{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T14:18:35Z","timestamp":1789395515024,"version":"build-2803163510"},"reference-count":36,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T00:00:00Z","timestamp":1789344000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Cocoa Research Association Ltd."}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Pathogens"],"abstract":"<jats:p>Cacao leafroll virus (CaLRV; species Polerovirus CALRV, family Solemoviridae), previously referred to as cacao polerovirus, infects cacao (Theobroma cacao) germplasm in the Americas. Screening of 287 cacao accessions with three published multiplex RT-PCR assays detected CaLRV in nine accessions but produced reproducibly discordant results for the accession LCT EEN 412. Metatranscriptomic sequencing generated three near-complete CaLRV genome sequences, CaLRV-TSAN792, CaLRV-POUND15A, and CaLRV-LCTEEN412 (5985\u20135989 nt), from accessions originating from Brazil, Trinidad and Tobago, and Ecuador, respectively. The divergent CaLRV-LCTEEN412 assembly was verified by RT-PCR, cloning, and nanopore sequencing. In addition, analysis of five public cacao RNA-seq datasets yielded one near-complete CaLRV-PA16 assembly included in whole-genome analysis and four fragmented assemblies that informed primer design. Comparison of the four sequences characterised here with six previously reported genomes showed that nine shared 98.8\u201399.7% pairwise nucleotide identity, whereas CaLRV-LCTEEN412 shared 85.0\u201385.2%. Primer-binding-site mismatches in CaLRV-LCTEEN412 were consistent with its discordant detection by published assays. Two new multiplex RT-PCR assays were therefore designed from the expanded alignment and evaluated using four CaLRV-positive cacao accessions, including LCT EEN 412. These results expand known CaLRV genomic diversity and provide dual-target tools that reduce dependence on a single locus and enable recognition of target discordance.<\/jats:p>","DOI":"10.3390\/pathogens15090975","type":"journal-article","created":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:05:26Z","timestamp":1789391126000},"page":"975","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Expanded Genomic Diversity of Cacao Leafroll Virus Enables Improved Detection Using Multiplex RT-PCR"],"prefix":"10.3390","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9367-6741","authenticated-orcid":false,"given":"Ihsan","family":"Ullah","sequence":"first","affiliation":[{"name":"School of Agriculture, Policy and Development, University of Reading, Reading RG6 6EU, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Muhammad","family":"Kamran","sequence":"additional","affiliation":[{"name":"Sugarcane Research Institute, Ayub Agricultural Research Institute, Faisalabad 38850, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7597-9423","authenticated-orcid":false,"given":"Andrew J.","family":"Daymond","sequence":"additional","affiliation":[{"name":"School of Agriculture, Policy and Development, University of Reading, Reading RG6 6EU, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2147-665X","authenticated-orcid":false,"given":"Jim M.","family":"Dunwell","sequence":"additional","affiliation":[{"name":"School of Agriculture, Policy and Development, University of Reading, Reading RG6 6EU, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,14]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1634","DOI":"10.1094\/PHYTO-97-12-1634","article-title":"Cacao diseases: Important threats to chocolate production worldwide","volume":"97","author":"Ploetz","year":"2007","journal-title":"Phytopathology"},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Ullah, I., Kamran, M., and Dunwell, J.M. (2023). Identification of a novel polerovirus in cocoa (Theobroma cacao) germplasm and development of molecular methods for use in diagnostics. Pathogens, 12.","DOI":"10.3390\/pathogens12111284"},{"key":"ref_3","unstructured":"End, M.J., Daymond, A.J., and Hadley, P. (2024). Technical Guidelines for the Safe Movement of Cacao Germplasm, Bioversity International. Available online: https:\/\/www.cacaonet.org\/fileadmin\/templates\/CacaoNet\/Uploads\/publications\/Safe_Movement_Guidelines_2024_ENGLISH_VERSION.pdf."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"veab102","DOI":"10.1093\/ve\/veab102","article-title":"Polerovirus genomic variation","volume":"7","author":"LaTourrette","year":"2021","journal-title":"Virus Evol."},{"key":"ref_5","unstructured":"International Committee on Taxonomy of Viruses (2026, July 30). Genus Polerovirus. ICTV Report on Virus Classification and Taxon Nomenclature. Available online: https:\/\/ictv.global\/report\/chapter\/solemoviridae\/solemoviridae\/polerovirus."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"975","DOI":"10.1094\/PDIS-06-22-1449-PDN","article-title":"A previously undescribed polerovirus (Solemoviridae) infecting Theobroma cacao germplasm","volume":"107","author":"Adegbola","year":"2023","journal-title":"Plant Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1007\/s00705-024-06013-7","article-title":"Complete genome characterization of cacao leafroll virus, a newly described cacao-infecting polerovirus","volume":"169","author":"Adegbola","year":"2024","journal-title":"Arch. Virol."},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"The Galaxy Community (2024). The Galaxy platform for accessible, reproducible, and collaborative data analyses: 2024 update. Nucleic Acids Res., 52, W83\u2013W94.","DOI":"10.1093\/nar\/gkae410"},{"key":"ref_9","unstructured":"Andrews, S. (2010). FastQC: A Quality Control Tool for High Throughput Sequence Data, Babraham Bioinformatics. Available online: https:\/\/www.bioinformatics.babraham.ac.uk\/projects\/fastqc\/."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"3047","DOI":"10.1093\/bioinformatics\/btw354","article-title":"MultiQC: Summarize analysis results for multiple tools and samples in a single report","volume":"32","author":"Ewels","year":"2016","journal-title":"Bioinformatics"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2114","DOI":"10.1093\/bioinformatics\/btu170","article-title":"Trimmomatic: A flexible trimmer for Illumina sequence data","volume":"30","author":"Bolger","year":"2014","journal-title":"Bioinformatics"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3211","DOI":"10.1093\/bioinformatics\/bts611","article-title":"SortMeRNA: Fast and accurate filtering of ribosomal RNAs in metatranscriptomic data","volume":"28","author":"Kopylova","year":"2012","journal-title":"Bioinformatics"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1038\/nmeth.1923","article-title":"Fast gapped-read alignment with Bowtie 2","volume":"9","author":"Langmead","year":"2012","journal-title":"Nat. Methods"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"644","DOI":"10.1038\/nbt.1883","article-title":"Full-length transcriptome assembly from RNA-Seq data without a reference genome","volume":"29","author":"Grabherr","year":"2011","journal-title":"Nat. Biotechnol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1038\/s41592-021-01101-x","article-title":"Sensitive protein alignments at tree-of-life scale using DIAMOND","volume":"18","author":"Buchfink","year":"2021","journal-title":"Nat. Methods"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"giab008","DOI":"10.1093\/gigascience\/giab008","article-title":"Twelve years of SAMtools and BCFtools","volume":"10","author":"Danecek","year":"2021","journal-title":"GigaScience"},{"key":"ref_17","unstructured":"National Center for Biotechnology Information (2026, July 30). BioProject PRJNA558793: Theobroma cacao Disease Resistance: Discovery and Functional Characterization of Genes Regulating Plant Immunity in Perennial Crops, Available online: https:\/\/www.ncbi.nlm.nih.gov\/bioproject\/PRJNA558793."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1792","DOI":"10.1093\/nar\/gkh340","article-title":"MUSCLE: Multiple sequence alignment with high accuracy and high throughput","volume":"32","author":"Edgar","year":"2004","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"msae263","DOI":"10.1093\/molbev\/msae263","article-title":"MEGA12: Molecular Evolutionary Genetic Analysis version 12 for adaptive and green computing","volume":"41","author":"Kumar","year":"2024","journal-title":"Mol. Biol. Evol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1007\/s00239-025-10287-z","article-title":"MEGA 12.1: Cross-platform release for macOS and Linux operating systems","volume":"94","author":"Stecher","year":"2026","journal-title":"J. Mol. Evol."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Muhire, B.M., Varsani, A., and Martin, D.P. (2014). SDT: A virus classification tool based on pairwise sequence alignment and identity calculation. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0108277"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"3096","DOI":"10.1093\/bioinformatics\/btl474","article-title":"GARD: A genetic algorithm for recombination detection","volume":"22","author":"Posada","year":"2006","journal-title":"Bioinformatics"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"773","DOI":"10.1093\/molbev\/msx335","article-title":"Datamonkey 2.0: A modern web application for characterizing selective and other evolutionary processes","volume":"35","author":"Weaver","year":"2018","journal-title":"Mol. Biol. Evol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1155","DOI":"10.1007\/s00705-008-0083-0","article-title":"Complete sequence analysis reveals two distinct poleroviruses infecting cucurbits in China","volume":"153","author":"Xiang","year":"2008","journal-title":"Arch. Virol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"W71","DOI":"10.1093\/nar\/gkm306","article-title":"Primer3Plus, an enhanced web interface to Primer3","volume":"35","author":"Untergasser","year":"2007","journal-title":"Nucleic Acids Res."},{"key":"ref_26","unstructured":"International Cocoa Quarantine Centre, Reading (2025). Operations Manual of the International Cocoa Quarantine Centre, Reading (ICQC,R), University of Reading. Available online: https:\/\/www.icgd.reading.ac.uk\/icqc\/documents.php."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"e2102914118","DOI":"10.1073\/pnas.2102914118","article-title":"Genomic structural variants constrain and facilitate adaptation in natural populations of Theobroma cacao, the chocolate tree","volume":"118","author":"Hamala","year":"2021","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"107","DOI":"10.1016\/j.jviromet.2016.09.004","article-title":"PCR bias associated with conserved primer binding sites, used to determine genotype diversity within Citrus tristeza virus populations","volume":"237","author":"Read","year":"2016","journal-title":"J. Virol. Methods"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"518","DOI":"10.1094\/PDIS-05-21-0949-RE","article-title":"Comparison of high throughput sequencing to standard protocols for virus detection in berry crops","volume":"106","author":"Villamor","year":"2022","journal-title":"Plant Dis."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"109","DOI":"10.2353\/jmoldx.2010.090035","article-title":"The effect of primer-template mismatches on the detection and quantification of nucleic acids using the 5\u2032 nuclease assay","volume":"12","author":"Stadhouders","year":"2010","journal-title":"J. Mol. Diagn."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1007\/s12560-019-09387-z","article-title":"Missing the match might not cost you the game: Primer-template mismatches studied in different hepatitis A virus variants","volume":"11","author":"Persson","year":"2019","journal-title":"Food Environ. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1223","DOI":"10.1094\/PHYTO-96-1223","article-title":"Simultaneous detection of nine grapevine viruses by multiplex reverse transcription-polymerase chain reaction with coamplification of a plant RNA as internal control","volume":"96","author":"Gambino","year":"2006","journal-title":"Phytopathology"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"42825","DOI":"10.1038\/srep42825","article-title":"Interference between variants of peach latent mosaic viroid reveals novel features of its fitness landscape: Implications for detection","volume":"7","author":"Serra","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"5419","DOI":"10.1128\/JVI.02312-08","article-title":"The C terminus of the polerovirus P5 readthrough domain limits virus infection to the phloem","volume":"83","author":"Peter","year":"2009","journal-title":"J. Virol."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/S0166-0934(00)00256-1","article-title":"Mechanical transmission of poleroviruses","volume":"91","author":"Hoffmann","year":"2001","journal-title":"J. Virol. Methods"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Vanneste, K., Garlant, L., Broeders, S., Van Gucht, S., and Roosens, N.H.C. (2018). Application of whole genome data for in silico evaluation of primers and probes routinely employed for the detection of viral species by RT-qPCR using dengue virus as a case study. BMC Bioinform., 19.","DOI":"10.1186\/s12859-018-2313-0"}],"container-title":["Pathogens"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/9\/975\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,14]],"date-time":"2026-09-14T13:25:52Z","timestamp":1789392352000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-0817\/15\/9\/975"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,14]]},"references-count":36,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["pathogens15090975"],"URL":"https:\/\/doi.org\/10.3390\/pathogens15090975","relation":{},"ISSN":["2076-0817"],"issn-type":[{"value":"2076-0817","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,14]]}}}