Identification of the coding-complete genome of Cycas necrotic stunt virus in transcriptomic data sets of alfalfa (Medicago sativa)

Authors: Jiang, Peng; Shao, Jonathan; Nemchinov, Lev

Submitted to: Microbiology Resource Announcements
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/23/2019
Publication Date: 9/19/2019
Citation: Jiang, P., Shao, J.Y., Nemchinov, L.G. 2019. Identification of the coding-complete genome of Cycas necrotic stunt virus in transcriptomic data sets of alfalfa (Medicago sativa). Microbiology Resource Announcements. 8:e00981-19. https://doi.org/10.1128/MRA.00981-19.
DOI: https://doi.org/10.1128/MRA.00981-19

Interpretive Summary: Evidence is presented demonstrating that alfalfa can be a natural host species for a new strain of Cycas necrotic stunt virus (CNSV), for which a name CNSV-A (alfalfa) is proposed. Prior to this report, the virus has not been identified in alfalfa. The research is important because the biology of this emerging virus in alfalfa and its geographic distribution are currently unknown and may potentially be of economic significance to the alfalfa industry. Data reported in this study can be used by plant pathologists for the diagnostics and identification of this viral strain in alfalfa.

Technical Abstract: Cycas necrotic stunt virus (CNSV), genus Nepovirus, family Secoviridae, was first detected in the gymnosperm Cycas revoluta in Japan. The severely affected plants deteriorated and subsequently died. CNSV and resembling viruses were also isolated from gladiolus (Gladiolus spp), peony (Paeonia lactiflora Pall.), Easter lily (Lilium longiflorum), aucuba (Aucuba japonica), daphne (Daphne odora) and allium (Allium fistlosum). Prior to this report, the virus has not been identified in alfalfa. Publicly available transcriptomic datasets became a valuable tool for discovery of new pathogens, particularly viruses. In this study, CNSV sequences were identified in alfalfa datasets retrieved from the NCBI Sequence Read Archive. A total of 14,402 CNSV sequences were found in 157,755,564 raw Illumina reads (0.009%) from SRR7751381, SRR7751384, and SRR7751386. Sequencing reads from SRR7751381 unmapped to the reference genomes of Medicago sativa (CADL v. 0.95P) and Medicago truncatula, were aligned to the NCBI viral genome database. Alignments were performed using BBMap (v. 37.66), Ngen (version 15.2.0 (130) and Bowtie 2 (v.2.3.4) with sensitive settings. Reads mapped to the viral sequences were sequestered and re-assembled de-novo using SPAdes (v.3.12.0) with k-mers 21-81. The final coverage was 57.47 and 80.37 reads for RNA1 and RNA2 of the assembled viral genome. The assembled virus had a bipartite single-stranded positive sense RNA genome. RNA 1 was 7,632 nucleotide-long and encoded a single polyprotein (P1) with conserved motifs for RNA helicase and RNA-dependent RNA polymerase (RdRP), as predicted by Pfam database (https://pfam.xfam.org). Other putative domains included those characteristic for the genus Nepovirus. RNA 2 was 4,637 nt-long and translated into a polyprotein 2 (P2) with predicted domains for movement and coat proteins (CP). The 5' and 3' untranslated regions (UTRs) were nearly identical between RNA1 and RNA2. The 3' UTRs of both RNAs incorporated an 80 nt-long fragment with no apparent homology to known CNSV strains and high percent identity to the 3' UTRs of other secoviruses, suggesting possible recombination events. The P1 consisted of 2338 amino acids (aa) and showed 94.3% aa identity to the P1 of CNSV (NP620619), while P2 was 1356 aa-long and had 91.3% aa identity to the P2 of CNSV (NP620620). The predicted CP region of P2 shared ~ 97.8% identity with the CP of CNSV (NP620620) and predicted RdRP region was 94.1% identical to the RdRP of the CNSV (NP620619). Based on these observations, the virus represents a new strain of the CNSV adapted to alfalfa, for which we propose the name CNSV-A. Phylogenic analyses based on the RdRP and CP genes of the CNSV-A and other nepoviruses, grouped the alfalfa strain together with CNSV isolated from other species, indicating their origin from the same ancestral virus. Further investigation is required to determine symptomatology of the virus, its geographic distribution and economic importance to the alfalfa industry.