Wir danken den Gutachtern für ihre durchdachten Vorschläge, die dieses Manuskript zu verbessern half. Diese Arbeit wurde durch Stipendien an MVC (NS21072, HD23315), ACW (GM61139, S10RR017970) und IM (AI073898, GM056927) von der NIH unterstützt. MK wurde zum Teil durch ein NIH Ausbildungsförderung (5T32 AI007180) unterstützt.

<ol>
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K., Tansky, C. S., Thompson, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+latent+herpes+simplex+virus+type+1+genome+copy+number+in+individual+neurons+is+virus+strain+specific+and+correlates+with+reactivation.">The latent herpes simplex virus type 1 genome copy number in individual neurons is virus strain specific and correlates with reactivation.</a> J. Virol. 72, 5343-5350 (1998).</li><li>Thompson, R. L., Cook, M. L., Devi-Rao, G., Wagner, E. K., Stevens, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+and+molecular+analysis+of+the+avirulent+wild-type+herpes+simplex+virus+type+1+strain+KOS.">Functional and molecular analysis of the avirulent wild-type herpes simplex virus type 1 strain KOS.</a> J. Virol. 58, 203-211 (1986).</li><li>Wilcox, C. L., Smith, R. L., Freed, C. 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Keine Interessenskonflikte erklärt.

Diese primäre Neuron Kultur und Infektion System bietet eine einfache und effektive Methode, um die molekularen Mechanismen HSV-1-Latenz und Reaktivierung zu erkunden. Das System treu rekapituliert die anerkannten Markenzeichen der Latenz in beiden Infektionen beim Menschen und bei Live-Tiermodellen definiert. Wenn das Virus in den SCG Kulturen latent ist, können infektiöse Partikel und lytischen Genprodukte nicht nachgewiesen werden. Die einzige virales Genprodukt in latent infizierten Neuronen, wir...

<table border="1"><tbody><tr><td> Reagens </td><td> Firma </td><td> Katalog # </td><td> Kommentare </td></tr><tr><td> 70μm Nylon-Filter (Zellsieb) </td><td> BD Biosciences </td><td> 352350 </td><td></td></tr><tr><td> 1x Hanks Balanced Salt Solution (HBSS - / -) </td><td> Invitrogen </td><td> 14175 </td><td> W / O CaCl 2 und MgCl 2 </td></tr><tr><td> 1x Minimum Essential Medium (MEM) </td><td> Invitrogen </td><td> 11095-080 </td><td></td></tr><tr><td> 5-Fluoro-2&#39;-desoxyuridin </td><td> Sigma </td><td> F0503 </td><td> Vorbereitung 20 mM Lager in 1X MEM; Lagerung bei -20 ° C </td></tr><tr><td> 96-Loch-flache Boden gut TC Platten </td><td> Corning </td><td> 3599 </td><td></td></tr><tr><td> Acyclovir </td><td> &lt;em&gt; Calbiochem </td><td> 114798 </td><td> Vorbereitung 31 mM Stammlösung in DMSO; Lagerung bei -20 ° C </td></tr><tr><td> Aphidicolin </td><td> Calbiochem </td><td> 178273 </td><td> Vorbereitung 10 mM Stammlösung in DMSO; Lagerung bei -20 ° C </td></tr><tr><td> B-27 Supplement </td><td> Invitrogen </td><td> 17504-44 </td><td></td></tr><tr><td> Collagenase </td><td> Sigma </td><td> C2674 </td><td> Vorbereitung 10 mg / ml in HBSS Lager - / -; Lagerung bei -20 ° C </td></tr><tr><td> D-(+)-Glucose </td><td> Sigma </td><td> G6152 </td><td> Vorbereitung 40% Lager in H 2 O; Filter sterilisieren und bei 4 ° C lagern </td></tr><tr><td> L-Glutamin </td><td> Invitrogen </td><td> 25030-081 </td><td></td></tr><tr><td> Laminin </td><td> Sigma </td><td> L2020 </td><td> Herstellung von 1 mg / ml Stammlösung in H 2 O; quick-freeze 20 ulaliquats &amp; Speicher bei -80 ° C; verdünnte bis 2 pg / ml konz arbeiten. in sterilem H 2 O </td></tr><tr><td> Leibovit&#39;z L-15-Medien </td><td> Invitrogen </td><td> 11415 </td><td></td></tr><tr><td> Nerve Growth Factor </td><td> Harlan Laboratories </td><td> BT.5017 </td><td> Vorbereitung 50 ug / ml in HBSS Lager - / -; Speicher bei -80 ° C </td></tr><tr><td> Neurobasalmedium </td><td> Invitrogen </td><td> 12348 </td><td></td></tr><tr><td> Phosphonoessigsäure (PAA) </td><td> Sigma </td><td> P6909 </td><td> Vorbereitung 75 mg / ml Stammlösung in H 2 O; Lagerung bei -20 ° C </td></tr><tr><td> Poly-D-Lysin Hydrobromid </td><td> Sigma </td><td> P0899 </td><td> Es werden 20 mg / ml Stammlösung in H 2 O; Lagerung bei -20 ° C </td></tr><tr><td> Rattenschwanz Collagen </td><td> Millipore </td><td> 08-115 </td><td>Konzentration variiert mit viel Versorgung; lagern bei 4 ° C und verdünnt auf 0,66 mg / ml konz arbeiten. in sterilem H 2 O </td></tr><tr><td> Trichostatin A </td><td> Sigma </td><td> T8552 </td><td> Vorbereitung 1 mM Stammlösung in DMSO; Lagerung bei -20 ° C </td></tr><tr><td> 2,5% Trypsin </td><td> Invitrogen </td><td> 15090-04 </td><td></td></tr></tbody></table>

a primary neuron culture system for the study of herpes simplex virus latency and reactivation

Herpes simplex Virus Typ-1 (HSV-1) stellt eine lebenslange latente Infektion in peripheren Neuronen. Dieses latente Reservoir ist die Quelle der wiederkehrenden Ereignissen, die Reaktivierung Übertragung zu gewährleisten und dazu beitragen, klinischen Erkrankung. Aktuelle antivirale Medikamente haben kaum Auswirkungen auf das latente Reservoir und es gibt keine Impfstoffe. Während die molekularen Details der Virusvermehrung gut charakterisiert sind, bleiben Mechanismen, die Latenz in den Neuronen schwer fassbar. Unser gegenwärtiges Verständnis von Latenzzeiten von In-vivo-Studien mit kleinen Tiermodellen, die zur Definition der virale Gen Anforderungen und die Rolle der Immunreaktionen wurden unentbehrlich abgeleitet. Jedoch ist es unmöglich, spezifische Wirkungen auf die Virus-Neuron-Verhältnis von mehr allgemeinen Folgen der Infektion durch Immun oder nicht-neuronalen Zellen Unterstützung in lebenden Tieren vermittelt unterscheiden. Darüber hinaus ist Tierversuchen kostspielig, zeitaufwendig und im Hinblick auf die begrenzten verfügbaren Optionen für die Manipulation von HostVerfahren. Um diese Einschränkungen zu überwinden, wird ein Neuron-System nur, dass dringend benötigte reproduziert die in vivo Eigenschaften von Latenz und Reaktivierung, sondern bietet die Vorteile von Gewebekulturen in Bezug auf die Homogenität und Zugänglichkeit. Hier präsentieren wir ein in vitro Modell unter Verwendung von kultivierten primären sympathischen Neuronen von Ratten überlegen Halsganglien (SCG) (Abbildung 1) zu HSV-1-Latenz und Reaktivierung, die passt zu studieren meisten, wenn nicht alle gewünschten Kriterien. Nach Beseitigung nicht-neuronalen Zellen, sind nahezu homogen TrkA + Neuronenkulturen mit HSV-1 in Anwesenheit von Acyclovir (ACV) die lytische Vermehrung zu unterdrücken infiziert. Nach der Entfernung ACV, nicht-produktive HSV-1-Infektionen, die treu zeigen akzeptiert Markenzeichen der Latenz werden effizient etabliert. Insbesondere werden lytischen mRNAs, Proteine, und das infektiöse Virus nicht nachweisbar, auch in Abwesenheit der Auswahl, aber Latenz-assoziierten Transkript (LAT) exprimierenIonen besteht in neuronalen Kerne. Virale Genome mit einer durchschnittlichen Anzahl von 25 Kopien pro Neuron gehalten und können zur produktiv zu stören PI3-Kinase / Akt Signalisierung oder Rücknahme eines Nerven Wachstumsfaktor 1 zu replizieren. Rekombinantes HSV-1-Codierung EGFP fusioniert mit dem lytischen Proteins US11 eine funktionale Echtzeit-Marker für die Replikation durch die Reaktivierung, die leicht quantifiziert wird. Zusätzlich zu den chemischen Behandlungen können genetische Methoden wie RNA-Interferenz oder Gentransfer über lentiviralen Vektoren erfolgreich auf das System ermöglicht mechanistische Studien, die sehr schwierig, wenn nicht unmöglich, bei Tieren angewendet werden. Zusammenfassend bietet die SCG-basierten HSV-1-Latenz / Reaktivierung System eine leistungsstarke, notwendiges Werkzeug, um die molekularen Mechanismen, die HSV1 Latenz und Reaktivierung in Neuronen, ein langjähriger Rätsel, dessen Lösung in der Virologie können neue Einblicke in die Entwicklung neuer Therapien anbieten zu entwirren, dass Ziel ter latent Herpesvirus Reservoir.

Watch this Scientific Journal Video about A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation at JoVE.com

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Das Protokoll beschreibt eine effiziente und reproduzierbare Modellsystem zur Untersuchung Herpes-simplex-Virus Typ 1 (HSV-1) Latenz und Reaktivierung. Der Test verwendet homogene sympathischen Neuronen Kulturen und ermöglicht die molekulare Analyse der Virus-Neuron-Wechselwirkungen unter Verwendung einer Vielzahl von Anwendungen, wie RNA-Interferenz und Expression von rekombinanten Proteinen.

Eine primäre Neuron Culture System zur Erforschung des Herpes Simplex Virus Latenz und Reaktivierung

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

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Research

JoVE Journal

Immunology and Infection

17.2K Aufrufe.  New York University School of Medicine. Das Protokoll beschreibt eine effiziente und reproduzierbare Modellsystem zur Untersuchung Herpes-simplex-Virus Typ 1 (HSV-1) Latenz und Reaktivierung. Der Test verwendet homogene sympathischen Neuronen Kulturen und ermöglicht die molekulare Analyse der Virus-Neuron-Wechselwirkungen unter Verwendung einer Vielzahl von Anwendungen, wie RNA-Interferenz und Expression von rekombinanten Proteinen.

Serie: A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

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 ...

Forschung

Immunologie und Infektion

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Ex vivo Infektion von murinen Epidermis mit Herpes Simplex Virus Typ 1

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes ...

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Entwicklung eines Systems Hepatitis-B-Virus Reporter den frühen Stadien des Replikationszyklus zu überwachen

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

Abgeleitet von Maus und Mensch primäre gastrische Epithelzellen Monolayer Kultur for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Ein Ex-Vivo Huhn Primärkultur Bursitis-Zelle Modell zur Bursitis Infektionskrankheit Virus Pathogenese

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Zeitliche Analyse der nuklearen-cytoplasmatische Translokation eines Herpes-Simplex-Virus 1-Proteins Immunofluorescent konfokalen Mikroskopie

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

In Vitro ELISA-Test zur Bewertung der Wirksamkeit von Tollwut-Impfstoffen

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

Reinigung von hochertragreichen extrazellulären Vesikelpräparaten weg vom Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

siRNA Electroporation to Modulate Autophagy in Herpes Simplex Virus Type 1-Infected Monocyte-Derived Dendritic Cells

Herpes simplex virus type-1 (HSV-1) induces autophagy in both, immature dendritic cells (iDCs) as well as mature dendritic cells (mDCs), whereas autophagic flux is only observed in iDCs. To gain mechanistic insights, we developed efficient strategies to interfere with HSV-1-induced autophagic turnover. An inhibitor-based strategy, to modulate HSV-1-induced autophagy, constitutes the first choice, since it is an easy and fast method. To circumvent potential unspecific off-target effects of such compounds, we developed an alternative siRNA-based strategy, to modulate autophagic turnover in iDCs upon HSV-1 infection. Indeed, electroporation of iDCs with FIP200-specific siRNA prior to HSV-1 infection is a very specific and successful method to ablate FIP200 protein expression and thereby to inhibit autophagic flux. Both presented methods result in the efficient inhibition of HSV-1-induced autophagic turnover in iDCs, whereby the siRNA-based technique is more target specific. An additional siRNA-based approach was developed to selectively silence the protein expression of KIF1B and KIF2A, facilitating autophagic turnover upon HSV-1 infection in mDCs. In conclusion, the technique of siRNA electroporation represents a promising strategy, to selectively ablate the expression of distinct proteins and to analyze their influence upon an HSV-1 infection.

In this study, we present inhibitor- and siRNA-based strategies to interfere with autophagic flux in Herpes simplex virus type-1 (HSV-1)-infected monocyte-derived dendritic cells.