The family Betaflexiviridae comprises twelve different genera , including Vitivirus, Trichovirus and Foveavirus. After in-silico and in-vitro analyses of trichoviruses and foveaviruses, we did not find evidence for cross-reaction by the universal assay. A single test for all known grapevine vitiviruses can be a useful tool for improving efficiency and reducing costs of large-scale surveys. Potentially, this generic assay may detect novel Vitivirus species in grapevine and other hosts given its unbiased nature. Similar assays have been developed for carlaviruses, nepoviruses and different members of the family Betaflexiviridae. Grapevine is clonally propagated, consequently, to prevent the spread of vitiviruses, it is critical to use virus tested material. The assay developed here will be made available to diagnostic labs and will facilitate the production of certified virus-tested propagation material and the effective control of vitiviruses.As people age, their risk for cognitive decline increases. An estimated 40% of people 65 years and older have age-associated memory impairment characterized by self perception of memory loss and a standardized memory test score demonstrating lower objective memory performance compared with young adults. While genetic factors play an important role in age-related memory decline, such nongenetic lifestyle factors as diet and exercise contribute as well. In particular, a growing body of evidence indicates that accumulation of oxidative damage to macromolecules increases progressively during the aging process and contributes to neurodegeneration in Alzheimer’s disease. Epidemiological studies have suggested a link between antioxidant consumption and cognitive protection,blueberry packing boxes and studies using a rodent model of Alzheimer’s disease suggest that tau phosphorylation occurs as a compensatory response to oxidative stress. Several clinical trials of antioxidant use in subjects with normal aging or Alzheimer’s disease suggest memory benefits, while others yielded negative results.

While many fruits are rich in various antioxidants, including ascorbic acid, carotenoids, and phenolics, commonly consumed fruits show large differences in antioxidant capacity, as determined by the ferric reducing/antioxidant power assay. In particular, there is special interest in the pomegranate polyphenols, which have been studied extensively in animal models of Alzheimer’s disease. The emerging data on pomegranate fruits and their inherent polyphenols suggest positive benefits ranging from neuroprotective effects to staving off effects of senescent neurodegeneration in animal models of Alzheimer’s disease. Previously, we found that pomegranate juice has the highest antioxidant capacity among fruit juices using five different in vitro antioxidant assays. In comparison, apple juice, which is low in antioxidant capacity, was less effective than pomegranate juice in affecting antioxidant capacity based on FRAP assays in 26 elderly subjects consuming pomegranate juice or apple juice over a 4-week period. Over expression of antioxidant enzymes or supplementation of some antioxidants appears effective in extending the life span in several animal models, and a few studies have specifically found memory-enhancing effects of polyphenols in mice . However, evidence for memory enhancement from polyphenols in human-controlled trials is limited. One preliminary study found beneficial effects of grape juice on memory in five older adults with ageassociated memory impairment compared to seven placebo controls; to our knowledge this small study is the only placebo-controlled human trial of polyphenols that assessed memory change. Therefore, more well-controlled studies are needed to determine potential beneficial effects of antioxidants that occur naturally in various food sources and to elucidate the possible underlying mechanisms. Moreover, studies that examine possible brain mechanisms of polyphenol treatment in humans are limited. One potential method for identifying mechanisms of recovery in drug trials is functional magnetic resonance imaging .

While structural MRI studies measure long-term changes in metrics, such as cortical thickness and volume , fMRI measures blood flow changes in response to memory challenge, which has proven to be effective in determining brain differences among subjects with Alzheimer’s disease, mild cognitive impairment, or non-demented subjects at risk for Alzheimer’s disease. Further, fMRI has proven to be sensitive in measuring changes in neural activation patterns among older subjects in response to memory-enhancing drugs, such as donepezil and memantine, with results showing increased activation or restoration of resting state brain networks following treatment. One investigation used fMRI to examine the effects of antioxidants in young healthy controls and demonstrated increased fMRI activity to cognitive challenge with flavonoid administration; however, no studies to date have examined fMRI effects of antioxidant therapy in older adults with or without memory complaints. To address this knowledge gap, we explored potential neuroprotective effects derived from polyphenols in pomegranate juice in older volunteers with mild memory complaints in a placebo-controlled, randomized, and double blind trial and measured three aspects: metabolites of pomegranate juice using blood biomarkers; effects of pomegranate juice on memory performance; and evidence of functional MRI changes during memory activation. We hypothesized that pomegranate juice compared to placebo would increase metabolites, improve memory, and increase task-related brain activation in the left and right hemispheres for verbal and nonverbal memory tasks, respectively. Together these metrics provide preliminary evidence of bio-availability, clinical efficacy, and brain mechanisms of pomegranate juice in older adults with memory complaints.Thirty-two volunteers were initially enrolled into the study out of 39 who were screened; of these, 28 completed the clinical trial . Subjects were recruited via advertisements through local newspapers, flyers, posters, lectures, and word-of-mouth. We specifically recruited nondemented, older right-handed subjects with self-reported age-related memory complaints. The subjects did not carry a diagnosis of mild cognitive impairment and were screened using the mini-mental state examination . Subjects randomized to placebo versus control groups did not differ in mean age, percentage of women, mean MMSE scores, or baseline memory performance .

Potential subjects were excluded who had documented neurological, psychiatric, or major medical conditions, including heart disease, diabetes, and gastrointestinal disease, that might affect cognitive function or interfere with study procedures. We also excluded subjects who routinely ate more than three servings per day of fruits and vegetables or were taking vitamin supplements, any medication, or dietary supplement that interferes with the absorption of polyphenols. Potential subjects participating in regular vigorous exercises other than ordinary daily walks or who were unwilling to maintain a sedentary lifestyle during the 28-day study were excluded, as were those for whom MRI was contraindicated . Seven potential subjects were screened out based on one or more of these exclusion criteria. Subjects were instructed that they would be removed from the study if they failed to follow the prescribed low polyphenol diet during the one-week run-in period based on self-report in-person interview with a study dietitian. The study was approved by the UCLA Institutional Review Board; all subjects gave informed consent to participate in all procedures and were reconsented for followup evaluations. Funding for this study was provided by the UCLA Center for Human Nutrition from a “various donors” account. The investigators were blind to the contributors to this fund, and there was no communication or input from donors or commercial entities at any stage of the study.Following phone screening, volunteers participated in an initial clinical evaluation,package of blueberries which included an explanation of the study procedures, consent, medical screening, MMSE, and routine CBC and chemistry panel. Prior to the intervention phase, each subject met with a registered dietitian and was instructed on a low polyphenol diet, which required that they restrict their intake of several fruits and vegetables, onions, tea, chocolate, and dried beans, for one week prior to the baseline visit and for the duration for the study. At baseline, subjects underwent neuroimaging and neurocognitive testing . We also acquired blood samples to perform the antioxidant assay and to verify compliance with the protocol. Subjects were then randomized to either the pomegranate or placebo groups; investigators and subjects were blind to group membership. After these procedures, subjects were given a one-week supply of 8-ounce containers of pomegranate or placebo juice with instructions to consume 8 ounces of juice daily. The pomegranate drink was the commercial Pom Wonderful product. The flavor-matched drink contained sugar, citric acid, and food color to match the calorie, taste, and color of the juice. Each week, subjects returned their empty juice containers to ensure compliance, picked up their juice supply for the following week, and met briefly with the dietitian to ensure compliance with the low polyphenol diet. At the end of the study , subjects again underwent neuroimaging, cognitive assessment, and blood draws. A placebo drink using sugars and natural colorings and flavoring to imitate the taste and appearance of pomegranate juice was used. The research personnel were blinded as to the assignment of subjects to placebo or pomegranate juice.Each subject underwent fMRI and brief memory testing immediately prior to beginning the trial and following 28 days of pomegranate juice or placebo. Subjects received a memory testing battery and underwent functional MRI scanning on the same day. Memory tests administered were the Buschke-Fuld selective reminding task, from which we calculated two performance measures, total items recalled and consistent long term retrieval; an experimental unrelated word pair associates learning task, based on the Wechsler memory scale paired associates learning in which we used identical procedures to that used in the fMRI scan but with different stimuli. We also obtained behavioral data from the MRI tasks administered in the scanner .

Of the 28 subjects completing the study, 26 had behavioral data on both occasions . Subjects were administered one of two alternate forms of each task, with the order of forms counterbalanced across subjects, to avoid practice effects. To assess changes in verbal memory, we compared performance on total number of items recalled on the Buschke-Fuld selective reminding task and on the consistent long term retrieval score . The TR score emphasizes immediate recall, while CLTR depends upon memory consolidation processes. Because memory performance in this age range is highly variable, we used time 2 minus time 1 difference scores as our dependent variables. At baseline, there were no differences between groups on either TR performance or CLTR . The spatial navigation task involved learning and recalling routes on a virtual map, while subjects play the role of a “taxi driver.” During the encoding phase, subjects watched and rewatched two sets of video clips. In each video, subjects first saw the name of the imaginary town, such as “Johnsberg,” then for 15 second blocks they saw a first-person view of “driving” to a virtual town laid out in 5×5 block grids, to a storefront destination with unique characteristics for specific storefronts, stopping for 3 seconds before proceeding to a new store within the same town starting from a different location. After each city, subjects performed a 30-second distractor task where they pressed a left or right button in response to a probe. This was repeated while subjects learned 6 cities in total. The fMRI data were then collected during memory retrieval in three conditions. Subjects were asked to remember one of three characteristics about each storefront for the separate tasks. Two conditions focused on the physical features of the environment . In condition 1, participants were asked to identify an appearance change of the store , such as signs, colors, and doors. In condition 2, they were asked to identify the spatial location change of the store within the town, relative to other buildings , respectively. Condition 3 required subjects to identify the store for a specific item that might be purchase there, requiring the participant to encode the association between an item and a spatial location. For this source task, subjects had to recall whether the store was in the first or second town . Only the latter condition tapped into the associative memory processes. The order of the three conditions was counterbalanced across subjects.All functional data were analyzed using FSL version 4.1.4 . Preprocessing included motion correction to the mean image, spatial smoothing , and high-pass temporal filtering .Twenty three subjects were able to complete the visual memory fMRI task without significant head motion, while for the verbal memory task, eight pomegranate juice and nine placebo subjects were able to complete both scanning sessions with high quality images. Each functional scan was registered to its corresponding coplanar high-resolution image with rigid body transformations and to the MNI152 standard brain using nonlinear transformation . Additionally, a high-pass temporal filtering of 100 sec was applied to the functional images. Individual preprocessing and statistical analysis of each subject’s functional scan was run using FSL FMRI Expert Analysis Tool ; FEAT was then used to analyze data at the group level.