Efficient capturing and sensitive detection of hepatitis A virus from solid foods (green onion, strawberry, and mussel) using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles and real-time RT-PCR.

Hepatitis A virus (HAV) continues to be a public health concern and has caused large foodborne outbreaks and economic losses worldwide. Rapid detection of HAV in foods can help to confirm the source of outbreaks in a timely manner and prevent more people getting infected. In order to efficiently detect HAV at low levels of contamination in foods, rapid and easy-to-use techniques are required to separate and concentrate viral particles to a small volume. In the current study, HAV particles were eluted from green onion, strawberry, and mussel using glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and suspended viral particles were captured using protamine-coated magnetic nanoparticles (PMNPs). This process caused a selective concentration of the viral particles, which could be followed by quantitative real-time RT-PCR analysis. Results showed that pH, NaCl concentration, and PMNP amount used for the capturing had significant effects on the recovery efficiency of HAV (P < 0.05). The highest recovery rate was obtained at pH 9.0, 0.14 M NaCl, and 50 µL of PMNPs. The optimized PMNP capturing method enabled the rapid capture and concentration of HAV. A sensitive real-time RT-PCR test was developed with detection limits of 8.3 × 100 PFU/15 g, 8.3 × 101 PFU/50 g, and 8.3 × 100 PFU/5 g of HAV in green onion, strawberry, and mussel, respectively. In conclusion, the PMNP method is rapid and convenient in capturing HAV from complex solid food samples and can generate concentrated HAV sample solutions suitable for high-sensitivity real time RT-PCR detection of the virus.

Prediction of the molecular boundary and functionality of novel viral AlkB domains using homology modelling and principal component analysis.

Alkylation B (AlkB) proteins are ubiquitous among diverse cellular organisms, where they act to reverse the damage in DNA and RNA due to methylation, such as 1-methyladenine and 3-methylcytosine. This process is found in virtually all forms of life, with the notable exception of archaea and yeast. This protein family is so significant to all forms of life that it was recently discovered that an AlkB domain is encoded as part of the replicase (poly)protein in a small subset of single-stranded, positive-sense RNA viruses, mainly belonging to the families Alphaflexiviridae, Betaflexiviridae and Closteroviridae. Interestingly, these AlkB-containing viruses are mostly important pathogens of woody perennials such as fruit crops, and are responsible for significant economic losses. As a newly identified protein domain in RNA viruses, the origin and molecular boundary of the viral AlkB domain, as well as its function in viral replication, virus-host interactions and infection are unknown. This is due to the limited sequence conservation of viral AlkB domains, especially at the N-terminal region corresponding to the nucleotide recognition lid. Here we apply several independent analytical approaches (homology modelling, principal component analysis and the Shannon diversity index) for the first time, to better understand this viral domain. We conclude that a functional AlkB domain in these viruses comprises approximately 150-170 amino acids. Although the exact function of the viral AlkB domain remains unknown, we hypothesize that it counteracts a host defence mechanism that is unique in these perennial plants and was acquired to enhance the long-term survival of these RNA viruses that infect perennial plants. Interestingly, a majority of these viruses have a tissue tropism for the phloem. Furthermore, we identified several additional amino acid residues that are uniquely conserved among viral AlkBs. This work helps to provide a foundation for further investigation of the function of viral AlkBs and critical residues involved in AlkB function.

Concentration of hepatitis A virus in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles.

Hepatitis A virus (HAV) continues to be the leading cause of viral hepatitis. HAV outbreaks have been linked to the consumption of milk, but methods for HAV detection in milk are very limited. We developed a method to concentrate HAV in milk using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles (PMNPs). In this study, protamine was covalently coated on the surface of the MNPs (20-30 nm) by a three-step chemical reaction. The successful linkage of protamine to the MNPs was confirmed by Fourier transform infrared spectroscopy (FTIR), zeta potential, and transmission electron microscopy (TEM). When used for concentrating HAV from 40 mL of milk, 50 µL of PMNPs were added to the sample and mixed for 20 min by gentle rotation, followed by a magnet capture for 30 min. The captured PMNPs were washed with glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and HAV RNA was extracted using the QIAamp MinElute Virus Spin Kit and quantified by real-time RT-PCR. The method showed a detection limit of 8.3 × 100 PFU of HAV in milk. The whole concentration procedure could be completed in approximately 50 min. The developed method was simple, inexpensive, and easy-to-perform.

Metagenomic Analysis of Riesling Grapevine Reveals a Complex Virome Including Two New and Divergent Variants of Grapevine leafroll-associated virus 3.

The virome of a major white wine grape of cultivar Riesling showing decline and leafroll disease symptoms was analyzed through high-throughput sequencing (HTS) using total RNAs as templates and the Illumina HiSeq 2500 platform. Analysis of HTS data revealed the presence of five viruses and three viroids in the infected vine. These viruses are Grapevine leafroll-associated virus 1 (GLRaV-1) and GLRaV-3 (genus Ampelovirus, family Closteroviridae) and three viruses of the family Betaflexiviridae (namely, Grapevine virus A [GVA], Grapevine virus B, and Grapevine rupestris stem pitting-associated virus [GRSPaV]). We also show that multiple distinct strains of three viruses (GLRaV-3, GVA, and GRSPaV) were present in this diseased grapevine. The complete genomes of two novel and highly divergent isolates of GLRaV-3 were determined using the draft genomes derived from HTS data and two independent rapid amplification of cDNA ends (RACE) strategies to obtain sequences at both the 5' and the 3' termini of the viral genomes. Questionable genome regions of both isolates were also verified through cloning of reverse transcription polymerase chain reaction products and Sanger sequencing. These two isolates are vastly divergent from all other isolates of GLRaV-3 whose genome sequences are available in GenBank. Isolate ON8415A has up to 76% nucleotide sequence identities to other isolates representing existing variant groups. We also revealed high degrees of variation in both length and sequence in the terminal untranslated regions (UTRs) of GLRaV-3 variants. The 5'-UTR of most GLRaV-3 isolates whose complete genomes have been sequenced contain tandem repeats of 65 nucleotides, a highly unusual feature rarely observed in (+)single-stranded RNA viruses. Mechanisms for the biogenesis of these tandem repeats and their function in virus replication and pathogenesis require investigation. Findings of this research add to the genetic diversity, evolutionary biology, and diagnostics of GLRaV-3 that afflicts the global grape wine industry.

Survey for major viruses in commercial Vitis vinifera wine grapes in Ontario.

BACKGROUND: In recent years, the Ontario grape and wine industry has experienced outbreaks of viral diseases across the province. Little is known about the prevalence of viruses and viral diseases in Ontario. Since 2015, we have conducted large-scale surveys for major viruses in commercial wine grapes in order to obtain a comprehensive understanding of the prevalence and severity of viral diseases in Ontario. METHODS: A total of 657 composite leaf samples representing 3285 vines collected from 137 vine blocks of 33 vineyards from three appellations: Niagara Peninsula, Lake Erie North Shore and Prince Edward County. These samples covered six major red cultivars and five major white grape cultivars. Using a multiplex RT-PCR format, we tested these samples for 17 viruses including those involved in all major viral diseases of the grapevine, such as five grapevine leafroll-associated viruses (GLRaV-1, 2, 3, 4, 7), grapevine red blotch virus (GRBV), grapevine Pinot gris virus (GPGV), grapevine rupestris stem sitting-associated virus (GRSPaV), grapevine virus A (GVA), grapevine virus B (GVB), grapevine fleck virus (GFkV), arabis mosaic virus (ArMV), tomato ringspot virus (ToRSV), trapevine fanleaf virus (GFLV), among others. RESULTS: Fourteen of the 17 viruses were detected from these samples and the predominant viruses are GRSPaV, GLRaV-3, GFkV, GPGV and GRBaV with an incidence of 84.0, 47.9, 21.8, 21.6 and 18.3%, respectively. As expected, mixed infections with multiple viruses are common. 95.6% of the samples included in the survey were infected with at least one virus; 67% of the samples with 2-4 viruses and 4.7% of the samples with 5-6 viruses. The major grape cultivars all tested positive for these major viruses. The results also suggested that the use of infected planting material may have been one of the chief factors responsible for the recent outbreaks of viral diseases across the province. CONCLUSIONS: This is the first such comprehensive survey for grapevine viruses in Ontario and one of the most extensive surveys ever conducted in Canada. The recent outbreaks of viral diseases in Ontario vineyards were likely caused by GLRaV-3, GRBV and GPGV. Findings from this survey provides a baseline for the grape and wine industry in developing strategies for managing grapevine viral diseases in Ontario vineyards.

Subcellular localization and membrane association of the replicase protein of grapevine rupestris stem pitting-associated virus, family Betaflexiviridae.

As a member of the newly established Betaflexiviridae family, grapevine rupestris stem pitting-associated virus (GRSPaV) has an RNA genome containing five ORFs. ORF1 encodes a putative replicase polyprotein typical of the alphavirus superfamily of positive-strand ssRNA viruses. Several viruses of this superfamily have been demonstrated to replicate in structures designated viral replication complexes associated with intracellular membranes. However, structure and cellular localization of the replicase complex have not been studied for members of Betaflexiviridae, a family of mostly woody plant viruses. As a first step towards the elucidation of the replication complex of GRSPaV, we investigated the subcellular localization of full-length and truncated versions of its replicase polyprotein via fluorescent tagging, followed by fluorescence microscopy. We found that the replicase polyprotein formed distinctive punctate bodies in both Nicotiana benthamiana leaf cells and tobacco protoplasts. We further mapped a region of 76 amino acids in the methyl-transferase domain responsible for the formation of these punctate structures. The punctate structures are distributed in close proximity to the endoplasmic reticulum network. Membrane flotation and biochemical analyses demonstrate that the N-terminal region responsible for punctate structure formation associated with cellular membrane is likely through an amphipathic α helix serving as an in-plane anchor. The identity of this membrane is yet to be determined. This is, to our knowledge, the first report on the localization and membrane association of the replicase proteins of a member of the family Betaflexiviridae.

A highly effective and versatile technology for the isolation of RNAs from grapevines and other woody perennials for use in virus diagnostics.

BACKGROUND: Isolation of pure RNA from woody perennials, especially fruit crops such as grapevine rich in complex secondary metabolites, has remained very challenging. Lack of effective RNA isolation technology has resulted in difficulties in viral diagnosis and discovery as well as studies on many biological processes of these highly important woody plants. It is imperative to develop and refine methodologies with which large amounts of pure nucleic acids can be readily isolated from woody perennials. METHODS: We compared five commonly used RNA isolation kits in isolating total RNA from twelve species of woody perennials. We made modifications to select RNA isolation systems to simplify and improve their efficiency in RNA isolation. The yield and quality of isolated RNAs were assessed via gel electrophoresis and spectrophotometric measurement. We also performed RT-PCR and RT-qPCR to detect several major viruses from grapevines. RESULTS: Two of the kits were shown to be the best in both the yield and quality of the isolated RNA from all twelve woody species. Using disposable extraction bags for tissue homogenization not only improved the yield without affecting quality, but also made the RNA isolation technology simpler, less costly, and suitable for adoption by many potential users with facility limitations. This system was successfully applied to a wide range of woody plants, including fruit crops, ornamentals and timber trees. Inclusion of polyvinylpyrrolidone in the extraction buffer drastically improved the performance of the system in isolating total RNA from old grapevine leaves collected later in the season. This modification made our system highly effective in isolating quality RNA from grapevine leaves throughout the entire growing season. We further demonstrated that the resulting nucleic acid preparations are suitable for detection of several major grapevine viruses with RNA or DNA genomes using PCR, RT-PCR and qPCR as well as for assays on plant microRNAs. CONCLUSIONS: This improved RNA isolation system would have wide applications in viral diagnostics and discovery, studies on gene expression and regulation, transcriptomics, and small RNA biology in grapevines. We believe this system will also be useful in diverse applications pertaining to research on many other woody perennials and recalcitrant plant species.

The triple gene block movement proteins of a grape virus in the genus Foveavirus confer limited cell-to-cell spread of a mutant Potato virus X.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a member of the genus Foveavirus in the family Betaflexiviridae. The genome of GRSPaV encodes five proteins, among which are three movement proteins designated the triple gene block (TGB) proteins. The TGB proteins of GRSPaV are highly similar to their counterparts in Potato virus X (PVX), as reflected in size, modular structure, conservation of critical amino acid sequence motifs, as well as similar cellular localization. Based on these similarities, we predicted that the TGB proteins of these two viruses would be interchangeable. To test this hypothesis, we replaced the entire or partial sequence of PVX TGB with the corresponding regions from GRSPaV, creating chimeric viruses that contain the PVX backbone and different sequences from GRSPaV TGB. These chimeric constructs were delivered into plants of Nicotiana benthamiana through agro-infiltration to test whether they were capable of cell-to-cell and systemic movement. To our surprise, viruses derived from pPVX.GFP(CH3) bearing GRSPaV TGB in place of PVX TGB lost the ability to move either cell-to-cell or systemically. Interestingly, another chimeric virus resulting from pPVX.GFP(HY2) containing four TGB genes (TGB1 from PVX and TGB1-3 from GRSPaV), exhibited limited cell-to-cell, but not systemic, movement. Our data question the notion that analogous movement proteins encoded by even distantly related viruses are functionally interchangeable and can be replaced by each other. These data suggest that other factors, besides the TGB proteins, may be required for successful intercellular and/or systemic movement of progeny viruses. This is the first experimental demonstration that the GRSPaV TGB function as movement proteins in the context of a chimeric virus and that four TGB genes were required to support the intercellular movement of the chimeric virus.

Molecular and Metagenomic Analyses Reveal High Prevalence and Complexity of Viral Infections in French-American Hybrids and North American Grapes.

French-American hybrids and North American grape species play a significant role in Canada's grape and wine industry. Unfortunately, the occurrence of viruses and viral diseases among these locally important non-vinifera grapes remains understudied. We report here the results from a large-scale survey to assess the prevalence of 14 viruses among 533 composite samples representing 2665 vines from seven French-American hybrid wine grape cultivars, two North American juice grape cultivars (Concord and Niagara), and the table grape cultivar Sovereign coronation. Based on reverse transcription polymerase chain reaction (RT-PCR) assays, ten viruses were detected. Grapevine rupestris stem pitting-associated virus, grapevine leafroll-associated virus 3, grapevine Pinot gris virus and grapevine red blotch virus were detected with the highest frequency. As expected, mixed infections were common; 62% of the samples contained two or more viruses. Overall, hybrid wine grapes were infected with more viruses and a higher prevalence of individual viruses than juice and table grapes. To validate these findings and to refine the virome of these non-European grapes, high-throughput sequencing (HTS) analyses of five composite samples representing each category of grapevine cultivars was performed. Results from HTS agreed with those from RT-PCR. Importantly, Vidal, a widely grown white-wine grape with international recognition due to its use in the award-winning icewine, is host to 14 viruses, four of which comprise multiple and distinct genetic variants. This comprehensive survey represents the most extensive examination of viruses among French-American hybrids and North American grapes to date.

Rapid Detection of Hepatitis A Virus in Foods Using a Bioluminescent Assay in Real-Time (BART) and Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Technology.

Foodborne hepatitis A infections have been considered as a major threat for public health worldwide. Increased incidences of hepatitis A virus (HAV) infection has been associated with growing global trade of food products. Rapid and sensitive detection of HAV in foods is very essential for investigating the outbreaks. Real-time RT-PCR has been most widely used for the detection of HAV by far. However, the technology relies on fluorescence determination of the amplicon and requires sophisticated, high-cost instruments and trained personnel, limiting its use in low resource settings. In this study, a robust, affordable, and simple assay, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay in combination with a bioluminescence-based determination of amplification in real-time (BART), was developed for the detection of HAV in different food matrices, including green onion, strawberry, mussel, and milk. The efficiencies of a one-step RT-LAMP-BART and a two-step RT-LAMP-BART were investigated for the detection of HAV in different food matrices and was compared with that of real-time RT-PCR. The sensitivity of the RT-LAMP-BART assay was significantly affected by Mg2+ concentration (P < 0.05), in addition to primer quality. The optimal Mg2+ concentration was 2 mM for one-step RT-LAMP-BART and 4 mM for two-step RT-LAMP-BART. Compared with cartridge-purified primers, HPLC-purified primers could greatly improve the sensitivity of the RT-LAMP-BART assay (P < 0.05). For detecting HAV in different food matrices, the performance of two-step RT-LAMP-BART was comparable with that of real-time RT-PCR and was better than that of one-step RT-LAMP-BART. The detection limit of the two-step RT-LAMP-BART for HAV in green onion, strawberry, mussel, and milk was 8.3 × 100 PFU/15 g, 8.3 × 101 PFU/50 g, 8.3 × 100 PFU/5 g, and 8.3 × 100 PFU/40 mL, respectively. The developed RT-LAMP-BART was an effective, simple, sensitive, and robust method for foodborne HAV detection.

Optimization of a Protocol for Launching Grapevine Infection with the Biologically Active cDNA Clones of a Virus.

Grapevine leafroll disease (GLRD) is the most globally prevalent and destructive disease complex responsible for significant reductions in grape yield and quality as well as wine production. GLRD is associated with several positive-strand RNA viruses of the family Closteroviridae, designated as grapevine leafroll-associated viruses (GLRaVs). However, the specific etiological role of any of these GLRaVs in GLRD has not been demonstrated. Even though GLRaV-3 is considered the chief GLRD agent, little is known about the molecular, cellular, and pathological properties of this virus. Such a knowledge gap is due to multiple factors, including the unavailability of biologically active virus cDNA clones and the lack of reliable experimental systems for launching grapevine infection using such clones. In this work, we tested four methods for inoculating tissue-cultured grapevine plantlets with cDNA clones of GLRaV-3: (i) vacuum agro-infiltration; (ii) agro-pricking; (iii) agro-drenching; and (iv) agro-injection. We showed that vacuum agro-infiltration was the most effective of these methods. Furthermore, we examined the impacts of different experimental conditions on the survival and infectivity rate of grapevines after infiltration. To verify the infectivity rate for different treatments, we used RT-PCR, RT-qPCR, and Western blotting. We found that humidity plays a critical role in the survival of plantlets after agro-infiltration and that the use of RNA silencing suppressor and dormancy treatment both had strong effects on the infection rates. To our knowledge, the experimental protocol reported herein is the most effective system for launching the infection of grapevine using cDNA clones of grapevine viruses featuring up to a 70% infection rate. This system has strong potential to facilitate grapevine virology research including the fulfillment of Koch's postulates for GLRD and other major virus diseases as well as identifying the molecular, cellular, and pathological properties of GLRaVs and, potentially, other important grapevine viruses.

Transcriptomic Analyses of Grapevine Leafroll-Associated Virus 3 Infection in Leaves and Berries of 'Cabernet Franc'.

Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting global grape and wine production. GLRaV-3 is the chief agent associated with grapevine leafroll disease (GLRD), the most prevalent and economically destructive grapevine viral disease complex. Response of grapevine to GLRaV-3 infection at the gene expression level is poorly characterized, limiting the understanding of GLRaV-3 pathogenesis and viral-associated symptom development. In this research, we used RNA-Seq to profile the changes in global gene expression of Cabernet franc, a premium red wine grape, analyzing leaf and berry tissues at three key different developmental stages. We have identified 1457 differentially expressed genes (DEGs) in leaves and 1181 DEGs in berries. The expression profiles of a subset of DEGs were validated through RT-qPCR, including those involved in photosynthesis (VvPSBP1), carbohydrate partitioning (VvSUT2, VvHT5, VvGBSS1, and VvSUS), flavonoid biosynthesis (VvUFGT, VvLAR1, and VvFLS), defense response (VvPR-10.3, and VvPR-10.7), and mitochondrial activities (ETFB, TIM13, and NDUFA1). GLRaV-3 infection altered source-sink relationship between leaves and berries. Photosynthesis and photosynthate assimilation were inhibited in mature leaves while increased in young berries. The expression of genes involved in anthocyanin biosynthesis increased in GLRaV-3-infected leaves, correlating with interveinal tissue reddening, a hallmark of GLRD symptoms. Notably, we identified changes in gene expression that suggest a compromised sugar export and increased sugar retrieval in GLRaV-3-infected leaves. Genes associated with mitochondria were down-regulated in both leaves and berries of Cabernet franc infected with GLRaV-3. Results of the present study suggest that GLRaV-3 infection may disrupt mitochondrial function in grapevine leaves, leading to repressed sugar export and accumulation of sugar in mature leaf tissues. The excessive sugar accumulation in GLRaV-3-infected leaves may trigger downstream GLRD symptom development and negatively impact berry quality. We propose a working model to account for the molecular events underlying the pathogenesis of GLRaV-3 and symptom development.

Concerning the Etiology of Syrah Decline: A Fresh Perspective on an Old and Complex Issue Facing the Global Grape and Wine Industry.

Syrah decline, first identified in Southern France in the 1990s, has become a major concern in the global grape and wine industry. This disease mainly affects Syrah (Shiraz) grapevines. Characteristic symptoms include the bright and uniform reddening of leaves throughout the canopy in late summer or early fall; the appearance of abnormalities on the trunk, mainly at the graft union (swelling, pits, grooves, and necrosis); and a reduction in vine vigor, yield and berry quality. Diseased vines may die a few years after disease onset. Damages to the vine are even more pronounced in cool climate regions such as Ontario (Canada), where the affected vines are subjected to very cold and prolonged winters, leading to large numbers of vine deaths. Despite the extensive efforts of the global grape research community over the past few decades, the etiology of this disease remains unclear. In this study, we conducted extensive analyses of viruses in declining Syrah vines identified in commercial vineyards in the Niagara region (Ontario, Canada) through high-throughput sequencing, PCR, RT-PCR and the profiling of genetic variants of select viruses. Multiple viruses and viral strains, as well as three viroids, were identified. However, an unequivocal causal relationship cannot be established between Syrah decline and any of these viruses, although the possibility that certain virus or genetic variants, or both in combination, may contribute to the disease cannot be excluded. Gleaning all information that is available to date, we feel that the traditional approach and an insistence on finding a single cause for such a complex disorder in a woody perennial fruit crop involving grafting will prove to be futile. We hope that this study offers new conceptual perspectives on the etiology of this economically important but enigmatic disease complex that affects the global grape and wine industry.

Virus and Virus-like Pathogens in the Grapevine Virus Collection of Croatian Autochthonous Grapevine Cultivars.

Grapevine collections play an important role, especially in the study of viruses and virus-like pathogens. In 2009, after an initial ELISA screening for eight viruses (arabis mosaic virus, grapevine fanleaf virus, grapevine fleck virus, grapevine leafroll-associated viruses 1, 2, and 3, and grapevine viruses A and B), a collection of 368 grapevine accessions representing 14 different Croatian autochthonous cultivars and containing single or mixed infection of viruses was established to further characterize the viral pathogens. Subsequently, Western blot, RT-PCR, cloning, and sequencing revealed that grapevine rupestris stem pitting-associated virus was frequently found in accessions of the collection, with isolates showing substantial genetic diversity in the helicase and coat protein regions. High-throughput sequencing of 22 grapevine accessions provides additional insight into the viruses and viroids present in the collection and confirms the fact that Croatian autochthonous grapevine cultivars have high infection rates and high virome diversity. The recent spread of "flavescence dorée" phytoplasma in Europe has not spared the collection. After the first symptoms observed in 2020 and 2021, the presence of phytoplasma was confirmed by LAMP in six grapevine accessions and some of them were lost. Single or multiple viruses and viroids, as well as own rooted grapevines in the collection, make the plants susceptible to various abiotic factors, which, together with the recent occurrence of "flavescence dorée", makes the maintenance of the collection a challenge. Future efforts will be directed towards renewing the collection, as 56% of the original collection has been lost in the last 13 years.

Genome-wide screening of novel RT-qPCR reference genes for study of GLRaV-3 infection in wine grapes and refinement of an RNA isolation protocol for grape berries.

BACKGROUND: Grapevine, as an essential fruit crop with high economic values, has been the focus of molecular studies in diverse areas. Two challenges exist in the grapevine research field: (i) the lack of a rapid, user-friendly and effective RNA isolation protocol for mature dark-skinned berries and, (ii) the lack of validated reference genes that are stable for quantification of gene expression across desired experimental conditions. Successful isolation of RNA with sufficient yield and quality is essential for downstream analyses involving nucleic acids. However, ripe berries of dark-skinned grape cultivars are notoriously challenging in RNA isolation due to high contents of polyphenolics, polysaccharides, RNase and water. RESULTS: We have optimized an RNA isolation protocol through modulating two factors at the lysis step that could impact results of RNA isolation - 2-ME concentration and berry mass. By finding the optimal combination among the two factors, our refined protocol was highly effective in isolating total RNA with high yield and quality from whole mature berries of an array of dark-skinned wine grape cultivars. Our protocol takes a much shorter time to complete, is highly effective, and eliminates the requirement for hazardous organic solvents. We have also shown that the resulting RNA preps were suitable for multiple downstream analyses, including the detection of viruses and amplification of grapevine genes using reverse transcription-polymerase chain reaction (RT-PCR), gene expression analysis via quantitative reverse transcription PCR (RT-qPCR), and RNA Sequencing (RNA-Seq). By using RNA-Seq data derived from Cabernet Franc, we have identified seven novel reference gene candidates (CYSP, NDUFS8, YLS8, EIF5A2, Gluc, GDT1, and EF-Hand) with stable expression across two tissue types, three developmental stages and status of infection with grapevine leafroll-associated virus 3 (GLRaV-3). We evaluated the stability of these candidate genes together with two conventional reference genes (actin and NAD5) using geNorm, NormFinder and BestKeeper. We found that the novel reference gene candidates outperformed both actin and NAD5. The three most stable reference genes were CYSP, NDUFS8 and YSL8, whereas actin and NAD5 were among the least stable. We further tested if there would be a difference in RT-qPCR quantification results when the most stable (CYSP) and the least stable (actin and NAD5) genes were used for normalization. We concluded that both actin and NAD5 led to erroneous RT-qPCR results in determining the statistical significance and fold-change values of gene expressional change. CONCLUSIONS: We have formulated a rapid, safe and highly effective protocol for isolating RNA from recalcitrant berry tissue of wine grapes. The resulting RNA is of high quality and suitable for RT-qPCR and RNA-Seq. We have identified and validated a set of novel reference genes based on RNA-Seq dataset. We have shown that these new reference genes are superior over actin and NAD5, two of the conventional reference genes commonly used in early studies.

Probing into the Effects of Grapevine Leafroll-Associated Viruses on the Physiology, Fruit Quality and Gene Expression of Grapes.

Grapevine leafroll is one of the most widespread and highly destructive grapevine diseases that is responsible for great economic losses to the grape and wine industries throughout the world. Six distinct viruses have been implicated in this disease complex. They belong to three genera, all in the family Closteroviridae. For the sake of convenience, these viruses are named as grapevine leafroll-associated viruses (GLRaV-1, -2, -3, -4, -7, and -13). However, their etiological role in the disease has yet to be established. Furthermore, how infections with each GLRaV induce the characteristic disease symptoms remains unresolved. Here, we first provide a brief overview on each of these GLRaVs with a focus on genome structure, expression strategies and gene functions, where available. We then provide a review on the effects of GLRaV infection on the physiology, fruit quality, fruit chemical composition, and gene expression of grapevine based on the limited information so far reported in the literature. We outline key methodologies that have been used to study how GLRaV infections alter gene expression in the grapevine host at the transcriptomic level. Finally, we present a working model as an initial attempt to explain how infections with GLRaVs lead to the characteristic symptoms of grapevine leafroll disease: leaf discoloration and downward rolling. It is our hope that this review will serve as a starting point for grapevine virology and the related research community to tackle this vastly important and yet virtually uncharted territory in virus-host interactions involving woody and perennial fruit crops.

Metatranscriptomic Sequencing Suggests the Presence of Novel RNA Viruses in Rice Transmitted by Brown Planthopper.

Viral pathogens are a major threat to stable crop production. Using a backcross strategy, we find that integrating a dominant brown planthopper (BPH) resistance gene Bph3 into a high-yield and BPH-susceptible indica rice variety significantly enhances BPH resistance. However, when Bph3-carrying backcross lines are infested with BPH, these BPH-resistant lines exhibit sterile characteristics, displaying panicle enclosure and failure of seed production at their mature stage. As we suspected, BPH-mediated viral infections could cause the observed sterile symptoms, and we characterized rice-infecting viruses using deep metatranscriptomic sequencing. Our analyses revealed eight novel virus species and five known viruses, including a highly divergent virus clustered within a currently unclassified family. Additionally, we characterized rice plant antiviral responses using small RNA sequencing. The results revealed abundant virus-derived small interfering RNAs in sterile rice plants, providing evidence for Dicer-like and Argonaute-mediated immune responses in rice plants. Together, our results provide insights into the diversity of viruses in rice plants, and our findings suggest that multiple virus infections occur in rice plants.

Subcellular localization of the triple gene block proteins encoded by a Foveavirus infecting grapevines.

Grapevine rupestris stem pitting-associated virus (GRSPaV; Foveavirus; Flexiviridae) contains a positive-sense, ssRNA genome. GRSPaV occurs worldwide in grapes and is involved in the Rugose Wood disease complex. The GRSPaV genome contains the triple gene block (TGB), a genetic module present in several genera of plant RNA viruses. TGB encodes three proteins (TGBp1, TGBp2 and TGBp3) that are believed to work together to achieve intra- and inter-cellular transport of virions in infected plants. To reveal the subcellular localization of each TGB protein and to examine the impact that different fusion positions may have on the behavior of the native protein, we made a series of expression constructs and expressed the corresponding protein fusions in Nicotiana tabacum BY-2 cells and protoplasts. We demonstrated that TGBp1 had both a cytosolic and nuclear distribution. Two TGBp1 fusions (GFP fused at the N- or C-terminus) differ in subcellular distribution. Through the use of truncation mutants, we mapped TGBp1 regions responsible for the formation of two distinct types of aggregates. Sequence analyses predicted two and one transmembrane domains in TGBp2 and TGBp3, respectively. GFP fusions at either terminus of TGBp2 revealed identical localization to the ER network and ER-derived structures. In contrast, the two TGBp3 fusions to mRFP differed in localization. This is the first report on the subcellular localization of the viral proteins of a member of the Foveavirus genus.

Characterization of grapevine leafroll-associated virus 3 genetic variants and application towards RT-qPCR assay design.

Grapevine leafroll-associated virus 3 (GLRaV-3) is the most widely prevalent and economically important of the complex of RNA viruses associated with grapevine leafroll disease (GLD). Phylogenetic studies have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species. In addition, new divergent variants have been discovered recently around the world. Accurate identification of the virus is an essential component in the management and control of GLRaV-3; however, the diversity of GLRaV-3, coupled with the limited sequence information, have complicated the development of a reliable detection assay. In this study, GLRaV-3 sequence data available in GenBank and those generated at Foundation Plant Services, University of California-Davis, was used to develop a new RT-qPCR assay with the capacity to detect all known GLRaV-3 variants. The new assay, referred to as FPST, was challenged against samples that included plants infected with different GLRaV-3 variants and originating from 46 countries. The FPST assay detected all known GLRaV-3 variants, including the highly divergent variants, by amplifying a small highly conserved region in the 3' untranslated terminal region (UTR) of the virus genome. The reliability of the new RT-qPCR assay was confirmed by an enzyme linked immunosorbent assay (ELISA) that can detect all known GLRaV-3 variants characterized to date. Additionally, three new GLRaV-3 divergent variants, represented by four isolates, were identified using a hierarchical testing process involving the FPST assay, GLRaV-3 variant-specific assays and high-throughput sequencing analysis. These variants were distantly related to groups I, II, III, V, VI, VII and IX, but much similar to GLRaV-3 variants with no assigned group; thus, they may represent new clades. Finally, based on the phylogenetic analysis, a new GLRaV-3 subclade is proposed and named as group X.

Molecular analysis of double-stranded RNAs reveals complex infection of grapevines with multiple viruses.

The table grape variety "Waltham Cross" was infected with Leafroll and Shiraz Disease. To reveal specific viruses that are associated with the diseased plants, we used an RT-PCR-based strategy to determine partial genome sequences of these viruses. Upon cloning and sequencing of the RT-PCR products, we detected seven groups of viral variants that are related to four species of the Closteroviridae: Grapevine leafroll-associated virus 1, 2, 3, and 5, in addition to Rupestris stem pitting-associated virus. The population composition of GLRaV-2 and GLRaV-3-like viruses is complex and consists of two or three distinct groups of viral variants. Based on the consensus sequence of several GLRaV-2 strains, we designed a pair of broad-spectrum primers (GLR2-4 and GLR2-5) and used them to detect a range of GLRaV-2 variants from "Waltham Cross". Moreover, we identified a novel group of viral variants from the diseased grapevines, which possess a stretch of 19 nucleotides inserted in the 3' non-coding region as compared to strain "PN" and "93/955" for which the complete genomes have been sequenced. In contrast, the population composition of GLRaV-1 and GLRaV-5-like virus seems to be more uniform and each consists of a single viral variant. Furthermore, the central 5.7kb genomic region encompassing ORF1b-ORF4 of the GLRaV-1 isolate detected in "Waltham Cross" was sequenced. The new isolate is designated GLRaV-1 "WC", which differs from GLRaV-1 "Type" by 16% in nucleotide sequence. The taxonomic standing of the GLRaV-5-like and GLRaV-3-like viruses detected in "Waltham Cross" is discussed.