Ce travail a été financé par le Fonds de recherche en islandais et l&#39;Université d&#39;Islande Fonds pour la recherche.

1. La transfection Le clone moléculaire est contenue dans deux plasmides, p8XSp5-EGFP et p67r, de 12 kb et 4,5 kb, respectivement 9,10. <ol><li> Couper des quantités équimolaires de deux plasmides, soit 4,4 mg d&#39;p8XSp5-EGFP et 1,6 mg d&#39;p67r par XbaI et ligaturer avant la transfection. </li><li> Pour la ligature, utilisez une unité de Weiss de ligase dans une réaction de 50 ul et incuber à 16 ° C pendant la nuit. </li><li> Lorsque la ligature est terminée, incuber le mélange de ligature à 65 ° C pendant 15 min pour l&#39;inactivation de la ligase. Cette étape peut être omise. </li><li> Deux jours avant la transfection, les graines de 10 6 cellules SCP dans un flacon de culture de tissu T25, et cultiver des cellules pour deux jours dans 5 ml de DMEM + 10% de sérum d&#39;agneau et la glutamine 2 mM, sans antibiotiques pour une monocouche de 90% de confluence. Si les cellules n&#39;ont pas atteint 90% de confluence, après deux jours, laissez les cellules à croître, une journée supplémentaire. </li></ol> Remarque: Le sérum d&#39;agneau peut être remplacé par du sérum de veau fœtal (FBS). Nous utilisons régulièrement l&#39;agneau du sérum, car certaines souches de MVV sont inhibées par FBS; cette souche MVV n&#39;est pas inhibée par FBS, cependant. <ol start="5"><li> Lorsque les cellules sont prêtes pour la transfection, le changement moyen du DMEM avec du sérum de 1% (sérum ou d&#39;agneau FBS) sans antibiotiques. Nous utilisons Lipofectamin 2000 (Invitrogen) pour la transfection. </li><li> Diluer l&#39;ADN dans 500μl milieu Opti-MEM. </li><li> Diluer 20 pi Lipofectamin 2000 dans 500μl milieu Opti-MEM et laisser à température ambiante (TA) pendant 5 min. </li><li> Après l&#39;incubation de 5 min, mélanger l&#39;ADN dilué avec Lipofectamine diluée 2000 (volume total = 1000 pl). Mélanger doucement et incuber pendant 20-30 min à température ambiante. </li><li> Ajouter le mélange de transfection (1 ml) à la fiole contenant des cellules T25 SCP et moyennes entreprises (5 ml). Mélanger en berçant doucement. </li><li> Incuber à 37 ° C et 5% de CO 2 durant la nuit (18 - 24 h). </li><li> Changer moyennes le jour suivant pour DMEM avec du sérum de 1%, 2 mM de glutamine, pénicilline et streptomycine 100IU 100IU. </li><li> Surveiller la production de la GFP dans les cellules par microscopie fluorescente SCP inversé. Faible efficacité de la transfection peut s&#39;attendre avec ces cellules primaires, généralement de 5 - 15%. </li><li> Incuber encore à 37 ° C, 5% de CO 2 pendant plusieurs jours afin de permettre la propagation du virus dans le ballon. Il prend habituellement 7-14 jours pour une infection complet. </li><li> Récolte du virus par aliquotage le surnageant dans des tubes Eppendorf. Spin dans une microcentrifugeuse à 3000 rpm pendant 5 min pour éliminer les débris cellulaires. Transférer le surnageant contenant le virus à de nouveaux tubes. Conserver à -80 ° C. </li></ol> 2. Titrage du virus <ol><li> Graine 3x10 4 cellules SCP dans 100 pl de milieu DMEM supplémenté avec 10% de sérum, 2 mM de glutamine, pénicilline et streptomycine 100IU/ml 100IU/ml, par puits dans une plaque à 96 puits. Incuber à 37 ° C, 5% de CO 2. </li><li> Si les cellules sont confluentes le lendemain, changement de milieu de DMEM avec 1% de sérum, 2 mM de glutamine, pénicilline et streptomycine 100IU/ml 100IU/ml, 100 ul de chaque puits. </li><li> Diluer le virus de la façon suivante: <ol><li> Ajouter 180 pl de DMEM avec du sérum de 1%, 2 mM de glutamine, pénicilline et streptomycine 100IU/ml 100IU/ml par puits à 4 x 10 puits dans un fond de 96 puits rond ou V-bottom assiette. </li><li> Pour des dilutions en série décuplé en quatre exemplaires, ajouter 20 ul de virus à chaque 4wells dans la première colonne de la plaque, et en utilisant une pipette à quatre canaux, pipette haut et bas pour s&#39;assurer que les échantillons soient bien mélangés. Changement des embouts de pipette et transférer 20 ul de la 4 prochaines puits et ainsi de suite. Laissez les derniers puits sans virus pour un contrôle négatif. </li></ol></li><li> Ajouter 100 ml de la dilution du virus aux cellules SCP et moyennes entreprises. </li><li> Incuber à 37 ° C et 5% de CO 2. Observer la fluorescence de la GFP et les effets cytopathiques pendant deux semaines. Effets cytopathiques sont constitués de cellules deviennent arrondies et moins transparents que les cellules normales avec les processus qui s&#39;étend, en terminant par les cellules apparaissant multinucléaire et la mort cellulaire. </li><li> Calculer le virus de titre (DICT50) en utilisant la méthode de Reed Muench 11. </li></ol> 3. Analyse FACS des cellules infectées Note: Pour le suivi rapide de la réplication du virus, les cellules infectées peuvent être examinés par cytométrie en flux. <ol><li> Trypsiniser cellules infectées (environ 10 5), laver avec du PBS 1x et de remettre en suspension dans 500μl de PBS. </li><li> Ajouter 500 ul solution de paraformaldéhyde à 4% (2% en concentration finale), incuber à température ambiante pendant 30 min. </li><li> Pellet cellules dans une microcentrifugeuse à 3000 rpm pendant 5 min. </li><li> Laver avec du PBS 2x5 min et d&#39;analyser sur un analyseur FACScan. Comptez 10 000 événements. </li></ol> 4. Les résultats représentatifs: Ce virus devrait être pleinement infectieuses et de répliquer à un titre de 10 juin à 10 juillet DICT 50 / ml. Les cellules infectées sont fluorescentes et peuvent être détecté par cytométrie en flux et microscopie à fluorescence. Typiquement, la GFP peut être détecté dans plus de 60% des cellules dans les cultures de cellules infectées en utilisant la cytométrie de flux (Fig. 1). De même, lorsque visualisé par microscopie à fluorescence, la majorité des cellules sont fluorescentes (figure 2A et B). <img src="/files/ftp_upload/3483/3483fig1.jpg" alt="Figure 1" /> Figure 1. Analyse FACScan de cellules infectées par le SCP KV1772-EGFP virus après 7 jours d&#39;infection. <img src="/files/ftp_upload/3483/3483fig2.jpg" alt="Figure 2" /> Figure 2. Microscopie à contraste de phase de cellules infectées par le SCP KV1772-EGFP après 7 jours d&#39;incubation (A). Mêmes cellules visualisées par microscopie à fluorescence (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. Virol. 66, 2679-2688 (1992).</li><li>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=Biological+and+genetic+differences+between+lung-+and+brain-derived+isolates+of+maedi-visna+virus.">Biological and genetic differences between lung- and brain-derived isolates of maedi-visna virus.</a> Virus Genes. 16, 281-293 (1998).</li><li>Georgsson, G., Houwers, D. J., Palsson, P. A., 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=Expression+of+viral+antigens+in+the+central+nervous+system+of+visna-infected+sheep:+an+immunohistochemical+study+on+experimental+visna+induced+by+virus+strains+of+increased+neurovirulence.">Expression of viral antigens in the central nervous system of visna-infected sheep: an immunohistochemical study on experimental visna induced by virus strains of increased neurovirulence.</a> Acta Neuropathol. 77, 299-306 (1989).</li><li>Turelli, P., Guiguen, F., Mornex, J. 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>

Aucun conflit d&#39;intérêt déclaré.

La GFP exprimer clone moléculaire de MVV présentés ici devraient être utiles pour analyser les interactions de la cellule hôte et la pathogénicité du MVV fois in vitro et in vivo. Le virus obtenu après 7-14 jours de la réplication a incontestablement acquis une certaine mutations dues à l&#39;imprécision de la transcriptase inverse. Cependant, il ya des limites à l&#39;utilisation de ce clone comme un vecteur seul cycle. La première est que l&#39;utilisation du clone est restreinte aux cellules de moutons e...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </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 (MVV) est un lentivirus de moutons, provoquant lentement progressive pneumonie interstitielle et d&#39;encéphalite 1. Les cellules cibles primaires du MVV in vivo sont considérés comme des 2 lignée des monocytes. Certaines souches de MVV peuvent se reproduire dans d&#39;autres types cellulaires, toutefois 3,4. La protéine fluorescente verte est un marqueur couramment utilisé pour étudier les lentivirus dans des cellules vivantes. Nous avons inséré le gène EGFP dans le gène codant pour dUTPase du MVV. Le gène dUTPase est bien conservée dans la plupart des souches de lentivirus des ovins et caprins et a été montré pour être important dans la réplication du CAEV 5. Toutefois, dUTPase a été montré pour être dispensable pour la réplication du clone moléculaire de MVV utilisées dans cette étude in vitro et in vivo 6. Réplication MVV est strictement limitée aux cellules de mouton ou de chèvre d&#39;origine. Nous utilisons une lignée de cellules primaires de la choroïde de mouton du plexus (cellules SCP) pour la transfection et la propagation du virus 7. La MVV fluorescente est entièrement infectieuses et expression de l&#39;EGFP est stable pendant au moins six passages 8. Il ya une bonne corrélation entre les mesures de DICT 50 et EGFP. Ce virus devrait donc être utile pour la détection rapide des cellules infectées dans les études du tropisme cellulaire et la pathogénicité in vitro et 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

Nous décrivons un clone moléculaire du maedi-visna qui exprime la GFP et est entièrement infectieuses. La réplication de ce virus peut être détecté en utilisant la microscopie par fluorescence et la cytométrie de flux.

Détection de multiplication et un clone moléculaire infectieux du virus Maedi-visna qui exprime Green Fluorescent Protein

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 vues.  University of Iceland. Nous décrivons un clone moléculaire du maedi-visna qui exprime la GFP et est entièrement infectieuses. La réplication de ce virus peut être détecté en utilisant la microscopie par fluorescence et la cytométrie de flux.

Vidéo: 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.

Marquage spécifique du VIH-1 des cellules positives à l&#39;aide d&#39;un vecteur Rev-dépendante lentiviraux exprimant la protéine fluorescente verte

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, ...

Recherche

Immunologie et infection

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.

La détection de virus infectieux à partir terrain recueillies par les moustiques Assay culture cellulaire Vero

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.

Visualisation virus de la dengue grâce à Alexa Fluor Étiquetage

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.

Imagerie de cellules vivantes de Alphaherpes Virus Transport antérograde et la propagation

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.

Modélisation du cycle de vie du virus Ebola Sous biosécurité de niveau 2 Conditions de particules de type viral contenant Tetracistronic minigénomes

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-immunoprécipitation de la protéine souris Mx1 avec le virus Influenza A nucléoprotéine

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.

Mise en place d&#39;un programme d&#39;installation à haut débit pour le criblage de petites molécules qui modulent c-di-GMP Signalisation dans<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 système infectieuse Culture cellulaire et la<em&gt; In vitro</em&gt; Effet prophylactique des interférons

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.

Un<em&gt; In Vitro</em&gt; Ensemble de liaison de caséine qui prédit la pénétration des drogues dans les lésions tuberculeuses

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.

Le sauvetage et la caractérisation du virus recombinant à partir d&#39;un nouveau clone infectieux du virus Zika du monde

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