GENETIC DIVERSITY AND MOLECULAR BIOLOGY OF GRAPEVINE LEAFROLL-ASSOCIATED VIRUSES

Grapevine leafroll disease (GLRD) is an economically important virus disease of wine grapes (Vitis vinifera L.). Ten grapevine leafroll-associated viruses (GLRaVs, family Closteroviridae), designated as GLRaV-1 to -10 in the order of their discovery, have been reported in GLRD-infected grapevines. This research was focused on GLRaV-2 and GLRaV-3 that are frequently detected in Washington State vineyards. Genetic diversity among GLRaV-2 isolates collected largely from Washington vineyards was analyzed by sequencing the entire coat protein (CP) and a portion of the heat-shock protein-70 homolog (HSP70h). Phylogenetic analysis of CP and HSP70h sequences with corresponding Genbank sequences revealed segregation of global isolates of GLRaV-2 into six lineages (`PN', `93/955', `H4', `PV20', `BD' and `RG'), with virus isolates from Washington distributed in `PN', `H4' and `RG' lineages. The ratio of genetic diversity at nonsynonymous and synonymous sites computed separately for CP and HSP70h indicated diversifying selection pressures on the two viral genes. A molecular diagnostic assay, based on reverse transcription-polymerase chain reaction and restriction fragment length polymorphism, was developed to differentiate GLRaV-2 isolates in `PN', `H4' and `RG' lineages currently documented in Washington vineyards.The genome of the Washington isolate of GLRaV-3 was determined to be 18,498 nucleotides (nt) long with an unusually long 5'-nontranslated region of 737 nt and encoding eleven open reading frames. In Northern blots probed with gene-specific riboprobes, four 3'-coterminal subgenomic RNAs (sgRNAs) specific to CP, p21, p20A, and p20B genes were detected in total RNA extracted from a wine grape cultivar naturally infected with GLRaV-3. An analysis of their leader sequences indicated differences in transcriptional regulation of sgRNAs between GLRaV-3 and other closteroviruses. A multi-step cloning strategy was used to assemble a full-length cDNA clone of GLRaV-3 and demonstrated that in vitro RNA transcripts generated from the cDNA clone can replicate in Nicotiana benthamiana protoplasts. Similarly, the full-length cDNA clone of GLRaV-3 cloned into a binary vector showed replication when introduced into N. benthamiana leaves via agroinfiltration and formed authentic virion particles. The infectious cDNA clone of GLRaV-3 will help applying reverse genetics to study the molecular biology of grapevine-infecting closteroviruses and the etiology of GLRD

Soil-borne wheat mosaic virus infectious clone and manipulation for gene-carrying capacity

A full-length infectious cDNA clone of soil-borne wheat mosaic virus (SBWMV; genus Furovirus; family Virgaviridae) was developed for agrobacterium delivery. The cloned virus can be agroinfiltrated to Nicotiana benthamiana for subsequent infection of wheat (Triticum aestivum, L.). The utility of the virus as a vector for gene silencing and expression was assessed through sequence insertions in multiple sites of RNA2. Virus-induced photobleaching was observed in N. benthamiana but not in wheat, despite the stability of the inserts. The SBWMV infectious clone can be used for further studies to investigate the biology of SBWMV through mutagenesis

3'-coterminal subgenomic RNAs and putative <it>cis</it>-acting elements of <it>Grapevine leafroll-associated virus 3 </it>reveals 'unique' features of gene expression strategy in the genus <it>Ampelovirus</it>

Abstract Background The family Closteroviridae comprises genera with monopartite genomes, Closterovirus and Ampelovirus, and with bipartite and tripartite genomes, Crinivirus. By contrast to closteroviruses in the genera Closterovirus and Crinivirus, much less is known about the molecular biology of viruses in the genus Ampelovirus, although they cause serious diseases in agriculturally important perennial crops like grapevines, pineapple, cherries and plums. Results The gene expression and cis-acting elements of Grapevine leafroll-associated virus 3 (GLRaV-3; genus Ampelovirus) was examined and compared to that of other members of the family Closteroviridae. Six putative 3'-coterminal subgenomic (sg) RNAs were abundantly present in grapevine (Vitis vinifera) infected with GLRaV-3. The sgRNAs for coat protein (CP), p21, p20A and p20B were confirmed using gene-specific riboprobes in Northern blot analysis. The 5'-termini of sgRNAs specific to CP, p21, p20A and p20B were mapped in the 18,498 nucleotide (nt) virus genome and their leader sequences determined to be 48, 23, 95 and 125 nt, respectively. No conserved motifs were found around the transcription start site or in the leader sequence of these sgRNAs. The predicted secondary structure analysis of sequences around the start site failed to reveal any conserved motifs among the four sgRNAs. The GLRaV-3 isolate from Washington had a 737 nt long 5' nontranslated region (NTR) with a tandem repeat of 65 nt sequence and differed in sequence and predicted secondary structure with a South Africa isolate. Comparison of the dissimilar sequences of the 5'NTRs did not reveal any common predicted structures. The 3'NTR was shorter and more conserved. The lack of similarity among the cis-acting elements of the diverse viruses in the family Closteroviridae is another measure of the complexity of their evolution. Conclusions The results indicate that transcription regulation of GLRaV-3 sgRNAs appears to be different from members of the genus Closterovirus. An analysis of the genome sequence confirmed that GLRaV-3 has an unusually long 5'NTR of 737 nt compared to other monopartite members of the family Closteroviridae, with distinct differences in the sequence and predicted secondary structure when compared to the corresponding region of the GLRaV-3 isolate from South Africa.</p

Transformation of an edible crop with the pagA gene of Bacillus anthracis

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First reports of Beet curly top virus, Citrus yellow vein-associated virus, and Hop latent viroid in industrial hemp (Cannabis sativa) in Washington State.

In 2021 and 2022, virus-like symptoms were observed in several cultivars of industrial hemp (Cannabis sativa) in two fields in central Washington, USA. Affected plants had a range of symptoms at different developmental stages, with young plants having severe stunting with shortened internodes and reduced flower mass. Young leaves of infected plants also showed light green to total yellowing, and twirling with twisting margins (Fig. S1). Infections of older plants caused less foliar symptoms that consisted of mosaic, mottling, and mild chlorosis on a few branches with tacoing of older leaves. To assess if symptomatic hemp plants were infected with Beet curly top virus (BCTV) as reported earlier (Giladi et al., 2020; Chiginsky et al., 2021), symptomatic leaves were collected from 38 plants, and the extracted total nucleic acids tested by PCR to amplify a 496-base pair (bp) fragment specific to BCTV coat protein (CP) using primers BCTV2-F 5'-GTGGATCAATTTCCAG-ACAATTATC-3' and BCTV2-R 5'-CCCATAAGAGCCATATCA-AACTTC-3' (Strausbaugh et al. 2008). BCTV was found in 37 of the 38 plants. To further assess the virome of symptomatic hemp plants, total RNA was extracted from symptomatic leaves of four plants using Spectrum total RNA isolation kits (Sigma-Aldrich, St. Louis, MO) and subjected to high-throughput sequencing on an Illumina Novaseq platform in paired-end mode (University of Utah, Salt Lake City, UT). The raw reads (33 to 40 million per sample) were trimmed based on quality and ambiguity and resulting paired-end reads of ≈142 bp length were assembled de novo into a pool of contigs (CLC Genomics Workbench 21, Qiagen Inc.). Virus sequences were identified through BLASTn analysis in GenBank (https://www.ncbi.nlm.nih.gov/blast). One contig of 2,929 nucleotides (nt) obtained from one sample (accession no. OQ068391) showed 99.3% identity with BCTV-Wor strain reported from sugar beet in Idaho (accession no. KX867055 Strausbaugh et al., 2017). Another contig of 1,715 nt from a second sample (accession no. OQ068392) shared 97.3% identity with BCTV-CO strain (accession no. KX867022). Two contig sequences of 2,876 nt (accession no. OQ068388) and 1,399 nt (accession no. OQ068389) obtained from the 3rd and 4th samples showed 97.2% and 98.3% identity, respectively, with Citrus yellow vein-associated virus (CYVaV, accession no. MT893740.1) reported in industrial hemp from Colorado (Chiginsky et al., 2021). Contigs of 256 nt sequence (accession no. OQ068390) obtained from the 3rd and 4th samples also showed 99-100% identity with Hop Latent viroid (HLVd) sequences in GenBank (accessions OK143457 and X07397). These results indicated single infections of BCTV strains and co-infection of CYVaV and HLVd in individual plants. To confirm theagents, symptomatic leaves were collected from 28 randomly selected hemp plants and tested by PCR/RT-PCR using primers specific to BCTV (Strausbaugh et al., 2008), CYVaV (Kwon et al., 2021) and HLVd (Matoušek et al., 2001). Amplicons specific to BCTV (496 bp), CYVaV (658 bp) and HLVd (256 bp) were detected in 28, 25, and 2 samples, respectively. BCTV CP sequences obtained by Sanger sequencing from seven samples showed 100% sequence identity with BCTV-CO and BCTV-Wor strains in six and one samples, respectively. Similarly, sequences of CYVaV- and HLVd-specific amplicons showed 100% identity with corresponding sequences in GenBank. To the best of our knowledge, this is the first report of two strains of BCTV (BCTV-CO and BCTV-Wor), CYVaV, and HLVd infecting industrial hemp in Washington state