An efficient and high fidelity method for amplification, cloning and sequencing of complete tospovirus genomic RNA segments

Abstract

Tospoviruses (genus Tospovirus, family Bunyaviridae) are responsible for major losses in an extensive range of crops worldwide. New species of these single-stranded, ambisense RNA viruses regularly emerge and have been shown to maintain heterogeneous populations with individual isolates having quite variable biological and virulence characteristics. Most tospovirus phylogenetic studies have focused on analysis of a single gene, most often the nucleocapsid protein gene. Complete genomic RNA segment amplification as a single fragment would facilitate more detailed analyses of genome-wide sequence variability, but obtaining such sequences for a large number of tospovirus isolates using traditional methods of amplification and cloning of small overlapping fragments is tedious, time consuming and expensive. In this study, protocols were optimized to amplify, clone and sequence full-length M- and S-RNA genome segments of Tomato spotted wilt virus and Impatiens necrotic spot virus. The strategy presented here is straightforward, scalable and offers several advantages over the previously commonplace and overlapping amplicon-based approach. Use of whole genome segments, instead of individual gene sequences or defined portions of genome segments, will facilitate a better understanding of the underlying molecular diversity of tospoviruses in mixed infections.

Complete Genome Segment Sequences of Tomato Chlorotic Spot Virus from Peanut in Haiti

“…The quality-filtered reads were assembled de novo into contigs using the default parameters in CLC Genomics Workbench 8.0. To validate the sequence data from HTS data, individual genomic segments were amplified with segment-specific primers, cloned, sequenced, and assembled as previously described (10).…”

Integrating Local Lesion Assays with Conventional RT-PCR for Detection of Interspecies Tospovirus Reassortants and Mixed Tospovirus Infections

“…Use of the complete N gene or other virus gene sequences for these techniques does not eliminate confounding by reassortants and mixed infections because it, too, is based on detection of a single virus gene. A method for amplification, cloning, and sequencing of complete tospovirus M and S RNA sequences was recently demonstrated (Marshall et al 2017). Full genome sequencing has also been described (Lian et al 2013).…”