67 90
6
371
57
148
7
354
86
7
390
46
139
8
680
91
8
665
47
9
319
92
10
95
98
9
94
11
228
93
10
248
12
102
95
11
105
50
13
211
82
12
209
56
14
138
87
15
92
94
16
199
87
17
236
83
18
562
97
123
19
572
AcMNPV
OpMNPV
PlxyGV
3
245
87
39
1
274
34
6
274
35
376
50
146
374
51
16
351
41
449
24
137
455
24
120
308
23
23
690
24
21
657
23
26
544
25
56
130
106
35
128
103
25
62
129
198
39
127
192
37
53
159
28
6
210
42
6
204
39
32
270
27
79
104
45
82
104
45
126
551
68
124
550
66
78
Virus Genes (2013) 47:133–151
141
Table 2 contined ORF no.
Homologous (ORF number, size, aa identity %) MacoNPV-A
SeMNPV
AcMNPV
19
372
72
20
141
87
21
269
79
22
211
92
23
110
76
24
214
69
25 26
558
93
27
337
98
16
337
87
28
117
91
15
154
60
29
215
96
14
216
30
350
93
13
363
31
262
95
25
32
179
94
33
516
95
34
177
35 36
OpMNPV
PlxyGV
2
328
22
105
553
37
127
323
57
125
324
53
64
14
266
40
13
243
43
54
13
327
31
12
320
25
267
74
64
302
59
69
321
57
26
165
60
9
160
30
27
523
72
15
506
49
14
489
47
96
28
190
46
213
91
29
213
40
17
164
32
16
207
31
848
89
30
886
45
37
150
64
68
161
35
145
77
42
110
93
46
38
173
83
31
241
26
39
168
93
32
164
45
24
169
35
40 41
113 290
97 93
33 34
112 281
48 40
21
319
23
19
298
33
42
419
96
35
413
72
22
382
59
20
382
43
529
94
36
526
56
119
530
49
44
81
97
37
80
47
45
373
73
38
404
47
46
238
90
40
241
43
47
395
90
41
413
46
48
110
94
42
81
53
49
389
95
43
386
60
50
135
96
44
140
47
51
313
95
45
313
68
52
338
72
53
321
97
46
335
77
54
113
85
47
103
56
55
120
91
56 57
204 151
89 98
48
151
81
58
118
87
49
130
23
59
203
94
50
214
64
60
158
90
51
142
42
61
483
88
52
529
34
62
216
97
53
222
74
63
359
96
54
364
56
21
141
43
107
436
53
55
251
36
12
98
38
57
37
368
47
7
536
34
106
378
40
20
235
30
119
529
50
120
82
33
32
181
28
27
205
31
133
419
39
131
424
39
18
353
26
18
355
22
131
252
32
129
297
34
31
151
74
29
152
71
115
204
54
115
205
43
29
181
40
107
243
56
107
256
59
40
206
49
123
142
Virus Genes (2013) 47:133–151
Table 2 contined ORF no.
Homologous (ORF number, size, aa identity %) MacoNPV-A
64
157
87
65
142
93
66
329
82
67
276
96
68
174
90
69
672
70 71
103 356
72 73
SeMNPV
55
143
AcMNPV
OpMNPV
PlxyGV
62 2
328
25
56
283
67
98
57
723
46
46
704
41
50
682
97 96
58 59
114 356
59 76
108 109
105 390
33 42
108 109
108 390
547
84
61
556
40
104
691
31
105
624
32
377
98
62
375
78
103
387
51
104
411
74
102
90
63
106
58
102
122
31
75
365
93
64
388
67
101
361
43
102
76
76
62
77
273
97
66
279
75
99
265
58
78
301
96
67
300
68
98
320
45
79
847
82
80
360
85
2
328
51
81
142
97
82
172
99
69
170
74
96
173
51
83
1212
96
70
1222
73
95
1221
43
96
1223
37
72
1124
28
84
216
95
71
216
80
94
228
46
95
229
40
74
214
51
85 86
161 252
99 95
72 73
157 252
75 78
93 92
161 259
50 51
94 93
159 282
46 47
75 76
156 250
34 34
87
168
91
88
454
93
74
466
61
90
464
45
91
457
38
78
432
34
89
325
80
75
326
57
89
347
42
90
351
47
79
320
34
90
279
82
76
461
29
88
264
22
89
249
22
91
809
93
77
813
55
83
847
42
86
819
41
92
195
91
78
196
63
82
180
32
79
240
65
81
233
54
84
218
94
333
98
80
331
81
80
409
57
83
367
95
67
93
81
127
48
78
109
35
81
105
35
96
380
98
82
372
90
97
50
92
98
364
83
99
244
97
87
250
100
252
89
88
317
101 102
275 121
93 96
89 90
103
385
82
104
740
105
998
106
93
36
263
52
41
30
682
39
35 42
43
414
32
44
63
377
40
354
38
66
366
26
100
263
51
69
247
49
99
313
42
70
340
41
97
172
50
71
161
39
84
533
28
85
139
34
50
86
191
47
50
87
283
31
89
346
30
96
128
34
93
979
37
91
81
44
77
379
68
80
374
64
3
53
46
136
53
47
65
136
240
35
132
230
28
49
71
249
33
74
236
32
299 133
61 74
69 68
262 192
45 47
73
131
46
91
422
49
67
385
27
72
373
25
80
92
704
31
66
808
25
95
93
1063
73
65
984
46
70
985
41
129
99
94
129
81
75
133
26
78
130
27
107
85
100
95
85
84
76
84
42
79
84
38
108
249
86
96
113
46
109
181
88
123
Virus Genes (2013) 47:133–151
143
Table 2 contined ORF no.
Homologous (ORF number, size, aa identity %) MacoNPV-A
SeMNPV
AcMNPV
110
156
97
111
215
88
112
357
86
113
235
78
114
505
97
97
495
85
115
195
99
98
195
116 117
819 179
83 96
99
118
90
100
119
166
82
120
159
95
121
89
122
OpMNPV
PlxyGV
2
328
40
2
328
34
62
516
65
65
489
64
99
494
53
88
61
214
62
64
208
59
100
138
41
719
26
134
803
42
100
89
71
60
87
52
63
90
41
101
195
82
59
172
55
62
176
48
102
178
62
57
161
37
61
163
38
93
103
93
46
60
82
47
71
96
104
67
60
123
336
93
105
346
67
54
365
41
58
378
37
115
311
32
124
75
94
106
77
69
53a
78
48
57
80
25
125
75
92
126
311
74
107
344
42
127
157
95
108
137
63
53
139
49
56
146
49
112
137
24
128
166
89
109
162
44
52
194
22
129
276
83
110
313
41
27
286
28
35
268
43
130 131
388 878
87 98
111 112
415 906
34 73
51 50
318 876
22 61
54
884
55
109
838
52
132
152
81
134
666
89
114
685
46
46
704
30
50
682
28
30
682
29
135
397
97
115
400
75
40
401
55
45
399
52
51
386
46
136
349
86
137
193
83
117
191
26
138
230
95
118
261
84
38
216
63
22
209
60
52
207
41
139
120
91
119
104
64
37
112
39
23
125
33
46
96
33
140
287
93
120
317
52
36
275
33
24
261
28
141
100
94
123
80
94
35
77
78
25
93
81
142
181
93
124
187
68
34
215
36
26
209
38
143
119
95
125
135
52
26
129
32
42
127
32
144
328
96
126
328
48
25
316
25
43
300
25
61
263
23
145 146
143 81
96 98
127 128
163 136
79 70
28 29
173 71
37 41
40 39
138 75
30 35
147
267
97
129
278
64
136
240
34
132
230
32
148
85
95
130
88
68
137
94
36
133
92
48
149
657
95
131
653
64
138
645
53
134
644
52
49
578
43
150
85
93
151
526
91
132
714
53
147
582
33
145
560
33
152
191
96
133
200
55
146
201
34
144
197
36
153
92
93
134
92
68
145
95
45
142
95
48
12
98
37
133
123
144
Virus Genes (2013) 47:133–151
Table 2 contined ORF no.
Homologous (ORF number, size, aa identity %) MacoNPV-A
SeMNPV
AcMNPV
OpMNPV
PlxyGV
154
278
98
135
281
86
144
290
52
141
297
47
155
83
89
136
80
62
143
62
83
140
85
76
156
461
98
137
234
25
142
477
49
139
484
49
157
234
96
138
244
63
141
261
31
138
245
29
158
761
94
139
770
53
80
287
28
14
446
36
E; early promoter motif CGTGC, 210 bp upstream of start codon, L; late promoter motif (A/T/G)TAAG 120 bp upstream of start codon a
Number of amino acids encoded by ORF
b
Promoter motifs present upstream of ORF
102 ORFs with AcMNPV, 90 ORFs with OpMNPV and 51 ORFs with PlxyGV. Polyhedrin was the most conserved ORF between MabrNPV-K1 and other baculoviruses with the highest shared deduced amino acid identities to HearMNPV (100 %), MacoNPV-B (100 %), MacoNPV-A (98 %), SeMNPV (92 %), AcMNPV (90 %), HearSNPV-G4 (87 %) and OpMNPV (87 %). Almost all of the MabrNPV-K1 ORFs had very high amino acid sequence identity, from 91 to 100 %, with HearMNPV and MacoNPV-B, except for ORF19 (bro-a). Additionally, MabrNPV-K1 shared 100 % amino acid sequence identity with HearMNPV (47 ORFs) and MacoNPV-B (54 ORFs). These results demonstrate the close relationship between these 3 NPVs at the genome level. The gene arrangement and distribution of the MabrNPV-K1 genome was examined using gene parity plots [22], and the resulting analysis further supported this relationship. The gene order of the MabrNPV-K1 genome was almost collinear with that of HearMNPV, MacoNPV-B and MacoNPV-A but was less collinear with SeMNPV (Fig. 2). These NPVs, including MabrNPV-K1, are Group II NPVs, but not Group I NPVs, such as AcMNPV and OpMNPV, had a dispersed pattern when compared with MabrNPV-K1. Although HearSNPV-G4 is also a Group II NPV, it had a different gene arrangement than other Group II NPVs. The results of the gene parity plot analysis and the size and identity of the nucleotide sequence of its genome suggested that HearSNPV is very different from MabrNPV-K1, HearMNPV and the MacoNPVs. The genome sequences of HearSNPV were reported for three viral isolates, i.e. HearSNPV-G4, HearSNPV-C1 and HearSNPV-NNg1 [23–25]. These previous results suggested that HearSNPVs have a close ancestral relationship with SeMNPV, Lymantria dispar MNPV (LdMNPV) and H. zea SNPV (HzSNPV) at the gene level. However, a comparative analysis of HearSNPV and HearMNPV showed a significant difference between these NPVs, not only in biological properties but also in genome characteristics [13]. Although MabrNPV-K1 is closely related to
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HearMNPV, it was predicted to have low similarity with HearSNPV. In a gene parity plot analysis, the arrangement of 157 ORFs was shared between MabrNPV-K1 and HearMNPV, whereas 1 (ORF133) and 5 (ORF66, 73, 135, 139 and 144) ORFs were unique to MabrNPV-K1 and HearMNPV, respectively (Table 2). A comparison of MabrNPV-K1 with MacoNPV-A and MacoNPV-B showed a shared arrangement of 157 and 156 ORFs, respectively. MabrNPV-K1 had 3 (ORF25, 93 and 133) and 2 (ORF17 and 133) unique ORFs when compared to MacoNPV-A and -B, respectively. Fourteen (ORF8, 16, 20, 21, 23, 28, 31, 52, 61, 64, 81, 103, 143 and 151) and 11 (ORF17, 18, 54, 55, 56, 57, 58, 59, 60, 80 and 150) ORFs were unique in MacoNPV-A and -B, respectively, when compared to MabrNPV-K1. These results indicated that MabrNPV-K1 is more closely related to HearMNPV than to MacoNPVs at the gene level. The differences of these unique ORFs may be related to the novel characteristics of each virus and requires further study because the function of these ORFs is not known. The results of ORF analysis support the idea that MabrNPV-K1, HearMNPV and MacoNPVs not only share their host but also have a high degree of genomic homology [11, 12] and a close ancestral relationship. Unique MabrNPV-K1 ORFs MabrNPV-K1 ORF133 is small in size (72 a.a.) and is unique in MabrNPV-K1 when compared with other NPVs (Table 2; Fig. 2). BLAST comparisons of the amino acid sequences of this ORF showed no similarity to any known genes or ORFs of baculovirus or other microorganisms. The presence of MabrNPV-K1-specific ORF133 was confirmed by the analysis of its transcript using RT-PCR and RACE (Fig. 3). The RT-PCR for ?61 to ?216 from ?1 ATG of the ORF133 showed the presence of transcript for the gap between ORF132 and 134. To clarify this transcript is specific for ORF133, the transcription start sites were
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Fig. 2 Gene parity plots comparing ORFs and their order in MabrNPV-K1 with HearMNPV (a), HearSNPV-G4 (b), MacoNPV-B (c), MacoNPV-A (d), SeMNPV (e), AcMNPV (f), OpMNPV (g) and PlxyGV (h). Each dot represents an ORF, and the compared genomes appear on the axis corresponding to the virus in which they are present
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MabrNPV-K1, HearMNPV and MacoNPV-B was further investigated by a comparison of major functional genes. MabrNPV-K1 structural genes
Fig. 3 RT-PCR and 50 -RACE of MabrNPV-K1 unique ORF using total RNAs from infected M. brassicae larvae. RT-PCR was performed using ORF1 (lane 1) and ORF133 (lane 2 and 3) specific primers. 50 -RACE was performed using ORF133 specific primer (lane 4 and 5). A healthy M. brassciae larva was used as a negative control (lane 2 and 4). Lane M, DNA ladder
The MabrNPV-K1 genome contained homologues of 35 known structural protein genes of baculoviruses, including polh, orf1629, pk-1, odv-e56, fusion protein, p24, pkip, pif2, pif-1, calyx, pif-3, p13, odv-e66a, ovd-ec43, vp80, p6.9, 38k, vef, odv-e28, odv-e25, p33, vp39, cg30, vp91, tlp-20, gp41, desmoplakin, vp1054, odv-e66b, p10, p74, odv-ec27, odv-e18 and p49 (Table 2). MabrNPV-K1 lacked the envelope glycoprotein gp64, which is unique to Group I NPVs, and this is a common characteristic of Group II NPVs. All structural genes of MabrNPV-K1 had 99–100 % sequence identity with those of both HearMNPV and MacoNPV-B, except orf1629 of HearMNPV, which showed 96 % identity. These results suggest that MabrNPV-K1 may be very structurally similar to HearMNPV and MacoNPV-B and supported the close relationship between these viruses. MabrNPV-K1 genes with two homologues
determined by 50 RACE analysis. Transcription initiated from CATT located 66 nt upstream of the ?1 ATG of ORF133. This result confirmed the presence of specific transcript of ORF133. The CATT is known as one of early gene promoter motifs in baculovirus [26], but other consensus sequences for promoter were not found. As this ORF is unique to MabrNPV-K1, a functional investigation may help researchers further understand the novel characteristics of MabrNPV-K1. Phylogenetic analysis of MabrNPV-K1 Thirty-five core genes have been identified and are present in all sequenced baculovirus genomes [21], and these genes were also present in MabrNPV-K1 (Table 2). Among those core genes, pif-2, lef-8 and lef-9 were confirmed to be reliable baculovirus markers for phylogenetic analyses at the virus family level [27]. A combined phylogenetic analysis using pif-2, lef-8 and lef-9 indicated that MabrNPV-K1, HearMNPV, MacoNPV-A and MacoNPVB are phylogenetically grouped together (Fig. 4). Although these viruses were grouped together with other Group II NPVs, they were distinguishable from other NPVs. As the results of the ORF analysis show (Table 2; Fig. 2), HearSNPVs are distantly related to MabrNPV-K1. MabrNPV-K1 is most closely related to HearMNPV and MacoNPV-B, but MacoNPV-A has a relatively distant relationship to the other 3 NPVs. This result corresponded to our preceding results of ORF and gene parity plot analyses (Table 2; Fig. 2). Therefore, the relationship between
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A pair of genes, xe and odv-e66, was identified as a duplicated homologue of MabrNPV-K1 ORFs 14/68 and 69/134, HearMNPV ORFs 14/69 and 70/136, and MacoNPV-B ORFs 14/76 and 77/143 (Table 2). The MabrNPVK1 ORFs 14/68 shared 98 and 99 % identity with HearMNPV ORFs 14/69 and MacoNPV-B ORFs 14/76, respectively. The presence of xe was also shown in MacoNPV-A and reported in Leucania separate NPV [28]. This gene is unique to a few Group II NPVs, but its function has not been elucidated. The homologues of MabrNPV-K1 ORFs 69/134 were also present in the MacoNPV-A and SeMNPV genomes, but they showed relatively low similarity, 89 or 98 % of MacoNPV-A and 46 % of SeMNPV, when compared to the high similarity of 99 % for those of HearMNPV and MacoNPV-B. These results also supported the close relationship among MabrNPV-K1, HearMNPV and MacoNPV-B. Genes with auxiliary functions Auxiliary genes are not essential for viral replication, but they provide a virus with some selective advantages [29]. MabrNPV-K1 had 15 homologues of auxiliary genes, including pk-1, chitinase, cathepsin, gp37, egt, arif, fgf, alkexo, sod, vef, ctl, p94, ubiquitin, p10 and p49 (Table 2). All of these auxiliary genes in MabrNPV-K1 were 96–100 % identical in amino acid sequence to those of HearMNPV and MacoNPV-B, but the egt size of the predicted protein (516 a.a.) in MacoNPV-B was smaller than that in MabrNPV-K1 and HearMNPV. Among the auxiliary genes, the vef gene is a
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Fig. 4 Phylogenetic analysis of deduced amino acid sequence alignment of pif-2, lef-8 and lef-9 genes of 49 currently sequenced genomes. The evolutionary history was inferred using the NeighbourJoining method. Numbers at nodes indicate bootstrap scores for the
NJ analysis (2,000 replicates, NJ bootstrap). Evolutionary analyses were conducted in MEGA5 software. The location of MabrNPV-K1 is expressed by the black circle
novel gene found in GVs and a few NPVs, and its protein product, VEF, is metalloprotease that is thought to enhance viral infectivity and is present in viral occlusion bodies [29]. The vef gene was identified not only in the MabrNPV-K1 genome (ORF79) but also in HearMNPV and MacoNPV-B. The vef gene in MabrNPV-K1 shares a high a.a. similarity with those of HearMNPV (99 %) and MacoNPV-B (99 %), and it has the same amino acid size (848 a.a.). The high similarities of auxiliary genes among MabrNPV-K1, HearMNPV and MacoNPV-B also supported their high degree of relatedness in the phylogenetic tree.
gene transcription that exist in other baculovirus genomes are also found in the MabrNPV-K1 genome, including lef 4-6, 811, 39K, p47 and vlf-1 [30]. All of these genes are 99–100 % identical in a.a. sequences with those of both HearMNPV and MacoNPV-B, with the exception of HearMNPV lef10 (98 %). Two other conserved genes that may be involved in DNA replication, dbp and p6.9, were found in MabrNPV-K1, ORFs 144 and 76, respectively. These 2 genes are also 99–100 % identical to those of both HearMNPV and MacoNPV-B. All of these results suggest that MabrNPV-K1, HearMNPV and MacoNPV-B have similar mechanisms in DNA replication and transcription.
DNA replication and transcription regulatory genes Inhibitors of apoptosis (IAPs) There are several reports about DNA replication and transcription genes in baculovirus [30]. Among those genes, 6 genes have been reported as essential DNA replication factors, including lef-1, lef-2, lef-3, dnapol, helicase and i.e.-1, and each of these is also found in the MabrNPV-K1 genome (Table 2). These genes are highly conserved in MabrNPV-K1 with respect to those of both HearMNPV and MacoNPV-B (99–100 %) and have less similarity with those of MacoNPVA (82–96 %), which supports the distant relationship of MacoNPV-A with other 3 NPVs. The genes implicated in late
Apoptosis or programmed cell death is an important virushost interaction process. Programmed cell death can be a mechanism to defend against the establishment of infections and is triggered early in baculovirus infection [7]. Baculoviruses have the p35/p49 family and the iap family of genes. P35 blocks diverse pathways of apoptosis, and its homologues are limited to a few Group I NPVs closely related to AcMNPV, and p49 is a variant that is found in Group II NPV genomes. In contrast, iaps have been found
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in all family members of Baculoviridae sequenced to date, and up to 5 iap homologues are found in baculovirus genomes. Homologues of iap-2 are found in the genomes of all Group I and most Group II NPVs, and, as with all iap genes, more distantly related genes are found in many organisms [31]. MabrNPV-K1 possesses two iaps, which are iap-2 and iap-3 homologues. The iap-2 and iap-3 gene products share 99 % identity with the HearMNPV and MacoNPV-B homologues, whereas MabrNPV-K1 iap-2 was 49 and 33 % identical to those of SeMNPV and AcMNPV, respectively. In addition, iap-3 was 41 and 28 % identical to that of SeMNPV and AcMNPV. The iap-3 is one of the baculovirus genes that affect viral host range and prevent apoptosis in baculovirus infected insects [32]. Thus, the high similarity of iaps between MabrNPV-K1, HearMNPV and MacoNPV-B suggests that they may have an overlapping host range and share some hosts. Baculovirus repeated ORFs (bro genes) The bro genes have been identified in most lepidopteran and dipteran NPV and some of the GV genomes sequenced to date [31, 33]. Although they were originally reported in baculoviruses, homologues of bro genes have been identified in other insect dsDNA viruses, including the entomopoxviruses. The presence of bro is important for NPV, although the copy number may vary according to the NPVs and its function remains unclear [31]. In addition, the absence of bro genes was also reported in a few baculovirus genomes, including SeMNPV and PlxyGV. Although their function is not clear, the proteins produced by bro genes contain a conserved N-terminal DNA binding domain (BRO-N) associated with a highly variable C-terminal domain (BRO-C), and these are purported to be involved in host transcriptional regulation and DNA replication [33]. The function of bro genes was also proposed to enhance the late phase of virus replication and CRM1dependent nuclear export shuttle proteins. The MabrNPVK1 genome sequence contains six bro genes, which have been named bro-a (ORF19), bro-b (ORF66), bro-d (ORF80), bro-e (ORF112), bro-f (ORF113) and bro-g (ORF117) based on their order in the genome (Fig. 1). Most of the MabrNPV-K1 bro genes have between 91 and 99 % identity with their respective homologues in HearMNPV and MacoNPV-B. However, bro-b shares only 78 % identity with its MacoNPV-B homologue, and bro-a shares 78 and 75 % identity with its respective homologues in HearMNPV and MacoNPV-B. In addition, the size of bro-a was much longer (488 a.a.) than the 332 a.a. of HearMNPV and 372 a.a. of MacoNPV-B. The loss, duplication and acquisition of bro or bro-like genes are all considered to be the result of recombination by horizontal gene transfer as the viruses adapted to their
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hosts. The high similarity of bro genes among these 3 viruses suggested that they may be derived from same ancestor. Homologous repeat regions (hrs) An important feature of all of the baculovirus genomes is the dispersal of at least four hrs located throughout the genome [34]. A single hr consists of palindromic and flanked repeated sequences. According to various pieces of evidence, hrs may function as replication origins of baculovirus [35] and enhancers of early gene expression [36], but their functional significance is still unclear. In addition, non-hr origins of replication have been identified in some NPVs [37]. There are four hrs (hr1, hr2, hr3 and hr4) in the MabrNPV-K1 genome, and they are located at similar sites as those in the HearMNPV and MacoNPV-B genomes (Fig. 5). The hrs are distributed around the MabrNPV-K1 genome with 15, 60, 121 and 133 kb regions separating hrs 1, 2, 3 and 4, respectively, and these account for 3.3 % of the genome. The hrs contain 14–17 direct repeats that are 40–50 bp in length with variable lengths of intervening sequence between the repeats. The genomic positions of MabrNPV-K1 region hr1–hr4 are analogous to the genomic positions in HearMNPV, MacoNPV-A and MacoNPVB hr1–hr4. MabrNPV-K1 hr1–hr4 are flanked by similar ORFs as those in the HearMNPV and MacoNPV-B genomes (Table 2). A previous report found that hrs share higher similarity within a virus strain than between species, and this evidence further indicates that hrs play a fundamental role in the viral life cycle and replication process because they appear to be tightly linked to functional conservation [38]. The high similarity of hrs among MabrNPV-K1, HearMNPV, MacoNPV-A and MacoNPV-B supports the possibility that these viruses have a common ancestor. The slight difference in genome among these viruses may be the result of DNA rearrangements occurring during viral recombination or replication slippage because the repetitive nature of hrs would facilitate this phenomenon [34]. Comparison of MabrNPV-K1, HearMNPV and MacoNPV-B As an overall comparison of these results supported a close relationship between MabrNPV-K1, HearMNPV and MacoNPV-B, the differences of these 3 NPVs were compared in detail. The most significant difference among MabrNPV-K1, HearMNPV and MacoNPV-B was that of genome size. The MabrNPV-K1 genome was 1,486 bp smaller than the HearMNPV genome and 5,772 bp smaller than the MacoNPV-B genome (Table 1). MabrNPV-K1
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Fig. 5 Comparisons of the hrs distribution. Linearised genome map shows relative genomic locations of hrs. The insertions in ORFs are expressed via a bar with striped lines
contained one (ORF133) and two (ORF17 and 133) novel ORFs that are absent in HearMNPV and MacoNPV-B, respectively, whereas HearMNPV and MacoNPV-B contained five and eleven novel ORFs, respectively, that are absent in MabrNPV-K1 (Table 2). MabrNPV-K1 shared
21 ORFs (ORF5, ORF9, ORF16, ORF17, ORF21, ORF22, ORF23, ORF24, ORF52, ORF55, ORF56, ORF64, ORF81, ORF93, ORF98, ORF109, ORF110, ORF111, ORF125, ORF132, ORF136, ORF142 and ORF150) with HearMNPV and MacoNPV-B that have unknown functions, and
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the homologues of these genes were not present in the other baculoviruses, with the exception of MacoNPV strain A (Table 2). In addition, there were 157 ORFs in common among MabrNPV-K1, HearMNPV and MacoNPV-B, and their gene order was almost identical (Fig. 2). Almost all of the common ORFs among MabrNPV-K1, HearMNPV and MacoNPV-B had over 90–100 % a.a. sequence identity, and only 2 ORFs (bro-a and bro-b) showed 75–78 % sequence identity (Table 2). On average, the amino acid sequence identity level of the 157 ORFs was 98 %. In particular, MabrNPV-K1 shared 47 ORFs and 54 ORFS with 100 % sequence identity for HearMNPV and MacoNPV-B, respectively. In addition, the genes involved in DNA replication and transcription had high similarity (99–100 %), and the relative locations of the hrs in the genomes were identical among MabrNPV-K1, HearMNPV and MacoNPV-B (Fig. 5). In previous studies, the MabrNPV, HearMNPV and MacoNPV had a high degree of genomic homology and had almost identical biological and biochemical characteristics, including cross infection among M. brassicae, H. armigera and M. configurata [9–12]. From these studies, it was suggested that these viruses should be considered variants of a single virus species. Although the baculovirus host range likely involves a complicated array of viral and host factors, there are difficulties in naming a baculovirus simply based on the host from which it was originally isolated. Our results demonstrating the high similarity of genomes among MabrNPV-K1, HearMNPV and MacoNPV-B strongly suggest that these three viruses are not only closely related to each other in overall characteristics but are also among the same virus strains, rather than of different virus species. Acknowledgments This work was supported by a Grant from the Next-Generation BioGreen 21 Program (No. PJ008036), Rural Development Administration, Republic of Korea.
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