ICTV Virus Taxonomy Profile: Closteroviridae.

Viruses in the family Closteroviridae have a mono-, bi- or tripartite positive-sense RNA genome of 13-19 kb, and non-enveloped, filamentous particles 650-2200 nm long and 12 nm in diameter. They infect plants, mainly dicots, many of which are fruit crops. This is a summary of the ICTV Report on the family Closteroviridae, which is available at ictv.global/report/closteroviridae.

Elucidating the Subcellular Localization of GLRaV-3 Proteins Encoded by the Unique Gene Block in N. benthamiana Suggests Implications on Plant Host Suppression.

Grapevine leafroll-associated virus 3 (GLRaV-3) is a formidable threat to the stability of the global grape and wine industries. It is the primary etiological agent of grapevine leafroll disease (GLD) and significantly impairs vine health, fruit quality, and yield. GLRaV-3 is a member of the genus Ampelovirus, Closteroviridae family. Viral genes within the 3' proximal unique gene blocks (UGB) remain highly variable and poorly understood. The UGBs of Closteroviridae viruses include diverse open reading frames (ORFs) that have been shown to contribute to viral functions such as the suppression of the host RNA silencing defense response and systemic viral spread. This study investigates the role of GLRaV-3 ORF8, ORF9, and ORF10, which encode the proteins p21, p20A, and p20B, respectively. These genes represent largely unexplored facets of the GLRaV-3 genome. Here, we visualize the subcellular localization of wildtype and mutagenized GLRaV-3 ORFs 8, 9, and 10, transiently expressed in Nicotiana benthamiana. Our results indicate that p21 localizes to the cytosol, p20A associates with microtubules, and p20B is trafficked into the nucleus to carry out the suppression of host RNA silencing. The findings presented herein provide a foundation for future research aimed at the characterization of the functions of these ORFs. In the long run, it would also facilitate the development of innovative strategies to understand GLRaV-3, mitigate its spread, and impacts on grapevines and the global wine industry.

Single berry reconstitution prior to RNA-sequencing reveals novel insights into transcriptomic remodeling by leafroll virus infections in grapevines.

Leafroll viruses are among the most devastating pathogens in viticulture and are responsible for major economic losses in the wine industry. However, the molecular interactions underlying the effects on fruit quality deterioration are not well understood. The few molecular studies conducted on berries from infected vines, associated quality decreases with the repression of key genes in sugar transport and anthocyanin biosynthesis. Sampling protocols in these studies did however not account for berry heterogeneity and potential virus induced phenological shifts, which could have biased the molecular information. In the present study, we adopted an innovative individual berry sampling protocol to produce homogeneous batches for RNA extraction, thereby circumventing berry heterogeneity and compensating for virus induced phenological shifts. This way a characterization of the transcriptomic modulation by viral infections was possible and explain why our results differ significantly from previously reported repression of anthocyanin biosynthesis and sugar metabolism. The present study provides new insights into the berry transcriptome modulation by leafroll infection, highlighting the virus induced upregulation of plant innate immunity as well as an increased responsiveness of the early ripening berry to biotic stressors. The study furthermore emphasizes the importance of sampling protocols in physiological studies on grapevine berry metabolism.

Development of infectious cDNA clones of Grapevine leafroll-associated virus 3 and analyses of the 5' non-translated region for replication and virion formation.

Infectious cDNA clones were developed for Grapevine leafroll-associated virus 3 (GLRaV-3, genus Ampelovirus, family Closteroviridae). In vitro RNA transcripts generated from cDNA clones showed replication via the production of 3'-coterminal subgenomic (sg) mRNAs in Nicotiana benthamiana protoplasts. The detection of sgRNAs and the recovery of progeny recombinant virions from N. benthamiana leaves agroinfiltrated with full-length cDNA clones confirmed RNA replication and virion formation. The 5' non-translated region (5' NTR) of GLRaV-3 was exchangeable between genetic variants and complement the corresponding cognate RNA functions in trans. Mutational analysis of the 5' NTR in minireplicon cDNA clones showed that the conserved 40 nucleotides at the 5'-terminus were indispensable for replication, compared to downstream variable portion of the 5' NTR. Some of the functional mutations in the 5' NTR were tolerated in full-length cDNA clones and produced sgRNAs and virions in N. benthamiana leaves, whereas other mutations affected replication and virion formation.

Genome analysis and detection of a Chilean isolate of Grapevine leafroll associated virus-3.

The complete genome of the Chilean isolate Cl-766 of Grapevine leafroll-associated virus-3 (GLRaV-3) has been sequenced. This is the first genome sequence obtained from a GLRaV-3 isolate of the Southern hemisphere. The genomic RNA of 17,919 nucleotides contains 13 open reading frames (ORFs) with 5' and 3' untranslated regions (UTR) of 158 and 277 nucleotides, respectively. Comparison with NY1, the only isolate with complete genomic sequence available today, shows 97.6% nucleotide identity between the two isolates. Examination of the genome variability shows that most of the genetic diversity is concentrated in ORF1a. Three additional isolates from different geographic regions of Chile were partially sequenced as well, one which showed sequence divergence with respect to the other local and foreign isolates, indicative of different evolutionary constrains. Immunodetection systems were developed using monoclonal and polyclonal antibodies produced against the recombinant major coat protein of GLRaV-3, providing sensitive and specific detection using a triple antibody sandwich-enzyme linked immunosorbent assay (TAS-ELISA) and an immunocapture-reverse transcription-polymerase chain reaction (IC-RT-PCR) assay.