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. 2021 Jan 11;11:414. doi: 10.1038/s41598-020-80117-3

Genome sequence and organization of the Mythimna (formerly Pseudaletia) unipuncta granulovirus Hawaiian strain

Yinü Li 1,#, Xingjian Liu 1,#, Ping Tang 3, Huan Zhang 2, Qilian Qin 2,✉, Zhifang Zhang 1,✉
PMCID: PMC7801670  PMID: 33432025

Abstract

Purified occlusion bodies (OBs) of Mythimna (formerly Pseudaletia) unipuncta (the true armyworm) granulovirus Hawaiian strain (MyunGV-A) were observed, showing typical GV morphological characteristics under scanning and transmission electron microscopy (EM). The genome of MyunGV-A was completely sequenced and analysed. The genome is 176,677 bp in size, with a G+C content of 39.79%. It contains 183 open reading frames (ORFs) encoding 50 or more amino acids with minimal overlap. Comparison of MyunGV-A with TnGV, XcGV, and HearGV genomes revealed extensive sequence similarity and collinearity, and the four genomes contain the same nine homologous regions (hrs) with conserved structures and locations. Three unique genes, 12 baculovirus repeated ORF (bro), 2 helicase, and 3 enhancin genes, were identified. In particular, two repeated genes (ORF39 and 49) are present in the genome, in reverse and complementarily orientations. Twenty-four OB proteins were identified from the putative protein database of MyunGV-A. In addition, MyunGV-A belongs to the Betabaculovirus group and is most closely related to TnGV (99% amino acid identity) according to a phylogenetic tree based on the combined amino acid sequences of 38 core gene contents.

Subject terms: Systems virology, Sequence annotation

Introduction

Baculoviruses are a large family of rod-shaped, invertebrate-infecting viruses with large circular, covalently closed, double-stranded DNA genomes of between 80 and 180 kb. This family was initially taxonomically subdivided into nucleopolyhedroviruses (NPVs) or granuloviruses (GVs) based on viral occlusion morphology1. However, when an increasing number of genome sequences became available, it was clear that lepidopteran NPVs and GVs are more closely related to each other than to dipteran and hymenopteran NPVs. Therefore, a new taxonomic division that follows the evolution of the host more closely2 was accepted by the International Committee on Taxonomy of Virus (ICTV). In the 10th report of the ICTV (online, 2019), the family Baculoviridae was still divided into four genera: Alphabaculovirus, Betabaculovirus, Deltabaculovirus and Gammabaculovirus (https://talk.ictvonline.org/ictv-reports/ictv_online_report/). To date, 85 baculovirus genomes have been sequenced (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?opt=virus&taxid=10442), including 55 from Alphabaculovirus (lepidopteran NPVs), 26 from Betabaculovirus (lepidopteran GVs), 1 from Deltabaculovirus (dipteran NPVs) and 3 from Gammabaculovirus (hymenopteran NPVs).

Betabaculoviruses are granuloviruses (GVs) infecting only lepidopteran hosts, whereas alphabaculoviruses, deltabaculoviruses and gammabaculoviruses are nucleopolyhedroviruses (NPVs) isolated from a wider range of hosts, including lepidopterans, dipterans and hymenopterans.

Lepidopteran NPVs are further divided into two groups, I and II, based on gene content3. Notably, the budded virus (BV) fusion protein in Group I NPVs is GP64, whereas Group II NPVs lack gp64 and utilize the F protein4. GVs are classified into three types according to tissue tropism5. Type I GVs, such as Xestia c-nigrum GV (XcGV), kill hosts at a slow speed by only infecting the midgut epithelium and fat body tissue6. Type II GVs, such as Cydia pomonella GV (CpGV), kill hosts at a rapid speed, similar to typical lepidopteran NPVs, by infecting most of the host’s major tissues7. Type III GVs infect only the midgut epithelium. Only one GV, Harrisina brillians GV (HabrGV)8, has been identified as Type III. Phylogenetic analysis on the basis of conserved genes of GVs does not show certain monophyletic origins for these different types of pathogenesis9.

Mythimna unipuncta granulovirus (MyunGV-A), originally described as Pseudaletia unipuncta granulovirus (PsunGV) based on an isolated Hawaiian population of Mythimna (Pseudaletia) unipuncta10, was identified as PsunGV by the ICTV in 2002. Until 2017, PsunGV was proposed to be renamed MyunGV-A by the ICTV to reflect the fact that the new species MyunGV-B is the second distinct betabaculovirus to be isolated from the host Mythimna (Pseudoletia) unipuncta.

MyunGV-A (PsunGV-H) was first discovered by synergistic factors (described later as enhancin)10. Subsequent studies on MyunGV-A mostly focused on the mechanisms of enhancement and the enhancin gene. The enhancin of MyunGV-A can interact with viral particles and increase the binding of viral particles to insect midgut microvilli, thereby dramatically promoting the oral infectivity of Mythimna unipuncta NPV and decreasing the larval survival time11. The enhancin of MyunGV-A comprising 901 amino acids have been purified and characterized12. Overall, high-throughput sequencing of baculovirus genomes appears to be essential for analysing the molecular mechanisms of baculovirus infection and understanding baculovirus genome evolution. In this study, the morphological characteristics of MyunGV-A were observed by electron microscopy (EM). We present the complete sequence and organization of the MyunGV-A genome and compare it with other baculoviruses by genomic and phylogenetic analysis. A total of 24 OB proteins of MyunGV-A were identified.

Materials and methods

Virus preparation and DNA extraction

MyunGV-A (PsunGV-H) was obtained from Tanada Y. and kept at the Institute of Zoology, Chinese Academy of Sciences13. The virus was propagated in laboratory stocks of healthy second-instar M. separate larvae by per os infection. The occlusion bodies (OBs) produced in larval cadavers were purified by a standard method14.

To extract viral DNA, the purified OBs were resuspended in 0.1 M sodium carbonate solution [0.1 M Na2CO3, 0.17 M NaCl, 0.01 M EDTA (pH 10.5)] and incubated at 37 °C for 1 h. The pH was adjusted to 7.0 with 0.1 M HCl. Sarcosyl 0.5% and proteinase K 0.25 mg/mL were added to the sample and incubated at 37 °C for 2 h and 65 °C for 2 h. Genomic DNA was extracted with an equal volume of phenol and chloroform. The DNA was precipitated with two volumes of 100% ethanol, washed with 70% ethanol, and dissolved in TE buffer [10 mM Tris–HCl (pH 8); 1 mM EDTA].

Electron microscopy observation

OBs of MyunGV-A were observed by scanning electron microscopy (SEM; Hitachi S3400N) and transmission electron microscopy (TEMl; JEOL JEM1230) according to standard methods15.

DNA sequencing and analysis

A random genomic library of MyunGV-A was constructed according to the “partial filling-in” method16. A total of 831 recombinant plasmids containing 1.5 to 5.0 kb viral DNA fragments were prepared for sequencing using a BigDye Terminator v3.1 (ABI) and a 3130XL Genetic analyser (ABI). The combined sequence generated from these clones represented sixfold genomic coverage. The gaps and ambiguities in the assembled sequence were resolved by PCR. All sequences were assembled into contigs using SeqMan from the DNASTAR 7.0 software package.

ORFs were defined using ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html). The criterion for defining an ORF was a size of 50 or more codons with minimal overlap. DNA and protein comparisons were performed using BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). For protein homology detection, we used the HHpred webserver for the translated ORFs17,18. Multiple alignments and percentage identities were obtained using ClustalW. Promoter motifs present upstream of the putative ORFs were screened as described previously19. Identity among homologous genes was determined with MegAlign software using ClustalW with default parameters. Homologous repeat regions (hrs) were analysed by Tandem Repeats Finder (https://tandem.bu.edu/trf/trf.html). GeneParityPlot analysis was performed as described by Hu et al.20.

Protein analysis of OBs of MyunGV-A

Fresh purified OBs of MyunGV-A suspended in ddH2O were incubated with an equal volume of lysis buffer (0.1 M Na2CO3, 0.17 M NaCl, 0.01 M EDTA, pH 10.6) at 4 °C for 1 h. The pH was adjusted to 8.0 with 0.1 M HCl. The samples were added to 10 mM Tris–HCl containing β-mercaptoethanol (0.2%) and sodium dodecyl sulfate (SDS) at 95 °C for 10 min. The proteins of MyunGV-An OBs were separated by SDS-PAGE using an 8% to 15% gradient gel. The protein bands were excised into 29 samples according to molecular weight from small to large for LC–MS/MS analysis (LCQ Deca Xp plus, ThermoFinnigan). LC–MS/MS analysis and protein identification were performed as described by Shi XF21. The raw files of MS spectra were searched against the putative protein database of MyunGV-A (NC_013772.1).

Phylogenetic analysis of MyunGV-A

The amino acid sequences encoded by the 38 core genes described for all members of family Baculoviridae22 of 82 complete baculovirus genomes (excluding 3 incomplete genomes) in the NCBI genome database (https://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?opt=virus&taxid=10442) were joined together according to a consistent order (ORF order of AcMNPV) and aligned using MAFFT with default parameters. A phylogenetic tree based on these sequences was constructed using MEGA 7 MEGA 7.0.1423. Maximum likelihood (ML) tree construction methods were used with 1000 bootstrap resamples. The GTR + G + I substitution model was used for ML analysis.

Results and discussion

Electron microscopy observation

SEM revealed that the purified OBs of MyunGV-A have elongated ellipse shapes, with a length of approximately 0.5 μm and a width of approximately 0.3 μm (Fig. 1A). TEM showed a single rod-shaped ODV of approximately 300 nm in length and 40 nm in width embedded in a granular OB (Fig. 1B,C). These are typical GV morphological characteristics.

Figure 1.

Figure 1

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A scanning electron micrograph of MyunGV-A (A) and transmission electron micrograph of MyunGV-A (B,C).

Sequence and genome characteristics of MyunGV-A

The size of the MyunGV-A genome is 176,677 bp (GenBank accession no. NC_013772), with a G+C content of 39.79%. MyunGV-A is the second largest GV sequenced to date, with XcGV (178,733 bp)6 being larger. Computer-assisted ORF analysis detected 372 ORFs of 50 or more codons and 9 homologous regions (hrs) in the MyunGV-A genome; 189 ORFs overlap significantly or are completely contained within other MyunGV-An ORFs. The deduced protein sequences of these 189 ORFs show no significant homology to protein sequences in GenBank. The remaining 183 ORFs and 9 h are shown in Table 1 according to location, orientation, size of the predicted amino acid sequence, potential baculovirus homologues, best matched baculovirus ORF and BLAST score (bits).

Table 1.

MyunGV-A (PsunGV-H) open reading frames (ORFs) and homologous repeat regions (hrs).

ORF Name Position Length (aa) Promoter Homologous ORF#/amino acid identity (%)
AcMNPV XcGV HearGV TnGV MyunGV-B CpGV
1 Granulin 1>747 248 L 8/55 1/100 1/100 1/99 1/98 1/87
2 1629 capsid 796<1518 240 L 9/38 2/72 2/72 2/98 2/44 2/39
3 pk 1499>2353 284 L 10/33 3/91 3/92 3/100 3/70 3/47
4 Unknown 2399<3274 291 L – 4/58 4/57 4/99 4/31 –
5 p10 3287>3646 119 L 137/33 5/90 5/92 5/100 5/65 –
6 Unknown 3682<4245 187 E – 7/90 6/89 6/100 6/73, 7/50 4/35
7 Unknown 4235>4495 86 L – 8/81 7/80 7/99 8/54 5/21
8 ie-1 4499<5923 474 E 147/27 9/77 8/76 8/100 9/58 7/28
9 Unknown 5946>6533 195 L 146/27 10/86 9/85 9/100 10/58 8/35
10 Unknown 6581<6880 99 L 145/39,150/44 11/97 10/96 10/100 11/83 9/53
11 odv-e18 6889<7140 83 L 143/44 12/90 11/90 – 12/81 14/61
12 p49 7144<8502 452 L 142/32 13/85 12/84 11/99 13/79 15/43
13 Unknown 8576<9265 229 E – 14/88 13/88 12/99 14/60 –
14 odv-e56 9279<10,340 353 L 148/38 15/83 14/86 13/99 15/73 18/57
15 Unknown 10,370>10,576 68 – – 16/81 15/78 14/100 16/58 19/35
16 pep 10,613<11,185 190 L 131/26 17/95 16/95 15/100 18/61 20/44
17 pep 11,262<11,723 153 L – 18/93 17/92 16/99 19/84 23/53
18 pep/p10 11,744<12,907 387 E/L – 19/94 18/94 17/100 20/80 22/49
19 Unknown 12,992>13,258 88 L 145/26,150/34 105/39,20/36 107/37,19/35 18/100 – –
20 p94 13,339>15,777 812 E/L 134/36 21/70 20/71 19/99 – –
21 Unknown 15,834<17,309 491 – – 22/73 160/45 20/99 137/41 –
22 Unknown 17,390<18,463 357 – – 23/55 22/56 21/99 23/44 –
23 Unknown 18,582<18,920 112 E/L – 24/75 23/75 22/100 24/59 –
24 Unknown 20,373>21,686 437 E – 25/61 24/60 23/99 26/24 29/56
25 Unknown 22,224>23,210 328 E/L – 26/56 25/57 24/98 28/39 30/22
26 efp 23,278>25,023 581 L 23/20 27/80 26/79 25/99 29/52 31/32
27 Unknown 25,152>25,628 158 E – 28/25 27/27 – – –
28 Unknown 26,027<26,737 236 L – 29/80 28/78 26/100 31/55 33/27
29 Unknown 26,756<27,334 192 L – 30/81 29/81 27/99 32/72 34/47
30 pif-3 27,364>27,948 194 L 115/41 32/91 30/92 28/100 33/66 35/46
31 Unknown 27,955>28,287 110 L – 33/53 31/52 – – –
32 Unknown 28,313>28,654 113 L – 34/99 32/99 29/100 35/85 39/49
33 lef-2 28,656>29,240 194 E/L 6/23 35/82 33/81 30/97 36/57 41/40
34 Cp35Ra 29,244>29,513 89 L – 36/87 34/87 31/100 37/43 42/36
35 Unknown 29,549<29,959 136 L – – – 32/100 – –
36 Unknown 30,049<30,369 106 E – 38/89 36/86 – 38/60 –
37 Unknown 30,494<30,952 152 E/L – 39/75 37/74 33/100 39/45 45/20
38 mp-nase 31,036<32,817 593 L – 40/81 38/77 34/99 40/53 46/31
39 Unknown 33,277>34,992 571 E – – 53/86,157/85 43/97 – –
40 Unknown 35,052<36,023 323 – – 42/60 39/58 – 41/25 –
41 p13 36,074>36,910 278 E/L – 43/91 40/91 35/100 42/66 47/50
42 Unknown 37,028>37,621 197 E/L – 44/59 41/61 36/100 – –
43 pif-2 37,638>38,804 388 L 22/51 45/93 42/93 37/100 43/82 48/55
hr1 – 38,812–39,431 – – – – – – – –
44 Unknown 39,435<39,677 80 L – 46/76 43/71 38/99 44/38 49/33
45 Unknown 39,702>43,343 1213 L – 47/82,48/51 44/52 39/99 45/43 50/29
46 Unknown 42,743<43,168 141 – – 49/48 – 40/100 – –
47 Unknown 43,330<44,112 260 L 106/44 50/84 45/83 41/100 46/68 52/53
48 pif-7 44,157>44,321 54 – 110/27 51/90 46/90 42/100 47/75 53/40
49 Unknown 44,456<46,171 571 E – – 53/86,157/85 43/97 – –
50 Ubiquitin 46,360<46,593 77 L 35/76 52/96 47/96 44/100 48/95 54/82
51 odv-ec43 46,695>47,756 353 L 109/30 53/83 48/83 45/99 50/67 55/44
52 Unknown 47,783>48,115 110 L 108/37 54/92 49/94 46/100 51/76 56/29
53 39 k 48,181<49,077 298 L 36/25 55/89 50/90 47/100 52/63 57/24
54 lef-11 49,058<49,345 95 E 37/28 56/89 51/87 – 53/78 58/53
hr2 – 49,441–49,811 – – – – – – – –
55 Unknown 49,955>50,803 282 E/L 57/72 52/71 48/100 57/55 –
56 bro-a/bro-f 50,984<52,480 498 E 60/28,131/30 54/31 49/100 – 63/29
57 Unknown 52,710<52,922 70 E 63/47 – 50/98 – –
58 Unknown 53,074>53,775 233 E/L 151/50 – – 64/74 –
59 Unknown 53,865>54,401 178 – – – – – – 64/34
60 Unknown 54,541<56,619 692 E/L – 64/64 59/64 51/64 – –
61 Unknown 56,674<58,119 481 – – 65/80 60/81 52/71 – –
62 bro 58,220<58,879 219 E/L 2/31 62/45 58/44 53/40 21/42 –
63 he65 59,144<60,775 543 E 105/37 67/82 62/81 54/99 – –
64 sod 60,936<61,397 153 L 31/57 68/89 63/89 55/99 58/74 59/59
65 cath 61,441<62,457 338 L 127/46 58/59 – 56/100 – 11/54
66 bro-a/bro-f 62,525<64,015 496 E 2/35 60/40,131/43 55/45 57/95 21/43 –
67 Unknown 64,111<64,281 56 L – 70/73 64/73 58/100 – –
hr3 – 64,322–64,816 – – – – – – – –
68 Unknown 64,845<65,033 62 – – – – 59/100 – –
69 Unknown 65,205<65,459 84 E – – – 60/94 – –
70 Unknown 65,594>66,247 217 E – 71/63 65/62 61/98 60/37 –
71 Unknown 66,293>67,327 344 E/L – 72/79 66/79 62/99 – –
72 Unknown 67,465>68,862 465 L – 73/82 67/79 63/99 61/48 –
73 Unknown 68,948<70,021 357 L – 74/69 68/70 64/99 65/40, 99/29 –
74 Unknown 70,125>70,412 95 L 79/38 75/87 69/86 65/100 66/63 65/36
hr4 – 70,464–71,122 – – – – – – – –
75 Unknown 72,110<72,367 85 – – – 70/77 66/98 – –
76 bro-b 72,366>73,178 270 E 2/23 76/84 71/83 67/99 67/61 –
77 p74 73,217>75,349 710 L 138/35 77/89 72/89 68/99 68/71 60/42
78 Unknown 75,346<75,681 111 L – – 73/68 69/99 – –
79 p47 75,764>76,948 394 E 40/42 78/91 74/91 70/100 70/74 68/57
80 Rep-like 76,992<77,480 162 E/L – – 75/65 71/100 – –
81 Rep-like 77,574<77,780 68 E – – 76/57 72/100 – –
82 Unknown 78,087>78,764 225 L 38/42 79/97 77/97 73/100 71/85 69/64
83 p24 capsid 78,786>79,301 171 L 129/23 80/88 78/88 74/100 72/70 71/51
84 p38.7 79,338<79,916 192 E/L 13/21 81/73 79/76 75/99 73/46 73/42
85 lef-1 79,917<80,633 238 E 14/31 82/87 80/87 76/99 74/78 74/50
86 p10 80,710>81,291 193 L – 83/79 81/77 77/100 75/51 –
87 pif-1 81,311>82,936 541 L 119/35 84/86 82/87 78/100 76/68 75/48
88 fgf-1 82,962<83,663 233 L – 85/68 83/67 79/99 77/45 76/36
89 Unknown 83,713<84,060 115 E/L – 86/71 84/71 – 78/43 –
90 Unknown 84,175>84,669 164 E/L 150/38 87/73 85/76 80/100 79/30, 80/59 79/29
91 lef-6 84,673<84,972 99 – 28/29 88/84 86/83 81/100 81/57 80/42
92 dbp 85,033<85,866 277 E 25/23 89/84 87/85 82/100 82/56 81/25
93 Unknown 85,972<86,184 70 E/L – – 88/82 83/100 83/61 –
94 Unknown 86,154<86,897 247 – – 90/73 89/75 84/100 84/52 82/35
95 p45 86,896>88,014 372 L 103/36 91/95 90/96 85/100 85/82 83/55
96 p12 88,043>88,408 121 L 102/21 92/80 91/78 86/99 86/59 84/39
97 p40 88,460>89,572 370 E/L 101/20 93/91 92/92 87/100 87/76 85/50
98 p6.9 89,629>89,811 60 L 100/– 94/93 93/90 – 88/79 86/56
99 lef-5 89,847<90,647 266 L 99/39 95/90 94/89 88/100 89/74 87/56
100 38 K 90,570>91,481 303 L 98/37 96/84 95/85 89/99 90/67 88/48
101 Unknown 91,502<91,975 157 L 96/32 97/92 96/92 90/100 91/82 89/49
102 Helicase-1 91,974>95,450 1158 L 95/26 98/89 97/88 91/100 92/78 90/37
103 odv-e25 95,536<96,195 219 L 94/36 99/96 98/95 94/100 93/84 91/65
104 Unknown 96,234<96,710 158 L 93/33 100/94 99/96 95/99 94/66 92/40
105 p33 96,821>97,576 251 L 92/36 101/95 100/94 96/100 95/82 93/54
106 ChaB 97,582<97,845 87 L 60/67 102/95 103/82 97/100 96/80 –
107 Unknown 97,880<98,107 75 E/L – – 104/74 98/98 – –
108 Chitinase 98,275>100,032 585 E/L 126/62 103/87 105/87 99/100 – 10/60
109 Unknown 100,414>100,755 113 E/L – 106/68 108/70 100/100 – –
110 gp37 100,824>101,570 248 E/L 64/45 107/82 109/83 101/99 – 13/44
111 Unknown 101,679>102,152 157 E – 108/78 110/84 102/100 – –
112 bro-c 102,340>103,416 358 E 2/20 109/76 101/68 103/98 – –
113 Unknown 103,455>103,679 74 – – – – – – –
114 lef-4 103,733<105,091 452 – 90/31 110/86 112/86 104/99 100/63 95/42
115 p39 capsid 105,143>106,126 327 L 89/28 111/80 113/80 105/99 101/77 96/38
116 odv-ec27 106,272>107,138 288 L 144/29 112/94 114/95 106/100 102/81 97/45
117 Unknown 107,471<108,685 404 E – 113/81 116/81 107/100 103/54 99/27
hr5 – 108,728–109,096 – – – – – – – –
118 bro-d 109,118<110,389 423 – 2/23 114/84 117/86 108/100 104/68 –
119 Unknown 110,368>111,543 391 – – 115/80 118/79 109/100 105/45 –
120 Unknown 111,652>112,023 123 E/L – 116/92 119/92 110/100 106/66 100/42
121 Unknown 112,066<112,614 182 E – 117/73 120/73 111/100 107/39 –
122 vp91 112,702<114,957 751 L 83/29 118/79 121/80 112/99 108/58 101/35
hr5a – 113,797–113,923 – – – – – – – –
123 tlp20 114,923>115,417 164 L 82/23 119/85 122/86 113/100 109/63 102/25
124 Unknown 115,438>116,001 187 L 81/47 120/96 123/96 114/100 110/80 103/57
125 gp41 116,058>116,930 290 E/L 80/32 121/91 124/91 115/100 111/71 104/51
126 Unknown 117,000>117,323 107 L 78/25 122/84 125/84 116/100 112/52 105/29
127 vlf-1 117,307>118,422 371 E/L 77/32 123/89 126/89 117/100 113/75 106/55
128 Unknown 118,437<118,973 178 E – 124/84 127/84 118/100 114/64 –
129 Unknown 119,015>119,272 85 L 76/34 125/97 128/98 119/100 115/88 107/58
130 Unknown 119,344>119,781 145 E/L 75/29 126/93 129/93 120/100 116/54 108/35
131 Unknown 119,833>120,150 105 L 150/29 20/33,105/31 130/36 121/99 – –
132 Unknown 120,192<120,629 145 E – 128/71 131/71 122/100 – –
133 Unknown 120,702<121,781 359 – – – – – – –
134 lef-7 121,991>122,959 322 E – 129/56 132/57 123/95 – –
135 bro-a/f 123,207>123,932 241 E 2/30 60/56,131/50 133/51,54/47 127/81 21/51 –
136 bro-a/f 124,050>124,505 151 E/L – 60/30,131/34 54/69,133/30 125/58 – –
137 bro-a/f 124,555>126,084 509 E/L 2/27 60/70,131/49 54/67 127/82 21/48 –
138 dna pol 126,186<129,470 1094 E 65/34 132/86 134/86 128/99 117/75 111/52
139 Desmoplakin 129,469>131,460 663 – 66/29 133/68 135/68 129/99 118/47 112/50
140 lef-3 131,553<132,590 345 E 67/28 134/64 136/65 130/99 119/48 113/25
141 pif-6 132,559>132,969 136 – 68/34 135/91 137/91 131/100 120/79 114/44
142 Unknown 133,028>133,543 171 – – 136/71 138/70 132/100 121/43 115/33
143 iap 133,631>134,503 290 – – 137/82 139/81 133/99 122/53 116/31
144 Unknown 134,625>136,646 673 E/L – 138/26 – 134/99 – –
145 lef-9 136,758>138,251 497 – 62/56 139/93 140/93 135/100 123/81 117/62
146 fp 138,304>138,516 70 E/L 61/31 140/91 141/92 136/100 124/83 118/45
147 Unknown 138,550>138,702 50 L – – – 137/100 – –
148 DNA ligase 138,757<140,415 552 E – 141/89 142/89 138/100 125/71 120/43
149 Unknown 140,592>140,831 79 E – 142/81 143/80 139/100 126/62 121/27
150 Unknown 140,892>141,092 66 – – 143/95 144/95 140/100 127/81 122/56
151 fgf 141,155<142,369 404 E 32/27 144/74 145/73 141/99 128/46 123/28
152 alk-exo 142,339>143,760 473 – 133/34 145/78 146/77 142/100 129/61 125/41
153 Helicase-2 143,830>145,203 457 L – 146/85 147/87 143/99 130/71 126/49
154 Unknown 145,314>146,315 333 E 112/30,113/40 147/80 148/79 144/99 131/61 –
155 lef-8 146,356<148,938 860 L 50/48 148/93 149/93 145/99 132/79 131/61
156 odv-e66 149,009<151,012 667 L 46/40 149/92 150/92 146/99 133/79 37/44
hr6 – 151,030–151,402 – – – – – – – –
157 Enhancin-1 151,411<153,897 828 L – 150/74 151/74 147/99 134/47 –
158 bro-f 154,072<155,493 473 E – 131/66 133/67 148/94 21/67 –
159 Enhancin-3 155,673>158,378 901 L – 154/80 153/80 149/99 135/35 –
160 Unknown 158,419>159,585 388 L – 155/89 154/90 150/99 – –
161 Unknown 159,833>160,012 59 – – 157/94 155/91 151/96 – –
162 bro-g 160,230<161,075 281 – 2/27 159/56 158/50 – – –
163 Unknown 161,223<161,435 70 E 111/42 160/70 – 152/86 136/63 –
164 Unknown 161,623>163,113 496 E/L – 161/77 160/76 153/100 137/51 –
165 Unknown 163,143<163,706 187 E – 162/72 161/72 154/97 138/40 –
hr7 – 163,796–164,430 – – – – – – – –
166 Unknown 164,387>164,587 66 – – – – – – –
167 bro-g 164,594>165,649 351 – – 159/64 159/82 155/92 – –
168 Unknown 165,772<165,939 55 – – – 162/83 156/100 139/56
169 Unknown 165,865>166,221 118 – – 165/91 163/94 157/100 – 132/35
170 Enhancin-4 166,267<168,840 857 L – 166/78 164/79 158/99 140/55 –
171 Unknown 169,109>169,438 109 L – 167/71 165/71 159/98 141/55
hr8 – 169,581–169,956 – – – – – – – –
172 Unknown 169,958<170,146 62 E – 170/87 168/87 161/100 142/38 133/53
173 Unknown 170,133>170,552 139 – 53/35 171/88 169/87 162/100 143/56 134/42
174 Unknown 170,556<171,677 373 L – 172/71 170/72 163/100 144/50 135/43
175 Unknown 171,705<171,908 67 L – 173/86 171/86 164/100 145/72 –
176 lef-10 171,886>172,098 70 L 53a/31 174/95 172/94 165/100 146/78 137/34
177 vp1054 171,977>172,948 323 – 54/31 175/92 173/91 166/100 147/78 138/49
178 Unknown 173,035>173,217 60 E/L – 176/91 174/91 167/100 148/72 –
179 Unknown 173,331>173,666 111 E – 177/73 175/72 168/100 149/42 –
180 fgf-1 173,710>174,633 307 – – 178/69 176/68 169/98 150/52 140/29
181 Unknown 174,743>175,375 210 E – 179/75 177/75 170/100 151/45 –
182 me53 175,416>176,330 304 E 139/20 180/88 178/87 171/100 152/63 143/34
183 Unknown 176,336>176,659 107 – – 181/85 179/84 172/100 153/63 –
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Putative MyunGV-A ORFs are listed in column 1 along with the gene homologues designated in column 2. Column 3 indicates ORF location and transcriptional direction on the MyunGV-A genome. Column 4 indicates the number of amino acids. Column 5 indicates the presences of early (E) and/or late (L) promoters located upstream of start codon of each ORF. E indicates a TATA sequence followed by a CAGT or CATT mRNA start site sequence 20–40 nucleotides downstream, with 180 bp upstream of the start codon. L indicates the presence of a (A/T/G)TAAG sequence. Column 6–11 list the homologous ORF and percent of amino acid identity from AcMNPV, XcGV, HearGV, TnGV, MyunGV-B and CpGV respectively.

The first nucleotide of the granulin start codon was defined as nucleotide 1, and the ORF encoding granulin was accordingly designated as the first ORF. The putative ORFs were numbered sequentially in this orientation. Ninety-nine ORFs are in the granulin-sense orientation and 84 in the opposite orientation. A total of 183 putative ORFs of MyunGV-A were searched for promotor motifs at 180 bp upstream of the initiation codon of each ORF; only 42 were found to have a canonical baculovirus early gene promoter motif (a TATA box followed by a CAGT or CATT motif 20 to 40 bp downstream)24,25. Seventy-five ORFs only possess a late promoter motif ((A/T/G) TAAG); 75 contain both early and late promoter motifs, which might allow transcription during both early and late stages of infection. Thirty-four lack any recognizable canonical promoter motif.

Comparison of MyunGV-An ORFs to other baculoviruses

Comparison of gene organization and homology between MyunGV-A and other baculovirus genomes provides insight into gene conservation and implications for the diversity of baculoviruses. MyunGV-A shares 88 ORFs with AcMNPV, 166 with XcGV, 169 with HearGV and TnGV, 139 with MyunGV-B and 104 with CpGV (Table 1). The average amino acid sequence identities of homologous ORFs between MyunGV-A and AcMNPV, XcGV, HearGV, TnGV, MyunGV-B and CpGV are 34%, 79%, 79%, 98%, 62% and 44%, respectively. A total of 180 ORFs were assigned a function or are homologous with other baculoviruses, of which three ORFs (68, 69 and 147) have homologues only with TnGV. ORF68 and ORF147 share 100% homology with TnGV but ORF69 94%. In addition, ORF69 has 37% homology with a kind of bacterium, Zooshikella ganghwensis. Three ORFs, ORF113, -133 and -166, were identified as unique to MyunGV-A.

GeneParityPlot analysis

The gene order of MyunGV-A was compared with that of AcMNPV, XcGV, HearGV, TnGV, MyunGV-B and CpGV by GnenParityPlots analysis (Fig. 2)20. The gene organization of MyunGV-A is distinctly different from that of AcMNPV, except for two reverse collinear gene clusters in which one is a 12-gene group including the core gene cluster of four genes, lef-5, 38K(ac98), ac96, and helicase, with relative positions that are conserved in baculovirus genomes26. In contrast, the gene order of MyunGV-A exhibits extensive collinearity with XcGV, HearGV, TnGV, MyunGV-B and CpGV, except for several genes in a different order that are almost bro or near bro, with the highest collinearity to TnGV. Interestingly, the arrangement of the MyunGV-A genome shows lower collinearity to MyunGV-B, a virus from the same host, than to XcGV, HearGV and TnGV.

Figure 2.

Figure 2

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GeneParityPlots analysis.

Homologous regions (hrs)

A typical feature of most baculovirus genomes is the presence of homologous regions (hrs) interspersed throughout the genome. The numbers of hrs in 82 complete baculovirus genomes range from none to 17, with 12 baculovirus genomes lacking typical hrs sequences (Table S1). In general, hrs are characterized by AT-rich and imperfect, reiterated palindromic sequences that may be replaced with direct repeats.

Eight major hr sequences (hr1-8) and one short hr sequence (hr5a) were identified in the MyunGV-A genome (Table 1). hr1-8 contains two to five direct imperfect repeats, each of approximately 120 bp, whereas hr5a does not contain multiple repeated sequences. It is interesting to note that hr5a is located in ORF122 (vp91), and the same situations exists in the XcGV and HearGV genomes. Six hrs were identified in the MyunGV-B genome lacking sequences corresponding to hr1 and hr5/5a of MyunGV-A27. No hrs were found in the TnGV genome deposited in 2018 (NC_038375.1), and there is no publication on the analysis of the sequence.

Although the nucleotide sequences of repeats vary between each hr, even in the same hr, two highly conserved 10 bp core sequences (TTAAT (G/A) TCGA) were found at the roughly same positions (approximately 35 bp) of each repeat6. In the MyunGV-A genome, the core sequences in each repeat of hr1, -2, -4, -7 and -8 are in the same directions, while those of hr3, -5, -5a and -6 are in opposite directions (Fig. 3).

Figure 3.

Figure 3

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Alignment of homologous regions in the MyunGV-A genome. The conserved 10 bp core sequences (TTAATG/ATCGA) are indicated by shaded boxed. The arrows indicate direction of core sequences.

Hrs have been reported to function in replication origins28,29 and serve as enhancers of transcription of early genes30. In addition, the number of hrs is connected to the replication efficiency or pathogenicity of a baculovirus. Deletion of one to five hrs of AcMNPV had little or no effect on virus infection, while deleting six or seven hrs resulted in 90% BV reduction. Deletion of all eight hrs caused 99.9% BV reduction and delay of early and late gene expression but did not completely inhibit virus production31.

Baculovirus repeated ORFs (bro genes)

Bro genes have been identified in most baculovirus genomes sequenced to date. The number of bro genes in different baculovirus genomes varies considerably. Thirteen of 82 complete baculovirus genomes have only one bro gene, though Lymantria dispar MNPV (LdMNPV) has 16 bro genes. Bro genes are entirely absent from 19 baculovirus genomes sequenced to date (Table S1).

In MyunGV-A, 12 bro genes were identified, of which 3 adjacent bro genes (ORF135, -136, -137) were found. BLAST results of amino acid sequences of these 3 bro genes in NCBI showed that ORF135 best matches with TnGV ORF127 (81%), ORF136 with HearGV ORF54 (69%), and ORF137 with TnGV ORF127 (82%). The TnGV genome have 4 adjacent bro genes (ORF124, -125, -126, -127), and the HearGV genome has 3 pairs of adjacent bro genes (hear54 and -55, hear101 and -102, hear158 and -159), but no adjacent bro genes were found in the XcGV and MyunGV-B genomes.

The exact function of bro genes is not yet clear, though their presence is very significant for baculoviruses. Studies on the function of bro genes have mostly focused on BmNPV and have found that BRO-A and C proteins can bind to DNA in infected cells32; BRO-A may be involved in influencing host DNA replication, similar to a laminin-binding protein33.

In addition, BmNPV BRO proteins act as nucleocytoplasmic shuttling proteins via the CRM1-mediated nuclear export pathway34. Recently, BmNPV BRO-B and E proteins associated with host T-cell intracellular antigen 1 homologue (BmTRN-1) were shown to be involved in the inhibitory regulation of certain mRNAs at the post-transcriptional level during infection35. The function of other baculovirus BRO proteins has seldom been reported.

Two repeat genes in MyunGV-A

Two repeat genes (ORF39 and ORF49), with amino acid sequence identities of 100%, were found in the MyunGV-A genome; the former is in the granulin-sense orientation and the latter in the opposite orientation.

There is no homologous gene with these two genes in the XcGV, MyunGV-B and CpGV genomes. Indeed, only one gene, ORF43, of the TnGV genome matches with them, and the amino acid sequence identity is 97%. Two genes, ORF53 and ORF157, in the HearGV genome are homologous, with amino acid sequence identities of 86% and 85%, respectively, and the amino acid sequence identity of ORF53 and ORF157 in the HearGV genome is 99%. One gene with two copies in one baculovirus genome was found in other baculovirus genomes, such as odv-e66, p26 and dbp of EcobNPV36 and odv-e66 and p26 of SfMNPV37.

BLAST results of amino acid sequences of these two homologous genes in MyunGV-A in NCBI suggested they match hr3 and hr4 of Heliothis virescens ascovirus 3e (amino acid sequence identities both 49%). In addition, they match the 70.4-kDa C-terminal Zn-finger DNA-binding domain of Spodoptera frugiperda ascovirus 1a (amino acid sequence identities of 48%), which suggests that their function may be associated with DNA binding.

ORFs with no homologues in other baculoviruses

Three ORFs, including ORF113, -133 and -166, were identified as having no homologues in other baculoviruses (Table 1). These three unique ORFs have no recognizable promoter. Protein homology analysis using HHpred showed that GP133 (aa 50–359) is a likely homologue of Mannan-binding lectin serine peptidase 1 (probability, 99.97%; E value, 1.1e-28). Mannan-binding lectin serine peptidase 1 plays a central role in the initiation of the complement lectin pathway38. This homology indicates that ORF133 might be related to the complement lectin pathway, which deserves further research. ORF113 encodes an 8.5-kDa protein with one transmembrane domain (aa 5–27, analysed by TMHMM server v2.0) at the N terminus of the protein with no similarity to any proteins in the nonredundant protein database. ORF166 encodes a 7.7-kDa protein with no similarity to any proteins in the nonredundant protein database.

The large gene in MyunGV-A

In most cases, helicase is the largest gene in baculovirus genomes; however, in the MyunGV-A genome, ORF45 encoding 1213 amino acids (longer than helicase-1, 1158) is the largest gene. Similar situations are present in the HearGV (ORF44, 1279 aa), TnGV (ORF39, 1213 aa) and MyunGV-B (ORF45, 1507 aa) genomes, though it is divided into two genes, ORF47 and ORF48, in XcGV6. Compared with XcGV, the MyunGV-A genome has an additional adenosine (A) at position40315, resulting in a reading frame shift. Protein homology analysis using HHpred and SWISS-MODEL showed no significant similarity to any other known sequences for Myun45.

Enhancins in MyunGV-A

It was first observed in Mythimna (formerly Pseudaletia) unipuncta that GV can increase the rate of infection and fatality of NPV and decrease the larval survival time when GV and NPV coinfect larvae10. Subsequent studies found that the factor responsible for synergistic interaction is a GV protein that shows a synergistic effect only when larvae are infected with NPV; it was identified as a synergistic factor (SF)39. The synergistic effect of viral enhancing factor (VEF) was also observed in TnGV40. The location and sequence of the VEF gene of TnGV have been identified41. This enhancing protein (enhancin) can disrupt the midgut peritrophic membrane (PM), thereby resulting in the more efficient passage of virions to host midgut cells12. Enhancin was identified as a metalloprotease via the discovery of a zinc-binding site as well as by inhibition with a metal chelator and reactivation with divalent ions42.

The MyunGV-A genome has three enhancin genes (Myun157, -159 and -170). Similarly, three enhancin genes were found in MyunGV-B and TnGV, but they show large diversity in amino acid sequence identity compared to MyunGV-A. MyunGV-B enhancins are only 35% to 55% identical to that of MyunGV-A but are as high as 99% identical to that of TnGV. Four enhancin genes were found in the XcGV and HearGV genomes, of which enhancin-1, -3, and -4 have high homology (amino acid sequence identities all above 74%) to three enhancin genes of MyunGV-A. The MyunGV-A enhancin gene (enhancin-3) encoding 901 amino acids has been sequenced and characterized12. The canonical sequence HEXXH, the zinc-binding site in most metalloproteases, was found in enhancing-3 but not in the other two enhancins. It is not clear why three enhancins are present in MyunGV-A, and the roles of these three enhancins in promoting NPV infection remain unclear.

Enhancins are found mainly in GVs and a few NPVs. They are localized within the granulin matrix in granuloviruses and released to increase virus pathogenicity by acting in the midgut. In contrast, LdMNPV enhancins are located within ODV envelopes and facilitate ODVs to pass the host defence barrier by acting directly on the peritrophic membrane as the nucleocapsids move through the barrier43. However, subsequent studies have indicated that LdMNPV enhancins have a function that may assist virus-host cell fusion beyond peritrophic membrane degradation44.

Protein analysis of OBs of MyunGV-A

To date, nine baculovirus proteomic studies have been performed with the intent of revealing infectious mechanisms and virus-host interactions, as follows: six alphabaculviruses—AcMNPV45,46, BmNPV47, HearSNPV48, HearNPV-G449, AgMNPV50 and ChchNPV51; two betabaculoviruses, ClanGV52 and PrGV53; and one deltabaculovirus, CuniNPV54. In this study, we performed an analysis of MyunGV-An OB proteins. For 29 samples, 24 proteins were identified from the putative protein database of MyunGV-A (NC_013772.1) (Table 2). Among the 24 proteins, 20 were detected with two or more peptides, and the other four were detected with one matching peptide. In addition, 15 of 24 identified proteins were detected in more than one sample. Granulin was found in 28 of the 29 samples (Table S2). The same situations were found for CuniNPV54, HearsNPV48 and AgMNPV50. A noticeable phenomenon was also observed, whereby the identified proteins were not distributed according to their molecular mass in SDS-PAGE gels. The reason was postulated to be incomplete denaturation of OBs and the breakdown of protein complexes or protein processing54.

Table 2.

Analysis of proteins identified from MyunGV-A.

ORF Protein ODV of GV ODV of NPV Characteristics/function
PrGV ClanGV AcMNPV HearNPV AgMNPV ChchNPV CuniNPV
1 (Ac8) Granulin 1 1 8 1 1 1 1 Occlusion bodies (OBs) matrix protein
11 (Ac143) ODV-e18 14 13 143 10 139 12 NA Core gene; Structural protein of ODV envelope
12 (Ac142) VP49 15 14 142 9 138 11 30 Core gene; a caspase inhibitor; Inhibiting diverse apoptotic stimuli
14 (Ac148) ODV-e56 16 15 148 15 144 7 102 Core gene; Structural protein of ODV envelope
16 (Cp20) PEP-1 20 19 NA 120 127 NA NA Additional ORFs conserved in GVs ; ORF16L family
17 (Cp23) PEP-2 22 36 NA NA NA – NA Additional ORFs conserved in GVs ; ORF16L family
18 (Cp22) PEP/P10 21 35 NA 21 133 – NA Additional ORFs conserved in GVs ; Similarities to P10 containing a baculovirus PEP C domain
29 (Cp34) Unknown 29 NA NA NA NA NA NA Unknown
32 (Cp39) Unknown - 39 NA NA NA NA NA Additional ORFs conserved in GVs
44 (Cp49) Unknown NA NA NA NA NA NA NA Unknown
48 (Ac110) PIF-7 – 20 – NA NA NA NA Unknown
51 (Ac109) ODV-ec43 46 44 109 94 107 99 69 Core gene; Structural protein of ODV envelope
64 (Ac31) SOD – 48 – 106 – 115 NA Metalloenzyme; Protecting the virus against superoxide radical induced in the environment by sunlight
65 (Ac127) Cathepsin – 11 127 – NA – NA Associated with liquefaction of insects at the end of infection; Promoting the release and spread of progeny virus
67 (Xc70) Unknown NA NA NA NA NA NA NA Unknown
103 (Ac94) ODV-e25 – 76 94 82 91 86 NA Core gene; Structural protein of ODV envelope
115 (Ac89) VP39 81 81 89 78 86 82 24 Core gene; Major capsid protein
120 (Cp100) Unknown 84 84 NA NA NA NA NA Additional ORFs conserved in GVs
125 (Ac80) GP41 88 89 80 73 79 78 33 Core gene; O-linked glycosylated ODV protein; BV production
156 (Ac40) ODV-e66 39/44 NA 46 96 114 101 NA Structural protein of ODV envelope
157 (Xc150) Enhancin-1 NA NA NA NA NA NA NA Metalloproteinases; Enhancing the oral infectivity of NPVs
159 (Xc154) Enhancin-3 NA NA NA NA NA NA NA Metalloproteinases; Enhancing the oral infectivity of NPVs
174 (Cp135) Unknown 114 116 NA NA NA NA NA Additional ORFs conserved in GVs
175 (Cp136) Unknown - 117 NA NA NA NA NA Additional ORFs conserved in GVs
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Dash (–), the protein was not detected. NA, ORF was not found in the baculovirus genome by BLASTP.

Of the 24 identified proteins, eight are encoded by core genes, including ORF11 (ODV-e18), ORF12 (VP49), ORF14 (ODV-e56), ORF48 (PIF-7), ORF51 (ODV-ec43), ORF103 (ODV-e25), ORF115 (VP39) and ORF125 (GP41); among them, VP39 is the major capsid protein, GP41 is a tegument protein only found in ODVs and is present in the nucleocapsid and the viral envelope as a structural protein of ODVs, and four proteins, including ODV-e18, ODV-e56, ODV-e25 and ODV-ec43, are ODV envelope proteins (Table 2). An ODV envelope protein, ODV-e66, was also identified.

For the 24 identified proteins, six are encoded by additional genes conserved in GVs, including ORF16, ORF17, ORF18, ORF120, ORF174 and ORF17555. Among them, proteins encoded by two contiguous ORFs (ORF16 and 17) belong to the CpGV ORF16 L family56, and the protein encoded by ORF18 is similar to P10, containing a baculovirus polyhedron envelope protein (PEP) C domain (pfam04513). In addition to structural proteins or those implicated in DNA replication and transcription, four important auxiliary proteins were identified, including SOD, cathepsin and two enhancins. Enhancin-1 and enhancin-3 were detected in our proteomic studies; enhancin-3 was present in 16 samples, while enhancin-1 was present in only 1 sample. Most baculovirus enhancins, including MyunGV-A, are located in the OB matrix, whereas LdMNPV enhancins were found to be associated with ODV envelopes43,57. In this study, we did not attempt to determine the specific location of enhancins.

Moreover, four proteins (Myun29, Myun32, Myun44 and Myun67) with unknown functions were detected (Table 2). An increasing number of baculovirus proteomic studies can provide valuable insight into baculovirus structure, infectious mechanisms and interactions with their hosts.

Phylogenetic analysis of MyunGV-A

A phylogenetic tree based on the combined amino acid sequences of 38 core genes from 82 complete baculovirus genomes (Table S1) classified MyunGV-A into clade “a” of Betabaculovirus, which clusters infecting the larvae of the Lepidopteran family Noctuidae. Within this clade, MyunGV-A is present into a subcluster together with TnGV, the closest neighbour, sharing a common hypothetical ancestor. XcGV and HearGV form another subcluster next to the MyunGV-A and TnGV subclusters. However, MyunGV-B, another granulovirus from the same host, groups into a subcluster with SpfrGV and slightly away from MyunGV-A across MolaGV (Fig. 4). This is consistent with the above comparison results of gene organization in which MyunGV-A is similar to TnGV, XcGV and HearGV, regardless of genome size, ORF number or gene order.

Figure 4.

Figure 4

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Phylogenetic tree of 82 baculoviruses with complete sequences. The phylogenetic tree was generated using MEGA X58 software and performed with the maximum likelihood method and JTT matrix-based model59. The result was visualized using iToL60.

Conclusion

The purified OBs of MyunGV-A show typical GV morphological characteristics under EM. The complete MyunGV-A (NC_013772.1) genome is 176,677 bases, with a G+C content of 39.79%, the second largest baculovirus genome to date. It contains 183 ORFs with a minimal size of 50 codons. The genome of MyunGV-A exhibits extensive sequence similarity and collinearity with TnGV, XcGV and HearGV. Three unique genes, 12 bro, 2 helicase and 3 enhancin genes, were identified. In particular, two repeated genes (ORF39 and 49) are present in the genome in reverse and complementarily orientations. Twenty-four OB proteins were identified from the putative protein database of MyunGV-A. According to our phylogenetic tree, MyunGV-A belongs to the Betabaculovirus group and is most closely related to TnGV.

Supplementary Information

Supplementary Tables. (323.8KB, pdf)

Acknowledgements

This work was supported by National Natural Sciences Foundation of China (No. 31872430 and 31670156), The State Key Laboratory of Integrated Management of Pest Insects and Rodents (Grant No. IPM1902) and The Agricultural Science and Technology Innovation Program.

Author contributions

Z.Z. and Q.Q. conceived the idea; Y.L. and Z.Z. designed research; Y.L., X.L., P.T., and H.Z. performed research; Y.L. and X.L. analyzed data; Y.L. and X.L. wrote the main manuscript text; All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Yinü Li and Xingjian Liu.

Contributor Information

Qilian Qin, Email: qinql@ioz.ac.cn.

Zhifang Zhang, Email: bri-zhangzhifang@caas.cn.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-020-80117-3.

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