RT-PCR analysis of Deformed wing virus in honeybees (Apis mellifera) and mites (Varroa destructor)

Deformed wing virus (DWV) is a honeybee viral pathogen either persisting as an inapparent infection or resulting in wing deformity. The occurrence of deformity is associated with the transmission of DWV through Varroa destructor during pupal stages. Such infections with DWV add to the pathology of V. destructor and play a major role in colony collapse in the course of varroosis. Using a recently developed RT-PCR protocol for the detection of DWV, individual bees and mites originating from hives differing in Varroa infestation levels and the occurrence of crippled bees were analysed. It was found that 100 % of both crippled and asymptomatic bees were positive for DWV. However, a significant difference in the spatial distribution of DWV between asymptomatic and crippled bees could be demonstrated: when analysing head, thorax and abdomen of crippled bees, all body parts were always strongly positive for viral sequences. In contrast, for asymptomatic bees viral sequences could be detected in RNA extracted from the thorax and/or abdomen but never in RNA extracted from the head. DWV replication was demonstrated in almost all DWV-positive body parts of infected bees. Analysing individual mites for the presence of DWV revealed that the percentage of DWV-positive mites differed between mite populations. In addition, it was demonstrated that DWV was able to replicate in some but not all mites. Interestingly, virus replication in mites was correlated with wing deformity. DWV was also detected in the larval food, implicating that in addition to transmission by V. destructor DWV is also transmitted by feeding.

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Study of Morphological Features in Pre-Imaginal Honey Bee Impaired by Varroa destructor by Means of Computer Tomography

Insects ◽

10.3390/insects12080717 ◽

2021 ◽

Vol 12 (8) ◽

pp. 717

Author(s):

Tamás Sipos ◽

Tamás Donkó ◽

Ildikó Jócsák ◽

Sándor Keszthelyi

Keyword(s):

Viral Infection ◽

Honey Bee ◽

Varroa Destructor ◽

Developmental Disorders ◽

Essential Element ◽

Total Body Length ◽

Pcr Analysis ◽

Brood Cell ◽

Deformed Wing Virus ◽

Total Body

The honey bee (Apis mellifera L. 1778) is an essential element in maintaining the diversity of the biosphere and food production. One of its most important parasites is Varroa destructor, Anderson and Trueman, 2000, which plays a role in the vectoring of deformed wing virus (DWV) in honey bee colonies. Our aim was to measure the potential morphometric changes in the pre-imaginal stage of A. mellifera caused by varroosis by means of computed tomography, hence supplying evidence for the presumable role that V. destructor plays as a virus vector. Based on our results, the developmental disorders in honey bees that ensued during the pre-imaginal stages were evident. The total-body length and abdomen length of parasitized specimens were shorter than those of their intact companions. In addition, the calculated quotients of the total-body/abdomen, head/thorax, and head/abdomen in parasitized samples were significantly altered upon infestation. In our view, these phenotypical disorders can also be traced to viral infection mediated by parasitism, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Capitalizing on a non-destructive method, our study reveals the deformation of the honey bee due to mite parasitism and the intermediary role this pest plays in viral infection, inside the brood cell.

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Abstract 175: The Role of ER-α, ER-ß, and AR in the FCG Model of Ischemic Brain Injury

Stroke ◽

10.1161/str.45.suppl_1.175 ◽

2014 ◽

Vol 45 (suppl_1) ◽

Author(s):

Kathryn C Bentivegna ◽

Bharti Manwani ◽

Sharon E Benashski ◽

Sarah Doran ◽

Sarah Pan ◽

...

Keyword(s):

Brain Injury ◽

Y Chromosome ◽

Sex Chromosome ◽

Chromosome Complement ◽

Pcr Analysis ◽

Sexually Dimorphic ◽

Ischemic Brain Injury ◽

Rt Pcr ◽

Ischemic Brain ◽

Significant Difference

Background: Ischemic stroke is a sexually dimorphic disease. Females are protected from ischemic brain injury throughout most of the lifespan, but the contribution of hormones in this “ischemia resistant” phenotype remains unclear. The “four core genotype” (FCG) model is used to differentiate the effect of the chromosomal complement (XX vs. XY) from an animal’s gonadal (estrogen vs. testosterone producing) sex through examination of five genotypes XYMwt, XY-M, XXM, XXF, and XYF. XY-M FCG mice are made by the translocation of Sry from the Y chromosome to an autosome. Stroke sex differences have been largely attributed to the neuroprotective effects of estrogen. However, it remains unclear whether hormonal receptors can be influenced at the chromosomal level, which could further explain sexual dimorphism in stroke phenotypes. We hypothesize that the sex chromosome complement and transgene Sry (the testis determining gene found on the Y chromosome) contribute to the sexually dimorphic stroke phenotype. Methods: FCG animals were gonadectomized at 3 weeks of age. Animals were subjected to stroke at 8-12 weeks of age. Cytosolic samples were evaluated for androgen receptor (AR) and estrogen receptor (ER-α and ER-β) levels in sham and stroke samples. RT-PCR for Sry on the stroke hemisphere (delta-delta-CT) was performed on the FCG mice and wild type males. Results: Western blot analysis showed that XXM and XXF mice had significant stroke induced reduction in AR expression (p<0.01). No significant difference in the expression of ER-α was seen in sham and stroke mice among genotypes. ER-β expression increased significantly in XXM post stroke (p<0.05), but not in the other genotypes. RT-PCR analysis showed approximately 90-fold increase of mRNA expression of Sry in the phenotypes with the exogenous Sry (XYM and XXM) compared to WTM mice with endogenous Sry (p<0.05). Conclusion: XXM and XYM mice show an increase in Sry expression compared to WTM suggesting insertional and positional effects of Sry transgene. AR expression in XXM and XXF mice is influenced at the chromosomal level, suggesting effects of X chromosome dosage on AR expression. Stroke induced ER-β expression in XXM mice may be an effect of the interaction between transgene Sry and other hormonal axes.

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Biological Efficacy of Modified Structure of Polymer Surfaces for Bone Substitute

Key Engineering Materials ◽

10.4028/www.scientific.net/kem.330-332.707 ◽

2007 ◽

Vol 330-332 ◽

pp. 707-710 ◽

Cited By ~ 2

Author(s):

Zhi Jun Pan ◽

Xin Huang ◽

Di Sheng Yang ◽

Hidero Unuma ◽

Wei Qi Yan

Keyword(s):

Bone Substitute ◽

Cellular Proliferation ◽

Cellular Responses ◽

Polymer Surfaces ◽

Pcr Analysis ◽

Rt Pcr ◽

Plla Scaffold ◽

Significant Difference ◽

Preliminary Study ◽

Potential Use

Hydroxyapatite (HA) coatings were introduced onto Poly L-lactic Acid (PLLA) polymer in a controlled manner by immobilized urease method with a shortened precipitation time. Osteoblastic-like cellular responses to the composite were examined in terms of cell proliferation, differentiation and cell morphology, as well as the expression of bone-associated genes. The cells exhibited higher cellular proliferation at 2 and 4 days on the HA/PLLA composite compared to PLLA scaffold, while no significant difference was observed later at 6 days. The alkaline phosphatase (ALP) activity by cells at 7 days was statistically higher on HA/PLLA scaffold than on PLLA. Moreover, the gene expression of ALP and osteocalcin (OC) was up regulated on HA/PLLA composite by RT-PCR analysis. The preliminary study suggested that the use of the controlled modification of hydroxyapatite coating on PLLA scaffold to produce HA/PLLA composite might enhance cellular activity, indicating the potential use for bone substitute in tissue engineering.

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Haemolymph removal by the parasite Varroa destructor can trigger the proliferation of the Deformed Wing Virus in mite infested bees (Apis mellifera), contributing to enhanced pathogen virulence

10.1101/257667 ◽

2018 ◽

Cited By ~ 6

Author(s):

Desiderato Annoscia ◽

Sam P. Brown ◽

Gennaro Di Prisco ◽

Emanuele De Paoli ◽

Simone Del Fabbro ◽

...

Keyword(s):

Honey Bee ◽

Varroa Destructor ◽

Viral Evolution ◽

Colony Losses ◽

Viral Pathogen ◽

Deformed Wing Virus ◽

Bee Colony ◽

Bee Health ◽

Honey Bee Health ◽

Mite Feeding

AbstractThe association between the Deformed Wing Virus and the parasitic mite Varroa destructor has been identified as a major cause of worldwide honey bee colony losses. The mite acts as a vector of the viral pathogen and can trigger its replication in infected bees. However, the mechanistic details underlying this tripartite interaction are still poorly defined, and, in particular, the causes of viral proliferation in mite infested bees.Here we develop and test a novel hypothesis - grounded in ecological predator-prey theory - that mite feeding destabilizes viral immune control through the removal of both viral ‘prey’ and immune ‘predators’, triggering uncontrolled viral replication. Consistent with this hypothesis, we show that experimental removal of increasing volumes of haemolymph from individual bees results in increasing viral densities. In contrast, we find no support for alternative proposed mechanisms of viral expansion via mite immune-suppression or within-host viral evolution.Overall, these results provide a new model for the mechanisms driving pathogen-parasite interactions in bees, which ultimately underpin honey bee health decline and colony losses.

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QSOX sulfhydryl oxidase in rat adenohypophysis: localization and regulation by estrogens

Journal of Endocrinology ◽

10.1677/joe.1.05842 ◽

2004 ◽

Vol 183 (2) ◽

pp. 353-363 ◽

Cited By ~ 24

Author(s):

A Tury ◽

G Mairet-Coello ◽

F Poncet ◽

C Jacquemard ◽

P Y Risold ◽

...

Keyword(s):

Secretory Granules ◽

Chronic Administration ◽

Anterior Lobe ◽

Ovariectomized Rats ◽

Pcr Analysis ◽

Cell Populations ◽

Sulfhydryl Oxidase ◽

Rt Pcr ◽

Almost All ◽

Quiescin Sulfhydryl Oxidase

The expression of the rat quiescin sulfhydryl oxidase (rQSOX) and its putative regulation by estrogens were investigated in the adenohypophysis. Immunohistochemical observations revealed that rQSOX protein is abundantly expressed throughout the anterior lobe of the pituitary, and can be found in almost all the different cell populations. However, as shown by double immunohisto-chemistry, the cells displaying the strongest rQSOX labeling belong to a subset of gonadotrophs. Immunoelectron microscopy showed that, in adenohypophyseal cells, the protein is linked to the membranes of the rough endoplasmic reticulum, the Golgi apparatus and to dense-core secretory granules. These results are consistent with the secretion of the protein and its presumed role in the extracellular matrix. According to its sulfhydryl oxidase function, rQSOX could also participate in the intracellular folding of secreted proteins or hormones like LH and FSH and act as an endogenous redox modulator of hormonal secretion. A semiquantitative RT-PCR analysis of rQSOX level across the estrous cycle and the fact that chronic administration of 17 β-estradiol to ovariectomized rats led to a sustained up-regulation of rQSOX in the pituitary suggest that rQSOX expression is controlled by sex hormone levels. Further investigations are needed in order to elucidate its precise roles in that gland and the mechanisms of its regulation.

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Vertical-transmission routes for deformed wing virus of honeybees (Apis mellifera)

Journal of General Virology ◽

10.1099/vir.0.83101-0 ◽

2007 ◽

Vol 88 (8) ◽

pp. 2329-2336 ◽

Cited By ~ 103

Author(s):

Constanze Yue ◽

Marion Schröder ◽

Sebastian Gisder ◽

Elke Genersch

Keyword(s):

Apis Mellifera ◽

Vertical Transmission ◽

Clinical Symptoms ◽

Viral Pathogen ◽

Deformed Wing Virus ◽

Colony Collapse ◽

Virgin Queens ◽

Viral Sequences

Deformed wing virus (DWV) is a viral pathogen of the European honeybee (Apis mellifera), associated with clinical symptoms and colony collapse when transmitted by the ectoparasitic mite Varroa destructor. In the absence of V. destructor, DWV infection does not result in visible symptoms, suggesting that mite-independent transmission results in covert infections. True covert infections are a known infection strategy for insect viruses, resulting in long-term persistence of the virus in the population. They are characterized by the absence of disease symptoms in the presence of the virus and by vertical transmission of the virus. To demonstrate vertical transmission and, hence, true covert infections for DWV, a detailed study was performed on the vertical-transmission routes of DWV. In total, 192 unfertilized eggs originating from eight virgin queens, and the same number of fertilized eggs from the same queens after artificial insemination with DWV-negative (three queens) or DWV-positive (five queens) semen, were analysed individually. The F0 queens and drones and F1 drones and workers were also analysed for viral RNA. By in situ hybridization, viral sequences were detected in the ovary of an F0 queen that had laid DWV-positive unfertilized eggs and was inseminated with DWV-positive semen. In conclusion, vertical transmission of DWV from queens and drones to drone and worker offspring through unfertilized and fertilized eggs, respectively, was demonstrated. Viral sequences in fertilized eggs can originate from the queen, as well as from drones via DWV-positive semen.

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Infection of a Lepidopteran Cell Line with Deformed Wing Virus

Viruses ◽

10.3390/v12070739 ◽

2020 ◽

Vol 12 (7) ◽

pp. 739 ◽

Cited By ~ 2

Author(s):

Tal Erez ◽

Nor Chejanovsky

Keyword(s):

Cell Line ◽

Honey Bee ◽

Cell Lines ◽

Cellular Level ◽

Pcr Analysis ◽

Virus Infections ◽

Rt Pcr ◽

Deformed Wing Virus ◽

Infected Cells ◽

Bee Virus

Many attempts to develop a reliable cell cultured-based system to study honey bee virus infections have encountered substantial difficulties. We investigated the ability of a cell line from a heterologous insect to sustain infection by a honey bee virus. For this purpose, we infected the Lepidopteran hemocytic cell line (P1) with Deformed wing virus (DWV). The genomic copies of DWV increased upon infection, as monitored by quantitative RT-PCR. Moreover, a tagged-primer-based RT-PCR analysis showed the presence of DWV negative-sense RNA in the cells, indicating virus replication. However, the DWV from infected cells was mildly infectious to P1 cells. Similar results were obtained when the virus was injected into Apis mellifera pupae. Thus, though the virus yields from the infected cells appeared to be very low, we show for the first time that DWV can replicate in a heterologous cell line. Given the availability of many other insect cell lines, our study paves the way for future exploration in this direction. In the absence of adequate A. mellifera cell lines, exploring the ability of alternative cell lines to enable honey bee virus infections could provide the means to study and understand the viral infectious cycle at the cellular level and facilitate obtaining purified isolates of these viruses.

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Abstract 13037: Tenascin-c Modulates Inflammatory Response and Aggravate Ventricular Remodeling After Myocardial Infarction in Mice Model

Circulation ◽

10.1161/circ.130.suppl_2.13037 ◽

2014 ◽

Vol 130 (suppl_2) ◽

Author(s):

Taizo Kimura ◽

Akira Sato ◽

Kazuko Tajiri ◽

Wang Zeng ◽

Satoshi Sakai ◽

...

Keyword(s):

Myocardial Infarction ◽

Acute Phase ◽

Ventricular Remodeling ◽

Biological Function ◽

Pcr Analysis ◽

Rt Pcr ◽

Knock Out ◽

Tenascin C ◽

Significant Difference ◽

Anti Inflammatory

Tenascin-C (TN-C), an extracellular matrix glycoprotein, transiently appeared in myocardial tissue after acute myocardial infarction (AMI). We have previously reported that AMI patients with higher serum TN-C levels had worse long-term prognosis, suggesting TN-C may play important roles during the development of ventricular remodeling. However, the biological function of TN-C in ventricular remodeling is not fully understood. In this study, using TN-C knock-out(KO) mice, we investigated the effects of TN-C on LV remodeling and the biological function of TN-C during the acute phase of inflammatory responses after myocardial infarction. The 8 to 10 weeks old male wild type (WT) and TN-C knock-out (KO) mice were divided into 4 groups of WT+Sham, KO+Sham, WT+MI and KO+MI. Mice 12 weeks post-MI, the survival rate of both WT+MI (48.3%,14 of 29 mice) and KO+MI (55.6%,15 of 27 mice) groups had no significant difference. However, TN-C KO group had the better cardiac function than WT had (LVEF, 19.02±6.31% vs 10.63±4.43%; p<0.001). Interstitial fibrosis at border area was significantly increased in the WT +MI group to compare with that of KO+MI, whereas the extent of fibrosis in the remote area revealed no significant difference between the two groups. By RT-PCR analysis, WT+MI group showed significantly higher expression of atrial natriuretic peptide at the border including infarcted areas than that of KO+MI at chronic MI phase. At acute phase, fluorescence activated cell sorting analysis showed that ratio of CD45+, CD11b+, Ly6c high pro-inflammatory monocyte were significantly decreased, whereas CD45+, F4/80+, CD206+, anti-inflammatory M2 macrophage were significantly increased in KO+MI compared with WT+MI group 7 days after MI. RT-PCR analysis showed that the expression of IL-10, an anti-inflammatory cytokine, was significantly higher in KO+MI than WT+MI. These findings suggest, TN-C aggravates the deterioration of LV function due to MI partly by modulating inflammation at acute phase.

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Quantitative Real-Time Reverse Transcription-PCR Analysis of Deformed Wing Virus Infection in the Honeybee (Apis mellifera L.)

Applied and Environmental Microbiology ◽

10.1128/aem.71.1.436-441.2005 ◽

2005 ◽

Vol 71 (1) ◽

pp. 436-441 ◽

Cited By ~ 162

Author(s):

Y. P. Chen ◽

J. A. Higgins ◽

M. F. Feldlaufer

Keyword(s):

Real Time ◽

Reverse Transcription ◽

Virus Titer ◽

Premature Death ◽

Pcr Analysis ◽

Life Stages ◽

Rt Pcr ◽

Deformed Wing Virus ◽

Modes Of Transmission ◽

Reverse Transcription Pcr

ABSTRACT Deformed wing virus (DWV) can cause wing deformity and premature death in adult honeybees, although like many other bee viruses, DWV generally persists as a latent infection with no apparent symptoms. Using reverse transcription (RT)-PCR and Southern hybridization, we detected DWV in all life stages of honeybees, including adults with and without deformed wings. We also found DWV in the parasitic mite Varroa destructor, suggesting that this mite may be involved in the transmission of DWV. However, the detection of the virus in life stages not normally associated with mite parasitism (i.e., eggs and larvae) suggests that there are other modes of transmission. The levels of DWV in different life stages of bees were investigated by using TaqMan real-time quantitative RT-PCR. The amounts of virus varied significantly in these different stages, and the highest levels occurred in pupae and in adult worker bees with deformed wings. The variability in virus titer may reflect the different abilities of bees to resist DWV infection and replication. The epidemiology of DWV is discussed, and factors such as mite infestation, malnutrition, and climate are also considered.

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Transformative Learning in Mastery-Oriented CS0 Course

Higher Education for the Future ◽

10.1177/23476311211007255 ◽

2021 ◽

pp. 234763112110072

Author(s):

Srinivasan Lakshminarayanan ◽

N. J. Rao ◽

G. K. Meghana

Keyword(s):

Transformative Learning ◽

First Year ◽

Guided Discovery ◽

Engineering Programs ◽

Transformative Experiences ◽

Significant Difference ◽

Cognitive Levels ◽

Almost All ◽

Core Course

The introductory programming course, commonly known as CS1 and offered as a core course in the first year in all engineering programs in India, is unique because it can address higher cognitive levels, metacognition and some aspects of the affective domain. It can provide much needed transformative experiences to students coming from a system of school education that is dominantly performance-driven. Unfortunately, the CS1 course, as practiced in almost all engineering programs, is also performance-driven because of a variety of compulsions. This paper suggests that the inclusion of a course CS0 can bring about transformative learning that can potentially make a significant difference in the quality of learning in all subsequent engineering courses. The suggested instruction design of this course takes the advantage of the unique features of a course in programming. The proposed CS0 course uses “extreme apprenticeship” and “guided discovery” methods of instruction. The effectiveness of these instruction methods was established through the use of the thematic analysis, a well-known qualitative research method, and the associated coding of transformative learning experiences and instruction components.

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