Detection of plant genes, gene expression and viral RNA from tissue prints on FTA cards

2005 ◽  
Vol 23 (4) ◽  
pp. 383-395 ◽  
Author(s):  
Yvette Roy ◽  
Annette Nassuth
Keyword(s):  
Gene Expression ◽  
Viral Rna ◽  
Fta Cards ◽  
Plant Genes ◽  
Tissue Prints

scholarly journals The [KIL-d] Element Specifically Regulates Viral Gene Expression in Yeast

Genetics ◽  
2000 ◽  
Vol 155 (2) ◽  
pp. 601-609 ◽  
Author(s):  
Zsolt Tallóczy ◽  
Rebecca Mazar ◽  
Denise E Georgopoulos ◽  
Fausto Ramos ◽  
Michael J Leibowitz
Keyword(s):  
Gene Expression ◽  
Phenotypic Expression ◽  
Viral Gene Expression ◽  
Virus Genome ◽  
Viral Rna ◽  
High Rate ◽  
Viral Gene ◽  
Double Stranded Rna ◽  
Yeast Prions ◽  
Killer Virus

Abstract The cytoplasmically inherited [KIL-d] element epigenetically regulates killer virus gene expression in Saccharomyces cerevisiae. [KIL-d] results in variegated defects in expression of the M double-stranded RNA viral segment in haploid cells that are “healed” in diploids. We report that the [KIL-d] element is spontaneously lost with a frequency of 10−4–10−5 and reappears with variegated phenotypic expression with a frequency of ≥10−3. This high rate of loss and higher rate of reappearance is unlike any known nucleic acid replicon but resembles the behavior of yeast prions. However, [KIL-d] is distinct from the known yeast prions in its relative guanidinium hydrochloride incurability and independence of Hsp104 protein for its maintenance. Despite its transmissibility by successive cytoplasmic transfers, multiple cytoplasmic nucleic acids have been proven not to carry the [KIL-d] trait. [KIL-d] epigenetically regulates the expression of the M double-stranded RNA satellite virus genome, but fails to alter the expression of M cDNA. This specificity remained even after a cycle of mating and meiosis. Due to its unique genetic properties and viral RNA specificity, [KIL-d] represents a new type of genetic element that interacts with a viral RNA genome.

scholarly journals Y2, the Smallest of the Sendai Virus C Proteins, Is Fully Capable of both Counteracting the Antiviral Action of Interferons and Inhibiting Viral RNA Synthesis

2001 ◽  
Vol 75 (8) ◽  
pp. 3802-3810 ◽  
Author(s):  
Atsushi Kato ◽  
Yukano Ohnishi ◽  
Masayoshi Kohase ◽  
Sakura Saito ◽  
Masato Tashiro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Rna Synthesis ◽  
Sendai Virus ◽  
Reporter Gene Expression ◽  
Viral Rna ◽  
Viral Transcription ◽  
Antiviral Action ◽  
Ifn Γ ◽  
C Proteins

ABSTRACT An open reading frame (ORF) overlapping the amino-terminal portion of the Sendai virus (SeV) P ORF in the +1 frame produces a nested set of carboxy-coterminal proteins, C′, C, Y1, and Y2, which are referred to collectively as the C proteins. The C proteins are extremely versatile triple-role players; they counteract the antiviral action of interferons (IFNs), inhibit viral RNA synthesis, and are involved in virus assembly. In this study, we established HeLa cell lines stably expressing the C, Y1, and Y2 proteins individually and examined the capacities of these cells to circumvent the antiviral action of alpha/beta IFN (IFN-α/β) and IFN-γ and to inhibit viral transcription. The assay protocols included monitoring of IFN-α/β-mediated signaling by interferon-stimulated response element-driven reporter gene expression and of the antiviral state induced by IFN-α/β and IFN-γ and measurement of reporter gene expression from an SeV minigenome, as well as quantification of SeV primary transcripts. When necessary, the activities measured were carefully normalized to the expression levels of the respective C proteins in cells. The data obtained clearly indicate that the smallest protein, Y2, was as active as the C and Y1 proteins in both counteracting the antiviral action of IFNs and inhibiting viral transcription. The data further show that intracellular transexpression of either C, Y1, or Y2 rendered HeLa cells moderately or only poorly permissive for not only wild-type SeV but also 4C(−) SeV, which expressed none of the four C proteins. On the basis of these findings, the roles of SeV C proteins in the natural life cycle are discussed.

scholarly journals Improved microarray gene expression profiling of virus-infected cells after removal of viral RNA

BMC Genomics ◽  
2008 ◽  
Vol 9 (1) ◽  
pp. 221 ◽  
Author(s):  
Matthijs Raaben ◽  
Penn Whitley ◽  
Diane Bouwmeester ◽  
Robert A Setterquist ◽  
Peter JM Rottier ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Profiling ◽  
Expression Profiling ◽  
Viral Rna ◽  
Microarray Gene Expression ◽  
Infected Cells ◽  
Microarray Gene

scholarly journals Absence of Symbiotic Leghemoglobins Alters Bacteroid and Plant Cell Differentiation During Development of Lotus japonicus Root Nodules

2009 ◽  
Vol 22 (7) ◽  
pp. 800-808 ◽  
Author(s):  
Thomas Ott ◽  
John Sullivan ◽  
Euan K. James ◽  
Emmanouil Flemetakis ◽  
Catrin Günther ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nitrogen Fixation ◽  
Cell Differentiation ◽  
Root Nodules ◽  
Lotus Japonicus ◽  
Nodule Development ◽  
Primary And Secondary Metabolism ◽  
Bacterial Differentiation ◽  
Plant Genes ◽  
Bacterial Genes

During development of legume root nodules, rhizobia and their host plant cells undergo profound differentiation, which is underpinned by massive changes in gene expression in both symbiotic partners. Oxygen concentrations in infected and surrounding uninfected cells drop precipitously during nodule development. To assess what effects this has on plant and bacterial cell differentiation and gene expression, we used a leghemoglobin-RNA-interference (LbRNAi) line of Lotus japonicus, which is devoid of leghemoglobins and has elevated levels of free-oxygen in its nodules. Bacteroids in LbRNAi nodules showed altered ultrastructure indicating changes in bacterial differentiation. Transcript analysis of 189 plant and 192 bacterial genes uncovered many genes in both the plant and bacteria that were differentially regulated during nodulation of LbRNAi plants compared with the wild type (containing Lb and able to fix nitrogen). These included fix and nif genes of the bacteria, which are involved in microaerobic respiration and nitrogen fixation, respectively, and plant genes involved in primary and secondary metabolism. Metabolite analysis revealed decreased levels of many amino acids in nodules of LbRNAi plants, consistent with the defect in symbiotic nitrogen fixation of this line.

scholarly journals Effects of the noncoding subgenomic RNA of red clover necrotic mosaic virus in virus infection

2021 ◽  
Author(s):  
Pulkit Kanodia ◽  
W. Allen Miller
Keyword(s):  
Gene Expression ◽  
Virus Infection ◽  
Mosaic Virus ◽  
Nicotiana Benthamiana ◽  
Red Clover ◽  
Plant Viruses ◽  
Viral Rna ◽  
Productive Infection ◽  
Deficient Mutant ◽  
Host Interactions

In recent years, a new class of viral noncoding subgenomic (ncsg)RNA has been identified. This RNA is generated as a stable degradation product via an exoribonuclease-resistant (xr) RNA structure, which blocks the progression of 5’→3’ exoribonuclease on viral RNAs in infected cells. Here, we assess the effects of the ncsgRNA of red clover necrotic mosaic virus (RCNMV), called SR1f, in infected plants. We demonstrate: (i) absence of SR1f reduces symptoms and decreases viral RNA accumulation in Nicotiana benthamiana and Arabidopsis thaliana plants; (ii) SR1f has an essential function other than suppression of RNA silencing; and (iii) the cytoplasmic exoribonuclease involved in mRNA turnover, XRN4, is not required for SR1f production or virus infection. A comparative transcriptomic analysis in N. benthamiana infected with wildtype RCNMV or an SR1f-deficient mutant RCNMV revealed that wt RCNMV infection, which produces SR1f and much higher levels of virus, has a greater and more significant impact on cellular gene expression than the SR1f-deficient mutant. Upregulated pathways include plant hormone signaling, plant-pathogen interaction, MAPK signaling, and several metabolic pathways, while photosynthesis-related genes were downregulated. We compare this to host genes known to participate in infection by other tombusvirids. Viral reads revealed a 10 to 100-fold ratio of positive to negative strand, and the abundance of reads of both strands mapping to the 3’ region of RCNMV RNA1 support the premature mechanism of synthesis for the coding sgRNA. These results provide a framework for future studies of the interactions and functions of noncoding RNAs of plant viruses. IMPORTANCE Knowledge of how RNA viruses manipulate host and viral gene expression is crucial to our understanding of infection and disease. Unlike viral protein-host interactions, little is known about the control of gene expression by viral RNA. Here we begin to address this question by investigating the noncoding subgenomic (ncsg)RNA of red clover necrotic mosaic virus (RCNMV), called SR1f. Similar exoribonuclease-resistant RNAs of flaviviruses are well-studied, but the roles of plant viral ncsgRNAs, and how they arise, are poorly understood. Surprisingly, we find the likely exonuclease candidate, XRN4, is not required to generate SR1f, and we assess the effects of SR1f on virus accumulation and symptom development. Finally, we compare the effects of infection by wildtype RCNMV vs an SR1f-deficient mutant on host gene expression in Nicotiana benthamiana , which reveals that ncsgRNAs such as SR1f are key players in virus-host interactions to facilitate productive infection.

scholarly journals Rapid assessment of West Nile virus circulation in a German zoo based on honey-baited FTA cards in combination with box gravid traps

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Noelle Fynmore ◽  
Renke Lühken ◽  
Heike Maisch ◽  
Tina Risch ◽  
Sabine Merz ◽  
...  
Keyword(s):  
West Nile Virus ◽  
Rapid Assessment ◽  
Gravid Female ◽  
Virus Detection ◽  
Turnaround Time ◽  
Viral Rna ◽  
Mass Trapping ◽  
Vector Surveillance ◽  
West Nile ◽  
Fta Cards

Abstract Background For over a decade, monitoring of West Nile virus (WNV) in Germany has consisted of a bird monitoring programme as well as a mosquito-based surveillance programme employing CO2-baited encephalitis vector surveillance (EVS) traps for mass trapping and screening of mosquitoes. In contrast to the EVS traps, the Reiter/Cummings type box gravid trap collects gravid female mosquitoes, which have already taken a blood meal, increasing the likelihood of being infected with pathogens. The traps can be equipped with a honey-baited Flinders Technology Associates® (FTA) card to encourage sugar feeding by the trapped mosquitoes. FTA cards contain nucleic acid preserving substances, which prevent the degradation of viral RNA in the expectorated mosquito saliva and allows for testing the card for flavivirus RNA. This study aimed to assess the suitability of the method for WNV surveillance in Germany as an alternative to previous methods, which are expensive, time-consuming, and predominantly target host-seeking populations less likely to be infected with WNV. Methods In the Thüringer Zoopark Erfurt, snowy owls (Nyctea scandiaca) and greater flamingos (Phoenicopterus roseus) died of WNV infections in July and August 2020. In response, five Reiter/Cummings type box gravid traps were positioned during the daytime on the 10th, 13th, and 16th of September in five different locations. The FTA cards and mosquitoes in the chamber were collected, kept in a cool chain, and further processed for virus detection using a modified generic flavivirus reverse transcription PCR. Results A total of 15 trappings during September collected a total of 259 female mosquitoes, 97% of which were Culex pipiens sensu lato, as well as 14 honey-baited FTA cards. Eight mosquitoes tested PCR-positive for WNV. Four FTA cards tested PCR-positive for mosquito-borne flaviviruses, two of which were confirmed as WNV, and the remaining two confirmed as Usutu virus. Conclusion The suitability of the FTA cards in preserving viral RNA in the field and rapid turnaround time from collection to result is combined with a simple, cost-effective, and highly specific trapping method to create an arbovirus surveillance system, which circumvents many of the difficulties of previous surveillance programmes that required the analysis of mosquitoes in the laboratory. Graphical Abstract

A Pilot Trial of Valproic Acid in Patients with Kaposi’s Sarcoma: A Multi-Center Trial of the AIDS Malignancy Consortium.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2279-2279 ◽  
Author(s):  
Mary Jo Lechowicz ◽  
Jeannette Lee ◽  
Dirk Dittmer ◽  
Susan Krown ◽  
Jonathan Said ◽  
...  
Keyword(s):  
Gene Expression ◽  
Valproic Acid ◽  
Copy Number ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Viral Rna ◽  
Short Term Treatment ◽  
Lytic Gene ◽  
Lytic Gene Expression ◽  
Viral Copy Number

Abstract PURPOSE: Although the use of highly active antiretroviral therapy (HAART) has led to improvements in the management of AIDS-associated Kaposi’s sarcoma (KS), KS may persist or progress despite HAART. It has been hypothesized that activation of KS-associated herpesvirus (KSHV, HHV8) lytic expression might render tumor cells susceptible to immune surveillance by cytotoxic T cells. Alternatively, it has been suggested that lytic induction could lead to tumor progression. In vitro, valproic acid (VA) and other histone deacetylase inhibitors induce KSHV lytic gene expression in primary effusion lymphoma cell lines. We investigated VA in AIDS/KS patients to assess its safety and its impact on lytic viral gene expression in tumor and viral copy number in blood. PATIENTS AND METHODS: VA was given orally to patients with AIDS and cutaneous KS on stable antiviral regimens; the dose was titrated to maintain trough concentrations between 50 and 100 mcg/mL. VA was given daily for 28 days followed by a rapid taper and patients were then followed for 6 months. Quantitative real time PCR was used to assess viral DNA in plasma and PBMC, and viral RNA in tumor specimens. Immunohistochemistry was used to assess viral antigen expression in tumor specimens. RESULTS: 18 patients were treated. 15/18 patients completed therapy; 3 patients discontinued therapy early, one secondary to grade 2 toxicity and 2 to patient preference. One patient showed a partial response and 17 showed stable disease at the completion of therapy. No patients progressed during treatment. There were no differences between KSHV copy number in plasma or PBMC before, during, or after therapy. Similarly, although serial biopsies in some patients showed an increase in lytic gene expression, these changes did not achieve statistical significance. However, in multivariate analyses, viral lytic RNA increased in tumor biopsies on day 8 as a function of VA level. There was no change in HIV viral load with VA treatment. CONCLUSION : VA was well tolerated in AIDS patients, was not associated with accelerated disease progression, but rarely induced tumor regression after short-term treatment. In patients who achieved the highest serum VA levels there was increased lytic viral RNA expression. These findings support investigation of more potent HDAC inhibitors over longer treatment courses in patients with AIDS-associated KS.

scholarly journals hsp72, a Host Determinant of Measles Virus Neurovirulence

2006 ◽  
Vol 80 (22) ◽  
pp. 11031-11039 ◽  
Author(s):  
Thomas Carsillo ◽  
Zachary Traylor ◽  
Changsun Choi ◽  
Stefan Niewiesk ◽  
Michael Oglesbee
Keyword(s):  
Gene Expression ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Protein Interactions ◽  
Measles Virus ◽  
Viral Rna ◽  
Viral Gene ◽  
Febrile Episodes

ABSTRACT Transient hyperthermia such as that experienced during febrile episodes increases expression of the major inducible 70-kDa heat shock protein (hsp72). Despite the relevance of febrile episodes to viral pathogenesis and the multiple in vitro roles of heat shock proteins in viral replication and gene expression, the in vivo significance of virus-heat shock protein interactions is unknown. The present work determined the in vivo relationship between hsp72 levels and neurovirulence of an hsp72-responsive virus using the mouse model of measles virus (MV) encephalitis. Transgenic C57BL/6 mice were created to constitutively overexpress hsp72 in neurons, and these mice were inoculated intracranially with Edmonston MV (Ed MV) at 42 h of age. The mean viral RNA burden in brain was approximately 2 orders of magnitude higher in transgenic animals than in nontransgenic animals 2 to 4 weeks postinfection, and this increased burden was associated with a fivefold increase in mortality. Mice were also challenged with an Ed MV variant exhibiting an attenuated in vitro response to hsp72-dependent stimulation of viral transcription (Ed N-522D). This virus exhibited an attenuated neuropathogenicity in transgenic mice, where mortality and viral RNA burdens were not significantly different from nontransgenic mice infected with either Ed N-522D or parent Ed MV. Collectively, these results indicate that hsp72 levels can serve as a host determinant of viral neurovirulence in C57BL/6 mice, reflecting the direct influence of hsp72 on viral gene expression.

scholarly journals Quercetin can reduce viral RNA level of O’nyong-nyong virus and resulting innate immune cytokine responses in cultured human synovial fibroblasts

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Axelle Septembre-Malaterre ◽  
Yosra Bedoui ◽  
Claude Giry ◽  
Philippe Gasque ◽  
Pascale Guiraud ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Secretion ◽  
Chikungunya Virus ◽  
Antiviral Treatment ◽  
Synovial Fibroblasts ◽  
Chronic Arthritis ◽  
Innate Immune ◽  
Viral Rna ◽  
Inflammatory Status ◽  
Cytokine Responses

AbstractO’nyong-nyong virus is an alphavirus closely related to chikungunya virus, causing arthralgia, rash and fever. Alphaviruses mainly target synovial fibroblasts and persists in the joints of patients, possibly leading to chronic arthritis. To date, no specific antiviral treatment is available for ONNV infection and induced-inflammation. Primary human synovial fibroblasts cells were used to assess infection by ONNV and the resulting cytokine responses. Phenolics (gallic acid, caffeic acid and chlorogenic acid, curcumin and quercetin) and a curcuminoids-rich extract from turmeric were tested for their antiviral and anti-inflammatory capacities. We showed that infection occurred in HSF cells and increased gene expression and protein secretion of two major proinflammatory CCL-2 and IL-1β markers. In ONNV-infected HSF cells (MOI 1), we found that non-cytotoxic concentrations of phenolics (10 µM) reduced the level of viral RNA (E1, E2, nsP1, nsP2) and downregulated CCL-2 and IL-1β expression and secretion. These results highlighted the high value of the flavonol quercetin to reduce viral RNA levels and inflammatory status induced by ONNV in HSF cells.

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