Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification.

Little cherry virus 2 (LChV2) (genus Ampelovirus) is the primary causal agent of little cherry disease (LCD) in sweet cherry (Prunus avium) in North America and other parts of the world. This mealybug-transmitted virus does not induce significant foliar symptoms in most sweet cherry cultivars, but does cause virus-infected trees to yield unevenly ripened small fruits with poor flavor. Most fruits from infected trees are unmarketable. In the present study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) technique was developed using LChV2 coat protein specific primers and probe. Detection of terminally labeled amplicons was achieved with a high affinity lateral flow strip. The RT-RPA is confirmed to be simple, fast, and specific. In comparison, although it retains the sensitivity of RT-PCR, it is a more cost-effective procedure. RT-RPA will be a very useful tool for detecting LChV2 from crude extracts in any growth stage of sweet cherry from field samples.

Genetic diversity of Grapevine virus A in Washington and California vineyards.

Grapevine virus A (GVA; genus Vitivirus, family Betaflexiviridae) has been implicated with the Kober stem grooving disorder of the rugose wood disease complex. In this study, 26 isolates of GVA recovered from wine grape (Vitis vinifera) cultivars from California and Washington were analyzed for their genetic diversity. An analysis of a portion of the RNA-dependent RNA polymerase (RdRp) and complete coat protein (CP) sequences revealed intra- and inter-isolate sequence diversity. Our results indicated that both RdRp and CP are under strong negative selection based on the normalized values for the ratio of nonsynonymous substitutions per nonsynonymous site to synonymous substitutions per synonymous site. A global phylogenetic analysis of CP sequences revealed segregation of virus isolates into four major clades with no geographic clustering. In contrast, the RdRp-based phylogenetic tree indicated segregation of GVA isolates from California and Washington into six clades, independent of geographic origin or cultivar. Phylogenetic network coupled with recombination analyses showed putative recombination events in both RdRp and CP sequence data sets, with more of these events located in the CP sequence. The preponderance of divergent variants of GVA co-replicating within individual grapevines could increase viral genotypic complexity with implications for phylogenetic analysis and evolutionary history of the virus. The knowledge of genetic diversity of GVA generated in this study will provide a foundation for elucidating the epidemiological characteristics of virus populations at different scales and implementing appropriate management strategies for minimizing the spread of genetic variants of the virus by vectors and via planting materials supplied to nurseries and grape growers.

Complete nucleotide sequence of a strain of cherry mottle leaf virus associated with peach wart disease in peach.

The complete nucleotide sequence and genome organization of a peach virus isolate from a naturally infected peach tree showing typical peach wart-like symptoms on the fruit surface was determined and compared to sequences of members of the family Betaflexiviridae. The genome consists of 7,987 nucleotides, excluding the poly-A tail, and has four open reading frames (ORFs). Analysis of the whole genome and putative proteins encoded by each ORF revealed greatest sequence similarity to a cherry isolate of cherry mottle leaf virus (CMLV). The two isolates have similar genome organizations and share 88 and 93 % homology in their corresponding products of the replicase and coat protein genes, respectively. CMLV has been reported from several Prunus spp. and may be associated with peach wart-like disease symptoms on peach fruit.

Complete nucleotide sequences and genome organization of a cherry isolate of cherry leaf roll virus.

The complete nucleotide sequence of cherry leaf roll virus (CLRV, genus Nepovirus) from a naturally infected cherry tree (Prunus avium cv. Bing) in North America was determined. RNA1 and RNA2 consist of 7,893 and 6,492 nucleotides, respectively, plus a poly-(A) tail. Each RNA encodes a single potential open reading frame. The first 657 nucleotides of RNA1 and RNA2 are 99% identical and include the 5'-UTR and the first 214 deduced amino acids of the polyproteins following the first of two in-frame start codons. Phylogenetic analysis reveals close relationships between CLRV and members of subgroup C of the genus Nepovirus.

Genome diversity and intra- and interspecies recombination events in Grapevine fanleaf virus.

ABSTRACT Grapevine fanleaf virus (GFLV) was documented in self-rooted vines of four grapevine (Vitis vinifera) cultivars in eastern Washington. GFLV was found as mixed infection in cvs. Pinot Noir, Chardonnay, and Cabernet Franc and as single infections in cv. Merlot. Fanleaf disease symptoms were only observed in the first two cultivars. The spatial distribution of GFLV-infected grapevines was random, suggesting primary spread through planting virus-infected cuttings rather than infield transmission. RNA1 sequences of Washington isolates showed 87 to 89% nucleotide sequence identity between them and with strain F13. RNA2 of Washington isolates was variable in size, showing 85 to 99% sequence identity between them and 81 to 92% with other isolates. As in other GFLV isolates, three conserved putative stem-loop structures were present in the 5' noncoding regions of both RNAs of Washington isolates. Phylogenetic incongruence of GFLV isolates from Washington in 2A(HP)- and 2B(MP)-based trees and identification of putative recombination events suggested that their genomic RNA2 originated from inter- and intraspecies recombination events between GFLV, Grapevine deformation virus, and Arabis mosaic virus. These results confirm interspecies recombination in RNA2 of grapevine-infecting nepoviruses as an important strategy for GFLV evolution.

Phloem unloading of potato virus X movement proteins is regulated by virus and host factors.

To determine the requirements for viral proteins exiting the phloem, transgenic plants expressing green fluorescent protein (GFP) fused to the Potato virus X (PVX) triple gene block (TGB)p1 and coat protein (CP) genes were prepared. The fused genes were transgenically expressed from the companion cell (CC)-specific Commelina yellow mottle virus (CoYMV) promoter. Transgenic plants were selected for evidence of GFP fluorescence in CC and sieve elements (SE) and proteins were determined to be phloem mobile based on their ability to translocate across a graft union into nontransgenic scions. Petioles and leaves were analyzed to determine the requirements for phloem unloading of the fluorescence proteins. In petioles, fluorescence spread throughout the photosynthetic vascular cells (chlorenchyma) but did not move into the cortex, indicating a specific barrier to proteins exiting the vasculature. In leaves, fluorescence was mainly restricted to the veins. However, in virus-infected plants or leaves treated with a cocktail of proteasome inhibitors, fluorescence spread into leaf mesophyll cells. These data indicate that PVX contributes factors which enable specific unloading of cognate viral proteins and that proteolysis may play a role in limiting proteins in the phloem and surrounding chlorenchyma.