Occurrence of Nine Grapevine Viruses in Commercial Vineyards of Mendoza, Argentina.

Grapevine is a widely grown fruit crop that is seriously affected by different viruses, reducing grape yield and quality, as well as threatening profitability. Vineyard disease management requires accurate identification of viral infections. This study aimed to survey the presence of ten grapevine viruses in four geographic sites in the Mendoza province of Argentina. Two hundred twenty-three composite cane samples from 1060 plants of six cultivars were collected from 26 blocks distributed across 11 vineyards. The cane samples were screened by RT-PCR for the following viruses: grapevine leafroll-associated viruses 1-4 (GLRaV 1, 2, 3, and 4), grapevine fanleaf virus (GFLV), grapevine fleck virus (GFkV), grapevine virus A (GVA) and B (GVB), grapevine rupestris stem pitting associated virus (GRSPaV), and arabis mosaic virus (ArMV). The results showed an uneven occurrence of viruses through the sampled regions, with GRSPaV being prevalent (71.1%), followed by GFLV (28.9%), GFkV (20.6%), and GLRaV-2 (14.7%). GVB was not detected. This study revealed a moderate prevalence of viruses associated with economically impactful diseases in the vineyards surveyed.

Virus classification based on in-depth sequence analyses and development of demarcation criteria using the Betaflexiviridae as a case study.

Currently, many viruses are classified based on their genome organization and nucleotide/amino acid sequence identities of their capsid and replication-associated proteins. Although biological traits such as vector specificities and host range are also considered, this later information is scarce for the majority of recently identified viruses, characterized only from genomic sequences. Accordingly, genomic sequences and derived information are being frequently used as the major, if not only, criteria for virus classification and this calls for a full review of the process. Herein, we critically addressed current issues concerning classification of viruses in the family Betaflexiviridae in the era of high-throughput sequencing and propose an updated set of demarcation criteria based on a process involving pairwise identity analyses and phylogenetics. The proposed framework has been designed to solve the majority of current conundrums in taxonomy and to facilitate future virus classification. Finally, the analyses performed herein, alongside the proposed approaches, could be used as a blueprint for virus classification at-large.

Subcellular localization and interactions among TGB proteins of cowpea mild mottle virus.

Cowpea mild mottle virus (CPMMV) is a flexuous filamentous virus that belongs to the genus Carlavirus (family Betaflexiviridae). The CPMMV genome contains six open reading frames (ORFs), among which the triple gene block (TGB), encoded by ORFs 2 to 4, has been reported to encode movement proteins for different viruses. The subcellular localization of the TGB proteins of CPMMV isolate CPMMV:BR:MG:09:2 was analysed by transient expression of each protein fused to a fluorophore. Overall, the accumulation pattern and interactions among CPMMV TGB proteins (TGBp) were similar to those of their counterparts from the potex-like group. Considering these similarities, we evaluated the potential interactions between the TGB proteins of CPMMV and of potato virus X, which could complement cell-to-cell movement. The TGBp2 and TGBp3 of PVX had an effect on CPMMV TGBp1, directing it to the plasmodesmata, but the reverse was not true.

Near-complete genome sequence and biological properties of an allexivirus found in Senna rizzinii in Brazil.

Senna rizzinii is a flowering shrub found mainly in the northeast region of Brazil. Here, we report the coding-complete genome sequence, particle morphology, mode of transmission, and the indicator host responses of an isolate of the putative allexivirus cassia mild mosaic virus (CaMMV) found in S. rizzinii. The virus was transmitted mechanically to Chenopodium amaranticolor, C. quinoa, Gomphrena globosa, which showed local lesions, and S. rizzinii, and S. occidentalis, which were infected systemically. It was also efficiently transmitted to S. rizzinii by grafting. Seed transmission was not observed. The near-complete genome sequence of the virus is 7829 nucleotides in length, containing six open reading frames (ORF), like other allexiviruses.

Complete genome sequence of a novel capillovirus infecting Hevea brasiliensis in China.

Tapping panel dryness (TPD) is a complex disorder that causes partial or complete cessation of latex drainage upon tapping of rubber trees (Hevea brasiliensis). In this work, we determined the complete genome sequences of a novel virus identified in a rubber tree with TPD syndrome in China. The genome of the virus consists of 6811 nt and possesses two overlapping open reading frames (ORF1 and ORF2), encoding a polyprotein and a movement protein, respectively. The polyprotein shares 37% amino acid sequence identity with cherry virus A (CVA, ARQ83874.1) over 99% coverage. The genome architecture is similar to that of members of the genus Capillovirus (family Betaflexiviridae). Phylogenetic analysis of the replicase proteins showed that the virus clustered together with members of the genus Capillovirus. The new virus is tentatively called "rubber tree virus 1" (RTV1). RTV1 is the first virus reported to infect rubber trees. This work lays a foundation for research into finding the potential causal agent of TPD in Hevea brasiliensis.

Molecular characterization of a new trichovirus from peach in Mexico.

The complete genome sequence of a trichovirus was obtained from peach samples collected from Mexico and found to be 7985 nucleotides long, excluding the poly(A) tail. Phylogenetic analysis using the complete nucleotide sequence revealed that the virus is a member of the genus Trichovirus and is closely related to peach mosaic virus (PcMV) and cherry mottle leaf virus (CMLV). The highest nucleotide sequence identity was 70% to both PcMV and CMLV, indicating that this virus, which we have tentatively named "peach virus M" (PeVM) should be considered a member of a new trichovirus species. We determined, for the first time, the initiation sites of the subgenomic RNAs (sgRNA) of a trichovirus. The sgRNAs for the movement and coat proteins started with the sequence 'GAA', while the smallest one, coding for the nucleotide-binding protein, started with the nucleotides 'GU'. In all cases, the sgRNAs leader ranged between 113 and 121 nt in length.

Molecular variants of Grapevine rupestris stem pitting-associated virus infecting grapevines (Vitis spp) in Brazil / Variantes moleculares de Grapevine rupestris stem pitting-associated virus infectando videiras (Vitis spp) no Brasil

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most common viruses of grapevine. It is involved in the graft-transmissible disease rupestris stem pitting of the rugose wood complex. The objective of the research was to perform the molecular characterization of the coat protein (CP) gene of sixteen Brazilian GRSPaV isolates aiming to determine the occurrence of molecular variants (strains) of this virus. Nine grapevine samples were evaluated, from which dsRNA was extracted. Nucleotide sequences were generated by Next generation sequencing (NGS). Fifteen complete sequences of the GRSPaV CP gene were obtained and phylogenetically analyzed. Multiple alignments of the sequences showed identities of nucleotides ranging from 82% to 99%, suggesting high variability among the CPs of Brazilian isolates. The study revealed that genetic variability of GRSPaV comprising three molecular variants is also present in Brazilian grapevine genotypes.(AU)

O GRSPaV é um dos vírus mais comuns da videira. Está associado à doença transmissível por enxertia denominada caneluras de rupestris que compõe o complexo do lenho rugoso. O objetivo do trabalho foi realizar a caracterização molecular do gene da proteína capsidial (CP) de 16 isolados brasileiros de GRSPaV visando determinar a ocorrência de variantes moleculares desse vírus. Nove amostras de videira foram avaliadas das quais foi extraído dsRNA. As sequências de nucleotídeos foram geradas pelo sequenciamento de nova geração (NGS). Quinze sequências completas do gene CP de GRSPaV foram obtidas e filogeneticamente analisadas. Os alinhamentos múltiplos entre as sequências mostraram identidades de nucleotídeos variando de 82% a 99%, sugerindo alta variabilidade entre as CPs de isolados brasileiros. O estudo revelou que a variabilidade genética de GRSPaV compreendendo três variantes moleculares também está presente nos genótipos de videira no Brasil.(AU)

A novel vitivirus-like sequence found in Arracacia xanthorrhiza plants by high throughput sequencing.

High throughput sequencing (HTS) is a very powerful tool for detecting and discovering novel viral-like sequences without prior knowledge of the sequence. Here we describe the complete genome of a new vitivirus-like sequence that was found in arracacha (Arracacia xanthorrhiza) plants using HTS technology. The complete genome sequence was validated by Sanger sequencing. The genomic organization of the new putative vitivirus resembles that of grapevine virus B (GVB) and grapevine virus D (GVD). The putative coat protein showed 41 to 49% identity with similar proteins of known vitiviruses, while the RNA-dependent RNA polymerase shared 52 to 55% identity with those encoded by grapevine vitiviruses. Based on the demarcation criteria for the genus Vitivirus, the virus described in this work, provisionally named as "Arracacha virus V", represents a novel species in this taxon.

A baculovirus-mediated strategy for full-length plant virus coat protein expression and purification.

BACKGROUND: Garlic production is severely affected by virus infection, causing a decrease in productivity and quality. There are no virus-free cultivars and garlic-infecting viruses are difficult to purify, which make specific antibody production very laborious. Since high quality antisera against plant viruses are important tools for serological detection, we have developed a method to express and purify full-length plant virus coat proteins using baculovirus expression system and insects as bioreactors. RESULTS: In this work, we have fused the full-length coat protein (cp) gene from the Garlic Mite-borne Filamentous Virus (GarMbFV) to the 3'-end of the Polyhedrin (polh) gene of the baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The recombinant baculovirus was amplified in insect cell culture and the virus was used to infect Spodoptera frugiperda larvae. Thus, the recombinant fused protein was easily purified from insect cadavers using sucrose gradient centrifugation and analyzed by Western Blotting. Interestingly, amorphous crystals were produced in the cytoplasm of cells infected with the recombinant virus containing the chimeric-protein gene but not in cells infected with the wild type and recombinant virus containing the hexa histidine tagged Polh. Moreover, the chimeric protein was used to immunize rats and generate antibodies against the target protein. The antiserum produced was able to detect plants infected with GarMbFV, which had been initially confirmed by RT-PCR. CONCLUSIONS: The expression of a plant virus full-length coat protein fused to the baculovirus Polyhedrin in recombinant baculovirus-infected insects was shown to produce high amounts of the recombinant protein which was easily purified and efficiently used to generate specific antibodies. Therefore, this strategy can potentially be used for the development of plant virus diagnostic kits for those viruses that are difficult to purify, are present in low titers or are present in mix infection in their plant hosts.

Recombinant expression of Garlic virus C (GARV-C) capsid protein in insect cells and its potential for the production of specific antibodies.

Garlic cultivars in Brazil are infected by a complex of viruses and for some virus species, such as the allexivirus, purification of the virions is sometimes cumbersume. To overcome this problem, recombinant expression of viral proteins in heterologous systems is an alternative method for producing antibodies. The capsid gene from Garlic virus C (GarV-C), an Allexivirus, was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) generating the recombinant virus vSynGarV-C. The recombinant protein expression was confirmed by SDS-PAGE and western-blot of extracts from recombinant virus infected insect cells, where a protein band of approximately 32KDa was observed only in extracts from recombinant infected cells. This protein corresponded to the predicted size of the capsid protein of the GarV-C. A rabbit polyclonal antibody was raised against this protein, shown to be specific for the GarV-C protein in western-blot and dot-Elisa, however with a low titer.

Detection of Banana mild mosaic virus and Banana virus X by polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR).

Viruses are important constraints to the movement and propagation of plant germplasm, especially for vegetatively propagated crops such as banana and plantain. Their control relies primarily on the use of virus-free plant material, whose production and certification requires sensitive and reliable detection methods. An existing polyvalent degenerate oligonucleotide RT-PCR (PDO-RT-PCR) assay was adapted to the detection of Banana mild mosaic virus (BanMMV) and Banana virus X, two Flexiviridae infecting Musa spp. PDO inosine-containing primers were found to be well suited to the detection of BanMMV, despite its high molecular diversity, but not to that of the highly conserved BVX, for which species-specific primers were designed. Sampling and sample processing steps were optimized in order to avoid nucleic acid purification prior to the reverse transcription step. A polyclonal anti-BanMMV antiserum was raised and successfully used for the immunocapture (IC) of BanMMV viral particles from leaf extracts, leading to the development of a PDO-IC-RT-nested PCR assay. Although the anti-BanMMV antiserum could to some extent recognize BVX viral particles, direct binding (DB) was shown to be a more efficient method for processing BVX-infected samples and a PDO-DB-RT-nested PCR assay was developed for the detection of BVX from leaf extracts.