Summary of functions of DHEA related to development and aging.
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Dehydroepiandrosterone (DHEA) is the principal carbon (C)-19 steroid produced by the adrenal gland in humans and mammals . DHEA and its sulfated derivative DHEAS are multifunctional steroids with actions in a wide variety of physiological systems, with effects on the brain , immune systems , and somatic growth and development [4, 5]. Although DHEA and DHEAS were identified more than 50 years ago, there remains some uncertainty as to their physiological significance, full mechanisms of action [6, 7, 8, 9], and their roles in human disease.\n
In humans, DHEA is a crucial precursor of sex steroid biosynthesis and exerts indirect endocrine and intracrine actions following conversion to androgens and estrogens. In addition, DHEA acts as a neurosteroid via its effects on neurotransmitter receptors in the brain. The potential health significance of DHEA in humans is highlighted by the observation that serum concentrations decrease steadily with age, approaching lowest concentrations around the time at which many diseases of aging, particularly neurocognitive decline, become apparent. The age-related decline in DHEA levels  has led to the suggestion that this is associated with a decrease in cognitive function as well as the increased rates of neuronal degeneration and dysfunction that occur during aging [11, 12]. Other studies have reported altered DHEA serum concentrations in patients with conditions such as schizophrenia , dementia , and Alzheimer’s disease (AD) [13, 15, 16, 17, 18]. Due to these associations, DHEAS has been widely publicized both in the lay press [19, 20] and in the scientific literature [21, 22] for their putative anti-aging and neuroprotective effects. This has sparked controversial speculation that DHEA treatment might be a remedy for neuropsychiatric and neurodegenerative disorders [7, 23, 24, 25, 26, 27] and, even more optimistically, that it is a hormone with the potential to increase the life span .\n
As promising as these speculations may seem, there are many contradictions about the roles of DHEA in normal and degenerative brain function. This is especially evident when comparing preclinical and clinical data. For example, studies in animals show a myriad of neuroprotective and trophic effects of DHEAS in development and disease, while clinical studies show inconsistent, and sometimes highly conflicting, results. Clinical studies of neurodegenerative diseases have variously reported increased or decreased DHEAS concentrations in serum, cerebrospinal fluid, and brain tissue, leading to doubt as to the role of DHEA in the neuropathology of aging. It has been suggested that the incongruity in measured DHEAS concentrations may lie in the methodological differences used to sample DHEAS; however, it is possible that these changes are indicative of a more nuanced and multifaceted role. There is consistent evidence that DHEA is neuroprotective with respect to oxidative stress, neuroinflammation, and excitotoxicity, and thus it is possible that DHEA assists the defense of the brain and has a beneficial effect on cognition in healthy brains. Therefore, it is the aim of this review to briefly discuss the physiology of DHEA and its synthesis and secretion during development and aging and to discuss the relationship between alterations in DHEA concentrations and cognition. We further discuss the possible role of DHEAS in a variety of disease states, including AD, and acute illnesses such as schizophrenia, with focus on the fact that these conditions are characterized by imbalances in oxidative stress, neuroinflammation, and excitotoxicity.\n
In humans, DHEA is one of the most abundant hormones synthesized and secreted by the adrenal cortex. This C19 steroid displays an episodic and diurnal rhythm of synthesis and release that parallels that of cortisol [29, 30]. The major synthetic pathways for DHEA and DHEAS are shown in Figure 1. The
DHEAS is the precursor of approximately 50% of androgens in adult men, 75% of active estrogens in premenopausal women, and almost 100% of active estrogens after menopause . DHEA has a 3- to 10-fold predominance of androgenic over estrogenic activity , and although a small portion of the circulating pool of DHEA is of gonadal origin in men and women, the majority of DHEA, and virtually all DHEAS, is produced by the adrenal cortex . However, DHEA is also synthesized in the brain, from cholesterol and other hormonal precursors, primarily by astrocytes and oligodendrocytes; indeed, much higher concentrations of DHEAS are found in the brain than in the serum, suggesting that the DHEAS is primarily synthesized
The specific receptors that bind DHEA as a ligand have been of great interest for over 20 years. The biological actions of DHEA and its metabolites are mediated through androgen receptors or estrogen receptors, which belong to the nuclear receptor steroid-receptor subfamily . DHEA has been found to exert both agonistic and antagonistic effects on the androgen receptor, and it acts as an agonist at both the estrogen receptor-α and estrogen receptor-β sites, with a binding preference for estrogen receptor-β [40, 41]. In the brain, DHEA is thought to affect neuronal excitability by modulating the
In humans, the patterns of DHEA synthesis and secretion change markedly throughout life. In the last months of gestation, the fetal adrenal can synthesize and release considerable amounts of DHEA and DHEAS, which together with estrogen and progesterone produced by the placenta play pivotal roles in the maintenance and endocrine control of pregnancy . Although the plasma concentrations of DHEAS remain high in the newborn, they decrease quickly as the fetal zone of the adrenal gland involutes after birth. From 1 to 6 years of age, the adrenal gland secretes very low concentrations of DHEAS and androstenedione . However at approximately 7–8 years of age, the adrenal zona reticularis increases the production of DHEAS and androstenedione, all of which are C19 steroids that exert androgenic activity in several tissues by converting into potent androgens . This pre-pubertal phenomenon is known as adrenarche, a biochemical, endocrine, and morphological event hypothesized to have evolved only in humans and higher primates. From an evolutionary point of view, adrenarche may be related to the highly coordinated events associated with human growth and organ maturation, particularly of the brain [53, 54, 55].\n
Following the onset of adrenarche, plasma concentrations of DHEAS differ between the sexes, with levels of DHEAS being about 2-fold higher in males than in females (Figure 2). This difference may reflect secretion of these androgens by the testes [10, 57], but it has also been proposed that the higher concentration of DHEAS in men may be attributable to steroid sulfatase, which degrades androgens. The gene for steroid sulfatase is located on the X chromosome, and in having only one copy of the gene, men may have less steroid sulfatase and consequently higher DHEAS concentrations .\n\n
Maximal plasma concentrations of DHEAS normally occur at 20–30 years of age (Figure 2), followed by a progressive decline in adrenal production in both males and females, until serum concentrations of DHEAS return to pre-adrenarche levels in persons over 80 years of age [59, 60]. The magnitude of this decline is such that serum levels of DHEAS in elderly adults are only around 10–20% of those in young adults [1, 61]. The diminution in adrenal androgens with aging is often termed ‘adrenopause.’ It has been suggested that adrenopause is associated with a generalized reduction in the 17,20 lyase activity of P450c17 in the zona reticularis of the adrenal gland . Interestingly, it has been shown that the zona reticularis of older men is reduced in size when compared to the adrenals of young men , suggesting that at least part of the age-associated decrease in adrenal androgens might relate to a reduction in the number of DHEA-secreting cells in the zona reticularis itself. Although the underlying mechanisms regarding this change must be further elucidated, the temporal association between falling DHEA concentrations and the onset of age-related diseases has led many investigators to suggest that some age-related neurological disorders such as AD and dementia may be partly attributable to the decrease in systemic DHEA concentrations .\n
The gradual decline in serum concentrations from the peak at 20–30 years of age has led to speculations that low DHEA concentrations could have a negative effect on cognitive function in later life. It has been hypothesized that rise in DHEA concentrations from 6 to 8 years until 20–30 years of age might be associated with the extended period of cortical maturation in humans . While numerous animal studies have shown that DHEA can modulate cognitive performance, the outcomes of such studies in humans are less clear. For example, one study reported that DHEA supplementation improves cognitive performance in young men , whereas other studies detected no benefit in an older group who were predominantly male and were HIV-1 seropositive . DHEA supplementation does not appear to improve cognition in the elderly .\n
A study evaluating the cognitive domains of working memory, executive function, and word processing speed in men and women aged between 60 and 88 years with low serum DHEAS concentrations found a positive association between serum DHEAS and working memory . However, the relationship was sex-specific, with a trend toward a better executive function in men only. Other studies in males have shown that increased endogenous androgen concentrations (following cessation of chemical castration in males) resulted in improved performance on the Cambridge Cognitive Examination (part of the Cambridge Examination for Mental Disorders of the Elderly, a global measure of cognition and memory) and verbal recall tests . A study in a population of older healthy women (aged 21–77 years) further indicated that women with high serum concentrations of DHEAS had increased performance on a variety of cognitive tests, including better verbal, visual, and spatial abilities; working memory; attention; concentration; and accuracy . In older men and women in an Italian cohort, low DHEAS levels were significant predictors of accelerated decline in Mini-Mental State Examination score during the 3-year follow-up period . Despite these associations, Mazat et al.  reported no significant role for serum DHEAS concentrations as a predictor of cognitive decline in an elderly population, while other studies conducted in frail elderly patients and nursing home residents found an inverse relationship between DHEAS levels and cognitive abilities [72, 73].\n
While the reasons for the conflicting data on DHEAS and cognition require further investigation, the changes in cognition are likely to be reflective of interactions with both the GABAergic and glutamatergic pathways, and possibly through the mediator brain-derived neurotrophic factor (BDNF). Neurosteroids have contrasting effects on GABAA receptors, which when activated result in chloride entry into the cell, hyperpolarization, and reduced membrane excitability . Reduced metabolites of progesterone and deoxycorticosterone have an agonistic effect on GABAA receptors, resulting in chloride ion movement into the cell. In contrast, DHEAS is a GABAA antagonist and thus increases the likelihood of membrane depolarization [48, 74]. Animal studies have shown that acute exposure to DHEAS may facilitate basal synaptic transmission in the CA1 region of the hippocampus through the non-competitive potentiation of GABAA receptors [75, 76, 77]. In terms of learning and memory, studies have shown that acute administration of DHEAS facilitates primed-burst potentiation, but not the induction of long-term potentiation , whereas long-term potentiation is stimulated by the chronic administration of DHEAS .\n
In addition to GABAA receptor modulation, neurosteroids have been found to interact in a structure-specific manner with glutamatergic NMDA receptors. DHEAS potentiates the neuronal response to NMDA in the rat hippocampus . These steroids also act as non-selective sigma-1 receptor antagonists , thus suppressing the activity of NMDA receptors, which are central to the process of excitotoxicity . In addition, DHEAS may reduce the cytoplasmic Ca2+-induced loss of mitochondrial membrane potential by preventing Ca2+ influx into the mitochondrial matrix . The neuroprotective effect of DHEA against NMDA-induced excitotoxicity may also involve the calcium/nitric oxide signaling pathway, since DHEA has been shown to inhibit NMDA-induced nitric oxide synthase activity and the production of nitric oxide in primary cultures of hippocampal neurons .\n
The potential of DHEAS to modulate the activity of NMDA receptors through a variety of mechanisms is likely to underpin their capacity to protect neurons from excitotoxicity when high levels of extracellular glutamate are present. Of note, glutamate excitotoxicity has been implicated in AD  (discussed further below), where a reduction in neurosteroid production may compromise the intrinsic defense mechanisms of the central nervous system (CNS). Another possible mechanism by which DHEAS could promote neurogenesis and neuronal survival in the CNS is through the mediation of the neurotrophin BDNF [86, 87].\n
BDNF is expressed in several areas of the CNS and is necessary for cell proliferation and differentiation [88, 89]. In addition, BDNF plays a vital role in neural plasticity, enhances long-term potentiation, and promotes learning and memory [90, 91]. As such, a mutation or deletion of the BDNF gene in mice results in learning deficits and long-term potentiation impairment [92, 93], as well as decreased learning and memory in behavioral paradigms . In humans, low plasma BDNF is associated with impairments in memory and general cognitive function in aging women .\n
A recent study investigated the effect of DHEA on cognition and learning in a rat model of vascular dementia  and found that DHEA treatment significantly preserved working and reference memory, which was accompanied by a significant increase in the levels of acetylcholine, norepinephrine, and dopamine in the brain. Of note was a significant increase in the hippocampal expression of BDNF after DHEA treatment . In a rodent model, Naert et al.,  showed that DHEAS treatment can lead to biphasic increases in BDNF in the hippocampus and amygdala, but decreased BDNF concentrations in the hypothalamus. It is interesting to note that glucocorticoids are also involved in BDNF regulation [27, 96], where stress has been found to decrease the expression of BDNF, leading to neuronal atrophy and degeneration in the hippocampus and the cortex, a process that may be common to both development and aging [97, 98]. These findings are important, considering, that BDNF expression is also altered in acute psychiatric disorders such as major depression [99, 100] and schizophrenia , as well as in neurodegenerative diseases such as AD .\n
AD is a chronic neurodegenerative disorder characterized by progressive memory loss and cognitive deterioration. It is the most common form of dementia, affecting about 50 million people worldwide , with the majority of cases in the elderly population, which presents global health and economic challenges . Currently, there are no disease-modifying therapies available to treat AD , and it represents a major unmet need in neurological research and patient management. The neuropathological hallmarks of AD include neurofibrillary tangles, which are formed when the neuronal cytoskeletal protein tau becomes hyperphosphorylated and precipitates, and also amyloid plaques, which are abnormal deposits of extracellular protein that accumulate after cleavage of the β-amyloid precursor protein . Other degenerative changes include cerebral amyloid angiopathy, glial inflammatory responses, and synaptic loss. These processes ultimately lead to neuronal atrophy, white matter loss, and a reduction in the volumes of the entorhinal, temporal, and frontal cortices as well as the hippocampus , followed by devastating clinical sequelae and resultant morbidity and mortality .\n
Sporadic AD is the predominant form of the disease, present in more than 95% of patients, and it usually occurs after 65 years of age . The etiology of sporadic AD is multifactorial and may be associated with a number of risk factors including advancing age [110, 111], increased oxidative stress [112, 113], autoimmunity , and excess glucocorticoids [115, 116, 117]. Although serum DHEA levels decrease with age, the majority of studies have reported that serum DHEAS levels in AD patients are even lower than in age-matched healthy controls. For instance, Yanase et al.  found that patients with AD or cerebrovascular dementia had lower concentrations of serum DHEAS and a lower DHEAS/DHEA ratio when compared to controls. Several other clinical studies have reported lower serum concentrations of DHEAS in patients with AD [14, 118, 119, 120], a reduction paralleled by decreases in the brain and cerebral spinal fluid [121, 122]. For instance, Weill-Engerer and colleagues  reported that not only are brain levels of DHEAS significantly lower in AD, but also the lower levels are inversely correlated with the presence of phosphorylated tau and β-amyloid. A few studies have not detected differences in serum DHEAS concentrations between AD patients and controls [120, 123], and there is one report that serum DHEAS levels are increased in mild-moderate AD . The reasons for these differences between studies have not yet been elucidated.\n
In contrast to the majority of studies, Naylor and colleagues  reported that cerebral spinal fluid levels of DHEA are significantly elevated in AD, as are tissue levels in the temporal cortex, with the extent of elevation being correlated with disease severity, as assessed by the burden of β-amyloid plaques. Similarly, Brown and colleagues  reported increased DHEA concentrations in the brains and cerebral spinal fluid of patients with AD when compared with controls, even though mean serum concentrations of DHEA did not differ. Interestingly, in this study, DHEA concentrations were highest in the hippocampus of AD patients, a region that does not express P450c17. Brown and colleagues speculated that the higher concentrations of DHEA in the hippocampus may have been produced by an as-yet-unknown pathway that involved the oxidation of an unknown precursor. This speculation has been given support by the finding that the addition of redox-active ferrous iron to serum samples causes a significant increase in the amount of detectable DHEA . It is also supported by the demonstration that oxidative stress associated with the presence of β-amyloid treatment induces DHEA synthesis in human and rodent cells
Another link to the pathogenesis and progression of AD comes from the anti-inflammatory properties of DHEA . Hence, the local production of DHEA in the AD brain may function, at least in part, to reduce the level of inflammation that would otherwise be injurious to neurons if left unchecked. Serum levels of DHEAS have been shown to negatively correlate with serum interleukin-6 (IL-6), to inhibit IL-6 secretion from human mononuclear cells , and to inhibit cytokine-stimulated, NF-κB–mediated transcription, partly through an antioxidant property . Interestingly, elevated levels of IL-6 are consistently detected in the brains of AD patients, but not in the brains of non-demented elderly persons . Several studies have suggested that an increase of circulating IL-6 in AD patients indicates immune activation and may be related to the pathophysiology of AD [136, 137, 138].\n
Perhaps the most intriguing link between DHEA and AD comes from its association with systemic stress and glucocorticoid production, which has lead to the hypothesis that chronic stress is an important factor in AD pathogenesis . Epidemiological evidence supports a role for stress in AD because elderly individuals prone to psychological distress are more likely to develop the disorder than age-matched, nonstressed individuals . Cortisol is the most prominent stress-related glucocorticoid in human serum. Serum cortisol levels are elevated in patients with AD , as are the levels of urinary cortisol . It is pertinent that the overactivation of GABAA receptors plays a central role in anxiety disorders and consequently these receptors are the principal targets of anxiolytic drugs for the treatment of affective disorders . Since DHEAS antagonizes GABAA receptors, they are thought to act as endogenous anxiolytics, and hence a reduction in the availability of DHEAS in aging or AD could contribute to increased anxiety and stimulate the chronic production of cortisol.\n
Animal experiments have shown that excess concentrations of glucocorticoids during prolonged periods of stress can have deleterious effects on the brain, especially in aged animals, and particularly affecting the hippocampus . Glucocorticoids exert several actions on the brain, including the stimulation of glutamatergic neurotransmission via the stimulation of glucocorticoid receptors (GR), which if left unchecked can lead to excitotoxicity. Several studies have shown that DHEA can protect against the effects of glucocorticoid-mediated neurotoxicity [144, 145]. The neuroprotective effects of DHEA have been modeled
DHEA may also attenuate the neurotoxic effects of cortisol by reducing the regeneration of active glucocorticoids. The 7α-hydroxylated metabolite of DHEA (7α-hydroxy-DHEA) has antiglucocorticoid effects in target tissues by competition with 11-keto glucocorticoids for access to 11β-hydroxysteroid dehydrogenase-1 . Enzyme kinetic data from yeast-expressed human 11β- hydroxysteroid dehydrogenase imply that 7α-hydroxysteroid substrates are preferred to cortisone by this enzyme . Therefore, in tissues such as the brain, 7α-hydroxy-DHEA may act as an endogenous inhibitor of 11β- hydroxysteroid dehydrogenase, thereby reducing the regeneration of active glucocorticoids . 7α-hydroxy-DHEA may have more potent bioactivity and stronger neuroprotective and antiglucocorticoid effects than DHEA itself . Interestingly, some investigators have hypothesized that the degree of metabolism of DHEA to 7α-hydroxy-DHEA is related to the pathology of AD [122, 151, 153, 154]. This is evident in the study by Yau et al. , which found that gene expression for cytochrome P4507b (which converts DHEA into 7α-hydroxy-DHEA) was significantly decreased in hippocampal dentate neurons from patients with AD when compared to controls . Another study found lower plasma 7α-hydroxy-DHEA concentrations in patients with AD when compared to controls .\n
Taken together, the preceding observations are generally supportive of the view that DHEAS levels in serum are reduced in AD when compared to those in healthy age-matched controls. Given that DHEAS reduces oxidative stress and neuroinflammation, protects against glutamate excitotoxicity, and minimizes the negative effects of cortisol on the brain, the reduced levels of serum DHEAS are likely to increase the vulnerability of the brain to these factors. While limited evidence suggests that the brain may compensate by increasing the local production of DHEAS, this may not be sufficient to slow the pathogenesis of the disease.\n
In addition to neurodegenerative diseases, there is evidence that low levels of circulating DHEA with normal levels of glucocorticoids (cortisol) place the developing brain at risk for a range of acute neuropsychiatric disorders, including major depressive disorder, bipolar disorder, and anxiety [155, 156, 157, 158]. It is further hypothesized that abnormalities of the hypothalamic-pituitary-adrenal (HPA) axis play a central role in the pathogenesis and etiology of schizophrenia [159, 160, 161]. Low ratios of DHEA to cortisol have been noted in patients with schizophrenia and are positively associated with the severity of depression, state and trait anxiety, anger, and hostility . DHEA augmentation in affected patients has been seen to attenuate the severity of some negative symptoms associated with this mental illness, including lack of volition and drive, and social withdrawal [16, 162].\n
Previous studies have found evidence of abnormal dopaminergic activity  and deficits in GABAergic and glutamatergic activity  in the brain tissue of patients with schizophrenia. Neuroactive steroids such as DHEA modulate the activity of these neurotransmitter systems, both directly and indirectly, and therefore may contribute to the pathophysiology of the illness [82, 165, 166, 167, 168]. A number of studies  have reported elevated plasma levels of DHEA and DHEAS in severely psychotic male subjects [170, 171], medicated patients with chronic schizophrenia , and nonmedicated first-episode patients [170, 173] compared with controls. Elevated DHEA levels have been detected in the
As a result of the positive modulatory effects of DHEA on NMDA receptors , in addition to its capacity to enhance learning and memory in rodent models , it may be speculated that an elevation of DHEA levels reflects a compensatory process in the schizophrenic brain. It is possible that subjects with schizophrenia may be physiologically resistant to DHEA action in some manner (potentially resulting in the increased synthesis of this neurosteroid) or that there is dysregulation in a feedback system involving the HPA axis . Specifically, DHEA increases following cortisol-releasing hormone  and adrenocorticotropic hormone  administration in humans, and persistent DHEA elevations may reflect a prolonged upregulation of this axis .\n
As noted earlier, DHEA can protect neurons from glutamate excitotoxicity, β-amyloid toxicity, and oxidative stress [49, 131], and furthermore, oxidative stress can lead to increased DHEA formation [84, 178]. Oxidative stressors may therefore stimulate DHEA levels in schizophrenic patients , in an adaptive change to other precipitating disease factors.\n
However, other studies have found no difference in DHEA levels between schizophrenic and control subjects , and some studies have reported significantly reduced plasma DHEA concentrations [179, 180, 181], particularly in the morning [180, 182, 183], as well as abnormal DHEA diurnal rhythms  in schizophrenics compared with matched controls. Furthermore, DHEA augmentation has been found to be effective in the management of depressive and anxiety symptoms of patients with schizophrenia , suggesting that higher levels of circulating DHEA in schizophrenic populations may be associated with superior functioning . The inconsistency between studies is understandable in view of the wide clinical polymorphism, variability of psychometric properties (distress and anxiety), drug treatment, and clinical responsiveness of schizophrenia patients to their antipsychotic treatment .\n
It may be difficult to interpret the significance of elevated or decreased DHEA levels in the absence of concentrations of other HPA axis hormones. Dysregulation of the HPA axis described in schizophrenia  includes increased basal cortisol levels , cortisol nonsuppression on the dexamethasone suppression test , increased adrenocorticotropic hormone and cortisol response to the dexamethasone/cortisol releasing hormone challenge test , and increases in glucocorticoid receptor mRNA as observed
There is also evidence for oligodendrocyte and myelin dysfunction in neuropathologies such as schizophrenia and bipolar affective disorder, where alterations in the cortisol/DHEA ratio have been observed [16, 17, 155]. Some key oligodendrocyte and myelination genes (such as proteolipid protein 1 and myelin-associated glycoprotein), and transcription factors that regulate the expression of these genes, are downregulated in brains of schizophrenia and bipolar subjects . Together, these studies indicate that common pathophysiological pathways may govern the disease phenotypes of schizophrenia, as well as other neurodegenerative diseases that specifically involve oligodendrocytes.\n
A significant body of preclinical research investigating the biological actions of DHEA have shown that this steroid, and its sulfated congener DHEAS, has a multifunctional role in a variety of physiological systems, including in the developing and aging brain. A summary of the actions of DHEA relevant to the discussion above is shown in Table 1. The present review has highlighted the involvement of DHEAS in glutamatergic and GABAergic neurotransmission, where this neurohormone acts as an important modulator of neuronal excitability. Consequently, perturbations in the level of DHEA can affect cognition and mood. DHEAS has also been shown to respond to stress and to modulate the effects of cortisol on the brain. Reductions in the availability of DHEAS can increase the likelihood of glutamate excitotoxicity as well as exacerbate the deleterious effects of cortisol. Evidence indicates that the brain is not dependent on serum levels of DHEA as it is able to synthesis DHEAS
|Agonistic and antagonistic effects on AR, agonist at ERα and ERβ [40, 41]\n|
|\n||Modulates the NMDA receptor [42, 43, 44]\n|
|\n||Positive allosteric modulator of the GABA-A receptor [46, 47, 48, 49]\n|
|\n||Nonselective sigma-1 receptor antagonist \n|
|\n||Selective antagonist of the GR \n|
|Maintenance and endocrine control of pregnancy \n|
|\n||Associated with human growth and organ maturation, particularly of the brain, during adrenarche [53, 54, 55]\n|
|\n||Promotes neurogenesis and neuronal survival in the CNS through the mediation of BDNF [86, 87]\n|
|DHEAS may facilitate basal synaptic transmission in the CA1 region of the hippocampus [75, 76, 77]\n|
|\n||Acute DHEAS administration facilitates primed-burst potentiation  and chronic administration of DHEAS stimulates LTP \n|
|\n||DHEA treatment significantly preserves working and reference memories and increases acetylcholine, norepinephrine, and dopamine concentrations in the rat brain \n|
|Reduces the cytoplasmic Ca2+-induced loss of mitochondrial membrane potential by preventing Ca2+ influx into the mitochondrial matrix \n|
|\n||Inhibits NMDA-induced nitric oxide synthase activity and the production of nitric oxide in primary cultures of hippocampal neurons \n|
|\n||Protect neurons from glutamate excitotoxicity, β-amyloid toxicity, and oxidative stress [49, 131]\n|
|Inhibits IL-6 secretion from human mononuclear cells \n|
|\n||Inhibits cytokine-stimulated, NF-κB–mediated transcription, partly through an antioxidant property \n|
|GR antagonist and can attenuate the translocation of stress-activated protein kinase-3 in rat hippocampal primary cultures \n|
|\n||Suppresses the nuclear localization of the GR in response to glutamate toxicity and inhibition of GR translocation into the nucleus \n|
|\n||Downregulation of the expression of glucocorticoid receptors \n|
|\n||Reduces the regeneration of active glucocorticoids \n|
The authors are grateful for support and many discussions from Dr. Udani Ratnayake, Dr. Stacey Ellery, Dr. Margie Castillo-Melendez, and Dr. Hayley Dickinson from The Ritchie Centre, Hudson Institute of Medical Research, and from Professor Jonathan Hirst, University of Newcastle, New South Wales, Australia. Tracey Quinn received support from an Australian Post-graduate Award (APA) postgraduate scholarship for some of the studies reported above. Tracey Quinn and David Walker are grateful for funding from National Health & Medical Research Council of Australia and Cerebral Palsy Alliance. We also acknowledge generous support from the Victorian Government Infrastructure Fund to the Hudson Institute of Medical Research.\n
Citrus belongs to family Rutaceae and holds an important position among fruits all around the globe. It is the most cultivated fruit in the world after grapes. Citrus is believed to be originated from southeastern Asian region . Northern hemisphere accounts for about 70% of the total citrus production and approximately 80 citrus species are native to India and other tropical and sub-tropical areas of Asia . Citrus being a perennial fruit tree is usually produced through vegetative propagation of scion on rootstock. Combination and compatibility of scion and rootstock can result in high yielding citrus plants. The United States, China, Brazil and the Mediterranean countries contribute two third of global citrus production and are regarded as major citrus producing countries . Citrus products and by-products provide the basis for local agricultural industries, which generate employment and raise income, and in many cases, this industry constitutes an important source of foreign revenue for developed and developing countries such as Pakistan.
A number of factors and certain conditions are collectively responsible for fluctuations in citrus production. Selection of rootstock, agronomic practices and management in citrus nurseries and orchards, propagation methods and biotic and abiotic factors contribute their share to some extent in reduced citrus production. Like other commercial crops, number of diseases, insect pests and genetic problems affect the citrus production. Diseases are one of the major limiting factors for the low citrus production and gives a serious threat to citrus industry. These diseases are caused by fungi, prokaryotes, nematodes, viroids, viruses and virus-like pathogens. Among these, viruses and virus-like pathogens play a major role in citrus decline. These pathogens incur varying degree of damages to citrus plants and make their life span shorter, causing low yield and deterioration of quality and ultimately loss of economy which leads towards the citrus decline .
Citrus decline is the matrix of all above mentioned factors and conditions. The common diseases, playing an important role in citrus decline are citrus gummosis caused by
Therefore, it is the time when citrus nurseries should operate on highly technical and scientific lines and start providing disease-free and certified plants to the growers. In the first instance, nurseries should be registered and indiscriminate multiplication and sale of uncertified citrus plants must end. For this purpose, the most imperative points such as the prevalence and detection of citrus viral diseases, selection of material, production of disease-free material and streamlined screening procedures are highlighted in this bulletin. If the guidelines are properly followed and certified bud-wood becomes available for producing disease-free citrus plants, the problem of citrus decline can be minimized.
Citrus pathology is the study of citrus diseases caused by biotic (pathogens) and abiotic factors. It is now being considered as a major part in the field of plant pathology. Being a major fruit crop in the world, citrus production always remains important for the citrus industry. Physiology, morphology, biochemistry and behavior of the citrus tree towards the prevailing climatic conditions are the key areas to be kept in mind while investigating the citrus diseases. Etiology of citrus diseases and their detection methods help to manage these diseases. A plenty of information regarding the diseases of citrus and their control has been published around the world.
Virus, viroids and virus-like diseases, however, infecting different citrus species could not receive due attention because of the lack of laboratories with proper facilities for their proper identification. These diseases are also known as ‘graft-transmissible diseases’ (GTDs) and the term used for the casual agents is ‘citrus graft-transmissible pathogens’ (CGTPS) . These are an emerging threat for citrus industry. Major viruses and virus-like pathogens include citrus tristeza virus (CTV), citrus yellow vein clearing virus (CYVCV), citrus variegation virus (CVV), concave gum, psorosis, cristacortis, ringspot, exocortis,
|Sr. No.||Citrus disease||Pathogen||Transmission||Incidence %||Host||Importance|
|1.||Citrus greening disease (CGD)|
|Bacterium-like organism||Psyllid: Diaphorina citri||20–90||Sweet orange, grapefruit, orange jessamine||Associated with citrus decline|
|2.||Citrus tristeza virus (CTV)||Closterovirus||Aphid species|
(Aphis gossypii, Toxoptera citricida)
Up to 48
|Sweet orange, lime, mandarin||Economically important|
|3.||Gummy bark (GB)||Virus probable||Grafting, mechanical||20–30||Mandarin, sweet orange||-do-|
|4.||Bud union crease (BUC)||Virus probable||Grafting, mechanical||20–30||Mandarin, sweet orange||-do-|
|5.||Cristacortis||Virus probable||Not known||10||All citrus species||-do-|
Up to 16
|8.||Citrus stubborn disease (CSD)||Prokaryote||Leaf hopper (||2–7||Sweet orange, grapefruit, periwinkle||-do-|
|9.||Yellow vein clearing (YVC)||Virus||Grafting, vector not known||2||Lemon, sour orange||Minor importance|
|10.||Ring spot/ Variegation||Virus||Not known||2–3||Sweet orange||Minor importance|
Although plant pathologists have put their efforts for the identification and management of virus and virus-like diseases of citrus but there are some areas need to be investigated. A comprehensive book has been written by Roistacher in 1991 regarding the detection of virus and virus-like diseases of citrus. These diseases reduce the citrus yield and ultimately result in the loss of low foreign exchange. Diseases caused by viruses and virus-like pathogens are infectious, contagious and devastating due to their systemic nature. They are transmitted through different means in nature; through vegetative propagation, by insect vectors and horticultural tools used for the routine activities in citrus orchards and nurseries. These diseases have a considerable economic importance because of their involvement in the citrus decline . Millions of citrus trees have been died due to CTV. The CGTPS usually have two types of effects either quick decline or long term losses. These diseases are very difficult to control or manage unless or until by the application of integrated management practices. The appropriate diagnosis or indexing method plays an important role for the management of CGTPS .
The major symptoms due to virus and virus-like pathogens are vein clearing, bark cracking, yellowing of leaves, leaf dropping, gummosis, mosaic, rugosity, bark scaling, stem pitting, dwarfing, chlorosis and mottling [10, 13]. The virus and virus-like diseases, infecting different citrus species in Pakistan, have been neglected for a long time due to lack of proper facilitations in the research laboratories and skilled personnel for their detection and characterization. A brief description is presented in Table 2 regarding the citrus species and viruses and virus-like diseases in Pakistan. Indexing facilities are very important for the diagnosis of plant pathogens. Similarly, unlike other pathogens viruses and virus-like pathogens are very sensitive to their indexing through different techniques. Pathogen detection system always played an important role in management of virus and virus-like pathogens. Proper indexing facilities help in the characterization and differentiation of different viruses and their isolates. Management of viruses and virus-like pathogens is only possible when appropriate indexing procedures and facilities are available.
|Citrus species||Virus||Viroid||Prokaryote||Virus-like symptoms||PS||DE|
|Sweet orange (Mosambi)||+||+||+||+||+||+||+||+||+||+||+||+|
|Sweet orange (Mosambi)||+||+||+||+||+||+||+||+||+||+||+||+|
Insect pests have always been key role players in the direct or indirect transmission of plant pathogens in agricultural and horticultural crops [14, 15, 16]. Citrus tristeza, cachexia-xyloporosis, greening or Huánglóngbìng, infectious variegation, vein enation, yellow vein clearing, exocortis and stubborn are the most conspicuous viral diseases of citrus all over the world including Pakistan [11, 17]. These diseases are usually graft-transmissible and phloem-restricted. Although these diseases along with other fungal, bacterial or mycoplasmic infections of citrus are usually spread through unhealthy mechanical intrusions and by the use of infected uncertified bud, scion or rootstock in plant propagation, many type of sap-feeding insect pests play important role in the transmission of these diseases such as leafhoppers, aphids, psyllids, whiteflies and thrips [17, 18, 19, 20].
Among the vector borne viral diseases of citrus, citrus tristeza (CTV) which is caused by a
Indexing is an indispensable procedure to produce and diagnose disease-free plants. Different techniques or combination of techniques have been applied in this regard and the effectiveness of each depends upon the facilities available. Generally indexing can be divided into two types.
Field indexing; also known as biological indexing including the mechanical inoculation through direct contact or vegetative propagation and/or through insect transmission.
Laboratory indexing; also known as quick indexing including serological, molecular and chemical assays.
Commonly used indexing methods are tissue grafting, budding, insect transmission for biological indexing and enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) for quick indexing strategies. Although all viruses and virus-like pathogens can be detected through PCR and its derivatives, polyacrylamide gel electrophoresis (PAGE) is commonly used for the detection of viroids.
Biological indexing is the inoculation or introduction of virus source (infected sample) into the indicator plants for detection and purification. It involves one of the common indexing methods such as vegetative propagation of infected scion (grafting/budding) to indicator plants, mechanical inoculation of indicator plants or transmission of virus through the insect vector (
|Sr. No.||Disease||Appropriate number of test plants||Indicator plants (for inoculation)||Favorable temperature||Symptoms on the indicator (indexed) plant|
|1.||Citrus tristeza||5||Mexican lime (C. aurantifolia), sweet orange on sour orange root stock, Duncan grapefruit||65–68° F|
|Vein clearing, stem pitting, leaf cupping, decline on sour orange stem pitting on grapefruit.|
|2.||Yellow vein clearing||5||Lemon (C. limon), sour orange (||Cool||Yellowing and clearing of veins|
|3.||Ringspot/ Infections variegation||4||Citron (C. medica), cowpea (Vigna unguiculata)||Cool||Ringspot, necrotic local lesions, distorted leaves.|
|4.||Psorosis||4||Sweet orange (C. sinensis)||Cool||Flecking on leaves|
|5.||Cachexia-xyloporosis||5||Mandarin (C. reticulata) Parsons special||Warm >95°F||Gum in bark, scion and at bud union|
|6.||Exocortis||5||Citron (||Warm||Tip browning, leaf epinasty|
|7.||Cristacortis||4||Grapefruit, sweet orange||Cool- warm||Flecking|
|8.||Concave gum||4||Sweet orange||Cool||Oak leaf pattern, narrowing of leaves|
|9.||Citrus greening disease||5||Mandarin (||Cool- warm||Leaf blotches, chlorosis|
|10.||Citrus stubborn disease||4||Sweet orange, grapefruit, periwinkle (Catharanthus roseus)||Warm||Stunted shoot, smelling of leaves, Zn deficiency like signs.|
|11.||Yellows (probably Aster)||5||Grapefruit, Periwinkle (C. roseus)||Warm||Chlorosis|
|12.||Bud union crease||5||Sweet orange||Warm||Brown line at bud union|
|13.||Gummy bark||5||Mandarin||Warm||Gum in the bark|
Detailed methodology for biological indexing has been described much in literature [28, 29, 30, 31]. Followings are the generalized and simplified steps to be kept in mind during the biological indexing on the basis of available literature.
Sow the seeds of test plants (usually Mexican lime or acid lime) in the sand in germinating tray. Transplant the seedlings in pots having potting media (sand, soil and moss @ 1:1:1 ratio) after 17–28 days of germination, depending on the germinating conditions.
Inoculate the seedlings at 4–6 leaf stage.
Keep the indicator plants in insect-free chambers before and after inoculation.
Pass the crude virus extract from double layer muslin cloth and then apply to the indicator plants with the help of forefinger and leave for 3–4 min. Remove the excess sap from indicator plants under tap water.
Observe the fasting period according to the nature of transmission (non-persistent, persistent or semi-persistent) before allowing them to be fed on virus source to acquire the virus.
For non-persistent transmission pre-acquisition fasting time is 30–90 min. Long fasting period enhances the chances of quick acquisition of virus from the infected source.
Transfer the insects on to the young leaves with virus symptoms/virus infected plants with the help of camel brush and allow them to feed for few min.
After few min, immediately transfer the viruliferous insects on to the indicator plants and keep the plants in insect-free chamber to avoid the contamination from other insects.
Maintain the insect population on indicator plants for at least 24 hr. and eradicate them after that through insecticides.
For persistent transmission and semi-persistent transmission pre-acquisition fast has no effect. In both cases long acquisition feeding period enhances the chance of transmission.
Maintain the insect population for a week and eradicate them with insecticide in case of semi-persistent transmission while maintain the insect population through transferring them on new indicator plants till they are alive.
Temperature range between 65 and 95°F helps the appearance of symptoms on indicator plants for viruses and virus-like pathogens. Observation time also varies from 3 to 16 months for different viruses, virus-like pathogens and viroids.
Laboratory indexing/advanced detection methods
There are rapid methods, highly specific, routinely applicable and some of which test large number of samples. These methods are summarized in Table 4. ELISA is the main laboratory indexing method used for the detection of CTV, PAGE for viroids and PCR for all diseases. Mother plants (plants recovered by nucellar embroyony
|Sr. No.||Methods||Tested for||Advantages and limitations||References|
|1.||Immunofluorescence, tissue staining Azure A, Light microscopic observations||Citrus tristeza virus, yellow vein virus, greening diseases||Simple, economical, limited number of samples, time consuming, non-specific||[29, 32]|
|2.||Gel immunodiffusion||Citrus tristeza virus||Economical, time consuming, require quality antiserum, where ELISA facilities are not available.|||
|3.||Enzyme-linked Immunosorbant Assay (ELISA and its variants)||Citrus tristeza virus and some prokaryotes||Rapid, economical, specific, routinely applied for large number of samples, quantitative, sensitive|||
|4.||Electron microscopy (EM)||Citrus tristeza viru and other viruses||Quick for elongated viruses (CTV, CYVV), requires proper facilities|||
|5.||Immunosorbant electron microscopy (ISEM)- Decoraton Technique||Citrus tristeza virus||Quick, specific, require antiserum and proper facilities, limited sampling|||
|6.||Polyacrylamide Gel Electrophoresis (PAGE)||Citrus viroids (Exocortis, Cachexia)||Excellent for viroid detection and characterization, requires purification of viroids and proper conditions and facilities|||
|7.||Molecular hybridization (RNA/DNA Probes), Polymerase Chain Reaction (PCR)||Virus and virus like diseases, CTV.||Highly sensitive, routinely applicable, time consuming, require primers and equipment facilities|||
Serology involves the quick indexing of plant viruses, based on the antibody–antigen reaction. Enzyme-linked immunosorbent assay (ELISA) is one of the widely used in detection of plant viruses. It is relatively cheap and can test large number of samples.
ELISA with its derivatives, direct (DAS-ELISA) and indirect (DAC-ELISA), is the main serological indexing tool used for most of the citrus viruses at large scale samples.
Followings are some general steps followed during the ELISA based detection or indexing .
Wash the plate with washing buffer for 3 times with the interval of 5 min.
Add the antigen (virus sap extracted from infected samples) into the wells of ELISA plate and incubate as above.
Repeat the washing and coat the ELISA plate with enzyme-labeled antibodies and incubate as above.
Repeat the washing step and add the substrate followed by incubation for 30 to 90 min for visual observation of color change and read the micro-plate through ELISA reader/spectrophotometer for quantitative data.
Wash the ELISA plate as in DAS-ELISA.
Add the primary antibody and incubate.
Add the secondary antibody and incubate.
Add the enzyme-labeled antibodies and incubate.
Add the substrate and then observe the color change after incubation and read the plate through ELISA reader for quantitative data.
Molecular detection of citrus viruses and virus-like diseases has revolutionized the subject and provided the platform to detect the early stages of infection to reduce the economic losses. The molecular hybridization techniques supplemented with nucleic acid amplification methods based on PCR, in which high-throughput sequencing approaches can be adopted to identify the strains in relation to evolutionary history or phylogenetic assemblages [36, 37]. Although, nucleic acid based methods are highly sensitive and discriminatory allowing specific strain typing, but it bears the problems in reproducibility [38, 39]. Progressive efforts have been made to decrease the troubleshoots and hurdles to improve the amplification systems by improving the sensitivity and specificity of detection by limiting the high contents of plant related enzyme inhibitors. In contest, nested and multiplex PCR provides high sensitivity and make the possible to detect several targets in single assay . Moreover, highly sensitive technologies by conducting the amplification of nucleic acids in an isothermal reaction, nucleic acid sequence-based amplification (NASBA) and reverse transcription loop-mediated isothermal amplification (RT-LAMP) provides specific detection of viruses and virus-like diseases.
The addition of real-time PCR for high-throughput testing allows the automation of PCR by combing the fluorimeteric approaches to detect and quantify the targets simultaneously [41, 42]. The combination of different protocols including the serological techniques and molecular approaches will increase the accuracy and reliability of virus diagnostic. Furthermore, in future prospects, nucleic acid arrays and biosensors assisted by nanotechnology will open new corridors to revolutionize the detection of plant viruses and virus-like diseases.
Citrus tristeza virus (CTV) is the most dangerous citrus disease all over the world and is also known as quick decline disease reducing the population of citrus trees significantly [43, 44, 45]. However, the utilization of advanced diagnostic methods, such as, biological indexing, electron microscopy (EM), ELISA and PCR or reverse transcriptase PCR (RT-PCR) is providing promising detection of the virus particles and leading towards the management strategies of CTV . The application of conventional PCR is sensitive and specific under optimized and controlled conditions. However, sometimes, it is not possible to judge the amount of pathogens in the samples. Therefore, researchers have to employ other subsequent techniques for complete detection and quantification. Meanwhile, with real-time PCR approach, users can monitor the reaction and also the quantification of the specific pathogen in the sample. While setting up the real-time reaction for virus detection, it is the basic requirement to adapt the specific conditions of the detection system and instrument, and the characteristics of the reaction reagents and cycling procedures in which the most important are primer design, reaction components and conditions. The real-time PCR works well with small amplicons (5–200 bp), while standard PCR allows amplification of several hundred bases without sensitivity and specificity. Moreover, concentrations of MgCl2, primers, and dNTPs are usually higher than conventional PCR .
The new developing chemistries are setting up the protocols with different characteristics depending upon the target and assay requirements. In addition to the most widely working chemistries (SYBRGreen, TaqMan, Scorpion, Molecular Beacons), there are more novel chemicals or technologies such as Amplifluor; Locked Nucleic Acid (LNA) Probes, Sigma Proligo; Cycling Probe Technology (CPT), Takara; Light Upon eXtension (Lux) Fluorogenic Primers, Invitrogen Corporation; Plexor Technology, Promega [48, 49]. Real-time technology is being used also in multiplex formatting for the specific detection and strain identification for several viruses [50, 51, 52, 53, 54, 55]. Furthermore, real-time reaction in multiplex system is difficult to optimize due to different ratio between the targets and the reaction. The replacement of conventional PCR with real-time PCR is providing new horizons towards the multiple detection system of plant viruses especially of the citrus viruses and virus-like diseases.
After the discovery of viroid group of pathogens as an infectious agent to the plants, new aspects in virology were come in front of researchers to be addressed. Viroids are the smallest pathogens which consist of 246 to 401 nucleotides. They are low molecular weight, circular and single stranded RNAs. Viroids exist as free RNA because they lack protein coat . Since viroids do not code for protein and enzyme, they rely on host enzyme for protein synthesis system and replication. To date, 38 viroids have been identified and they are classified into 2 families
The major economic important viroids in different plants are coconut viroids (CCCVd), citrus viroids (Exocortis and cachexia and variants), Hop stunt viroid and Potato spindle tuber viroids . The origin of viroids is still questionable as they do not have natural host relationship [58, 59].
Citrus production is also affected by viroids. These are the emerging threat to citrus industry. To date, seven citrus viroids have been detected so far in citrus
All citrus viroids are classified in different genus under
|Australia, Argentina, Brazil, Japan, Taiwan, Corsica, China, India, Israel, Spain, Pakistan, South Africa USA, Uruguay, Iran||[8, 57, 60, 61, 62]|
|Israel, Japan, Australia, China, Uruguay, Pakistan, UAE, Iran, Spain||[8, 60, 61, 63, 64, 65]|
|Israel, Brazil, Uruguay||[8, 60, 62]|
|USA, Uruguay, Pakistan||[8, 60, 65, 66]|
|USA, Uruguay||[60, 67]|
|Spain, Iran||[61, 68]|
|Genus||Citrus viroid||Length (nucleotides)||Diseases|
|371||Citrus exocortis disease|
|299–302||Citrus cachexia disease.|
|284–286||Citrus bark cracking disease|
|318||Citrus leaf bending disease|
|294–297||Citrus dwarfing disease|
Collect the leave samples based on virus and viroids-like symptoms in the field. Bring the leaves samples to laboratory for processing and preservation until use as follows;
Collect the leave samples in sterile plastic bags and place in ice box.
In the laboratory, wash the samples first in 10% bleach followed by distilled water.
Dry the samples and put in the plastic bags.
Label the plastic bags and store them at −80°C until further use.
Extract the nucleic acids from leave samples using the TESLP buffer  as follows;
Grind the 2-3 g of leaves (about 10–12 leaves) using mortar and pestle with liquid nitrogen. The slurry needs to be transferred to 50 ml screw cap tubes.
Add 10 ml of TESLP buffer [0.13 M Tris–HCl (pH 8.9), 0.017 M EDTA (pH 7.0), 1 M LiCl, 0.83%SDS, 5%PVP] into the tube.
Add 16 μl of 2-mercapthoethanol into the mixture.
Incubate the mixture for 30 min at room temperature in the rotary shaker.
Centrifuge the mixture at 11000 rpm for 15 min.
The supernatant needs to be transferred to a new 50 ml screw tube.
Add phenol:chloroform:iso-amyl (PCA, 25:24:1) @ 3:2 and mix well using vortex followed by centrifugation for 15 min, 11,000 rpm at room temperature.
Transfer the supernatant into a new 15 ml screw tube and add CA (24:1) @ 4:3. The mixture needs to be mixed well using vortex and repeat the step 7.
The supernatant is obtained into a new 15 ml screw tube @ 1 volume of supernatant with 0.9 volumes of 90% isopropanol.
The tube is inverted 3–4 times to mix the components. Do not vortex or centrifuge.
The mixture is incubated at −80°C for 30–40 min (or -20°C for 3–4 hr. or overnight).
The mixture is centrifuged for 15 min, 11,000 rpm at room temperature.
The isopropanol is discarded and the pellet obtained is transferred into 1.5 ml micro centrifuge tube.
The pellet is washed with 1 ml of 70% ethanol followed by washing with 1 ml absolute ethanol until the clean pallet is obtained.
The pellet is suspended in 50 μl of sterile double distilled water.
The pellet is immediately used for RT-PCR or stored in -20°C until use.
Reverse Transcription Polymerase Chain Reaction :
The extracted RNA is used to run RT-PCR. Reverse Transcription process is carried out in two steps to synthesis cDNA as follows;
Experimental RNA = 5 μl
Reverse primer = 1 μl
Double distilled water = 2.5 μl
Total Volume = 8.5 μl
The reaction is incubated at 80°C for 12 min then immediately transferred to ice for 5 min.
AMV-RT = 1 μl
dNTPs = 2 μl
RNAse Inhibitor = 0.5 μl
MgCL2 = 4 μl
RT buffer = 4 μl
Total volume = 11.5 μl
The reaction is incubated at 55°C for 30 min. After 30 min, the process is stopped when it reaches to 10°C. The cDNA obtained is stored in −80°C freezer until use (or it can be used immediately).
The final volume of PCR should be 25 μl which consists of 12.5 μl of PCR master mix, 5 μl of cDNA, 5.5 μl of sterile double distilled water, 1 μl of forward primer and 1 μl of reverse primer.
The conditions for PCR amplification (35 cycles) are as follows:
94°C for 10 min
94°C for 30 seconds
60°C for 1 min
b. Annealing at 60°C for 10 seconds.
c. Extension at 72°C for 10 seconds and then 5 min.
The list of specific primers used is given in Table 7.
|Viroid||Type||Sequence||Target (Product size)|
The amplified RT-PCR product is separated using 2% agarose gel as follows ;
2% agarose gel is prepared with 1x TBE buffer
Samples are loaded in the gel and electricity is provided at 100 volts for 50 min.
The gel is stained with Ethidium bromide for 10 min and washed with distilled water for 5 min.
The gel is visualized under Trans UV and captured with Gel Doc XR system.
Positive PCR products with expected size are purified using MinElute® Gel Extraction Kit according to the standard protocol provided with Kit.
The expected size of band is excised from the agarose gel with a sterile, sharp scalpel.
The gel slice is put in a sterile 1.5 ml micro-centrifuge tube and weighed.
QC buffer, provided with the kit, is added @ 3:1 volume of gel.
The gel slice is incubated at 50°C for 10 min until the gel slice has completely dissolved.
The mixture is vortexed every 2–3 min to facilitate the dissolution of gel slices.
Then, 1 gel volume of isopropanol is added and mixed by inverting with pipette.
The MinElute spin column is placed into 2 ml collection tube.
The sample is transferred into the MinElute column and centrifuged at 13000 rpm for 1 min.
The flow-through is discarded and put back the column into the same collection tube. 750 μl of Buffer PE is added to MinElute column and let it stand for 1–2 min.
Centrifuged at 13000 rpm for 1 min and the flow-through is discarded.
The process is repeated to remove Buffer PE completely.
The ethanol residual left at the bottom of the column is discarded and MinElute column is placed into a sterile 1.5 ml micro-centrifuge tube.
30 μl of EB Buffer is added to the center of MinElute membrane to elute DNA. The mixture is let to stand for 1 min, and then is centrifuged at 13000 rpm for 1 min.
MinElute column is discarded and the tube is stored in - 20°C.
Positive PCR samples will be cloned using the TOPO TA cloning kit according to the standard protocol provided along with the Kit as follows;
4 μl purified PCR products.
1 μl vector (pCR2.1-TOPO).
1 μl Salt solution.
Incubate in PCR machine/ heat block at 25°C for 30 min.
Add 2 μl of ligation mixture into competent cell
Put 30 min in ice.
Put 30 sec in 42°C water bath (heat shock).
Put in ice for 5 min (immediately after heat shock).
Add 250 μl SOC medium to mixture-seal competent cell tube with parafilm.
Put at 200 rpm in 37°C incubator shaker for 1 h 30 min.
Warm the petri dish in incubator for 20–30 min.
Spread 40 μl X-gal on petri dish (LBA media).
After spread the X-gal, put the petri dish in incubator for 20–30 min.
Finally, spread the sample mix on petri dish and incubate overnight at 37°C.
2D PAGE is carried out to for the detection and to check the circularity of Viroid RNA. Following is the recipe and protocol for PAGE.
40% AB in 50 ml distilled water @ 19:1 ratio
|Ingredients||8% GEL||5% GEL|
|40% AB||6 ml||6.25 ml|
|10X TBE||3 ml||5 ml|
|dH2O||20.25 ml||37.7 8 ml|
|10% APS||750 μl||937.5 μl|
|TEMED||40 μl||43.75 μl|
|Total Volume||30 ml||50 ml|
Mix the gel with magnetic bar.
Wash the glass with KOH and dH2O.
KOH washing buffer includes 10 g KOH + 10 ml dH2O + 90 ml and 99% ETOH.
Rinse the glass with dH2O and let it dry.
Prepare the gel and cast into electrophoresis set.
Let the gel to polymerase for 30 min.
Pre-run empty gel for 20 min at 10 mA.
Pre-run sample for 10 min at 10 mA and then run sample for 1 hr. plus at 20 mA.
40% AB: 8.7 ml
Urea: 25.2 g
10XTBE: 5.25 ml
10% APS: 750 μl
TEMED: 45 μl
dH2O: 17.25 ml
Total volume: 52.5 ml
|ETOH (10%, V/V)||10 ml|
|Acetic Acid (5%, V/V)||5 ml|
|ETOH (10%, V/V)||10 ml|
|Acetic Acid (5%, V/V)||0.5 ml|
|Silver Nitrate||0.3 g|
|3 mM NaBH4||0.023 g|
|0.375 M NaOH||3 g|
Fix the gel in fixer 1 for 10 min at room temperature in shaker.
Fix the gel in fixer 2 for 10 min at room temperature in shaker.
Dip the gel in silver stain solution for 1 hr. under dark condition.
Rinse with dH2O twice with the interval of 1 min.
Add developer solution in the end.
Stop developing by adding 5% acetic acid.
CTV belongs to the genus
|EPPO||Israel, Spain, Turkey||Present|
|France||Found but not established|
|Algeria, Cyprus, Egypt, Italy, Morocco, Tunisia||Scattered infection|
|Asia||Brunei, China, Georgia||Present|
|Indonesia. Iran, Japan||Present|
|Korea, Malaysia, Nepal||Present|
|Pakistan||Present (Scarce Information available)|
|Philippines, KSA, Sri Lanka, Taiwan, Thailand, Viet Nam, Yemen||Present|
|Africa||Cameroon, Chad, Ethopia, Gabon, Ghana, Kenya, Mauritious, Mozambique, Nigeria, South Africa, Tanzania, Zaire, Zambia, Zimbabwe||Present|
|North America||Bermuda, Mexico, USA||Present|
|Central America and Caribbean||Antigua, Barbuda, Bahamas, Belize, Costa Rica, El Salvador, Guatemala, Honduras, Jamaica, Netherlands Antilles, Nicaragua, Puerto Rico, St. Lucia, Trinidad, Tobago||Present|
|South America||Argentina, Bolivia, Brazil||Present (wide spread)|
|Chile||Found, not established|
|Colombia, Ecuador, Guyana, Peru, Paraguay, Suriname, Uruguay||Present|
|Oceania||American Samoa, Australia, Fiji, New Zealand||Present|
In Morocco, 14 diverse isolates were selected from samples during survey and then characterized on the basis of reaction pattern. Among these 14 isolates, four were severe and two were mild isolates. Isolates were also indexed against a series of monoclonal antibodies . DAS-ELISA was used to detect the CTV from the samples collected during a survey in Western and Midwestern development regions of Nepal . One hundred and eighty-eight samples were analyzed through biological indexing and DAS- ELISA to detect tristeza, psorosis and similar diseases like-symptoms including viroids in orange varieties in all the regions and the cachexia was detected as the most important and widespread disease . Biological indexing is still considered as an important tool using for the characterization of CTV isolates. Different strains were identified through symptoms expression on differential hosts, including Mexican lime and sweet orange. Moreover, they observed visual symptoms of different strains on Mexican lime and sweet orange through biological indexing followed by ELISA . Detection of CTV in Spain was compared by indexing using monoclonal and polyclonal antibodies .
Three microscopy procedures for detecting CTV were compared which provided additional alternatives for very rapid CTV indexing, including the use of EM, SSEM and light microscopy. In light microscopy, inclusions were found in young phloem tissues of all CTV-infected hosts examined. Similarly, in SSEM virus particles were found on grids prepared with antiserum and extracts from infected tissue. CTV particles could be detected in pooled samples representing one in 100. Similarly, virus particle fragments were observed infrequently in samples representing one infected plant in 1,000 samples .
Citrus is an important fruit crop of the world and has a great potential for local consumption, export purposes and industrial uses. Unfortunately, citrus orchards are facing the problem of low productivity due to citrus decline. This is mainly attributed, among other factors to the prevalence of graft-transmissible virus and virus-like diseases, unhygienic nursery operations and poor orchard management. However, most of the problems arise from nurseries. It is the time that the nurseries should operate on highly technical and scientific lines and should work on providing disease-free and certified plants to the citrus growers. To establish the disease free nurseries, indexing of virus and virus-like diseases are the major area that needs to be focused. Implication of traditional and modern high-throughput biological, serological and molecular indexing techniques, such as ELISA, RT-PCR, PAGE, should be put in practice for the detection and indexing of virus and virus-like diseases of citrus plants. Moreover, citrus nurseries should be registered and indiscriminate multiplication and sale of uncertified citrus plants should be prohibited.