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

<i>Dichorhavirus</i> is a recently accepted plant virus genus within the family <i>Rhabdoviridae</i>. Species assigned to the genus consist of bi-segmented, negative sense, single-stranded RNA viruses and are transmitted by <i>Brevipalpus</i> spp. Currently, there are five recognized species and two unclassified members in the genus <i>Dichorhavirus.</i> 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. The E-probe Diagnostic for Nucleic Acid Analysis (EDNA) was developed to reduce computational effort and then integrated within Microbe-Finder (MiFi^®) online platform to design and evaluate e-probes in raw High Throughput Sequencing (HTS) data. During this study, <i>Dichorhavirus</i> genomes were downloaded from public databases and e-probes were designed using the MiProbe incorporated into the 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 searched in the 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 <i>Dichorhavirus</i> genus, species, strain, and variant-specific e-probes were validated in vivo using HTS meta-transcriptomic libraries generated from <i>Dichorhavirus</i>-suspected citrus, orchid, and ornamentals. Through downstream analysis of HTS data, EDNA not only detected the known hosts of OFV but also discovered an unknown host leopard plant (<i>Farfugium japonicum</i>), and the possible existence of a new ornamental strain of OFV in nature.