scholarly journals Generation of a GFP Reporter Akabane Virus with Enhanced Fluorescence Intensity by Modification of Artificial Ambisense S Genome

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 634
Author(s):  
Akiko Takenaka-Uema ◽  
Shin Murakami ◽  
Nanako Ushio ◽  
Tomoya Kobayashi-Kitamura ◽  
Masashi Uema ◽  
...  
Keyword(s):  
Imaging Study ◽  
Green Fluorescence Protein ◽  
Histopathological Study ◽  
Akabane Virus ◽  
Gfp Reporter ◽  
The Central Nervous System ◽  
Infected Cells ◽  
Fluorescence Protein

We previously generated a recombinant reporter Akabane virus expressing enhanced green fluorescence protein (eGFP-AKAV), with an artificial S genome encoding eGFP in the ambisense RNA. Although the eGFP-AKAV was able to detect infected cells in in vivo histopathological study, its fluorescent signal was too weak to apply to in vivo imaging study. Here, we successfully generated a modified reporter, eGFP/38-AKAV, with 38-nucleotide deletion of the internal region of the 5′ untranslated region of S RNA. The eGFP/38-AKAV expressed higher intensity of eGFP fluorescence both in vitro and in vivo than the original eGFP-AKAV did. In addition, eGFP/38-AKAV was pathogenic in mice at a comparable level to that in wild-type AKAV. In the mice infected with eGFP/38-AKAV, the fluorescent signals, i.e., the virus-infected cells, were detected in the central nervous system using the whole-organ imaging. Our findings indicate that eGFP/38-AKAV could be used as a powerful tool to help elucidate the dynamics of AKAV in vivo.

scholarly journals The Feasibility of Pressurised Intraperitoneal Aerosolised Virotherapy (PIPAV) to Administer Oncolytic Adenoviruses

Pharmaceutics ◽  
2021 ◽  
Vol 13 (12) ◽  
pp. 2043
Author(s):  
Sophia J. Tate ◽  
Leen Van de Sande ◽  
Wim P. Ceelen ◽  
Jared Torkington ◽  
Alan L. Parker
Keyword(s):  
Anticancer Agents ◽  
Treatment Options ◽  
Intraperitoneal Administration ◽  
Green Fluorescence Protein ◽  
Firefly Luciferase ◽  
Oncolytic Adenoviruses ◽  
Poor Treatment ◽  
Fluorescence Protein

Background: The prognosis of patients with peritoneal metastases is poor. Treatment options are limited because systemically delivered chemotherapy is not usually effective in this type of disease. Pressurised intraperitoneal aerosolised chemotherapy (PIPAC) is a recently developed alternative technology for delivering intraperitoneal chemotherapy, potentially enhancing treatment efficacy. Here, we assess the feasibility of pressurised intraperitoneal aerosolised virotherapy (PIPAV) to deliver a different class of anticancer agents, oncolytic adenoviruses, in vitro and in vivo. Methods: Adenoviral vectors expressing reporter genes green fluorescence protein (Ad5.GFP) or firefly luciferase (Ad5.Luc) were subject to pressurised aerosolisation. The ability of the virus to survive PIPAV was assessed in vitro and in vivo by monitoring reporter gene activity. Wistar rats subjected to PIPAV were assessed for any adverse procedure related events. Results: In vitro transduction assays demonstrated that Ad5 retained viability following pressurised aerosolisation and could transduce permissive cells equally effectively as non-aerosolised control vector. PIPAV was well tolerated in rats, although minimal transduction was observed following intraperitoneal administration. Conclusions: PIPAV appears viable and well tolerated, though in vivo efficacy requires further optimisation.

scholarly journals Sugar Functionalized Synergistic Dendrimers for Biocompatible Delivery of Nucleic Acid Therapeutics

Polymers ◽  
2018 ◽  
Vol 10 (9) ◽  
pp. 1034 ◽  
Author(s):  
Shuqin Han ◽  
Tsogzolmaa Ganbold ◽  
Qingming Bao ◽  
Takashi Yoshida ◽  
Huricha Baigude
Keyword(s):  
Nucleic Acid ◽  
Sirna Delivery ◽  
Green Fluorescence Protein ◽  
Nucleic Acid Delivery ◽  
Cationic Polymers ◽  
Nucleic Acid Therapeutics ◽  
Fluorescence Protein ◽  
Interfering Rna

Sugars containing cationic polymers are potential carriers for in vitro and in vivo nucleic acid delivery. Monosaccharides such as glucose and galactose have been chemically conjugated to various materials of synergistic poly-lysine dendrimer systems for efficient and biocompatible delivery of short interfering RNA (siRNA). The synergistic dendrimers, which contain lipid conjugated glucose terminalized lysine dendrimers, have significantly lower adverse impact on cells while maintaining efficient cellular entry. Moreover, the synergistic dendrimers complexed to siRNA induced RNA interference (RNAi) in the cells and profoundly knocked down green fluorescence protein (GFP) as well as the endogenously expressing disease related gene Plk1. The new synergic dendrimers may be promising system for biocompatible and efficient siRNA delivery.

scholarly journals Coronavirus induction of class I major histocompatibility complex expression in murine astrocytes is virus strain specific.

1994 ◽  
Vol 180 (3) ◽  
pp. 1013-1023 ◽  
Author(s):  
W Gilmore ◽  
J Correale ◽  
L P Weiner
Keyword(s):  
Acute Infection ◽  
Demyelinating Diseases ◽  
Class I ◽  
Virus Infectivity ◽  
Cell Functions ◽  
The Central Nervous System ◽  
Infected Cells ◽  
The Individual

Neurotropic strains of mouse hepatitis viruses (MHV) such as MHV-A59 (A59) and MHV-4 (JHMV) cause acute and chronic encephalomyelitis and demyelination in susceptible strains of mice and rats. They are widely used as models of human demyelinating diseases such as multiple sclerosis (MS), in which immune mechanisms are thought to participate in the development of lesions in the central nervous system (CNS). The effects of MHV infection on target cell functions in the CNS are not well understood, but A59 has been shown to induce the expression of MHC class I molecules in glial cells after in vivo and in vitro infection. Changes in class I expression in infected cells may contribute to the immunopathogenesis of MHV infection in the CNS. In this communication, a large panel of MHV strains was tested for their ability to stimulate class I expression in primary astrocytes in vitro. The data show that the more hepatotropic strains, such as MHV-A59, MHV-1, MHV-2, MHV-3, MHV-D, MHV-K, and MHV-NuU, were potent inducers of class I expression in astrocytes during acute infection, measured by radioimmunoassay. The Kb molecule was preferentially expressed over Db. By contrast, JHMV and several viral strains derived from it did not stimulate the expression of class I molecules. Assays of virus infectivity indicated that the class I-inducing activity did not correlate with the ability of the individual viral strain to replicate in astrocytes. However, exposure of the viruses or the supernatants from infected astrocytes to ultraviolet light abolished the class I-inducing activity, indicating that infectious virus is required for class I expression. These data also suggest that class I expression was induced directly by virus infection, and not by the secretion of a soluble substance into the medium by infected astrocytes. Finally, analyses of A59/JHMV recombinant viral strains suggest that class I-inducing activity resides in one of the A59 structural genes.

Thiamine transport in the central nervous system

1976 ◽  
Vol 230 (4) ◽  
pp. 1101-1107 ◽  
Author(s):  
R Spector
Keyword(s):  
Choroid Plexus ◽  
Intraventricular Injection ◽  
Simple Diffusion ◽  
Thiamine Transport ◽  
Choroid Plexuses ◽  
The Central Nervous System ◽  
The Brain ◽  
Thiamine Phosphates

Total thiamine (free thiamine and thiamine phosphates) transport into the cerebrospinal fluid (CSF), brain, and choroid plexus and out of the CSF was measured in rabbits. In vivo, total thiamine transport into CSF, choroid plexus, and brain was saturable. At the normal plasma total thiamine concentration, less than 5% of total thiamine entry into CSF, choroid plexus, and brain was by simple diffusion. The relative turnovers of total thiamine in choroid plexus, whole brain, and CSF were 5, 2, and 14% per h, respectively, when measured by the penetration of 35S-labeled thiamine injected into blood. From the CSF, clearance of [35S]thiamine relative to mannitol was not saturable after the intraventricular injection of various concentrations of thiamine. However, a portion of the [35S]thiamine cleared from the CSF entered brain by a saturable mechanism. In vitro, choroid plexuses, isolated from rabbits and incubated in artificial CSF, accumulated [35S]thiamine against a concentration gradient by an active saturable process that did not depend on pyrophosphorylation of the [35S]thiamine. The [35S]thiamine accumulated within the choroid plexus in vitro was readily released. These results were interpreted as showing that the entry of total thiamine into the brain and CSF from blood is regulated by a saturable transport system, and that the locus of this system may be, in part, in the choroid plexus.

scholarly journals In Vivo and In Vitro Toxicodynamic Analyses of New Quinolone-and Nonsteroidal Anti-Inflammatory Drug-Induced Effects on the Central Nervous System

1999 ◽  
Vol 43 (5) ◽  
pp. 1091-1097 ◽  
Author(s):  
Hideki Kita ◽  
Hirotami Matsuo ◽  
Hitomi Takanaga ◽  
Junichi Kawakami ◽  
Koujirou Yamamoto ◽  
...  
Keyword(s):  
Brain Tissue ◽  
Threshold Concentration ◽  
Current Response ◽  
Drug Induced ◽  
Inhibition Ratio ◽  
New Quinolones ◽  
The Central Nervous System ◽  
Anti Inflammatory

ABSTRACT We investigated the correlation between an in vivo isobologram based on the concentrations of new quinolones (NQs) in brain tissue and the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) for the occurrence of convulsions in mice and an in vitro isobologram based on the concentrations of both drugs for changes in the γ-aminobutyric acid (GABA)-induced current response in Xenopus oocytes injected with mRNA from mouse brains in the presence of NQs and/or NSAIDs. After the administration of enoxacin (ENX) in the presence or absence of felbinac (FLB), ketoprofen (KTP), or flurbiprofen (FRP), a synergistic effect was observed in the isobologram based on the threshold concentration in brain tissue between mice with convulsions and those without convulsions. The three NSAIDs did not affect the pharmacokinetic behavior of ENX in the brain. However, the ENX-induced inhibition of the GABA response in the GABAA receptor expressed in Xenopus oocytes was enhanced in the presence of the three NSAIDs. The inhibition ratio profiles of the GABA responses for both drugs were analyzed with a newly developed toxicodynamic model. The inhibitory profiles for ENX in the presence of NSAIDs followed the order KTP (1.2 μM) > FRP (0.3 μM) > FLB (0.2 μM). These were 50- to 280-fold smaller than those observed in the absence of NSAIDs. The inhibition ratio (0.01 to 0.02) of the GABAA receptor in the presence of both drugs was well-fitted to the isobologram based on threshold concentrations of both drugs in brain tissue between mice with convulsions and those without convulsions, despite the presence of NSAIDs. In mice with convulsions, the inhibitory profiles of the threshold concentrations of both drugs in brain tissue of mice with convulsions and those without convulsions can be predicted quantitatively by using in vitro GABA response data and toxicodynamic model.

Studies of two temperature-sensitive mutants of Mengo virus

1979 ◽  
Vol 57 (6) ◽  
pp. 902-913 ◽  
Author(s):  
Patrick W. K. Lee ◽  
John S. Colter
Keyword(s):  
Rna Synthesis ◽  
Viral Rna ◽  
Temperature Sensitive ◽  
Supporting Evidence ◽  
Structural Polypeptide ◽  
Infected Cells ◽  
Mengo Virus ◽  
In Vitro Stability

Studies of the synthesis of viral ribonucleates and polypeptides in cells infected with two RNA−ts mutants of Mengo virus (ts 135 and ts 520) have shown that when ts 135 infected cells are shifted from the permissive (33 °C) to the nonpermissive (39 °C) temperature: (i) the synthesis of all three species of viral RNA (single stranded, replicative form, and replicative intermediate) is inhibited to about the same extent, and (ii) the posttranslational cleavage of structural polypeptide precursors A and B is partially blocked. Investigations of the in vivo and in vitro stability of the viral RNA replicase suggest that the RNA− phentotype reflects a temperature-sensitive defect in the enzyme. The second defect does not appear to result from the inhibition of viral RNA synthesis at 39 °C, since normal cleavage of polypeptides A and B occurs in wt Mengo-infected cells in which viral RNA synthesis is blocked by cordycepin, and at the nonpermissive temperature in ts 520 infected cells. Considered in toto, the evidence suggests that ts 135 is a double mutant.Subviral (53 S) particles have been shown to accumulate in ts 520 (but not ts 135) infected cells when cultures are shifted from 33 to 39 °C. This observation provides supporting evidence for the proposal that this recently discovered particle is an intermediate in the assembly pathway of Mengo virions.

scholarly journals Use of a Beet necrotic yellow vein virus RNA-5-derived replicon as a new tool for gene expression

2005 ◽  
Vol 86 (2) ◽  
pp. 463-467 ◽  
Author(s):  
Laure Schmidlin ◽  
Didier Link ◽  
Jérôme Mutterer ◽  
Hubert Guilley ◽  
David Gilmer
Keyword(s):  
Gene Expression ◽  
Recombinant Proteins ◽  
Expression System ◽  
Green Fluorescence Protein ◽  
Yellow Vein ◽  
Expression Levels ◽  
Virus Rna ◽  
New Gene ◽  
Infected Cells ◽  
Fluorescence Protein

A new gene-expression system based on RNA-5 of Beet necrotic yellow vein virus (BNYVV) was constructed to allow the expression of recombinant proteins in virally infected cells. Replication and expression levels of the RNA-5-based replicon containing the green fluorescence protein (GFP) gene were compared with those obtained with the well-characterized RNA-3-derived replicon (Rep-3). When RNA-3 and/or RNA-4 BNYVV RNAs were added to the inoculum, the expression levels of RNA-5-encoded GFP were considerably reduced. To a lesser extent, RNA-3-derived GFP expression was also affected by the presence of RNA-4 and -5. Both RNA-3- and RNA-5-derived molecules were able to express proteins within the same infected cells. Together with Rep-3, the RNA-5-derived replicon thus provides a new tool for the co-expression of different recombinant proteins. In Beta macrocarpa, Rep-5-GFP was able to move in systemic tissues in the presence of RNA-3 and thus provides a new expression system that is not restricted to the inoculated leaves.

scholarly journals Analysis of the Interaction of the Adenovirus L1 52/55-Kilodalton and IVa2 Proteins with the Packaging Sequence In Vivo and In Vitro

2005 ◽  
Vol 79 (4) ◽  
pp. 2366-2374 ◽  
Author(s):  
Pilar Perez-Romero ◽  
Ryan E. Tyler ◽  
Johanna R. Abend ◽  
Monica Dus ◽  
Michael J. Imperiale
Keyword(s):  
Viral Protein ◽  
Direct Interaction ◽  
Dna Interaction ◽  
The Other ◽  
Infected Cells ◽  
In Vitro Interaction ◽  
Packaging Sequence

ABSTRACT We previously showed that the adenovirus IVa2 and L1 52/55-kDa proteins interact in infected cells and the IVa2 protein is part of two virus-specific complexes (x and y) formed in vitro with repeated elements of the packaging sequence called the A1-A2 repeats. Here we demonstrate that both the IVa2 and L1 52/55-kDa proteins bind in vivo to the packaging sequence and that each protein-DNA interaction is independent of the other. There is a strong and direct interaction of the IVa2 protein with DNA in vitro. This interaction is observed when probes containing the A1-A2 or A4-A5 repeats are used, but it is not found by using an A5-A6 probe. Furthermore, we show that complex x is likely a heterodimer of IVa2 and an unknown viral protein, while complex y is a monomer or multimer of IVa2. No in vitro interaction of purified L1 52/55-kDa protein with the packaging sequence was found, suggesting that the L1 52/55-kDa protein-DNA interaction may be mediated by an intermediate protein. Results support roles for both the L1 52/55-kDa and IVa2 proteins in DNA encapsidation.

Detection of Circulating Immune Complexes in the Sera of Rabbits with Experimental Syphilis: Possible Role in Immunoregulation

1980 ◽  
Vol 29 (2) ◽  
pp. 575-582
Author(s):  
Robert E. Baughn ◽  
Kenneth S. K. Tung ◽  
Daniel M. Musher
Keyword(s):  
Immune Complexes ◽  
Proteolytic Enzymes ◽  
Suppressor Cell ◽  
Suppressive Effect ◽  
Circulating Immune Complexes ◽  
Time Period ◽  
Infected Cells ◽  
Disseminated Disease

The in vivo and in vitro immunoglobulin G plaque-forming cell responses to sheep erythrocytes (SRBC) are nearly obliterated during disseminated syphilitic infection (3 to 8 weeks post-intravenous injection) in rabbits. Splenic and lymph node cells obtained from infected rabbits during this time period were capable of suppressing the normal in vitro responses of uninfected, SRBC-primed cells. Cell-free washings of cells from infected animals were also suppressive. This finding coupled with the fact that treatment of infected cells with proteolytic enzymes abrogated the suppressive effect constitute arguments against involvement of a specific suppressor cell population. The incidence of elevated levels of circulating immune complexes in the sera of rabbits with disseminated disease was also significantly different from that of uninfected controls or infected rabbits before the onset or after the regression of lesions. When added to cultures of lymphocytes from uninfected, SRBC-sensitized rabbits, sera containing complexes caused dose-related suppression of the in vitro immunoglobulin responses. Unlike immune complexes, no correlation was found between the presence of mucopolysaccharide materials and the stage of infection or the ability of serum to suppress the immunoglobulin responses to SRBC.

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