Questo lavoro è stato finanziato dal Fondo di ricerca islandese e l&#39;Università di Fondo Islanda ricerca.

1. Trasfezione Il clone molecolare è contenuto in due plasmidi, p8XSp5-EGFP e p67r, di 12 kb e 4,5 kb rispettivamente. 9,10 <ol><li> Tagliare quantità equimolare di due plasmidi, ovvero 4,4 mg di p8XSp5-EGFP e 1,6 mg di p67r con XbaI e legare prima di trasfezione. </li><li> Per la legatura, usare 1 unità Weiss di ligasi in una reazione 50 microlitri e incubare a 16 ° C durante la notte. </li><li> Quando la legatura è completata, incubare la miscela legatura a 65 ° C per 15 minuti per l&#39;inattivazione della ligasi. Questo passaggio può essere omesso. </li><li> Due giorni prima della transfezione, semi di 10 6 cellule SCP in un pallone T25 di coltura tissutale, e far crescere le cellule per due giorni in 5 ml di DMEM + 10% siero di agnello e glutammina 2mM senza antibiotici per un monostrato di 90% di confluenza. Se le cellule non hanno raggiunto il 90% confluenza dopo due giorni, lasciare le cellule a crescere un giorno in più. </li></ol> Nota: Il siero di agnello può essere sostituito da siero bovino fetale (FBS). Noi abitualmente uso di agnello siero, dal momento che alcuni ceppi di MVV sono inibiti da FBS; questo ceppo MVV non è inibito da FBS, comunque. <ol start="5"><li> Quando le cellule sono pronti per la transfezione, il cambiamento medio DMEM con 1% di siero (siero o agnello FBS) senza antibiotici. Noi usiamo Lipofectamin 2000 (Invitrogen) per la trasfezione. </li><li> Diluire il DNA in 500μl Opti-MEM medium. </li><li> Diluire 20μl Lipofectamin 2000 a 500μl Opti-MEM medio e lasciare a temperatura ambiente (RT) per 5 min. </li><li> Dopo l&#39;incubazione 5 minuti, unire il DNA diluito con Lipofectamine diluito 2000 (volume totale = 1000 ml). Mescolare delicatamente e incubare per 20-30 minuti a temperatura ambiente. </li><li> Aggiungere la miscela di trasfezione (1 ml) al pallone T25 contenente cellule SCP e medie (5 ml). Miscelare delicatamente a dondolo. </li><li> Incubare a 37 ° C e 5% CO 2 durante la notte (18 - 24 h). </li><li> Cambia media il giorno successivo a DMEM con 1% di siero, 2mM glutamina, penicillina e streptomicina 100IU 100IU. </li><li> Monitorare la produzione di GFP nelle cellule SCP da invertita microscopia a fluorescenza. Bassa efficienza di trasfezione può essere previsto con queste pile, di solito 5 - 15%. </li><li> Mantenere ancora in incubazione a 37 ° C, 5% di CO 2 per diversi giorni per consentire la diffusione del virus nel pallone. Di solito ci vogliono 7-14 giorni per l&#39;infezione pieno. </li><li> Raccolto il virus aliquoting il surnatante in provette Eppendorf. Spin in una microcentrifuga a 3000 rpm per 5 minuti per rimuovere i detriti cellulari. Trasferire il supernatante contenente il virus ai nuovi tubi. Conservare a -80 ° C. </li></ol> 2. Titolazione di virus <ol><li> Semi di 3x10 4 celle SCP in 100 microlitri di media DMEM supplementato con 10% siero, 2mM glutammina, 100IU/ml penicillina e streptomicina 100IU/ml, per bene in una piastra da 96 pozzetti. Incubare a 37 ° C, 5% di CO 2. </li><li> Se le cellule sono confluenti il ​​giorno successivo, il cambiamento medio DMEM con 1% di siero, 2mM glutamina, penicillina e streptomicina 100IU/ml 100IU/ml, 100 l in ciascun pozzetto. </li><li> Diluire il virus nel modo seguente: <ol><li> Aggiungere 180 microlitri DMEM con 1% di siero, 2mM glutamina, penicillina e streptomicina 100IU/ml 100IU/ml per pozzetto di 4 x 10 pozzi in un fondo da 96 pozzetti rotondi o V-piastra inferiore. </li><li> Per le diluizioni seriali dieci volte in quadruplice copia, aggiungere 20 ml di virus a ciascuno dei 4wells nella prima colonna del piatto, e con un quattro canali pipette, pipetta su e giù per garantire che i campioni siano ben mescolati. Cambia puntali e trasferire 20 l per i prossimi 4 pozzi e così via. Lasciare i pozzi ultimi senza virus per un controllo negativo. </li></ol></li><li> Aggiungere 100 ml di diluizioni del virus alle cellule SCP e medie imprese. </li><li> Incubare a 37 ° C e 5% di CO 2. Osservare per fluorescenza GFP ed effetti citopatico per due settimane. Citopatico effetti consistono in cellule diventando arrotondata e meno trasparente rispetto a quelle normali con i processi si allunga, per finire con le cellule multinucleare appaiono e morte cellulare. </li><li> Calcolare virus titolo (TCID50) utilizzando il metodo Reed Muench 11. </li></ol> 3. Analisi FACS di cellule infette Nota: Per il monitoraggio rapido di replicazione del virus, le cellule infette può essere esaminata mediante citometria di flusso. <ol><li> Trypsinize cellule infette (ca 10 5), lavare con PBS 1x e risospendere in 500μl di PBS. </li><li> Aggiungere 500 ul di soluzione di paraformaldeide al 4% (2% di concentrazione finale), incubare a RT per 30 min. </li><li> Celle di pellet in una microcentrifuga a 3000 rpm per 5 min. </li><li> Lavare con PBS 2x5 min e analizzare su un analizzatore FACScan. Contano 10.000 eventi. </li></ol> 4. Rappresentante dei risultati: Questo virus dovrebbe essere pienamente infettive e replicare ad un titolo del 10 Giugno-10 Luglio TCID 50 / ml. Cellule infette sono fluorescenti e possono essere detperturbato mediante citometria a flusso e microscopia a fluorescenza. Tipicamente, GFP può essere rilevato in oltre il 60% delle cellule nelle colture di cellule infettate in citometria a flusso (Fig. 1). Allo stesso modo, quando visualizzata al microscopio a fluorescenza, la maggior parte delle cellule sono fluorescenti (Fig. 2A e B). <img src="/files/ftp_upload/3483/3483fig1.jpg" alt="Figura 1" /> Figura 1. FACScan analisi delle cellule infettate con KV1772 SCP-EGFP virus dopo 7 giorni di infezione. <img src="/files/ftp_upload/3483/3483fig2.jpg" alt="Figura 2" /> Figura 2. Microscopio a contrasto di fase di cellule infettate con KV1772 SCP-EGFP dopo 7 giorni di incubazione (A). Stesse cellule visualizzati al microscopio a fluorescenza (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>

Nessun conflitto di interessi dichiarati.

La GFP che esprimono clone molecolare di MVV qui presentato dovrebbe essere utile per analizzare le interazioni della cellula ospite e la patogenicità dei MVV sia in vitro che in vivo. Il virus ottiene dopo 7-14 giorni di replica ha indubbiamente acquisito alcune mutazioni a causa della inesattezza della trascrittasi inversa. Tuttavia, ci sono limitazioni per l&#39;utilizzo di questo clone come vettore singolo ciclo. Una è che l&#39;uso del clone è limitata alle cellule di pecora e capra origine. L&#39;efficienza di ...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </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) è un lentivirus di pecore, lentamente progressiva che causa la polmonite interstiziale e l&#39;encefalite 1. Le cellule bersaglio primario della MVV in vivo sono considerate dei 2 lignaggio monociti. Alcuni ceppi di MVV può replicare in altri tipi cellulari, ma 3,4. La proteina verde fluorescente è un marker comunemente usato per lo studio lentivirus nelle cellule viventi. Abbiamo inserito il gene EGFP nel gene per dUTPase di MVV. Il gene dUTPase è ben conservato in ceppi più lentivirus di pecore e capre e ha dimostrato di essere importante nella replicazione del CAEV 5. Tuttavia, dUTPase ha dimostrato di essere indispensabili per la replica del clone molecolare di MVV usato in questo studio sia in vitro che in vivo 6. Replica MVV è strettamente limitata alle cellule di pecora o capra origine. Noi usiamo una linea principale di cellule dal plesso coroideo di pecore (cellule SCP) per la trasfezione e la propagazione del virus 7. La MVV fluorescente è completamente infettive e di espressione EGFP è stabile per almeno 6 passaggi 8. C&#39;è una buona correlazione tra le misurazioni di TCID 50 e EGFP. Questo virus dovrebbe quindi essere utile per il rilevamento rapido delle cellule infette negli studi di tropismo cellulare e patogenicità in vitro e 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

Descriviamo un clone molecolare di Maedi-Visna virus che esprime GFP ed è completamente infettive. Replicazione di questo virus possono essere rilevati tramite microscopio a fluorescenza e citometria a flusso.

Di moltiplicazione e di Rilevamento di un clone infettive molecolare di Maedi-Visna Virus che esprime la proteina fluorescente verde

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 Views.  University of Iceland. Descriviamo un clone molecolare di Maedi-Visna virus che esprime GFP ed è completamente infettive. Replicazione di questo virus possono essere rilevati tramite microscopio a fluorescenza e citometria a flusso.

Video: 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.

Marcatura specifica di HIV-1 Cellule positive con un Rev-dipendente Vector lentivirali Esprimendo la proteina fluorescente verde

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, ...

Ricerca

Immunologia e infezioni

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.

Il rilevamento del virus infettivo da Campo-zanzare raccolte mediante test coltura cellulare 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.

Visualizzazione virus Dengue attraverso Alexa Fluor Etichettatura

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.

Imaging cellulare dal vivo di Alphaherpes Virus Trasporti anterograda e Diffusione

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.

Modellazione il ciclo di vita Ebola Virus Sotto livello di biosicurezza 2 condizioni con particelle virus-simili contenenti 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 ...

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-immunoprecipitazione della proteina mouse Mx1 con il virus nucleoproteina Influenza A

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.

Creazione di una configurazione high-throughput screening per piccole molecole che modulano c-di-GMP Segnalazione di<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 System infettiva coltura cellulare e la<em&gt; In Vitro</em&gt; Effetto profilattico di interferoni

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; Test di rilascio di Caseum che prevede la penetrazione di farmaci nelle lesioni della tubercolosi

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.

Salvataggio e caratterizzazione del virus ricombinante da un nuovo mondo Clone infettivo del virus Zika

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