High-throughput sequencing applied for the identification of viruses infecting grapevines in Brazil and genetic variability analysis

Abstract

The application of high-throughput sequencing technologies (HTS) enables the recovery of many nucleotide sequence fragments from diseased plants and may help in pathogen identification. This study was designed to identify viruses infecting 15 grapevine (Vitis spp.) samples collected from experimental fields and vine collections and assess the genetic variability of the identified viruses. The virus-enriched dsRNAs were extracted from bark scrapings and sequenced using an Illumina platform. The paired-end reads were analyzed, assembled contigs were generated and identified as related to viruses. Contigs of 14 viruses have been identified, some of them covering large extensions of viral genomes or resulting in assembly of near-complete or complete genomes. Grapevine virus infections are usually mixed and the HTS assays were suitable to identify ten viruses already reported that traditionally infect grapevines in Brazil, one that has been recently identified (Grapevine Syrah virus 1) and others (Grapevine Cabernet Sauvignon reovirus, Grapevine Red Globe virus and Grapevine vein clearing virus) not previously reported in this country. Nucleotide identities among Brazilian isolates identified by HTS and homologous grapevine virus sequences in GenBank were high, ranging from 77% to 99%. Genetic variability analysis of viral sequences obtained by HTS and sequences available in GenBank indicated that the coding regions in the different viral species are under purifying selection, and that recombination events occurred in the majority of the viral species analyzed. The coat protein genes, generally, had lower genetic variability than the replicase and movement protein genes.

Characterization and genetic variability of coat protein genes of Apple chlorotic leaf spot virus isolates from southern Brazil

“…The dataset comprising ACLSV Brazilian isolates had a π value of 0.07690 ± 0.00542, indicating lower genetic variability than the isolates from other countries. The π value detected in ACLSV (for five datasets) is in accordance with π values found in different viral species by several authors (Garcia-Arenal et al 2001;Lima et al 2013Lima et al , 2017Zanardo et al 2014;Fajardo et al 2017). The Watterson's estimator for the population-scaled mutation rate (θ-W) for Brazilian and Indian ACLSV isolates was in the order of 10 −2 , and for Chinese, Japanese and "world" isolates was in the order of 10 −1 ( Table 2), meaning that Chinese and Japanese isolates are more variable due to the greater number of mutations.…”

“…The Watterson's estimator for the population-scaled mutation rate (θ-W) for Brazilian and Indian ACLSV isolates was in the order of 10 −2 , and for Chinese, Japanese and "world" isolates was in the order of 10 −1 ( Table 2), meaning that Chinese and Japanese isolates are more variable due to the greater number of mutations. The θ-W values of 10 −1 are lower when compared with other coding regions and other viral species described in the literature (Fajardo et al 2017;Lima et al 2013;Moura et al 2018;Rocha et al 2013;Zanardo et al 2014).…”

Molecular variants of Grapevine rupestris stem pitting-associated virus infecting grapevines (Vitis spp.) in Brazil

“…CLC Genomics Workbench software was used for quality trimming and de novo contig assembly from the reads. All contigs were analyzed using NCBI's Blast program (http://www.ncbi.nlm.nih.gov/ blast) against the viral RefSeq databases and also all contigs identified as GRSPaV were individually analyzed using Blastn against the GenBank database ( FAJARDO et al, 2017).…”

Discovery and molecular characterization of a novel enamovirus, Grapevine enamovirus-1

“…After scaffolding of the fragmented S12-CG and S19-CF contigs, an identity of around 99% at the nucleotide level was verified for GEV-1 among these four samples. Mixed infections were present in all GEV-1 positive samples [1,2] and the virus communities in these samples included 11 pathogens: Grapevine Cabernet Sauvignon reovirus (GCSV), Grapevine vein clearing virus (GVCV), Grapevine Red Globe virus (GRGV), Grapevine leafroll-associated virus 2 and 3 (GLRaV-2, -3), Grapevine rupestris stem pitting-associated virus (GRSPaV), Grapevine virus A (GVA), Grapevine virus B (GVB), Grapevine fleck virus (GFkV), Grapevine rupestris vein feathering virus (GRVFV), and Grapevine yellow speckle viroid 1 (GYSVd-1). Additionally, GEV-1 was detected on a Malvasia Longa vine by RT-PCR during the virus indexing of this cultivar.…”

“…A limited survey was performed on 17 grapevine samples subjected to high-throughput sequencing (HTS). These plants were collected from three different grapevine collections from the south (11), northeast (2), and southeast (4) regions of Brazil, and the symptoms in the V. vinifera hosts were downward rolling of leaves and reddening or yellowing, whereas other genotypes were asymptomatic [1,2]. Following a typical metagenomic pipeline using HTS, we were able to identify a new putative Enamovirus member, tentatively named Grapevine enamovirus-1 (GEV-1), infecting distinct grapevine cultivars in Brazil.…”