A novel emaravirus identified in maple with leaf mottle symptoms by deep sequencing

The full-length genome of a novel Emaravirus has been identified and characterized from sycamore maple (Acer pseudoplatanus) - a tree species of significant importance in urban and forest areas - showing leaf mottle symptoms. RNA-Seq was performed using RNA preparations from a symptomatic and a symptomless maple tree. Purified double-stranded cDNA from each sample were used for RNA-Seq analysis on the Illumina HiSeq2500system and 14-198 MB data/sample of 100 bp-long paired-end sequence reads were generated. The sequence assembly and analysis revealed the presence of six RNA segments in the symptomatic sample (RNA1: 7,075 nt-long encoding the viral replicase; RNA2: 2,289 nt-long encoding the glycoprotein precursor; RNA3: 1,525 nt-long encoding the nucleocapsid protein; RNA4: 1,533 nt-long encoding the putative movement protein; RNA5: 1,825 nt-long encoding a hypothetical protein P5; RNA6: 1,179 nt-long encoding a hypothetical protein P6). Two independent HTS sequencing runs from the same symptomatic maple tree detected the same genome segments. For one of these sequencing runs the cDNA library was prepared using a primer targeting the conserved genome terminal region, known to be shared between emaraviruses genome segments and a high amount of sequence data was generated. We suggest, therefore, that the six identified genome segments represent the complete genome of a novel emaravirus from maple, which we tentatively name maple mottle-associated virus (MaMaV). RT-PCR assays were performed on symptomatic and non-symptomatic leaves of A. pseudoplatanus trees coming growing on two different locations in Berlin. MaMaV was only detected from symptomatic trees and all six RNAs were generally simultaneously detected. Non-symptomatic samples were consistently negative for MaMaV. These results suggest that MaMaV might be the symptom inducing virus in the sampled trees. In the present state of the art, this is the first time an Emaravirus is described from maple and is fully genetically characterized.

Two Novel Negative-Sense RNA Viruses Infecting Grapevine Are Members of a Newly Proposed Genus within the Family Phenuiviridae

Two novel negative-stranded (ns)RNA viruses were identified by high throughput sequencing in grapevine. The genomes of both viruses, named grapevine Muscat rose virus (GMRV) and grapevine Garan dmak virus (GGDV), comprise three segments with each containing a unique gene. Based on sequence identity and presence of typical domains/motifs, the proteins encoded by the two viruses were predicted to be: RNA-dependent RNA polymerase (RdRp), nucleocapsid protein (NP), and putative movement protein (MP). These proteins showed the highest identities with orthologs in the recently discovered apple rubbery wood viruses 1 and 2, members of a tentative genus (Rubodvirus) within the family Phenuiviridae. The three segments of GMRV and GGDV share almost identical sequences at their 5′ and 3′ termini, which are also complementary to each other and may form a panhandle structure. Phylogenetics based on RdRp, NP and MP placed GMRV and GGDV in the same cluster with rubodviruses. Grapevine collections were screened for the presence of both novel viruses via RT-PCR, identifying infected plants. GMRV and GGDV were successfully graft-transmitted, thus, they are the first nsRNA viruses identified and transmitted in grapevine. Lastly, different evolutionary scenarios of nsRNA viruses are discussed.

Pineapple bacilliform CO virus: Diversity, Detection, Distribution, and Transmission

Members of the genus Badnavirus (family Caulimovirdae) have been identified in dicots and monocots worldwide. The genome of a pineapple badnavirus, designated Pineapple bacilliform CO virus-HI1 (PBCOV-HI1), and nine genomic variants (A through H) were isolated and sequenced from pineapple, Ananas comosus, in Hawaii. The 7,451-nucleotide genome of PBCOV-HI1 possesses three open reading frames (ORFs) encoding putative proteins of 20 (ORF1), 15 (ORF2), and 211 (ORF3) kDa. ORF3 encodes a polyprotein that includes a putative movement protein and viral aspartyl proteinase, reverse transcriptase, and RNase H regions. Three distinct groups of putative endogenous pineapple pararetroviral sequences and Metaviridae-like retrotransposons encoding long terminal repeat, reverse-transcriptase, RNase H, and integrase regions were also identified from the pineapple genome. Detection assays were developed to distinguish PBCOV-HI1 and genomic variants, putative endogenous pararetrovirus sequences, and Ananas Metaviridae sequences also identified in pineapple. PBCOV-HI1 incidences in two commercially grown pineapple hybrids, PRI 73-114 and PRI 73-50, was 34 to 68%. PBCOV-HI1 was transmitted by gray pineapple mealybugs, Dysmicoccus neobrevipes, to pineapple.

A Host-Factor Interaction and Localization Map for a Plant-Adapted Rhabdovirus Implicates Cytoplasm-Tethered Transcription Activators in Cell-to-Cell Movement

To identify host factors that play critical roles in processes, including cell-to-cell movement of plant-adapted rhabdoviruses, we constructed and validated a high-resolution Nicotiana benthamiana yeast two-hybrid library. The library was screened with the putative movement protein (sc4), nucleocapsid (N), and matrix (M) proteins of Sonchus yellow net virus (SYNV). This resulted in identification of 31 potential host factors. Steady-state localization studies using autofluorescent protein fusions to full-length clones of interactors were conducted in transgenic N. benthamiana marker lines. Bimolecular fluorescence complementation assays were used to validate two-hybrid interactions. The sc4 interactor, sc4i21, localized to microtubules. The N interactor, Ni67, localized to punctuate loci on the endoplasmic reticulum. These two proteins are 84% identical homologues of the Arabidopsis phloem-associated transcription activator AtVOZ1, and contain functional nuclear localization signals. Sc4i17 is a microtubule-associated motor protein. The M interactor, Mi7, is a nuclear-localized transcription factor. Combined with a binary interaction map for SYNV proteins, our data support a model in which the SYNV nucleocapsids are exported from the nucleus and moved cell-to-cell by transcription activators tethered in the cytoplasm.

First Report of Grapevine Virus Sequences Highly Similar to Grapevine Syrah virus-1 from Washington Vineyards

Grapevine Syrah virus-1 (GSyV-1), a tentative member of the genus Marafivirus in the family Tymoviridae, has recently been found in a declining Syrah grapevine in California vineyards (1). To determine if GSyV-1 is present in grapevines grown in Washington State vineyards, extracts prepared from individual grapevines of six cultivars (Merlot, Chardonnay, Pinot Noir, Lemberger, Cabernet Sauvignon, and Syrah/Shiraz) were tested by single-tube reverse transcription (RT)-PCR using the primer pair GSyV-1 Det-F (5′-CAAGCCATCCGTGCATCTGG-3′) and GSyV-1 Det-R (5′-GCCGATTTGGAACCCGATGG-3′). The primer GSyV-1 Det-F is identical to nucleotides (nt) 1125 to 1144 and GSyV-1 Det-R complementary to nt 1401 to 1420 of the GSyV-1 genome (GenBank Accession No. NC_012484) in the putative movement protein encoding gene (1). DNA fragment of approximately 296 base pairs (bp) was amplified only from 7 of 60 and 2 of 20 individual grapevines of cv. Syrah/Shiraz and Chardonnay, respectively, obtained from geographically separate vineyards. The 296-bp fragments from three Syrah/Shiraz and two Chardonnay grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones derived from each DNA fragment were sequenced from both orientations and the sequences edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparison of four of these sequences (Accession Nos. GU372349–52) showed 99 to 100% amino acid (aa) sequence identity among themselves and with corresponding sequences of GSyV-1. Because of the lack of antibodies, an additional 611-bp fragment specific to the capsid protein (CP) gene of GSyV-1 was amplified from six isolates (five from cv. Syrah/Shiraz, and one from cv. Chardonnay) (Accession Nos. GU372353–66) using primers GSyV-1-F (5′-TGTCGACGCTCCAATGTCTGA-3′) and GSyV-1-R (5′-CATTGCTGCGCTTTGGAGGCTTTA-3′). GSyV-1-F is identical to nt 5775 to 5795 and GSyV-1-R is complementary to nt 6385 to 6408 of the GSyV-1 genome. The amplicons were cloned and sequenced as described above. Comparison of these sequences among themselves and with corresponding sequences of GSyV-1 showed 96 to 99% aa sequence identity, further complementing the results obtained above. To our knowledge, this is the first report of the occurrence of viral sequences closely related to GSyV-1 in Washington vineyards. Together with other reports (1,2), this study suggests that viruses similar to GSyV-1 could be widely distributed in wine grape cultivars across grape-growing regions. References: (1) M. Rwahnih et al. Virology 387:395, 2009. (2) S. Sabanadzovic. Virology 394:1, 2009.

A potential nuclear envelope-targeting domain and an arginine-rich RNA binding element identified in the putative movement protein of the GAV strain of Barley yellow dwarf virus

In this study, the structural elements in the putative movement protein (MP) of the GAV strain of Barley yellow dwarf virus (BYDV-GAV) were investigated. The GFP fusion protein of BYDV-GAV MP was found to be associated with the nuclear envelope (NE) in transgenic Arabidopsis thaliana (L.) cells. Serial deletion mapping demonstrated that the predicted α-helical domain located at the N-terminus of BYDV-GAV MP was required and sufficient for NE targeting in onion epidermal cells. This α-helical domain does not contain any sequence elements similar to known nuclear localisation signals or bear any significant resemblance to previously characterised NE-targeting structure, indicating that it may represent a novel NE-targeting domain in plant cells. Deletion mutagenesis showed that the C-terminal end of BYDV-GAV MP possessed an element required for its RNA binding activity in vitro. Further analysis revealed that the arginine amino acids within the last 11 residues of the C-terminal end were crucial for the binding of BYDV-GAV MP to RNA. This C-terminal element enriched in basic residues was also present in the MPs of other BYDV strains and the polerovirus Potato leaf roll virus (PLRV), suggesting the conservation of a RNA binding element in the MPs from both luteoviruses and poleroviruses. The data in this work present an initial characterisation of a novel plant NE-targeting domain and a RNA binding element on BYDV-GAV MP. Further studies are underway to investigate the function of these elements in the biology of natural BYDV-GAV infection.

RNA3 nucleotide sequence analyses ofpeanut stunt virusMi strain

Nucleotide sequence of full-length cDNA ofpeanut stunt virus(PSV) Mi strain RNA3 was determined and compared with those of PSV-ER and -J (subgroup I) and PSV-W (subgroup II), strains ofcucumber mosaic virus(CMV) andtomato aspermy virus(TAV). PSV-Mi RNA3 consists of 2170 nt and has two open reading frames, encoding a putative movement protein (3a protein) and a coat protein (CP). PSV-Mi RNA3 is 77.7% and 78.5% identical to those of PSV-ER and -J, whereas it shares 76.6% identity with PSV-W. Nucleotide identity of3aandcpgenes between PSV strains Mi and ER, J and W was 78.3–79.3% and 74.4–77.8%, respectively. Amino acid identity of 3a and CP between PSV-Mi and -ER, -J and -W was 73.9–77.4% and 64.8–77.5%, respectively. RNA3 of PSV-Mi (GenBank accession no. AY775057) had a varied intercistronic and 5′-untranslated region compared with those of PSV strains ER, J and W. Results indicate that PSV-Mi represents a new PSV subgroup from China, designated as subgroup III.