The genome sequence of a novel small RNA virus, tentatively named Diabrotica undecimpunctata virus 1 (DuV1), was discovered from the transcriptome of the southern corn rootworm, Diabrotica undecimpunctata howardi Barber. DuV1 has a positive-sense, single-stranded RNA genome that encodes a single polyprotein of 3,401 amino acids with limited similarity to other viruses

Bonning, Bryony C.

Darlington, Molly

Liu, Sijun

Valencia-Jiménez, Arnubio

Vélez, Ana M.

English

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University of Nebraska - Lincoln 
DigitalCommons@University of Nebraska - Lincoln 
Faculty Publications: Department of 
Entomology Entomology, Department of 
2020 
Genome Sequence of a Small RNA Virus of the Southern Corn 
Rootworm, Diabrotica undecimpunctata howardi Barber 
(Coleoptera: Chrysomelidae) 
Sijun Liu 
Iowa State University 
Arnubio Valencia-Jiménez 
Universidad de Caldas & University of Nebraska—Lincoln 
Molly Darlington 
University of Nebraska—Lincoln 
Ana M. Vélez 
University of Nebraska—Lincoln 
Bryony C. Bonning 
University of Florida, Gainesville, bbonning@ufl.edu 
Follow this and additional works at: https://digitalcommons.unl.edu/entomologyfacpub 
 Part of the Entomology Commons 
Liu, Sijun; Valencia-Jiménez, Arnubio; Darlington, Molly; Vélez, Ana M.; and Bonning, Bryony C., "Genome 
Sequence of a Small RNA Virus of the Southern Corn Rootworm, Diabrotica undecimpunctata howardi 
Barber (Coleoptera: Chrysomelidae)" (2020). Faculty Publications: Department of Entomology. 863. 
https://digitalcommons.unl.edu/entomologyfacpub/863 
This Article is brought to you for free and open access by the Entomology, Department of at 
DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: 
Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. 
Genome Sequence of a Small RNA Virus of the Southern Corn
Rootworm, Diabrotica undecimpunctata howardi Barber
(Coleoptera: Chrysomelidae)
Sijun Liu,a Arnubio Valencia-Jiménez,b,c Molly Darlington,c Ana M. Vélez,c Bryony C. Bonningd
aDepartment of Entomology, Iowa State University, Ames, Iowa, USA
bDepartamento de Producción Agropecuaria, Universidad de Caldas, Manizales, Colombia
cDepartment of Entomology, University of Nebraska—Lincoln, Lincoln, Nebraska, USA
dDepartment of Entomology and Nematology, University of Florida, Gainesville, Florida, USA
ABSTRACT The genome sequence of a novel small RNA virus, tentatively named
Diabrotica undecimpunctata virus 1 (DuV1), was discovered from the transcriptome of
the southern corn rootworm, Diabrotica undecimpunctata howardi Barber. DuV1 has
a positive-sense, single-stranded RNA genome that encodes a single polyprotein of
3,401 amino acids with limited similarity to other viruses.
The southern corn rootworm (SCR), Diabrotica undecimpunctata howardi Barber, is apolyphagous pest that impacts multiple crops in the southeastern United States,
southern Canada, and northern Mexico (1). While several small RNA viruses have been
reported from other rootworm species (2–4), this is the first report of a virus genome
sequence from the transcriptome of D. undecimpunctata.
SCR were supplied by Crop Characteristics (Farmington, MN). Total RNA was isolated
from three biological replicates of multiple developmental stages (eggs; first-, second-, and
third-instar larvae; pupae; adult females; and adult males) using the RNeasy minikit (Qiagen,
Valencia, CA, USA). RNA extractions from each stage-specific sample were used for cDNA
library construction with poly(A) selection. The NEBNext Ultra RNA library prep kit for
Illumina was used for library preparation. cDNA libraries were paired-end sequenced
(150-nucleotide [nt] reads) on a HiSeq 2500 platform (Illumina, San Diego, CA). High-quality
sequencing reads (48.2 million reads for males, 41.5 million reads for females; mean quality
score, 37.5) were assembled by using the Trinity assembler v2.6.6 (5). Default parameters
were used for all software unless otherwise noted. Putative viral sequences were initially
identified by BLASTx analysis (6) against a local viral sequence database with the following
cutoff values: contig length, 300 nt; protein sequence length, 50 amino acids [aa]; and
E value, 0.01. Here, we describe one of two near-complete viral genome sequences
identified from this analysis.
A 10,483-nt contig with a GC content of 47% was assembled from the sequencing
reads from both female and male adult SCR and was named Diabrotica undecimpunc-
tata virus 1 (DuV1). A total of 1,070 reads from males and 1,078 from females mapped
to viral sequences with a coverage of 15.3 and 15.4, respectively. Only a few short
fragments were identified from larval samples. A 10,203-nt coding region was flanked
by a 5= untranslated region (UTR) of at least 139 nt and a 3= UTR of at least 141 nt. The
predicted molecular mass of the polyprotein is 378.7 kDa. The N-terminal half of the
hypothetical protein contains putative nonstructural proteins, with the C-terminal half
comprised of putative capsid proteins. The nonstructural protein sequence contained
six conserved domains, four of which overlapped, i.e., Macro_Poa1p_like domain (aa 46
to 151; NCBI accession no. cd02901); A1pp, an appr-1-p processing enzyme (aa 48 to
149; SMART accession no. SM00506); Macro, an ADP-ribose binding module (aa 89 to
Citation Liu S, Valencia-Jiménez A, Darlington
M, Vélez AM, Bonning BC. 2020. Genome
sequence of a small RNA virus of the southern
corn rootworm, Diabrotica undecimpunctata
howardi Barber (Coleoptera: Chrysomelidae).
Microbiol Resour Announc 9:e00379-20.
https://doi.org/10.1128/MRA.00379-20.
Editor Kenneth M. Stedman, Portland State
University
Copyright © 2020 Liu et al. This is an open-
access article distributed under the terms of
the Creative Commons Attribution 4.0
International license.
Address correspondence to Bryony C. Bonning,
bbonning@ufl.edu.
Received 10 April 2020
Accepted 28 May 2020
Published 25 June 2020
GENOME SEQUENCES
crossm
Volume 9 Issue 26 e00379-20 mra.asm.org 1
149; NCBI accession no. pfam01661); and Ymd, an O-acetyl-ADP-ribose deacetylase
(regulator of RNase III) (aa 63 to 174; NCBI accession no. COG2110). An RNA_helicase
domain was located between aa 812 and 913 (NCBI accession no. pfam00910), and an
RdRP domain was located between aa 1934 and 2367 (EMBL-EBI accession no.
pfam00680, NCBI accession no. cd01699). Two cysteine protease sequence motifs,
GxCG and GxHxxG, were identified between aa 1734 and 1737 (GDCG) and aa 1755 and
1760 (GIHVAG). No conserved domains were found in the putative viral capsid region.
The genome organization of DuV1 encoding a single polyprotein with N-terminal nonstruc-
tural proteins is similar to that of Diabrotica virgifera virgifera virus 2 (DvvV2) (3). However, the
highest BLASTp score (6, 7) was an unclassified virus, Wuhan house centipede virus 3 (GenBank
accession no. YP_009342325.1) (8), with only 36% sequence coverage and 31% amino acid
identity. Similar sequences were limited to nonstructural proteins. A phylogenetic tree
based on the RdRP sequences of DuV1 and selected related RNA viruses highlights the
novelty of this virus (Fig. 1). Based on this analysis, DuV1 represents a novel virus.
Data availability. The genome sequence of DuV1 is available in GenBank under
accession no. MN646770. Raw reads for SRA accession no. SRX8018653 to SRX8018656
are available at BioProject no. PRJNA615920.
ACKNOWLEDGMENTS
This research was supported by the USDA National Institute of Food and Agriculture,
Hatch project IOW05450; by State of Iowa funds; University of Nebraska—Lincoln funds;
the 2016 DuPont Young Professor Grant awarded to A.M.V.; and by the University of
Florida Institute of Food and Agricultural Sciences.
REFERENCES
1. Krysan JL. 1986. Introduction: biology, distribution, and identification of
pest Diabrotica. In Krysan JLM, Thomas A (ed), Methods for the study of
pest Diabrotica. Springer, New York, NY.
2. Liu S, Chen Y, Sappington TW, Bonning BC. 2017. Genome sequence of a
novel positive-sense, single-stranded RNA virus isolated from western
corn rootworm, Diabrotica virgifera virgifera LeConte. Genome Announc
5:e00366-17. https://doi.org/10.1128/genomeA.00366-17.
3. Liu S, Chen Y, Sappington TW, Bonning BC. 2017. Genome sequence of
FIG 1 Phylogenetic analysis of DuV1 RdRP. The RdRP sequences identified by an RdRP domain search on BLAST
annotation were extracted from the NCBI database. Sequences were aligned by use of MAFFT v7.4.07 with the AUTO
strategy. The phylogenetic tree was constructed using the maximum likelihood (ML) method. ModelFinder implemented
in IQ-TREE was used to choose the best partitioning scheme and models. ML analysis was performed using IQ-TREE with
10,000 Ultrafast bootstraps (9). Constructed trees were uploaded to Interactive Tree of Life (http://itol.embl.de) for
visualization and annotation. The selected RNA viruses and their NCBI genome accession numbers are as follows: Beihai
picorna-like virus 72, APG76793.1; Citrus mosaic sadwavirus, AWY04239.1; Dinocampus coccinellae paralysis virus,
YP_009111311.1; Diabrotica virgifera virgifera virus 2, YP_009352233.1; Hubei orthoptera virus 3, YP_009336506.1; Hubei
picorna-like virus 53, YP_009336820.1; Hubei tetragnatha maxillosa virus 4, YP_009336535.1; Moyer virus, AOC55061.1;
Nylanderia fulva virus 1, YP_009268643.1; Solenopsis invicta virus 3, ACZ65579.1; Tomato chocolate virus, ACU01024.1;
Wuhan house centipede virus 3, YP_009342325.1; and Wuhan insect virus 12, YP_009345893.1.
Liu et al.
Volume 9 Issue 26 e00379-20 mra.asm.org 2
Diabrotica virgifera virgifera virus 2, a novel small RNA virus of the
western corn rootworm, Diabrotica virgifera virgifera LeConte. Genome
Announc 5:e00365-17. https://doi.org/10.1128/genomeA.00365-17.
4. Liu S, Chen Y, Sappington TW, Bonning BC. 2017. Genome sequence of
the first coleopteran iflavirus isolated from western corn rootworm, Dia-
brotica virgifera virgifera LeConte. Genome Announc 5:e01530-16. https://
doi.org/10.1128/genomeA.01530-16.
5. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J,
Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, Pochet
N, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R,
Leduc RD, Friedman N, Regev A. 2013. De novo transcript sequence
reconstruction from RNA-seq using the Trinity platform for reference
generation and analysis. Nat Protoc 8:1494 –1512. https://doi.org/10
.1038/nprot.2013.084.
6. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman
DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein
database search programs. Nucleic Acids Res 25:3389 –3402. https://doi
.org/10.1093/nar/25.17.3389.
7. Altschul SF, Wootton JC, Gertz EM, Agarwala R, Morgulis A, Schaffer AA,
Yu YK. 2005. Protein database searches using compositionally adjusted
substitution matrices. FEBS J 272:5101–5109. https://doi.org/10.1111/j
.1742-4658.2005.04945.x.
8. Shi M, Lin XD, Tian JH, Chen LJ, Chen X, Li CX, Qin XC, Li J, Cao JP, Eden
JS, Buchmann J, Wang W, Xu J, Holmes EC, Zhang YZ. 2016. Redefining
the invertebrate RNA virosphere. Nature 540:539 –543. https://doi.org/10
.1038/nature20167.
9. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. 2018.
UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol
35:518 –522. https://doi.org/10.1093/molbev/msx281.
Microbiology Resource Announcement
Volume 9 Issue 26 e00379-20 mra.asm.org 3


Genome Sequence of a Small RNA Virus of the Southern Corn Rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae)

DigitalCommons@University of Nebraska

University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Faculty Publications: Department of Entomology Entomology, Department of 2020 Genome Sequence of a Small RNA Virus of the Southern Corn Rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae) Sijun Liu Iowa State University Arnubio Valencia-Jiménez Universidad de Caldas & University of Nebraska—Lincoln Molly Darlington University of Nebraska—Lincoln Ana M. Vélez University of Nebraska—Lincoln Bryony C. Bonning University of Florida, Gainesville, bbonning@ufl.edu Follow this and additional works at: https://digitalcommons.unl.edu/entomologyfacpub  Part of the Entomology Commons Liu, Sijun; Valencia-Jiménez, Arnubio; Darlington, Molly; Vélez, Ana M.; and Bonning, Bryony C., "Genome Sequence of a Small RNA Virus of the Southern Corn Rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae)" (2020). Faculty Publications: Department of Entomology. 863. https://digitalcommons.unl.edu/entomologyfacpub/863 This Article is brought to you for free and open access by the Entomology, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Faculty Publications: Department of Entomology by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. Genome Sequence of a Small RNA Virus of the Southern CornRootworm, Diabrotica undecimpunctata howardi Barber(Coleoptera: Chrysomelidae)Sijun Liu,a Arnubio Valencia-Jiménez,b,c Molly Darlington,c Ana M. Vélez,c Bryony C. BonningdaDepartment of Entomology, Iowa State University, Ames, Iowa, USAbDepartamento de Producción Agropecuaria, Universidad de Caldas, Manizales, ColombiacDepartment of Entomology, University of Nebraska—Lincoln, Lincoln, Nebraska, USAdDepartment of Entomology and Nematology, University of Florida, Gainesville, Florida, USAABSTRACT The genome sequence of a novel small RNA virus, tentatively namedDiabrotica undecimpunctata virus 1 (DuV1), was discovered from the transcriptome ofthe southern corn rootworm, Diabrotica undecimpunctata howardi Barber. DuV1 hasa positive-sense, single-stranded RNA genome that encodes a single polyprotein of3,401 amino acids with limited similarity to other viruses.The southern corn rootworm (SCR), Diabrotica undecimpunctata howardi Barber, is apolyphagous pest that impacts multiple crops in the southeastern United States,southern Canada, and northern Mexico (1). While several small RNA viruses have beenreported from other rootworm species (2–4), this is the first report of a virus genomesequence from the transcriptome of D. undecimpunctata.SCR were supplied by Crop Characteristics (Farmington, MN). Total RNA was isolatedfrom three biological replicates of multiple developmental stages (eggs; first-, second-, andthird-instar larvae; pupae; adult females; and adult males) using the RNeasy minikit (Qiagen,Valencia, CA, USA). RNA extractions from each stage-specific sample were used for cDNAlibrary construction with poly(A) selection. The NEBNext Ultra RNA library prep kit forIllumina was used for library preparation. cDNA libraries were paired-end sequenced(150-nucleotide [nt] reads) on a HiSeq 2500 platform (Illumina, San Diego, CA). High-qualitysequencing reads (48.2 million reads for males, 41.5 million reads for females; mean qualityscore, 37.5) were assembled by using the Trinity assembler v2.6.6 (5). Default parameterswere used for all software unless otherwise noted. Putative viral sequences were initiallyidentified by BLASTx analysis (6) against a local viral sequence database with the followingcutoff values: contig length,300 nt; protein sequence length,50 amino acids [aa]; andE value, 0.01. Here, we describe one of two near-complete viral genome sequencesidentified from this analysis.A 10,483-nt contig with a GC content of 47% was assembled from the sequencingreads from both female and male adult SCR and was named Diabrotica undecimpunc-tata virus 1 (DuV1). A total of 1,070 reads from males and 1,078 from females mappedto viral sequences with a coverage of 15.3 and 15.4, respectively. Only a few shortfragments were identified from larval samples. A 10,203-nt coding region was flankedby a 5= untranslated region (UTR) of at least 139 nt and a 3= UTR of at least 141 nt. Thepredicted molecular mass of the polyprotein is 378.7 kDa. The N-terminal half of thehypothetical protein contains putative nonstructural proteins, with the C-terminal halfcomprised of putative capsid proteins. The nonstructural protein sequence containedsix conserved domains, four of which overlapped, i.e., Macro_Poa1p_like domain (aa 46to 151; NCBI accession no. cd02901); A1pp, an appr-1-p processing enzyme (aa 48 to149; SMART accession no. SM00506); Macro, an ADP-ribose binding module (aa 89 toCitation Liu S, Valencia-Jiménez A, DarlingtonM, Vélez AM, Bonning BC. 2020. Genomesequence of a small RNA virus of the southerncorn rootworm, Diabrotica undecimpunctatahowardi Barber (Coleoptera: Chrysomelidae).Microbiol Resour Announc 9:e00379-20.https://doi.org/10.1128/MRA.00379-20.Editor Kenneth M. Stedman, Portland StateUniversityCopyright © 2020 Liu et al. This is an open-access article distributed under the terms ofthe Creative Commons Attribution 4.0International license.Address correspondence to Bryony C. Bonning,bbonning@ufl.edu.Received 10 April 2020Accepted 28 May 2020Published 25 June 2020GENOME SEQUENCEScrossmVolume 9 Issue 26 e00379-20 mra.asm.org 1149; NCBI accession no. pfam01661); and Ymd, an O-acetyl-ADP-ribose deacetylase(regulator of RNase III) (aa 63 to 174; NCBI accession no. COG2110). An RNA_helicasedomain was located between aa 812 and 913 (NCBI accession no. pfam00910), and anRdRP domain was located between aa 1934 and 2367 (EMBL-EBI accession no.pfam00680, NCBI accession no. cd01699). Two cysteine protease sequence motifs,GxCG and GxHxxG, were identified between aa 1734 and 1737 (GDCG) and aa 1755 and1760 (GIHVAG). No conserved domains were found in the putative viral capsid region.The genome organization of DuV1 encoding a single polyprotein with N-terminal nonstruc-tural proteins is similar to that of Diabrotica virgifera virgifera virus 2 (DvvV2) (3). However, thehighest BLASTp score (6, 7) was an unclassified virus,Wuhan house centipede virus 3 (GenBankaccession no. YP_009342325.1) (8), with only 36% sequence coverage and 31% amino acididentity. Similar sequences were limited to nonstructural proteins. A phylogenetic treebased on the RdRP sequences of DuV1 and selected related RNA viruses highlights thenovelty of this virus (Fig. 1). Based on this analysis, DuV1 represents a novel virus.Data availability. The genome sequence of DuV1 is available in GenBank underaccession no. MN646770. Raw reads for SRA accession no. SRX8018653 to SRX8018656are available at BioProject no. PRJNA615920.ACKNOWLEDGMENTSThis research was supported by the USDA National Institute of Food and Agriculture,Hatch project IOW05450; by State of Iowa funds; University of Nebraska—Lincoln funds;the 2016 DuPont Young Professor Grant awarded to A.M.V.; and by the University ofFlorida Institute of Food and Agricultural Sciences.REFERENCES1. Krysan JL. 1986. Introduction: biology, distribution, and identification ofpest Diabrotica. In Krysan JLM, Thomas A (ed), Methods for the study ofpest Diabrotica. Springer, New York, NY.2. Liu S, Chen Y, Sappington TW, Bonning BC. 2017. Genome sequence of anovel positive-sense, single-stranded RNA virus isolated from westerncorn rootworm, Diabrotica virgifera virgifera LeConte. Genome Announc5:e00366-17. https://doi.org/10.1128/genomeA.00366-17.3. Liu S, Chen Y, Sappington TW, Bonning BC. 2017. Genome sequence ofFIG 1 Phylogenetic analysis of DuV1 RdRP. The RdRP sequences identified by an RdRP domain search on BLASTannotation were extracted from the NCBI database. Sequences were aligned by use of MAFFT v7.4.07 with the AUTOstrategy. The phylogenetic tree was constructed using themaximum likelihood (ML) method. ModelFinder implementedin IQ-TREE was used to choose the best partitioning scheme and models. ML analysis was performed using IQ-TREE with10,000 Ultrafast bootstraps (9). Constructed trees were uploaded to Interactive Tree of Life (http://itol.embl.de) forvisualization and annotation. The selected RNA viruses and their NCBI genome accession numbers are as follows: Beihaipicorna-like virus 72, APG76793.1; Citrus mosaic sadwavirus, AWY04239.1; Dinocampus coccinellae paralysis virus,YP_009111311.1; Diabrotica virgifera virgifera virus 2, YP_009352233.1; Hubei orthoptera virus 3, YP_009336506.1; Hubeipicorna-like virus 53, YP_009336820.1; Hubei tetragnatha maxillosa virus 4, YP_009336535.1; Moyer virus, AOC55061.1;Nylanderia fulva virus 1, YP_009268643.1; Solenopsis invicta virus 3, ACZ65579.1; Tomato chocolate virus, ACU01024.1;Wuhan house centipede virus 3, YP_009342325.1; and Wuhan insect virus 12, YP_009345893.1.Liu et al.Volume 9 Issue 26 e00379-20 mra.asm.org 2Diabrotica virgifera virgifera virus 2, a novel small RNA virus of thewestern corn rootworm, Diabrotica virgifera virgifera LeConte. GenomeAnnounc 5:e00365-17. https://doi.org/10.1128/genomeA.00365-17.4. Liu S, Chen Y, Sappington TW, Bonning BC. 2017. Genome sequence ofthe first coleopteran iflavirus isolated from western corn rootworm, Dia-brotica virgifera virgifera LeConte. Genome Announc 5:e01530-16. https://doi.org/10.1128/genomeA.01530-16.5. Haas BJ, Papanicolaou A, Yassour M, Grabherr M, Blood PD, Bowden J,Couger MB, Eccles D, Li B, Lieber M, MacManes MD, Ott M, Orvis J, PochetN, Strozzi F, Weeks N, Westerman R, William T, Dewey CN, Henschel R,Leduc RD, Friedman N, Regev A. 2013. De novo transcript sequencereconstruction from RNA-seq using the Trinity platform for referencegeneration and analysis. Nat Protoc 8:1494–1512. https://doi.org/10.1038/nprot.2013.084.6. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, LipmanDJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of proteindatabase search programs. Nucleic Acids Res 25:3389–3402. https://doi.org/10.1093/nar/25.17.3389.7. Altschul SF, Wootton JC, Gertz EM, Agarwala R, Morgulis A, Schaffer AA,Yu YK. 2005. Protein database searches using compositionally adjustedsubstitution matrices. FEBS J 272:5101–5109. https://doi.org/10.1111/j.1742-4658.2005.04945.x.8. Shi M, Lin XD, Tian JH, Chen LJ, Chen X, Li CX, Qin XC, Li J, Cao JP, EdenJS, Buchmann J, Wang W, Xu J, Holmes EC, Zhang YZ. 2016. Redefiningthe invertebrate RNA virosphere. Nature 540:539–543. https://doi.org/10.1038/nature20167.9. Hoang DT, Chernomor O, von Haeseler A, Minh BQ, Vinh LS. 2018.UFBoot2: improving the ultrafast bootstrap approximation. Mol Biol Evol35:518–522. https://doi.org/10.1093/molbev/msx281.Microbiology Resource AnnouncementVolume 9 Issue 26 e00379-20 mra.asm.org 3

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Genome Sequence of a Small RNA Virus of the Southern Corn Rootworm, Diabrotica undecimpunctata howardi Barber (Coleoptera: Chrysomelidae)

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