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Article ; Online: Complete genome characterization of cacao leafroll virus, a newly described cacao-infecting polerovirus.

Adegbola, Raphael O / Keith, Cory V / Gutierrez, Osman / Goenaga, Ricardo / Brown, Judith K

2024 Volume 169, Issue 4, Page(s) 83

Abstract: The complete genome sequence of cacao leafroll virus (CaLRV; family Solemoviridae, genus Polerovirus) was determined by high-throughput sequencing of total RNA isolated from symptomatic cacao Theobroma cacao L. plants (n = 4). The CaLRV genome sequences ... ...

Abstract	The complete genome sequence of cacao leafroll virus (CaLRV; family Solemoviridae, genus Polerovirus) was determined by high-throughput sequencing of total RNA isolated from symptomatic cacao Theobroma cacao L. plants (n = 4). The CaLRV genome sequences ranged from 5,976 to 5,997 nucleotides (nt) in length and contained seven open reading frames (ORFs). Nucleotide and amino acid (aa) sequence comparisons showed that, among selected well-characterized poleroviruses, the CaLRV genome shared the highest nt sequence identity of 62% with that of potato leafroll virus (PLRV, NC_076505). A comparison of the predicted aa sequence of the CaLRV coat protein indicated that cotton leafroll dwarf virus (CLRDV, NC_014545) and melon aphid-borne yellows virus (MABYV, NC_010809) were the closest relatives, sharing 57% aa sequence identity. Bayesian phylogenetic analysis based on complete genome sequences showed that CaLRV grouped with well-characterized poleroviruses that cause diseases of cereal and vegetable crops. During the course of publishing this work, the nearly complete genome sequence of a member of the same polerovirus species, referred to as "cacao polerovirus" (OR605721), with which CaLRV shares 99% nt sequence identity, was reported.
MeSH term(s)	Genome, Viral ; Cacao ; Phylogeny ; Bayes Theorem ; Luteoviridae ; Plant Diseases ; Open Reading Frames
Language	English
Publishing date	2024-03-23
Publishing country	Austria
Document type	Journal Article
ZDB-ID	7491-3
ISSN	1432-8798 ; 0304-8608
ISSN (online)	1432-8798
ISSN	0304-8608
DOI	10.1007/s00705-024-06013-7
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Article ; Online: A Previously Undescribed Polerovirus (Solemoviridae) Infecting Theobroma cacao Germplasm

Adegbola, Raphael O. / Keith, Cory V. / Gutierrez, Osman A. / Goenaga, Ricardo / Brown, Judith K.

Plant Disease. 2023 Mar. 01, v. 107, no. 3 p.-975

2023

Abstract: Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al. 2018). Virus-like symptoms consisting of discoloration, leaf distortion ... ...

Abstract	Cacao Theobroma cacao L. (Malvaceae) is an economically important crop cultivated in tropical climates for the bean from which chocolate and other products are made (Zarrillo et al. 2018). Virus-like symptoms consisting of discoloration, leaf distortion with downward rolling of leaves, and yellow speckling or mottling, were observed in imported cacao germplasm at the USDA-ARS-SHRS cacao quarantine facilities in the fall of 2020. Total RNA was isolated from leaves collected from four symptomatic plants using the silica RNA extraction method (Rott and Jelkmann 2001). Ribosomal RNA (rRNA)-depleted RNA samples were used for cDNA library construction, followed by high throughput sequencing on an Illumina NovaSeq 6000 platform (Novogene, Sacramento, CA). Quality-filtered, 150-bp paired-ended reads (2,601,293 to 3,104,474) were assembled de novo using SPAdes v.3.14.1 (Nurk et al. 2013). The contigs (200,799 to 276,851) were queried against the NCBI virus reference sequence (RefSeq) database using the discontiguous MegaBLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi?). The resultant contigs (n = 1,344) were 150- to 1,463-nucleotides (nt) in length (k-mer coverage from 6.3× to 26,721.7×) and shared their highest nt identity with potato leafroll virus (PLRV; NC_001747; genus Polerovirus; family Solemoviridae), at 69.1 to 72.8%. The contigs pooled from the four samples were assembled into 15 scaffolds. BLASTn analyses of the 15 scaffolds against the RefSeq database indicated the best matches were to 13 other polerovirus species, with top hits to cereal yellow dwarf virus-RPV (D10206) and pepper vein yellows virus (LC528383), having similarity scores of 66.2 and 100%, respectively. The 15 scaffolds matched to the 5′ terminal end, ORF1-2, ORF3, ORF4, and ORF3-5 of the different polerovirus genomes. For confirmatory sequencing, total RNA was subjected to reverse transcription using SuperScript IV (Invitrogen, Carlsbad, CA), followed by RT-PCR amplification with general polerovirus primers PoconF/PoconcpR (Xiang et al. 2008) expected to yield an amplicon of ∼1,400 bp located at the 3′ end of the RNA-dependent, RNA polymerase (RdRp), including the complete coat protein (CP) and movement protein (MP) genes. Amplicons were ligated to pGEM-T Easy vector (Promega, Madison, WI) and sequenced bidirectionally by Sanger sequencing (Eton Bio, Research Triangle Park, NC). BLASTn analysis of the polerovirus-like nt sequences (GenBank accession nos. ON745771 to ON745774) indicated the closest relatives were potato leafroll virus (OK058524) and cucumber aphid-borne yellows virus (FJ460218), at 71 and 73%, respectively. The CP amino acid (aa) sequence shared the greatest similarity to cereal yellow dwarf virus RPV (NP_840023), at 53%, and the MP aa sequence shared the greatest aa similarity to wheat yellow leaf dwarf virus-GPV (YP_003029842), at 38%. These results provide robust support for the association of a previously undescribed polerovirus with symptomatic cacao trees, herein named cacao leafroll virus (Solemoviridae; Polerovirus). Although Koch’s postulates have not been completed to confirm causality, the presence of this virus in cacao germplasm undermines efforts to distribute pathogen-free germplasm and may pose a risk to cacao production in trees established from virus-infected plant material. To our knowledge, this is the first report of a polerovirus infecting cacao trees. All trees of these accessions at the quarantined facility in Miami, FL, have been destroyed.
Keywords	Cereal yellow dwarf virus-RPV ; DNA-directed RNA polymerase ; Potato leafroll virus ; Solemoviridae ; Theobroma cacao ; algorithms ; amino acids ; beans ; cDNA libraries ; chocolate ; coat proteins ; cucumbers ; databases ; discoloration ; genome ; germplasm ; leaves ; pepper ; quarantine ; reverse transcription ; ribosomal RNA ; risk ; silica ; viruses ; wheat ; cacao ; polerovirus ; Theobroma cacao L.
Language	English
Dates of publication	2023-0301
Size	p. -975.
Publishing place	The American Phytopathological Society
Document type	Article ; Online
ZDB-ID	754182-x
ISSN	0191-2917
ISSN	0191-2917
DOI	10.1094/PDIS-06-22-1449-PDN
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Article ; Online: Complete Genome Segment Sequences of Tomato Chlorotic Spot Virus from Peanut in Haiti.

Adegbola, Raphael O / Kemerait, Robert C / Adkins, Scott / Naidu, Rayapati A

Microbiology resource announcements

2019 Volume 8, Issue 18

Abstract: Tomato chlorotic spot virus (TCSV) is emerging as a significant constraint to vegetable and legume crops in the Americas. The complete genome sequence of a TCSV isolate naturally infecting peanut ( ...

Abstract	Tomato chlorotic spot virus (TCSV) is emerging as a significant constraint to vegetable and legume crops in the Americas. The complete genome sequence of a TCSV isolate naturally infecting peanut (
Language	English
Publishing date	2019-05-02
Publishing country	United States
Document type	Journal Article
ISSN	2576-098X
ISSN (online)	2576-098X
DOI	10.1128/MRA.00306-19
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Article ; Online: Cotton Leafroll Dwarf Virus US Genomes Comprise Divergent Subpopulations and Harbor Extensive Variability.

Ramos-Sobrinho, Roberto / Adegbola, Raphael O / Lawrence, Kathy / Schrimsher, Drew W / Isakeit, Thomas / Alabi, Olufemi J / Brown, Judith K

Viruses

2021 Volume 13, Issue 11

Abstract: Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates ...

Abstract	Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates in the US, complete genomes of the virus were obtained from infected cotton plants displaying mild to severe symptoms from AL, Florida, and Texas. Eight CLRDV genomes were determined, ranging in size from 5865 to 5867 bp, and shared highest nucleotide identity with other CLRDV isolates in the US, at 95.9-98.7%. Open reading frame (ORF) 0, encoding the P0 silencing suppressor, was the most variable gene, sharing 88.5-99.6% and 81.2-89.3% amino acid similarity with CLRDV isolates reported in cotton growing states in the US and in Argentina and Brazil in South America, respectively. Based on Bayesian analysis, the complete CLRDV genomes from cotton in the US formed a monophyletic group comprising three relatively divergent sister clades, whereas CLRDV genotypes from South America clustered as closely related sister-groups, separate from US isolates, patterns reminiscent of phylogeographical structuring. The CLRDV isolates exhibited a complex pattern of recombination, with most breakpoints evident in ORFs 2 and 3, and ORF5. Despite extensive nucleotide diversity among all available CLRDV genomes, purifying selection (
Language	English
Publishing date	2021-11-05
Publishing country	Switzerland
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2516098-9
ISSN	1999-4915 ; 1999-4915
ISSN (online)	1999-4915
ISSN	1999-4915
DOI	10.3390/v13112230
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Article: Cotton Leafroll Dwarf Virus US Genomes Comprise Divergent Subpopulations and Harbor Extensive Variability

Ramos-Sobrinho, Roberto / Adegbola, Raphael O. / Lawrence, Kathy / Schrimsher, Drew W. / Isakeit, Thomas / Alabi, Olufemi J. / Brown, Judith K.

Viruses. 2021 Nov. 05, v. 13, no. 11

2021

Abstract	Cotton leafroll dwarf virus (CLRDV) was first reported in the United States (US) in 2017 from cotton plants in Alabama (AL) and has become widespread in cotton-growing states of the southern US. To investigate the genomic variability among CLRDV isolates in the US, complete genomes of the virus were obtained from infected cotton plants displaying mild to severe symptoms from AL, Florida, and Texas. Eight CLRDV genomes were determined, ranging in size from 5865 to 5867 bp, and shared highest nucleotide identity with other CLRDV isolates in the US, at 95.9–98.7%. Open reading frame (ORF) 0, encoding the P0 silencing suppressor, was the most variable gene, sharing 88.5–99.6% and 81.2–89.3% amino acid similarity with CLRDV isolates reported in cotton growing states in the US and in Argentina and Brazil in South America, respectively. Based on Bayesian analysis, the complete CLRDV genomes from cotton in the US formed a monophyletic group comprising three relatively divergent sister clades, whereas CLRDV genotypes from South America clustered as closely related sister-groups, separate from US isolates, patterns reminiscent of phylogeographical structuring. The CLRDV isolates exhibited a complex pattern of recombination, with most breakpoints evident in ORFs 2 and 3, and ORF5. Despite extensive nucleotide diversity among all available CLRDV genomes, purifying selection (dN/dS < 1) was implicated as the primary selective force acting on viral protein evolution.
Keywords	Bayesian theory ; Cotton leafroll dwarf virus ; amino acids ; cotton ; genes ; genetic variation ; monophyly ; phylogeography ; viruses ; Alabama ; Argentina ; Brazil ; Florida ; Texas
Language	English
Dates of publication	2021-1105
Publishing place	Multidisciplinary Digital Publishing Institute
Document type	Article
ZDB-ID	2516098-9
ISSN	1999-4915
ISSN	1999-4915
DOI	10.3390/v13112230
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Article ; Online: An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments.

Marshall, Spencer H / Adegbola, Raphael O / Adkins, Scott / Naidu, Rayapati A

Journal of virological methods

2017 Volume 242, Page(s) 22–26

Abstract: Tospoviruses (genus Tospovirus, family Bunyaviridae) are responsible for major losses in an extensive range of crops worldwide. New species of these single-stranded, ambisense RNA viruses regularly emerge and have been shown to maintain heterogeneous ... ...

Abstract	Tospoviruses (genus Tospovirus, family Bunyaviridae) are responsible for major losses in an extensive range of crops worldwide. New species of these single-stranded, ambisense RNA viruses regularly emerge and have been shown to maintain heterogeneous populations with individual isolates having quite variable biological and virulence characteristics. Most tospovirus phylogenetic studies have focused on analysis of a single gene, most often the nucleocapsid protein gene. Complete genomic RNA segment amplification as a single fragment would facilitate more detailed analyses of genome-wide sequence variability, but obtaining such sequences for a large number of tospovirus isolates using traditional methods of amplification and cloning of small overlapping fragments is tedious, time consuming and expensive. In this study, protocols were optimized to amplify, clone and sequence full-length M- and S-RNA genome segments of Tomato spotted wilt virus and Impatiens necrotic spot virus. The strategy presented here is straightforward, scalable and offers several advantages over the previously commonplace and overlapping amplicon-based approach. Use of whole genome segments, instead of individual gene sequences or defined portions of genome segments, will facilitate a better understanding of the underlying molecular diversity of tospoviruses in mixed infections.
MeSH term(s)	Bunyaviridae/genetics ; Cloning, Molecular/methods ; Genome, Viral ; Nucleic Acid Amplification Techniques ; Nucleocapsid/genetics ; Phylogeny ; RNA, Viral/genetics ; RNA, Viral/isolation & purification ; Sequence Analysis, DNA/methods ; Tospovirus/genetics ; Tospovirus/isolation & purification
Chemical Substances	RNA, Viral
Language	English
Publishing date	2017-04
Publishing country	Netherlands
Document type	Journal Article
ZDB-ID	8013-5
ISSN	1879-0984 ; 0166-0934
ISSN (online)	1879-0984
ISSN	0166-0934
DOI	10.1016/j.jviromet.2016.12.018
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Article ; Online: Sequence analysis of the medium and small RNAs of impatiens necrotic spot virus reveals segment reassortment but not recombination.

Adegbola, Raphael O / Marshall, Spencer H / Batuman, Ozgur / Ullman, Diane E / Gilbertson, Robert L / Adkins, Scott / Naidu, Rayapati A

Archives of virology

2019 Volume 164, Issue 11, Page(s) 2829–2836

Abstract: The complete sequence of the medium (M) and small (S) RNA genome segments were determined for twelve isolates of impatiens necrotic spot virus from eight plant species. The M- and S-RNAs of these isolates shared 97-99% and 93-98% nucleotide sequence ... ...

Abstract	The complete sequence of the medium (M) and small (S) RNA genome segments were determined for twelve isolates of impatiens necrotic spot virus from eight plant species. The M- and S-RNAs of these isolates shared 97-99% and 93-98% nucleotide sequence identity, respectively, with the corresponding full-length sequences available in public databases. Phylogenetic analysis based on the M- or S-RNA sequences showed incongruence in the phylogenetic position of some isolates, suggesting intraspecies segment reassortment. The lack of phylogenetic discordance in individual and concatenated sequences of individual genes encoded by M- or S-RNAs suggests that segment reassortment rather than recombination is driving evolution of these INSV isolates.
MeSH term(s)	Base Sequence ; Genome, Viral/genetics ; Plants/virology ; RNA, Viral/genetics ; Reassortant Viruses/genetics ; Sequence Analysis, RNA ; Tospovirus/genetics ; Tospovirus/isolation & purification
Chemical Substances	RNA, Viral
Language	English
Publishing date	2019-09-05
Publishing country	Austria
Document type	Journal Article
ZDB-ID	7491-3
ISSN	1432-8798 ; 0304-8608
ISSN (online)	1432-8798
ISSN	0304-8608
DOI	10.1007/s00705-019-04389-5
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Article: Sequence analysis of the medium and small RNAs of impatiens necrotic spot virus reveals segment reassortment but not recombination

Adegbola, Raphael O / Marshall, Spencer H / Batuman, Ozgur / Ullman, Diane E / Gilbertson, Robert L / Adkins, Scott / Naidu, Rayapati A

Archives of virology. 2019 Nov., v. 164, no. 11

2019

Abstract	The complete sequence of the medium (M) and small (S) RNA genome segments were determined for twelve isolates of impatiens necrotic spot virus from eight plant species. The M- and S-RNAs of these isolates shared 97–99% and 93–98% nucleotide sequence identity, respectively, with the corresponding full-length sequences available in public databases. Phylogenetic analysis based on the M- or S-RNA sequences showed incongruence in the phylogenetic position of some isolates, suggesting intraspecies segment reassortment. The lack of phylogenetic discordance in individual and concatenated sequences of individual genes encoded by M- or S-RNAs suggests that segment reassortment rather than recombination is driving evolution of these INSV isolates.
Keywords	Impatiens necrotic spot tospovirus ; RNA ; nucleotide sequences ; phylogeny ; sequence analysis ; virology
Language	English
Dates of publication	2019-11
Size	p. 2829-2836.
Publishing place	Springer Vienna
Document type	Article
Note	NAL-AP-2-clean
ZDB-ID	7491-3
ISSN	1432-8798 ; 0304-8608
ISSN (online)	1432-8798
ISSN	0304-8608
DOI	10.1007/s00705-019-04389-5
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