Diese Arbeit wurde von der isländischen Research Fund und der Universität von Island Research Fund gefördert.

1. Transfektion Die molekulare Klon ist in zwei Plasmiden, p8XSp5-EGFP und p67r, von 12 kb und 4,5 kb bzw. enthalten. 9,10 <ol><li> Cut äquimolaren Mengen der beiden Plasmide, dh 4,4 pg p8XSp5-eGFP und 1,6 ug p67r mit XbaI und ligieren vor der Transfektion. </li><li> Für die Ligation, verwenden Sie 1 Weiss Einheit Ligase in einem 50 ul Reaktions-und Inkubieren bei 16 ° C über Nacht. </li><li> Wenn Ligation abgeschlossen ist, inkubieren Ligationsmix bei 65 ° C für 15 min zur Inaktivierung der Ligase. Dieser Schritt kann weggelassen werden. </li><li> Zwei Tage vor der Transfektion, Saatgut 10 6 SCP Zellen in einer T25 Zellkulturflasche und wachsen Zellen für zwei Tage in 5 ml DMEM + 10% Lamm Serum und 2 mM Glutamin, ohne Antibiotika, um eine Monoschicht von 90% Konfluenz. Wenn die Zellen nicht 90% Konfluenz nach zwei Tagen erreicht, lassen die Zellen wachsen einem zusätzlichen Tag. </li></ol> Hinweis: Das Lamm Serum kann durch fötales Rinderserum (FBS) ersetzt werden. Wir verwenden routinemäßig Lamm Serum, da einige Stämme von MVV von FBS sind gesperrt; dieser MVV Stamm ist nicht gehemmt durch FBS jedoch. <ol start="5"><li> Wenn die Zellen bereit für die Transfektion sind, ändern Sie mittel-bis DMEM mit 1% Serum (Lamm Serum oder FBS) ohne Antibiotika. Wir verwenden Lipofectamin 2000 (Invitrogen) für die Transfektion. </li><li> Verdünnen Sie die DNA in 500μl Opti-MEM-Medium. </li><li> Verdünnen Sie 20 &amp;mgr; l Lipofectamin 2000 in 500μl Opti-MEM-Medium und lassen bei Raumtemperatur (RT) für 5 min. </li><li> Nach dem 5 min Inkubation, kombinieren die verdünnte DNA mit verdünnter Lipofectamine 2000 (Gesamtvolumen = 1000 ul). Vorsichtig mischen und inkubieren für 20-30 min bei RT. </li><li> Fügen Sie die Transfektion Mix (1 ml) der T25 Kolben mit SCP Zellen und Medium (5 ml). Mix von rocking sanft. </li><li> Bei 37 ° C und 5% CO 2 über Nacht (18 bis 24 h). </li><li> Ändern Medium am folgenden Tag um DMEM mit 1% Serum, 2 mM Glutamin, Penicillin und 100IU 100IU Streptomycin. </li><li> Überwachen der Herstellung von GFP in der SCP-Zellen durch invertierte Fluoreszenz-Mikroskopie. 15% - Niedrige Transfektionseffizienz können mit diesen primären Zellen, in der Regel 5 zu erwarten. </li><li> Inkubieren bei 37 ° C, 5% CO 2 für mehrere Tage, damit die Verbreitung des Virus in den Kolben. Es dauert in der Regel 7-14 Tage für volle Infektion. </li><li> Ernte des Virus durch Aliquotierung der Überstand in Eppendorf-Röhrchen. Spin in einer Mikrozentrifuge bei 3000 rpm für 5 min um Zelltrümmer zu entfernen. Übertragen Sie den Überstand mit dem Virus auf neue Röhren. Lagerung bei -80 ° C. </li></ol> 2. Titration von Viren <ol><li> Seed 3x10 4 SCP-Zellen in 100 ul DMEM Medium mit 10% Serum, 2 mM Glutamin, Penicillin und 100IU/ml 100IU/ml Streptomycin pro Well in einer 96-Well-Platte ergänzt. Bei 37 ° C, 5% CO 2. </li><li> Wenn die Zellen konfluent sind am folgenden Tag, ändern mittel-bis DMEM mit 1% Serum, 2 mM Glutamin, Penicillin und 100IU/ml 100IU/ml Streptomycin, 100 ul in jede Vertiefung. </li><li> Verdünnen Sie das Virus in folgender Weise: <ol><li> Add 180 ul DMEM mit 1% Serum, 2 mM Glutamin, Penicillin und 100IU/ml 100IU/ml Streptomycin pro Vertiefung zu 4 x 10 Wells in einer 96-Well-Rundboden-oder V-Bodenplatte. </li><li> Für die serielle Zehnfachverdünnungen in vierfacher Ausfertigung, mit 20 ul des Virus auf jeden 4wells in der ersten Spalte der Platte, und mit einem Vier-Kanal-Pipette Pipette auf und ab, um sicherzustellen, dass die Proben gründlich gemischt werden. Ändern Pipettenspitzen und Transfer 20 pl auf die nächsten 4 Brunnen und so weiter. Lassen Sie die letzten Brunnen ohne Virus für eine negative Kontrolle. </li></ol></li><li> 100 l des Virus Verdünnungen der SCP-Zellen und Medium. </li><li> Bei 37 ° C und 5% CO 2. Beachten Sie für die GFP-Fluoreszenz und zytopathischen Effekte für zwei Wochen. Zytopathischen Effekte bestehen aus Zellen immer gerundet und weniger transparent als normale Zellen mit Prozessen ausstreckt, endend mit mehrkernigen Zellen erscheinen und Zelltod. </li><li> Berechnen Virustiter (TCID50) mit der Reed Muench-Methode 11. </li></ol> 3. FACS-Analyse von infizierten Zellen Hinweis: Für die schnelle Kontrolle der Replikation des Virus, infizierten Zellen können mittels Durchflusszytometrie untersucht werden. <ol><li> Trypsinize infizierten Zellen (ca. 10 5), waschen 1x mit PBS und Resuspendieren in 500μl PBS. </li><li> Add 500 ul 4% Paraformaldehyd-Lösung (2% Endkonzentration) bei RT für 30 min inkubieren. </li><li> Pellet-Zellen in einer Mikrozentrifuge bei 3000 rpm für 5 min. </li><li> Waschen mit PBS 2x5 min und zu analysieren, auf einem FACScan-Analysator. Count 10.000 Veranstaltungen. </li></ol> 4. Repräsentative Ergebnisse: Dieser Virus sollte voll ansteckender und zu replizieren, um einen Titer von 10. Juni - 10 Juli TCID 50 / ml. Infizierte Zellen werden Fluoreszenz und kann det werdenected mittels Durchflusszytometrie und Fluoreszenzmikroskopie. In der Regel können GFP in mehr als 60% der Zellen in die infizierten Zellkulturen mittels Durchflusszytometrie (Abb. 1) festgestellt werden. Ebenso, wenn visualisiert durch Fluoreszenz-Mikroskopie, sind ein Großteil der Zellen fluoreszierende (Abb. 2A und B). <img src="/files/ftp_upload/3483/3483fig1.jpg" alt="Abbildung 1" /> Abbildung 1. FACScan Analyse der SCP-Zellen mit KV1772-eGFP Viren nach 7 Tagen der Infektion infiziert. <img src="/files/ftp_upload/3483/3483fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Phasenkontrast-Mikroskopie von SCP-Zellen mit KV1772-egfp nach 7 Tagen Inkubation (A) infiziert. Gleiche Zellen visualisiert mittels Fluoreszenzmikroskopie (B).

<ol>
	<li>Sigurdsson, B., Palsson, P. A., Grimsson, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna,+a+demyelinating+transmissable+disease+of+sheep.">Visna, a demyelinating transmissable disease of sheep.</a> J. Neuropathol. Exp. Neurol. 14, 389-403 (1957).</li><li>Gorrell, M. D., Brandon, M. R., Sheffer, D., Adams, R. J., Narayan, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovine+lentivirus+is+macrophagetropic+and+does+not+replicate+productively+in+T+lymphocytes.">Ovine lentivirus is macrophagetropic and does not replicate productively in T lymphocytes.</a> J. 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F., Vigne, R., Querat, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=dUTPase-minus+caprine+arthritis-encephalitis+virus+is+attenuated+for+pathogenesis+and+accumulates+G-to-A+substitutions.">dUTPase-minus caprine arthritis-encephalitis virus is attenuated for pathogenesis and accumulates G-to-A substitutions.</a> J. Virol. 71, 4522-4530 (1997).</li><li>Petursson, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visna+virus+dUTPase+is+dispensable+for+neuropathogenicity.">Visna virus dUTPase is dispensable for neuropathogenicity.</a> J. Virol. 72, 1657-1661 (1998).</li><li>Sigurdsson, B., Thormar, H., Palsson, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cultivation+of+visna+virus+in+tissue+culture.">Cultivation of visna virus in tissue culture.</a> Arch Gesamte Virusforsch. 10, 368-380 (1960).</li><li>Gudmundsdottir, H. S., Olafsdottir, K., Franzdottir, S. R., Andresdottir, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+characterization+of+an+infectious+molecular+clone+of+maedi-visna+virus+that+expresses+green+fluorescent+protein.">Construction and characterization of an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein.</a> J. Virol. Methods. 168, 98-102 (2010).</li><li>Andresson, O. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleotide+sequence+and+biological+properties+of+a+pathogenic+proviral+molecular+clone+of+neurovirulent+visna+virus.">Nucleotide sequence and biological properties of a pathogenic proviral molecular clone of neurovirulent visna virus.</a> Virology. 193, 89-105 (1993).</li><li>Skraban, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+occurring+mutations+within+39+amino+acids+in+the+envelope+glycoprotein+of+maedi-visna+virus+alter+the+neutralization+phenotype.">Naturally occurring mutations within 39 amino acids in the envelope glycoprotein of maedi-visna virus alter the neutralization phenotype.</a> J. Virol. 73, 8064-8072 (1999).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+method+of+estimating+fifty+percent+endpoints.">A simple method of estimating fifty percent endpoints.</a> American Journal of Hygiene. 27, 493-497 (1938).</li><li>Berkowitz, R. D., Ilves, H., Plavec, I., Veres, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+transfer+systems+derived+from+Visna+virus:+analysis+of+virus+production+and+infectivity.">Gene transfer systems derived from Visna virus: analysis of virus production and infectivity.</a> Virology. 279, 116-129 (2001).</li></ol>

Keine Interessenskonflikte erklärt.

Die GFP-exprimierenden molekularen Klon des MVV hier vorgestellten sollte für die Analyse der Host-Zell-Interaktionen und Pathogenität von MVV sowohl in vitro und in vivo. Das Virus nach 7-14 Tagen der Replikation erhalten hat zweifellos einige Mutationen aufgrund der Ungenauigkeit der reversen Transkriptase erworben. Allerdings gibt es Einschränkungen bei der Verwendung dieser Klon als Single-Cycle-Vektor. Einer ist, dass die Verwendung der Klon-Zellen von Schafen und Ziegen Herkunft beschränkt ist. Die Transfektio...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> DMEM </td><td> Gibco </td><td> 10938025 </td></tr><tr><td> Opti-MEM </td><td> Gibco </td><td> 51985018 </td></tr><tr><td> Lipofectamine 2000 </td><td> Invitrogen </td><td> 11668019 </td></tr></tbody></table>

propagating and detecting an infectious molecular clone of maedi-visna virus that expresses green fluorescent protein

Maedi-Visna Virus (MVV) ist ein Lentivirus von Schafen, was langsam fortschreitende interstitielle Pneumonie und Enzephalitis 1. Die primären Zielzellen von MVV in vivo gelten als der Monozyten-Linie 2 sein. Bestimmte Stämme von MVV können in andere Zelltypen zu replizieren, jedoch 3,4. Das grün fluoreszierende Protein ist ein häufig verwendeter Marker für die Untersuchung Lentiviren in lebenden Zellen. Wir haben die EGFP-Gen in das Gen für dUTPase der MVV eingefügt. Die dUTPase Gen ist in den meisten Lentivirus Stämme von Schafen und Ziegen erhalten und hat sich gezeigt, dass bei der Replikation von CAEV 5 wichtige. Allerdings hat dUTPase konnte gezeigt werden, entbehrlich für die Replikation des molekularen Klon des MVV in dieser Studie sowohl in vitro und in vivo 6 verwendet. MVV-Replikation ist streng auf Zellen von Schafen oder Ziegen Herkunft beschränkt. Wir verwenden eine primäre Zelllinie, die aus dem Plexus von Schafen (SCP-Zellen) für die Transfektion und Vermehrung der Virus 7. Die fluoreszierenden MVV ist voll Infektions-und EGFP-Expression über mindestens 6 Durchgänge 8 stabil. Es ist eine gute Korrelation zwischen den Messungen der TCID 50 und EGFP. Dieses Virus ist daher sinnvoll sein, für die schnelle Erkennung von infizierten Zellen in Studien Zelltropismus und Pathogenität in vitro und in vivo 8.

Watch this Scientific Journal Video about Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein at JoVE.com

Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Wir beschreiben einen molekularen Klon des Maedi-Visna Virus, das GFP exprimiert und ist voll ansteckend. Die Replikation von diesem Virus kann durch die Verwendung der Fluoreszenzmikroskopie und Durchflusszytometrie nachgewiesen werden.

Propagierung und Erkennen einer ansteckenden Molecular Clone von Maedi-Visna Virus, das Green Fluorescent Protein exprimiert

Maedi-visna virus (MVV) is a lentivirus of sheep, causing slowly progressive interstitial pneumonia and encephalitis1. The primary target cells of MVV in ...

propagating-detecting-an-infectious-molecular-clone-maedi-visna-virus

Research

JoVE Journal

Immunology and Infection

9.8K Aufrufe.  University of Iceland. Wir beschreiben einen molekularen Klon des Maedi-Visna Virus, das GFP exprimiert und ist voll ansteckend. Die Replikation von diesem Virus kann durch die Verwendung der Fluoreszenzmikroskopie und Durchflusszytometrie nachgewiesen werden.

Serie: Propagating and Detecting an Infectious Molecular Clone of Maedi-visna Virus that Expresses Green Fluorescent Protein

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

Spezielle Kennzeichnung von HIV-1 positiven Zellen mit Hilfe eines Rev-abhängigen Lentivirale Vector mit dem Ausdruck des Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Forschung

Immunologie und Infektion

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Nachweis von infektiösen Viren aus Field-gesammelt Moskitos von Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizing Dengue Virus durch Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

Live Cell Imaging von Alphaherpes Virus Anterograde Transport und Aufstrich

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Modellierung des Lebenszyklus von Ebola Virus unter Biosafety Level 2 Bedingungen mit Virus-ähnlichen Partikeln, die Tetracistronic Minigenome

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-Immunopräzipitation der Maus Mx1 Protein mit dem Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Einrichtung eines Setup-Hochdurchsatz-Screening für kleine Moleküle, die Modulate c-di-GMP-Signalisierung in<em&gt; Pseudomonas aeruginosa</em

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV is an RNA virus of the Flaviviridae family. ZIKV is mainly transmitted by mosquitoes, but can also be spread by maternal to fetal vertical transmission as well as sexual contact. To date, there are no reliable treatment or vaccine options available to protect those infected by the virus. The development of a reproducible, effective Zika virus infectious cell culture system is critical for studying the molecular mechanisms of ZIKV replication as well as drug and vaccine development. In this regard, a protocol describing a mammalian cell-based in vitro Zika virus culture system for viral production and growth analysis is reported here. Details on the formation of plaques by Zika virus on a cell monolayer and plaque assay for measuring viral titer are presented. Viral genome replication kinetics and double-stranded RNA genome replicatory intermediates are determined. This culture platform was utilized to screen against a library of a small set of cytokines resulting in the identification of interferon-&#945; (IFN-&#945;), IFN-&#946; and IFN-&#947; as potent inhibitors of Zika viral growth. In summary, an in vitro infectious Zika viral culture system and various virological assays are demonstrated in this study, which has the potential to greatly benefit the research community in elucidating further the mechanisms of viral pathogenesis and the evolution of viral virulence. Antiviral IFN-alpha can further be evaluated as a prophylactic, post-exposure prophylactic, and treatment option for Zika virus infections in high-risk populations, including infected pregnant women.

Zika Virus Infectious Cell Culture System and the In Vitro Prophylactic Effect of Interferons

Zika Virus (ZIKV), an emerging pathogen, is linked to fetal developmental abnormalities and microcephaly. The establishment of an effective infectious cell culture system is crucial for studies of ZIKV replication as well as vaccine and drug development. In this study, various virological assays pertaining to ZIKV are illustrated and discussed.

Zika Virus Infectious Zellkultursystem und die<em&gt; In Vitro</em&gt; Prophylaktische Wirkung von Interferonen

Zika Virus (ZIKV) is an emerging pathogen that is linked to fetal developmental abnormalities such as microcephaly, eye defects, and impaired growth. ZIKV ...

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in the caseous cores of cavities and lesions is especially crucial because they harbor subpopulations of drug-tolerant bacteria also commonly referred to as persisters. Existing methods for the measurement of drug penetration in tuberculosis lesions involve costly and time-consuming in vivo pharmacokinetic studies coupled to bioanalytical or imaging techniques. The in vitro measurement of drug binding to caseum macromolecules was proposed as an alternative to such techniques since this binding hinders the passive diffusion of drug molecules through caseum. Rapid equilibrium dialysis is a fast and reliable system for performing plasma protein and tissue binding studies. In this protocol, we used a rapid equilibrium dialysis (RED) device to measure drug binding to homogenates of caseum that is excised from the lesions and cavities of tuberculosis-infected rabbits. The protocol also describes how to generate a surrogate matrix from lipid loaded THP-1 macrophages to use in place of caseum. This caseum/surrogate binding assay is an important tool in tuberculosis drug discovery and can be adapted to help study drug distribution in lesions or abscesses caused by other diseases.

An In Vitro Caseum Binding Assay that Predicts Drug Penetration in Tuberculosis Lesions

Here we describe a rapid equilibrium dialysis (RED) method to measure drug binding to caseum from pulmonary tuberculosis lesions and cavities. The protocol is also used with a foamy macrophage-derived matrix that is an effective surrogate to caseum.

Ein<em&gt; In Vitro</em&gt; Caseum-Bindungsassay, der die Drug Penetration in Tuberkulose-Läsionen vorhersagt

The eradication of tuberculosis disease requires drug regimens that can penetrate the multiple layers of complex pulmonary lesions. Drug distribution in ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Rettung und Charakterisierung des rekombinanten Virus aus einer neuen Welt Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...