First report of Alstroemeria necrotic streak virus infecting greenhouse bell pepper (Capsicum annuum) in Canada.

Alstroemeria necrotic streak virus (ANSV) is an Orthotospovirus that has been isolated from symptomatic Alstroemeria plant in 2010 (Hassani-Mehraban et al. 2010). It has been shown to infect crops of bell pepper (Capsicum annuum) and tomato (Solanum lycopersicum) (Olaya et al. 2017) which are two of the three biggest greenhouse crops in Canada in terms of production volume and value (Statistic Canada. 2020). In July of 2022, the entire production of bell pepper (all plants) from a greenhouse in Québec was presenting necrotic rings and discoloration in fruit and seemingly healthy leaves. Samples from these infected bell pepper were found to be negative for twenty two common viruses infecting bell pepper by ELISA immunoassay by the Laboratoire d'expertise et de diagnostic en phytoprotection (LEDP) (Québec, Canada). To identify the causal agent, double-stranded RNA was extracted from leaf and fruit of one plant to form two separate samples (leaf and fruit) and used for cDNA library preparations with Nextera XT DNA Sample Prep kit (Illumina, USA). The libraries were sequenced using Illumina Miseq (Fall et al. 2020). The same dsRNA were also sequenced with MinION nanopore sequencing method as described previously (Javaran et al. 2021; Javaran et al. 2023). The obtained raw FASTQ data were processed following the methodology described in Fall et al. 2020 and Javaran et al. 2023. The Miseq sequencing yielded over 2 million reads per sample with a percentage of mapped viral reads ranging from 26.92 to 47.29% of the total number of reads. The leaf samples were positive to Bell pepper endornavirus (BPEV) with the full genome covered 16713 times and Alstroemeria necrotic streak virus (ANSV) with 98% of the genome covered 4929 times. The MinION sequencing yielded 1,028,460 reads and the same viruses were detected with 1288 long reads (mean length of 745bp) assigned to ANSV genome. Both viruses were detected in the leaf and fruit samples. The complete ANSV genome comprising three segments (L, M, and S) was assembled and deposited in GenBank: (OQ261731-OQ261733). These L, M and S segments shown 99% nt identity with an isolate from the Columbia (GenBank: MF469036, MF469037, MF469038). It is interesting that read coverage at near the 2000th position of the S segment, was very low. This phenomenon may suggest a cleavage site nearby by a viral or host factor. ANSV was mainly found in leaf samples and very low numbers of reads in fruit samples. The presence of ANSV was confirmed by RT-PCR using the primers specific to the ANSV nucleocapsid gene Tospo_S_F (5'- CAG AAT CAG GCT GCA TTT AAT TTC C-3') and Tospo_S_R (5'-CAA CGC TTC CTT TAG CAT TAG G-3') (Gallo et al. 2019). The sequences of ∼600 bp amplicons were determined using Sanger sequencing and showed 100% nt identity with Miseq-derived sequences of ANSV. The virus has previously been detected in Colombia (Hassani-Mehraban et al. 2010) and then in California in 2018 (Tian et al. 2020). This is to our knowledge the first detection of ANSV in Canada. Bell pepper is one of the most important crops in Canada and the ANSV vector, the western flower thrips (Frankliniella occidentalis), known to spread the tomato spotted wilt virus (TSWV) is established in Canada (Allen et al. 1986). The detection of ANSV in Canada is line with the hypothesis of an international spread of this virus (Tian et al. 2020) as is it not known to spread through seeds.

Grapevine Virology in the Third-Generation Sequencing Era: From Virus Detection to Viral Epitranscriptomics.

Among all economically important plant species in the world, grapevine (Vitis vinifera L.) is the most cultivated fruit plant. It has a significant impact on the economies of many countries through wine and fresh and dried fruit production. In recent years, the grape and wine industry has been facing outbreaks of known and emerging viral diseases across the world. Although high-throughput sequencing (HTS) has been used extensively in grapevine virology, the application and potential of third-generation sequencing have not been explored in understanding grapevine viruses and their impact on the grapevine. Nanopore sequencing, a third-generation technology, can be used for the direct sequencing of both RNA and DNA with minimal infrastructure. Compared to other HTS methods, the MinION nanopore platform is faster and more cost-effective and allows for long-read sequencing. Due to the size of the MinION device, it can be easily carried for field viral disease surveillance. This review article discusses grapevine viruses, the principle of third-generation sequencing platforms, and the application of nanopore sequencing technology in grapevine virus detection, virus-plant interactions, as well as the characterization of viral RNA modifications.

Transient expression of recombinant tissue plasminogen activator (rt-PA) gene in cucurbit plants using viral vector.

OBJECTIVE: To use a transient expression system to express a truncated human tissue plasminogen activator (K2S) gene in cucurbit plants. RESULTS: The recombinant tissue plasminogen activator protein (K2S form) was expressed in active form in cucurbit plants. Its molecular weight was 43 kDa. The plant-derived rt-PA was determined using goat anti-rabbit antibody by western blotting. Among the infected lines, the highest expression of rt-PA was 62 ng/100 mg per leaf tissue as measured by ELISA. The enzymatic activity of the plant-derived rt-PA was 0.8 IU/ml. CONCLUSIONS: The K25 form of rt-PA was expressed for the first time using the viral expression system. Plant-derived rt-PA showed similar potency to commercially-available PA.