Consumption of a 30% fructose diet for 8weeks resulted in a decrease in serotonin reuptake transporter protein levels in mouse duodenum. Interestingly, SERT-deficient mice are used as a relevant model for depression, suggesting a link between fructose consumption and psychological effect. Experimental evidence indicates that consumption of a high-fructose diet starting during juvenile life promotes a pro-inflammatory state involving both the Central and Peripheral Nervous Systems, and resulting in psychiatric-like disorders in adulthood. Obese phenotype in female rats caused by high fructose consumption is associated with an increase in IL-1β production, microglial reactivity and hyperphosphorylation of tau in the hippocampus, concomitant to neuronal loss and neurological dysfunction at 48 h post-stroke. Fructose-induced insulin resistance is closely associated with the two neuropathological biomarkers of Alzheimer’s disease namely senile plaques and neurofibrillary tangles. Under fructose induced hyperinsulinemic conditions, insulin competes with amyloid b protein for insulin-degrading enzyme, leading to the accumulation of Aβ and deposition of SPs. In addition, the fructose-induced impairment of the insulin receptor signaling culminates in loss of insulin-mediated activation of phosphoinositide 3-kinase /Akt pathway,nft hydroponic and subsequent dephosphorylation of glycogen synthase kinase-3b , which potentiates the phosphorylation and formation of NFTs. The significant correlation between Akt activity/ protein levels found in human Alzheimer’s disease indicates a timedependent and insulin-stimulated PI3-K signaling.
Experimental studies have demonstrated that fructose consumption aggravates the effects of brain trauma on molecular systems engaged in cell energy homeostasis and synaptic plasticity in the hippocampus. Fructose also aggravates the effects of brain trauma on spatial memory in association with a decrease in hippocampal insulin receptor signaling. High fructose consumption under the threshold for establishment of MetS exacerbates the disruptive effects of brain trauma on inflammation and lipid peroxidation in the liver. These effects seem to engage the neuroendocrine growth hormone system with increases of a metabolic/ inflammatory cascade and lipid peroxidation, and disruption of cell energy homeostasis and insulin signaling. Diet-induced metabolic disorders pose a risk for incidence of post-stroke depression, and exacerbate damage caused by ischemic stroke in cerebral vessels. These events result in an increase in BBB permeability and proinflammatory response that may exacerbate infarct volume. The period of fructose exposure seems a critical factor for the involvement of systemic metabolism and subsequent effects on brain. A minimum of 6 weeks of fructose in rodents is crucial for development of MetS with concomitant effects on brain function and cognition. Long-term high fructose consumption in rodents results in decrements in brain plasticity and learning and memory performance. Fructose alters brain molecular pathways involved in mitochondrial bio-energetics and plasma membrane homeostasis, neuronal signaling, and synaptic plasticity. Nevertheless, several studies indicate that a short period of fructose feeding for a duration insufficient to disrupt peripheral metabolism can also affect the brain by reducing cerebral blood flow, myelin basic protein, and the axonal growth-associated protein , concomitant with a decline in hippocampal weight.
Two weeks fructose diet induce inflammation, oxidative stress, impairment of insulin signaling as well as a significant decrease in mitochondrial function in the hippocampus . Although the greater amount of fructose is intuitively worse for systemic physiology, information derived from animal studies is not conclusive since animals adjust their own fructose consumption based on caloric contents. Although the BBB has low affinity for fructose, a short term fructose consumption seem to enable neuronal cells to metabolize fructose as evidenced by increased levels of GLUT5 in hippocampal microglia and cerebellar Purkinje cells . Short fructose consumption also produces insulin signaling alterations accompanied by neurite and synaptic reduction and astroglial activation in the rat hippocampus . Also, the hippocampus and hypothalamus contains the enzyme Ketohexokinase that degrades fructose reinforcing the possibility that fructose can be metabolized in the brain. The phylum Negarnaviricota, composed of viruses with negative-stranded RNA genome, includes species characterized by non-segmented or segmented genomes, the presence or absence of a membrane enveloping the capsid, and a diverse host range including plants and animals. Examples of nsRNA viruses associated with economically important diseases in plants are rose rosette virus, rice stripe virus, citrus psorosis virus, and blueberry mosaic associated virus. Historically, only a relatively small number of nsRNA viruses infecting plants as their primary host have been reporte. Recently, however, more novel viruses infecting plants have been discovered around the world. In the last few years, the use of high throughput sequencing technology has allowed the identification and characterization of new nsRNA viruses in pistachio, citrus, watermelon, and apple. Interestingly, most of these novel nsRNA viruses were classified under the family Phenuiviridae . To date, there are fifteen recognized genera integrating the family Phenuiviridae : Banyangvirus, Beidivirus, Goukovirus, Horwuvirus, Hudivirus, Hudovirus, Kabutovirus, Laulavirus, Mobuvirus, Phasivirus, Phlebovirus, Pidchovirus, Tenuivirus, Wenrivirus, and Wubeivirus.
Except for members of the genus Tenuivirus that are plant-infecting viruses, the members of the other genera infect vertebrates, including humans, and arthropods. Viruses belonging to the recently established genus Coguvirus also infect plants and, although this genus has not been included yet in the family Phenuiviridae, many structural and molecular features and phylogenetic relationships with other phenuiviruses support its classification in this family. With respect to nsRNA viruses, there is only one preliminary report of tomato spotted wilt virus in this host ; however, later studies failed to transmit TSWV to grapevine. During the screening of two selections of Garan dmak and Muscat rose grapevines via HTS, partial sequences with homology to apple rubbery wood viruses 1 and 2 were identified. ARWV1 and ARWV2 are two novel nsRNA viruses with tri-segmented genomes recently discovered in apple trees displaying rubbery wood symptoms in Canada. Based on sequence identity and phylogenetic analysis, ARWV1 and ARWV2 were proposed to be representative members of a new genus, tentatively named Rubodvirus, within the family Phenuiviridae. Here, we report the natural occurrence and discovery of two novel nsRNA viruses, named grapevine Muscat rose virus and grapevine Garan dmak virus , in grapevine plants. Even more, this is the first evidence for the natural occurrence of phenui-like viruses in grapevine. Later, reverse transcription PCR -based assays for the specific detection of both viruses were designed to investigate the prevalence in different grapevine populations. Phylogenetics and transmission of GMRV and GGDV were also investigated.HTS analysis was performed as described by Al Rwahnih et al.. Briefly, aliquots of TNA from source samples were subjected to rRNA depletion and complementary DNA library construction. Later, cDNA libraries were sequenced using the Illumina NextSeq 500 platform using a single-end 75-bp regime. Illumina reads were demultiplexed and adapter trimmed prior to analysis using Illumina bcl2fastq v2.20.0.422. Trimmed reads were subsequently de novo assembled into contigs using SPAdes v3.13. Generated contigs were compared against the viral database of the National Center for Biotechnology Information using tBLASTx. Novel virus contigs were initially identified by their conserved protein domains. Open reading frames were annotated using HMMER v3.1 to look for Pfam protein domains associated with viruses infecting land plants. In the case of Phenuiviridae-like contigs,nft system the large RNAs were annotated with the bunyavirus RNA-dependent RNA polymerase domain. The medium RNAs were annotated with the viral movement protein domain. The small RNAs were annotated with the Tenuivirus/Phlebovirus nucleocapsid protein domain. The association between the small and the medium RNAs was investigated by BLASTn sequence similarity of their 50 ends. These contig sequences were subsequently confirmed by BLASTx hits to the NCBI nucleotide database which produced top hits to different accessions of ARWV2 and ARWV1. To complete the 50 end of each RNA segment present in GMRV and GGDV, the SMARTer RACE 5 0 /3 0 Kit was employed following the instructions provided by the manufacturer. In the case of the 30 ends, the methodology described by Navarro et al.was used, which involved the addition of a poly tail to the RNA template.
A total of 1732 samples collected in three different grapevine populations were screened for GMRV and GGDV using the above-described assays. The NCGR , located in Winters, California, includes selections of table and wine grapevines originated from around the world; the Davis Virus Collection , which is integrated by plants infected with different viruses; and the FPS pipeline of foreign and domestic introductions. The last two mentioned collections belonging to the University of California-Davis. To determine the transmissibility of GMRV and GGDV, plants infected by these viruses were used as an inoculum source in a biological assay. Following the methodology described by Al Rwahnih et al., bud chips from the Garan dmak and Muscat rose grapevines were grafted onto Cabernet franc indicator plants . Grafted plants were maintained in an insect-proof greenhouse for one month to allow the graft to heal before planting in the field. One year later, indicator plants were sampled for virus detection. Likewise, non-graft Cabernet franc plants were used as negative controls. An ML phylogenetic tree was inferred using the RdRp core sequence of the two novel viruses from grapevine reported here, the two viruses recently identified in apple , representative members of all the genera in the family Phenuiviridae and the plant infecting nsRNA viruses in the genera Emaravirus, Orthotospovirus, and Coguvirus . In this tree, GMRV, GGDV, and the rubodviruses ARWV1 and ARWV2 form a monophyletic group with high bootstrap support . In addition, this tentative rubodvirus clade is contained in a superclade that also contains coguvirus and laulavirus clades. The coguvirus clade consists of two members and two tentative members of the genus Coguvirus infecting plants. The trivial laulavirus clade contains the arthropod-associated Laurel Lake virus . The same superclade was observed when the phylogenetic tree was generated from the putative NPs encoded byGGDV, GMRV, and different recognized/tentative members of the family Phenuiviridae ; with GGDV, GMRV, ARWV1 and ARWV2 as a monophyletic group, and the same phylogenetic relationship between the rubodviruses, coguviruses, and LLV. Interestingly, in the ML tree inferred using the MPs of diverse nsRNA viruses , rubodviruses and coguviruses are clustered in two distantly related clades.In the last few years, as a consequence of the increasing application of HTS, many novel nsRNA viruses have been identified, most of which are from invertebrates. Thus, the classification of these viruses has been recently reassessed, with plant-infecting viruses now classified in the order Bunyavirales, Serpentoviralesand Mononegavirales . Recently, the tentative genus Rubodvirus has been officially proposed to classify two novel nsRNA viruses from apple trees, with a suggested species demarcation criteria of <95% aa identity for the RdRp. In the present study, HTS allowed the identification of two novel plant-infecting viruses with segmented nsRNA genome, GMRV, and GGDV, which are also the first nsRNA viruses identified and transmitted in grapevine. Although these viruses infect the same host species, the aa sequence identity between the putative proteins encoded by their genomic RNAs is always below 75% , indicating that GMRV and GGDV are two different viruses. Their genomes encode proteins showing the highest sequence identity with ARWV1 and ARWV2. GGDV and GMRV also share other traits with these viruses, including the number of genomic components, limited to three nsRNAs, identical terminal nucleotides in the genomic RNAs, and the lack of any ORF coding for glycoproteins. Moreover, close phylogenetic relationships between these four viruses are supported by the ML phylogenetic trees reported here, in which, independently of the considered protein , they always clustered in the same clade, which is significantly separated from all the other nsRNA viruses included in the analyses. Altogether these data support the classification of GMRV and GGDV as two novel species in the tentative genus Rubodvirus. When the other bunyavirales are considered, the ML phylograms inferred from the RdRps or the NPs show a close phylogenetic relationship between rubodviruses and coguviruses, which together with the arthropod-infecting LLV, form a superclade nested at a basal node, in closer proximity to arthropod-infecting viruses than other plant-infecting viruses. These data are consistent with the hypothesis, previously advanced for the coguviruses, that all the members of this super clade evolved from a common ancestor virus infecting arthropods. In this evolutionary scenario, the acquisition of the MP gene appears to be the key step in the adaptation of the ancestor virus to plants, an event that likely happened through the typical modular genome evolution process proposed for most eukaryotic viruses. However, the clustering of rubodviruses and coguviruses in two distant clades, observed in the ML tree inferred with MPs , supports the independent acquisition of the MP gene by the ancestor of the viruses included in these two taxa.