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

Abstract

Dichorhavirus is a recently accepted plant virus genus 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 unclassified members in the genus 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. 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, Dichorhavirus 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 Dichorhavirus genus, species, strain, and variant-specific e-probes were validated in vivo using HTS meta-transcriptomic libraries generated from Dichorhavirus-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 (Farfugium japonicum), and the possible existence of a new ornamental strain of OFV in nature.

Keywords: Brevipalpus transmitted virus (BTV); EDNA; MiFi; e-probe; high-throughput sequencing; orchid fleck virus; virus detection; virus discovery.

Figure 1

Phylogenetic relationships of dichorhaviruses [orchid fleck virus (OFV), citrus leprosis virus N (CiLV-N), citrus chlorotic spot virus (CiCSV), citrus bright spot virus (CiBSV), clerodendrum chlorotic spot virus (ClCSV), and coffee ring spot virus (CoRSV)] using neighbor-joining methods based on (A) RNA1 and (B) RNA2 genome sequences. Phylogenies were supported by 1000 bootstrap replicates and bootstrap values greater than 50 are shown at the nodes. Genetic pieces of information associated with the NCBI accessions mentioned in the distinct subgroup clades were considered for e-probes design.

Figure 2

Multiple pairwise whole genome alignment with gene annotations and highlights for e-probe target regions of the dichorhaviruses used in this study. Alignments performed with the MiProbe v2 software revealed regions of genomic similarity between the targeted dichorhaviruses, and the links between viruses display local high-identity pairwise alignments. The number showing outside the outermost ring represents three subgroups of dichorhavirus; 1–26 represent subgroup 1 (OFV), 27–40 represent subgroup 2 (CiBSV and CiLV-N), and 41–56 represent subgroup 3 (CiCSV, ClCSV, and CoRSV). Odd and even numbers outside the circle represent Dichorhavirus RNA1 and RNA2 genome segments, respectively. From the outermost to innermost rings: (outermost ring) nucleotide positions of RNA1 and RNA2 of genomes of dichorhaviruses; (inner ring 1) green highlights corresponding to e-probes targeting exclusively specific virus species; (inner ring 2) gene annotations retrieved from NCBI reports for all viruses for both RNA1 and RNA2 segments; (innermost half-circle) red highlights corresponding to e-probes designed for orchid fleck virus (OFV) generic regions shared by multiple targets. As stated by the latest NCBI annotations of the studied accessions, hypothetical proteins and orphan ORFs were not considered for gene annotation display.

Figure 3

Distribution of rhabdovirus and other virus reads (OTH) among total virus reads detected in 31 RNASeq data, which were obtained from Brevipalpus-transmitted virus-suspected samples from four countries: (A) USA, (B) Mexico (C) Colombia and Costa Rica. Samples from each country are represented by individual colors in the bar diagrams. Single-segment nucleo-rhabdovirus (NuRh) reads in the samples are represented by purple, bi-segment Dichorhavirus (DIC) is represented by plum color, whereas other viruses are represented by green (Florida), light blue (California), dark blue (Hawaii), in the USA samples, blue color in Mexican samples, dark green in Colombia samples, and orange color in Costa Rica samples.