High throughput RNA sequencing discovers symptomatic and latent viruses: an example from ornamental Hibiscus

Abstract

Hibiscus rosa-sinensis L. (Hibiscus, Malvaceae) is an ornamental species grown widely in amenity plantings. We collected leaves on an urban roadside pavement (sidewalk) near a market in Guangzhou which showed multiple symptoms of leaf rolling, deformation and chlorosis. Initial evaluation by electron microscopy using negative staining of drip preparations revealed the presence of tobamovirus-like particles. Total RNA was extracted, and, unusually, without any RNA selection based on sequence, was used for cDNA library construction and high-throughput survey sequencing. From the 814 Mb of clean sequence data (from 2,712,161 paired reads of 150 bp) reads representing chloroplast, ribosomal, and mitochondrial genes were filtered out, eliminating 79.1% of reads. 1,135,848 x 150 bp of the sequence was retained and screened for viral sequences. Assembly of these sequences detected nine virus species from seven virus genera comprising three tobamoviruses, namely, Tobacco mosaic virus, Tobacco mild green mosaic virus and Hibiscus latent Singapore virus, Turnip mosaic virus (Potyvirus), Potato virus M (Carlavirus), Hibiscus chlorotic ringspot virus (Betacarmovirus), Fabavirus sp (Fabavirus), Cotton leaf curl Multan virus (Begomovirus) and a putative mitoviruses replicating in mitochondria, Chenopodium quinoa mitovirus 1. Mapping the reads to complete virus reference sequences showed high and uniform coverage of the genomes from 3,729x coverage for Turnip mosaic virus to 22x for Cotton leaf curl Multan virus. By comparison, nuclear reference genes actin showed 14x coverage and polyubiquitin 27x. Notable variants from reference sequences (SNPs) were identified. With the low cost of sequencing and potential for semi-automated bioinformatic pipelines, the whole-RNA approach has huge potential for identifying multiple undiagnosed viruses in ornamental plants, resulting in the ability to take preventive measures in production facilities against spread and to product quality for the mutual benefit of producers and consumers.

Meta-Transcriptomic Analysis Uncovers the Presence of Four Novel Viruses and Multiple Known Virus Genera in a Single Hibiscus rosa-sinensis Plant in Colombia

“…Three new carlaviruses (HiCaV-A, HiCaV-B, and HiCaV-C) and one possible distinct strain of HiCaV-C (HiCaV-D) were identified for the first time in H. rosa-sinensis. Even though there was a finding of a carlavirus sequence in an H. rosa-sinensis sample from Guangzhou province in China [ 33 ], which shared 79.6% nt identity with potato virus M (MT114149), no correlation was established between the presence of the Chinese hibiscus carlavirus sequence and the observed leaf rolling, deformation, or chlorosis symptoms in H. rosa-sinensis . Moreover, no aligned PVM-like sequence or raw HTS data are available in GenBank for the sequence comparison.…”

“…Moreover, no HLRSV sequence is available in GenBank to confirm the presence of a Nepovirus in hibiscus. A recent HTS study, on a Guangzhou H. rosa-sinensis sample from China, revealed the presence of nine virus species in a mixed infection [ 33 ]. Out of the nine, tobacco mild green mosaic virus ( Tobamovirus ), turnip mosaic virus ( Potyvirus ), potato virus M ( Carlavirus ), a fabavirus species ( Fabavirus ), and reads related to mitoviruses, were identified for the first time.…”