Name: influenza virus propagation in embryonated chicken eggs
Uploaded: 2015-03-19T13:00:00.000+00:00
Duration: 6 min 56 s
Description: Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

The present study was supported by the NIH grants HL120947 (P.C.), HL103868 (P.C.), and the American Heart Association Grant-in-Aid (P.C.)

HINWEIS: Allgemeine Hinweise: Führen Sie alle Verfahren, die Manipulation des Ei unter sterilen Bedingungen und steriler Technik sollte entsprechend verwendet werden. Vorreinigen gesamte Ausrüstung mit 70% Ethanol vor dem Gebrauch. Im allgemeinen sollte Influenza Virusinokulation und Ernte in einem BSL-2 Labor durchgeführt werden. Allerdings, wenn dieses Protokoll wird verwendet, um zu verbreiten viel mehr pathogenen Influenzaviren (zB Pandemie und Pre-Pandemiestämme, Stämme, die eine Gefahr für Geflügel und Vieh, hoch pathogenen aviären Influenza-Stämme, die 1918 H1N1-Stamm darstellen), höhere Biosicherheitsstufen und in einigen Fällen werden Biosicherheitsmaßnahmen und Genehmigungen erforderlich. Der lokale Institutional Biosafety Committee sollte alle Forschung mit Influenza-Virus zu genehmigen. 1. Vorbereitung der Eier für Impfung <ol><li> Erhalten Serum pathogenfreien (SPF) frisch befruchteten Hühnerkeimen aus einem geeigneten Anbieter. Typischerweise sind Hühnereier verwendet. Eier sollten unsed sofort nach Ankunft. </li><li> Nach der Ankunft statt Eier in einem befeuchteten Brutkasten mit einem automatischen Ei Turner, Eier regelmäßig zu drehen. Ist ein automatischer Ei Turner nicht verfügbar ist, von Hand drehen Eier mindestens 2-3 mal pro Tag (stellen Sie sicher, Handschuhe zu tragen und halten alles so steril wie möglich). </li><li> Eiern bei 37 ° C und 55-60% Luftfeuchtigkeit für 10-11 Tage. </li></ol> 2. Eierkerzen <ol><li> Verwenden Sie ein Ei candler, um das Ei vor einem hellen Licht in einem dunklen Raum zu untersuchen, um Eier für Unfruchtbarkeit, die durch Durchleuchten nach ca. 7 oder 8 Tagen Inkubation zu überprüfen. <ol><li> Wischen Sie die Eierdurchleuchter mit 70% Ethanol. Handschuhe häufig wechseln und Desinfektion mit 70% Ethanol, um das Risiko einer Kontamination mit Krankheitserregern zu minimieren. </li><li> Entfernen Sie die Eier aus dem Inkubator und sie in leeren Eierkartons. </li><li> Schalten Sie das Licht in den Raum. </li><li> Halten Sie das große Ende jedes Ei einzeln nacheinander gegen die candler. </li><li> Beobachten Sie das Ei zu determine, wenn es fruchtbar oder unfruchtbar. HINWEIS: Dünne Blutgefäße, die zu einer bohnenförmigen Embryos muss deutlich sichtbar sein. Unbefruchteten Eiern wird als kleiner Blutfleck mit einem sichtbaren Eigelb angezeigt. </li></ol></li><li> Werfen Sie unbefruchtete Eier, die sind, und gibt die lebensfähige Eier in den Inkubator. Lassen Sie keine Eier außerhalb des Inkubators für mehr als 30 min. Wenn der Status eines Eies nicht bestätigt werden kann, markieren Sie ihn und später beobachten sie. </li></ol> 3. Herstellung von Virus Inoculum <ol><li> Verdünne Influenzavirusbestand zu etwa 3. Oktober-4. Oktober pfu / ml in sterilem PBS, enthaltend 10 mM HEPES (pH 7,4). Critical Schritt: Verdünnen Sie die Virusstamm unmittelbar vor der Anwendung und halten die verdünnten Virus auf dem Eis die ganze Zeit. </li></ol> 4. Influenza Virus Impfung über die Allantois Routen <ol><li> Am Tag der Virusinokulation (Tag 10 oder 11), Kerze die Eier wieder und beseitigen tote Embryonen. Unterscheiden Sie Live-EmbryonenSuche nach Bewegung in das Ei. </li><li> Entfernen Sie drei Eier aus dem Inkubator, und ordnen Sie die Eier in einem separaten Halter mit dem Luftsack auf. </li><li> Markieren Sie den Luftraum der Eier mit einem wasserfesten Stift. </li><li> Oberhalb des Luftraums mit 70% Ethanol waschen die Eierschale. </li><li> Die Eier mit dem Luftraum in einem Eierkarton und in einen biologischen Sicherheit Haube (BSL-2). </li><li> Verwenden Sie eine sterile 18G Nadel ein kleines Loch in der Schale auf den Luftsack jedes Ei zu stanzen. Achten Sie darauf, um die Nadel zu tief einfügen, um zu vermeiden, stech den Embryo oder Eigelb. </li><li> Erstellung des verdünnten Influenza-Virus in eine sterile 1 ml-Spritze und fügen Sie eine 22G-Nadel. </li><li> Die Spritze mit Nadel vorsichtig voran in einem Winkel von 45 ° in die Allantoishöhle. </li><li> Injizieren 0,2 ml der verdünnten Influenza-Virus. </li><li> Wiederholen Sie die Injektion mit den anderen zwei Eier, und dann werfen Sie die Spritze und Nadel. </li><li> Dichten Sie das Loch mit einem Tropfen Klebstoff aus einer Klebepistole (oder geschmolzene Paraffinwachs). Be Achten Sie darauf, dass Kleber nicht im Inneren des Eis lecken. </li><li> Legen Sie die Eier in die Brutkasten mit der Luftraum deutete nach oben. </li><li> Wiederholen Sie die Schritte 4,2 bis 4,12, bis alle Eier wurden geimpft. </li></ol> 5. Inkubationszeit <ol><li> Die infizierten Eiern Inkubieren ohne Drehen bei 37 ° C und ca. 60% Luftfeuchtigkeit. </li><li> Wählen optimale Inkubationszeit in Abhängigkeit von der Influenza-Virus-Typ. Inkubieren Influenza A 48 h; inkubieren Influenza B und C für 72 Stunden. </li></ol> 6. Influenza Virus-Ernte <ol><li> Nach Inkubationszeit Chill die Eier bei 4 ° C für mindestens 4 Stunden (oder O / N), um den Embryo zu töten und verengen die Blutgefäße, um das Risiko einer Verunreinigung des infizierten Allantoisflüssigkeit mit Blut zu verringern. Tun dies, um die roten Blutzellen absorbieren das Virus, was die Ausbeute verringert verhindern. Alternativ Chill Eier für 30 min bei -20 ° C; aber dies erhöht die Gefahr einer Kontamination des Blutes. </li><li> Nachdem die Eier ausreichend gekühlt wurde, übertragen Sie die Eier auf einem Bio-Sicherheitshaube. Legen Sie das Ei in eine feste Halterung mit der Luftsack nach oben zeigt. Reinigen Sie das Ei Oberfläche mit 70% Ethanol. </li><li> Verwenden einer sterilen Schere, um die Eierschale über dem Luftsack zu öffnen. Entfernen Sie die Hülle um den Luftsack, während man aufpassen, nicht die Chorioallantoismembran zerstören. </li><li> Öffnen Sie die Chorioallantoismembran mit sterilem stumpfen Pinzette. Vorsichtig zur Seite legen den Embryo und der Dottersack mit einem kleinen Spatel oder Löffel. Darauf achten, dass das Eigelb bersten. </li><li> Mit einer 1 ml-Eppendorf-Pipette vorsichtig sammeln die Allantoisflüssigkeit. Wenn nötig, kippen Sie das Ei ein wenig zur Seite, um so viel Flüssigkeit wie möglich zu sammeln. </li><li> Kombinieren Sie die Allantoisflüssigkeit aller Eier in einen 50-ml-Kunststoff konischen Rohr. Während der Virusernte halten Röhrchen auf Eis zu allen Zeiten. Ein Ei Ausbeuten 5-10 ml eines leicht gelblichen Flüssigkeit. </li><li> Drehe das virushaltige Allantoisflüssigkeit bei 1.000 × g für 10 min bei 46; C bis Trümmer zu pelletieren. Übertragen Sie die klare Flüssigkeit in eine neue 50-ml-Röhrchen auf Eis gehalten. Entsorgen Sie Eier in eine BSL-2 Abfallbehälter. </li></ol> 7. Lagerung <ol><li> Die geklärt Allantoisflüssigkeit (zB 50, 100 oder 500 ul Aliquote) sofort aliquotiert und Snap-freeze in flüssigem Stickstoff. Übertragen gefrorenen Virusstocks bis -80 ° C Gefrierschrank für langfristige Lagerung. </li></ol>

<ol>
	<li>Centers for Disease Control and Prevention (CDC). <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimates+of+deaths+associated+with+seasonal+influenza+---+United+States,+1976-2007.">Estimates of deaths associated with seasonal influenza --- United States, 1976-2007.</a> MMWR. Morbidity and mortality weekly report. 59 (33), 1057-1062 (2010).</li><li>Neumann, G., Noda, T., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emergence+and+pandemic+potential+of+swine-origin+H1N1+influenza+virus.">Emergence and pandemic potential of swine-origin H1N1 influenza virus.</a> Nature. 459 (7249), 931-939 (2009).</li><li>Tumpey, T. M., Garcia-Sastre, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenicity+of+Influenza+Viruses+with+Genes+from+the+1918+Pandemic+Virus:+Functional+Roles+of+Alveolar+Macrophages+and+Neutrophils+in+Limiting+Virus+Replication+and+Mortality+in+Mice.">Pathogenicity of Influenza Viruses with Genes from the 1918 Pandemic Virus: Functional Roles of Alveolar Macrophages and Neutrophils in Limiting Virus Replication and Mortality in Mice.</a> J Virol. 79 (23), 14933-14944 (2005).</li><li>Doherty, P. C., Turner, S. J., Webby, R. G., Thomas, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+and+the+challenge+for+immunology.">Influenza and the challenge for immunology.</a> Nat Immu. 7 (5), 449-455 (2006).</li><li>Webster, R. G., Govorkova, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuing+challenges+in+influenza.">Continuing challenges in influenza.</a> Ann N Y Acad Sci. 1323 (1), 115-139 (2014).</li><li>Szretter, K. J., Balish, A. L., Katz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unit+15G.1+Influenza:+propagation,+quantification,+and+storage.+.">Unit 15G.1 Influenza: propagation, quantification, and storage. .</a> Current protocols in microbiology. , (2006).</li><li>Short, K. R., Kroeze, E. J. B. V., Fouchier, R. A. M., Kuiken, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+influenza-induced+acute+respiratory+distress+syndrome.">Pathogenesis of influenza-induced acute respiratory distress syndrome.</a> Lancet Infect Dis. 14 (4), 57-69 (2014).</li><li>Ito, T., Suzuki, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differences+in+sialic+acid-galactose+linkages+in+the+chicken+egg+amnion+and+allantois+influence+human+influenza+virus+receptor+specificity+and+variant+selection.">Differences in sialic acid-galactose linkages in the chicken egg amnion and allantois influence human influenza virus receptor specificity and variant selection.</a> J Virol. 71 (4), 3357-3362 (1997).</li><li>Robertson, J. S., Nicolson, C., Major, D., Robertson, E. W., Wood, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+amniotic+passage+in+the+egg-adaptation+of+human+influenza+virus+is+revealed+by+haemagglutinin+sequence+analyses.">The role of amniotic passage in the egg-adaptation of human influenza virus is revealed by haemagglutinin sequence analyses.</a> J Gen Virol. 74 (Pt 10), 2047-2051 (1993).</li><li>Gaush, C. R., Smith, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+and+plaque+assay+of+influenza+virus+in+an+established+line+of+canine+kidney+cells).">Replication and plaque assay of influenza virus in an established line of canine kidney cells).</a> Appl Microbiol. 16 (4), 588-594 (1968).</li></ol>

The authors have nothing to disclose.

Influenza verursacht einen großen globalen Krankheitslast, und die Arbeit geht weiter in das Verständnis der Pathogenese der Lungenschädigung 7. Erleichtert die Forschung in dieser tödlichen Krankheit, wurden verschiedene Verfahren entwickelt, um den Influenzavirus 6 ausbreiten. Hier beschreiben wir eine Technik, um Influenza-Virus in Hühnereiern produziert. Der Vorteil dieses Verfahrens ist, dass es in hohem Maße reproduzierbar ist und führt zu großen Mengen von hohem Titer Influenzaviruss...

Influenza A weiterhin eine große Bedrohung für die menschliche Gesundheit sein. Es ist eine potenziell verheerenden Erkrankung der Atemwege mit einem großen globalen Belastung verursacht bis zu 500.000 Todesfälle weltweit pro Jahr 1. Influenza-Viren sind in der Familie Orthomyxoviridae und führen 8 Minus-Sense einzelsträngige RNA in ihrem Genom 1,2. Die hohe Veränderlichkeit (dh antigene &quot;Drift&quot;) des Virusgenoms verhindert Langzeitimmunität. Außerdem ist Influenza zunehmend resistent gegen antivirale Medikamente 3. Das H1N1 2009-Grippe-Pandemie markiert alle herausfordernden Fragen mit dem Influenzaerkrankung (Pandemie-Stamm, anti-virale Resistenz, verzögert Impfstoffproduktion) verbunden. Die Impfstoff-Formulierung wird jährlich von der Weltgesundheitsorganisation für die wahrscheinlichste pathogenen Influenza-Stämme (je eine für H1N1, H3N2 und Influenza B) 4 bestimmt. Da diese Methode beruht auf Annahmen basierte Influenza-Stamms, pathogenen Stämmen werden gelegentlich falsch identifiziert und die Wirksamkeit des Impfstoffes sinkt dramatisch. Darüber hinaus kann das Auftreten von neuen Influenza-Stämme diese präventive Programme verursachen Pandemien 2 zu umgehen. Die Schaffung eines universellen Influenza-Impfstoff ist schwer 5. Daher müssen Erforschung Verständnis der Pathogenese der Lungenschädigung weiter. Um die Anlage der Laborforschung wurden verschiedene Verfahren für die virale Isolation und Vermehrung 6 entwickelt. Menschlichen Influenza-Viren können in einer Vielzahl von Säugetierzellsubstraten verstärkt werden. Jedoch werden die Erzeugung hoher Titer von Viren in großen Mengen am besten in embryonierten Eiern erreicht. Das folgende Protokoll beschreibt eine Technik für die Influenza A-Virusvermehrung und Speicherung von vorhandenen Virusstocks.

<table><tbody><tr><td>Phosphate buffered saline (PBS)</td><td>Cellgro&amp;nbsp;</td><td>21-040-CV</td><td></td></tr><tr><td>serum pathogen-free (SPF) fertilized chicken eggs&amp;nbsp;</td><td>VALO BioMedia</td><td></td><td></td></tr><tr><td>humidified egg incubator&amp;nbsp;</td><td>FARM iNNOVATORS</td><td>Model 2100</td><td></td></tr><tr><td>automatic egg turner</td><td>FARM iNNOVATORS</td><td>Model 3200</td><td></td></tr><tr><td>egg candler</td><td>FARM iNNOVATORS</td><td>Model 3300&amp;nbsp;</td><td></td></tr><tr><td>1 ml syringe</td><td>BD Bioscience</td><td>309659</td><td></td></tr><tr><td>18G needle</td><td>BD Bioscience</td><td>305196</td><td></td></tr><tr><td>20G / 22G needle</td><td>BD Bioscience</td><td>305176 / 305156</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Ethanol&gt;99.5%</td><td>Sigma-Aldrich</td><td>459844</td><td>diluted to 70% using water</td></tr><tr><td>glue gun &quot;Ad tech Hi Temp Project Pro&quot;</td><td>Ad tech, Adhesive Technologies, Inc.</td><td>Model 1105</td><td></td></tr><tr><td>glue &quot;Ad tech MINI SIZE, Multi Temp&quot;</td><td>Ad tech, Adhesive Technologies, Inc.</td><td>220-34ZIP30</td><td></td></tr><tr><td>MDCK cells</td><td>ATCC</td><td>CRL-2935</td><td></td></tr><tr><td>Washed Pooled Turkey Red Blood Cells, 10%</td><td>Lampire Biological Laboratories</td><td>724908</td><td></td></tr></tbody></table>

influenza virus propagation in embryonated chicken eggs

Influenza-Infektion ist mit rund 36.000 Tote und mehr als 200.000 Krankenhauseinweisungen pro Jahr in den Vereinigten Staaten verbunden. Die kontinuierliche Entstehung neuer Influenza-Virus-Stämme aufgrund von Mutation und Wiederzusammenstellung erschwert die Kontrolle des Virus und die ständige Entwicklung neuer Arzneimittel und Impfstoffe. Die Laboruntersuchung von Influenza erfordert ein zuverlässiges und kostengünstiges Verfahren für die Vermehrung des Virus. Hier wird ein umfassendes Protokoll für Influenza-A-Virusvermehrung in fruchtbare Hühnereiern, die konsequent liefert hohen Titer Virusbeständen. 11 Tage - Kurz gesagt, werden Serum pathogenfreien (SPF) befruchtete Hühnereier bei 37 ° C und 55-60% Luftfeuchtigkeit für 10 inkubiert. In diesem Zeitraum können die Entwicklung des Embryos einfach mit ein Ei candler überwacht werden. Virusinokulation erfolgt durch Injektion von Virusstocks in die Allantois-Höhle unter Verwendung einer Nadel durchgeführt. Nach 2 Tagen Inkubation bei 37 ° C, sind die Eiergekühlt für mindestens 4 Stunden bei 4 ° C. Die Eischale oberhalb der Luftsack und die Chorioallantoismembran werden dann vorsichtig geöffnet und die Allantoisflüssigkeit, die das Virus geerntet. Das Fluid wird von Bruchstücken durch Zentrifugation geklärt, in Aliquote aufgeteilt und bis -80 ° C zur Langzeitlagerung transferiert. Die große Menge (5-10 ml des virushaltigen Fluids pro Ei) und hoher Virustiter in der Regel mit diesem Protokoll erreicht hat die Verwendung von Eiern für die Viruspräparation unserer günstiges Verfahren zur in vitro-Studien, die größere Mengen, insbesondere des Virus in denen hohe Dosierungen des gleichen Virusstock erforderlich.

Mehrere Verfahren sind entwickelt worden, um Influenza-Virus zu titrieren. Eine Überlegung bei der Auswahl eines geeigneten Verfahrens ist, dass einige zu bestimmen Gesamtpartikel unabhängig von Lebensfähigkeit (zB Hämagglutinin-Assay), während andere auf die Infektiosität der Basis, die lebensfähigen Virionen bewertet wird (zB Plaque-Test) 6. Hier wurde der Virustiter von Allantoisflüssigkeit aus Hühnereiern mit einer Maus-adaptierten Influenzavirus (A / PR / 8/34) beimpft gesammel...

Watch this Scientific Journal Video about Influenza Virus Propagation in Embryonated Chicken Eggs at JoVE.com

Influenza Virus Propagation in Embryonated Chicken Eggs

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Influenza Virus Vermehrung in embryonierten Hühnereiern

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

influenza-virus-propagation-in-embryonated-chicken-eggs

Research

JoVE Journal

Immunology and Infection

42.9K Aufrufe.  Cedars-Sinai Medical Center. Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

Serie: Influenza Virus Propagation in Embryonated Chicken Eggs

Generation of Recombinant Influenza Virus from Plasmid DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally established in 1999 1,2 plasmid-based reverse genetic techniques to generate recombinant viruses have revolutionized the influenza research field because specific questions have been answered by genetically engineered, infectious, recombinant influenza viruses. Such studies include virus replication, function of viral proteins, the contribution of specific mutations in viral proteins in viral replication and/or pathogenesis and, also, viral vectors using recombinant influenza viruses expressing foreign proteins 3.

Rescue of influenza A viruses from plasmid DNA is a basic and essential experimental technique that allows influenza researchers to generate recombinant viruses to study multiple aspects in the biology of influenza virus, and to be used as potential vectors or vaccines.

Erzeugung von rekombinanten Influenza Virus von Plasmid-DNA

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

Forschung

Immunologie und Infektion

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Mit Bioluminescent Imaging, um Synergie von Untersuchen Streptococcus pneumoniae Und Influenza A Virus in Infant Mäuse

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

Rettung von rekombinanten Newcastle Disease Virus aus cDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Production of High-Titer Recombinant Newcastle Disease Virus from Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both as an oncolytic virus and a viral-vectored vaccine. NDV is an attractive therapeutic and prophylactic agent due to its well-established reverse genetics system, potent immunostimulatory properties, and excellent safety profile. When administered as an oncolytic virus or a viral-vectored vaccine, NDV elicits a robust antitumor or antigen-specific immune response, activating both the innate and adaptive arms of the immune system. 
Given these desirable characteristics, NDV has been evaluated in numerous clinical trials and is one of the most well-studied oncolytic viruses. Currently, there are two registered clinical trials involving NDV: one evaluating a recombinant NDV-vectored vaccine for SARS-CoV-2 (NCT04871737), and a second evaluating a recombinant NDV encoding Interleukin-12 in combination with Durvalumab, an antiPD-L1 antibody (NCT04613492). To facilitate further advancement of this highly promising viral vector, simplified methods for generating high-titer, in vivo-grade, recombinant NDV (rNDV) are needed. 
This paper describes a detailed procedure for amplifying rNDV in specified pathogen-free (SPF) embryonated chicken eggs and purifying rNDV from allantoic fluid, with improvements to reduce loss during purification. Also included are descriptions of the recommended quality control assays, which should be performed to confirm lack of contaminants and virus integrity. Overall, this detailed procedure enables the synthesis, purification, and storage of high-titer, in vivo-grade, recombinant, lentogenic, and mesogenic NDV for use in preclinical studies.

Here we provide a detailed procedure for production, purification, and quantification of high-titer recombinant Newcastle disease virus. This protocol consistently yields &#62; 6 &#215; 109 plaque-forming units/mL, providing virus quantities appropriate for in vivo animal studies. Additional quality control assays to ensure safety in vivo are described.

Herstellung des rekombinanten Newcastle-Disease-Virus mit hohem Titer aus Allantoic Fluid

Newcastle disease virus (NDV), also known as avian orthoavulavirus serotype-1, is a negative sense, single-stranded RNA virus that has been developed both ...

Krebsforschung

Using Chicken Embryo as a Powerful Tool in Assessment of Developmental Cardiotoxicities

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is well-defined, and it is relatively easy to achieve precise and timely exposure via multiple methods. Moreover, the process of heart development in chicken embryos is similar to mammals, also resulting in a four-chambered heart, making it a valuable alternative model in the assessment of developmental cardiotoxicities. In our lab, the chicken embryo model is routinely used in the assessment of developmental cardiotoxicities following exposure to various environmental pollutants, including per- and polyfluoroalkyl substances (PFAS), particulate matter (PMs), diesel exhaust (DE) and nano materials. The exposure time can be freely selected based on the need, from the beginning of development (embryonic day 0, ED0) all the way to the day prior to hatch. The major exposure methods include air-cell injection, direct microinjection, and air-cell inhalation (originally developed in our lab), and the currently available endpoints include cardiac function (electrocardiography), morphology (histological assessments) and molecular biological assessments (immunohistochemistry, qRT-PCR, western blotting, etc.). Of course, the chicken embryo model has its own limitations, such as limited availability of antibodies. Nevertheless, with more laboratories starting to utilize this model, it can be used to make significant contributions to the study of developmental cardiotoxicities.

Chicken embryos, as a classical developmental model, are used in our lab to assess developmental cardiotoxicities following exposure to various environmental contaminants. Exposure methods and morphological/functional assessment methods established are described in this manuscript.

Verwendung von Hühnerembryon als leistungsfähiges Werkzeug bei der Beurteilung von Entwicklungskardiotoxizitäten

Chicken embryos are a classical model in developmental studies. During the development of chicken embryos, the time window of heart development is ...

Entwicklungsbiologie

Single Cell Transfection in Chick Embryos

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a thorough analysis of the fates of single cells under normal and experimental conditions. To this end, transfection methods that target single progenitors are a prerequisite. We describe here a technically straightforward method for transfecting single cells in chicken tissues in-ovo, allowing reliable lineage tracing as well as genetic manipulation. Specific tissue domains are targeted within the somite or neural tube, and DNA is injected directly into the epithelium of interest, resulting in sporadic transfection of single cells. Using reporters, clonal populations may consequently be traced for up to three days, and behavior of genetically manipulated clonal populations can be compared with that of controls. This method takes advantage of the accessibility of the chick embryo along with emerging tools for genetic manipulation. We compare and discuss its advantages over the widely-used electroporation method, and possible applications and use in additional in-vivo models are also suggested. We advocate the use of this method as a significant addition and complement for existing lineage tracing and genetic interference tools.

Using fine tip micropipettes we inject plasmid DNA into subdomains of chicken somites or neural tubes. The concentration of the plasmid is adjusted to generate single transfected cells. We then allow the cells to develop into clonal populations.

Single Cell Transfektion in Hühnerembryonen

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

Biologie

In Ovo Intravascular Injection in Chicken Embryos

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering exogenous materials into chicken embryos has a great advantage for studying gene function, transgenic breeding, and chimera preparation during embryonic development. Here we show the method of in ovo intravascular injection whereby exogenous materials such as plasmid vectors or modified primordial germ cells (PGCs) can be transferred into donor chicken embryos at early developmental stages. The results show that the intravascular injection through the dorsal aorta and head allows injected materials to diffuse into the whole embryo through the blood circulatory system. In the presented protocol, the efficacy of exogenous plasmid and lentiviral vector introduction, and the colonization of injected exogenous PGCs in the recipient gonad, were determined by observing fluorescence in the embryos. This article describes detailed procedures of this method, thereby providing an excellent approach to studying gene function, embryo and developmental biology, and gonad-chimeric chicken production. In conclusion, this article will allow researchers to perform in ovo intravascular injection of exogenous materials into chicken embryos with great success and reproducibility.

In Ovo Intravascular Injection in Chicken Embryos

The overall goal of this paper is to describe how to perform in ovo intracellular injection of exogenous materials into chicken embryos. This approach is very useful to study the developmental biology of chicken embryos.

In Ovo Intravaskuläre Injektion in Hühnerembryonen

As a classical model system of embryo biology, the chicken embryo has been used to investigate embryonic development and differentiation. Delivering ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

Mit Zebrabärbling Modelle menschlicher Influenza-A-Virus-Infektionen Virostatika auf Bildschirm und Host-Immun-Zell-Antworten Charakterisieren

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

Herstellung von hochtiterinfektiösen Influenza-Pseudotyppartikeln mit Hülllykoproteinen aus hoch pathogenen H5N1- und Avian H7N9-Viren

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of interest. Currently, transgenic technology is becoming an indispensable tool for the study of Eimeria, the causative agents of coccidiosis in poultry and livestock. This protocol provides a detailed description of stable transfection in eimerian parasites: purification and nucleofection of sporozoites or second-generation merozoites, and in vivo propagation of transfected parasites. Using this protocol, we achieved transfection in several species of Eimeria. Taken together, nucleofection is a useful tool to facilitate genetic manipulation in eimerian parasites.

Nucleofection and In Vivo Propagation of Chicken Eimeria Parasites

Here, we provided a method to achieve stable transfection of chicken Eimeria parasites by nucleofecting sporozoites or second-generation merozoites. Genetically modified eimerian parasites expressing heterologous antigenic genes could be used as vaccine delivery vehicles.

Nukleofection und In Vivo Propagation von Hühner Eimeria Parasiten

Transfection is a technical process through which genetic material, such as DNA and double-stranded RNA, are delivered into cells to modify the gene of ...