Detection and in-vivo validation of Dichorhavirus e-probes in meta-transcriptomic data via Microbe Finder (MiFi®) discovers a novel host and a possible new strain of Orchid fleck virus

Authors: Roy, Avijit; Shao, Jonathan; ESPINDOLA, ANDRES - Oklahoma State University; RAMOS-LOPEZ, ALVARO - Oklahoma State University; OTERO-COLINA, GABRIEL - Campus Montecillo; RIVERA, YAZMIN - Animal And Plant Health Inspection Service (APHIS); MAVRODIEVA, VESSELA - Animal And Plant Health Inspection Service (APHIS); NAKHLA, MARK - Animal And Plant Health Inspection Service (APHIS); SCHNEIDER, WILLIAM - F1k9; CARDWELL, KITTY - Oklahoma State University

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/13/2025
Publication Date: 3/19/2025
Citation: Roy, A., Shao, J.Y., Espindola, A.S., Ramos-Lopez, A., Otero-Colina, G., Rivera, Y., Mavrodieva, V.A., Nakhla, M.K., Schneider, W.L., Cardwell, K. 2025. Detection and in-vivo validation of Dichorhavirus e-probes in meta-transcriptomic data via Microbe Finder (MiFi®) discovers a novel host and a possible new strain of Orchid fleck virus. Viruses. https://doi.org/10.3390/v17030441.
DOI: https://doi.org/10.3390/v17030441

Interpretive Summary: Citrus leprosis disease (CiLD), an exotic as well as reemerging disease for the United States, is associated with seven different viruses, and causes significant economic damage to the citrus crops of Central and South America. Apart from citrus, several ornamentals and orchids have been reported as hosts of some of these viruses. To prevent the introduction of these viruses at the port of entry, a rapid, specific, and sensitive method is needed to avoid any false positive or false negative diagnoses. In this study, we explored millions of reads of DNA sequences generated from 31 suspected plant samples for their ability to match with the newly curated short sequence/s (e-probe) specific to citrus leprosis viruses. A platform integrated with multiple complex software was utilized to detect the targeted short specific virus sequence from the tested data set. Newly developed e-probes for citrus leprosis viruses correctly diagnosed 29 tested samples with minimum error (6.5%) and led to the discovery of a new host (leopard plant) and a novel virus species. The results of the method were validated with two other gold standard diagnostic methods. Overall, this method is very specific, sensitive and can detect the targeted pathogen within one minute to 30 minutes dependent on the total number of pathogen reads in the sequencing data set. The same protocol can be used by other scientists to develop and validate e-probes related to any virus genera and discovery of new host, or new virus species. Implementation of the E-probe Diagnostic for Nucleic Acid Analysis (EDNA) method and updated host information for CiLD diagnosis should assist regulatory agencies in surveillance activities to monitor the distribution pattern of citrus leprosis-associated viruses in alternate hosts in countries where it is present and to prevent dissemination into citrus growing countries where there is no report of these viruses.

Technical Abstract: Dichorhavirus is one of the recently accepted plant virus genera within the family Rhabdoviridae. Species assigned to the genus consist of bi-segmented, negative sense, single-stranded RNA viruses and are transmitted by Brevipalpus spp. Currently, there are five recognized species and two un-classified members in the genus of Dichorhavirus. Four out of seven; orchid fleck virus (OFV), citrus leprosis virus N, citrus chlorotic spot virus, and citrus bright spot virus can infect citrus and produce leprosis disease-like symptoms. Previously, the E-probe Diagnostic for Nucleic Acid Analysis (EDNA) was developed to reduce computational effort and then integrated with Mi-crobe-Finder (MiFi®) online platform to design and evaluate e-probes in raw HTS data. During this study, Dichorhavirus genomes were downloaded from public databases and specific e-probes were designed using the MiProbe incorporated into MiFi® platform. Three different sizes of e-probes, 40, 60 and 80 nucleotides were developed and selected based on whole genome comparisons with near neighbor genomes. For curation, each e-probe was blasted against target and near neighbor virus sequences and the available NCBI nucleotide sequence database using BLASTn. All the e-probes that had hits with non-target species with =90% identities were removed. The sensitivity and specificity of Dichorhavirus genus, species, and strain-specific e-probes were validated in vivo using HTS meta-transcriptomic libraries generated from Dichorhavirus infected citrus, orchid and or-namentals. Through downstream analysis of HTS data, we not only detected the known hosts of OFV but also discovered an unknown host (leopard plant, Farfugium japonicum) and the possible existence of a new ornamental strain of OFV in nature.