Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes with the size of the RNAs combined ranging from 9 to 13.7 kb. They are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the family Secoviridae available at www.ictv.global/report/secoviridae.

Grapevine red blotch disease has been recognized since 2008 as affecting North American grape production. The presence of the newly described Grapevine red blotch-associated virus (GRBaV) is highly correlated with the disease. To more effectively detect and monitor the presence of the virus, a sample processing strategy and multiplex polymerase chain reaction assay were developed. A total of 42 of 113 vine samples collected in or received from seven of the United States were shown to harbor the virus, demonstrating the virus is widely distributed across North America. Phylogenetic analyses of a viral replication-associated protein (Rep) gene fragment from the 42 isolates of GRBaV demonstrated distinct clades of the virus (1 and 2), with clade 1 showing the greatest variability. The full-length genome of six virus isolates was sequenced, and phylogenetic analyses of 14 whole genomes recapitulated results seen for the Rep gene. A comparison of GRBaV genomes revealed evidence of recombination underlying some of the variation seen among GRBaV genomes within clade 1. Phylogenetic analyses of coat and replicase-associated protein sequences among single-stranded DNA viruses showed GRBaV to group within the family Geminiviridae. This grouping is distinct from members of the families Nanoviridae and Circoviridae, with limited significant affinities to both recognized genera and novel plant-infecting, gemini-like viruses.

A novel circular DNA virus sequence is reported from grapevine. The corresponding genomic organization, coding potential, and conserved origin of replication are similar to those of members of the family Geminiviridae , but the genome of 3,206 nucleotides is 4% larger than the largest reported geminiviral genome and shares only 50% overall sequence identity.

Grapevine red blotch virus (GRBV) has a monopartite single-stranded DNA genome and is the type species of the genus Grablovirus in the family Geminiviridae. To address the etiological role of GRBV in the recently recognized red blotch disease of grapevine, infectious GRBV clones were engineered from the genome of each of the two previously identified phylogenetic clades for Agrobacterium tumefaciens-mediated inoculations of tissue culture-grown Vitis spp. plants. Following agroinoculation and one or two dormancy cycles, systemic GRBV infection was detected by multiplex polymerase chain reaction (PCR) in Vitis vinifera exhibiting foliar disease symptoms but not in asymptomatic vines. Infected rootstock genotype SO4 (V. berlandieri × V. riparia) exhibited leaf chlorosis and cupping, while infection was asymptomatic in agroinoculated 110R (V. berlandieri × V. rupestris), 3309C (V. riparia × V. rupestris), and V. rupestris. Spliced GRBV transcripts of the replicase-associated protein coding region accumulated in leaves of agroinfected vines, as shown by reverse-transcription PCR; this was consistent with systemic infection resulting from virus replication. Additionally, a virus progeny identical in nucleotide sequence to the infectious GRBV clones was recovered from agroinfected vines by rolling circle amplification, cloning, and sequencing. Concomitantly, subjecting naturally infected grapevines to microshoot tip culture resulted in an asymptomatic plant progeny that tested negative for GRBV in multiplex PCR. Altogether, our agroinoculation and therapeutic experiments fulfilled Koch’s postulates and revealed the causative role of GRBV in red blotch disease.

The adoption of Oxford Nanopore Technologies (ONT) sequencing as a tool in plant virology has been relatively slow despite its promise in more recent years to yield large quantities of long nucleotide sequences in real time without the need for prior amplification. The portability of the MinION and Flongle platforms combined with lowering costs and continued improvements in read accuracy make ONT an attractive method for both low- and high-scale virus diagnostics. Here, we provide a detailed step-by-step protocol using the ONT Flongle platform that we have developed for the routine application on a range of symptomatic post-entry quarantine and domestic surveillance plant samples. The aim of this methods paper is to highlight ONT’s feasibility as a valuable component to the diagnostician’s toolkit and to hopefully stimulate other laboratories towards the eventual goal of integrating high-throughput sequencing technologies as validated plant virus diagnostic methods in their own right.