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.)

NOTA: Observaciones generales: Llevar a cabo todos los procedimientos que implican la manipulación del huevo en condiciones estériles, y una técnica estéril debería utilizarse en consecuencia. Pre-limpie todo el equipo con etanol al 70% antes de su uso. En general, la inoculación del virus de la influenza y la cosecha deben llevarse a cabo en un laboratorio BSL-2. Sin embargo, si este protocolo se utiliza para propagar mucho más patógeno virus de la gripe (por ejemplo, las cepas pandémicas y prepandémicas, cepas que representan un peligro para las aves de corral y el ganado, las cepas de influenza aviar altamente patógena, la cepa H1N1 1918), los niveles de bioseguridad más altos y en algunos casos, se requieren precauciones de bioseguridad y autorizaciones. El Comité Institucional de Bioseguridad local debe aprobar toda la investigación con virus de la gripe. 1. Preparación de los huevos para la inoculación <ol><li> Obtener huevos de aves de suero libres de patógenos (SPF) recién fecundado de un proveedor adecuado. Típicamente, se utilizan huevos de gallina. Los huevos deben ser nosotrosed inmediatamente a su llegada. </li><li> Al lugar de llegada huevos en una incubadora de huevos humidificado con un tornero de huevo automático para girar los huevos con regularidad. Si un tornero de huevo automático no está disponible, gire manualmente los huevos por lo menos 2-3 veces al día (asegúrate de usar guantes y mantener todo lo más estéril posible). </li><li> Incubar huevos a 37 ° C y 55-60% de humedad durante 10-11 días. </li></ol> Candling 2. Huevo <ol><li> Utilice un candler huevo para examinar el huevo contra una luz brillante en un cuarto oscuro para comprobar los huevos para la infertilidad por candling después de unos 7 u 8 días de incubación. <ol><li> Limpie la candler huevo con etanol al 70%. Cambiar los guantes a menudo y desinfectar con etanol al 70% para reducir al mínimo el riesgo de contaminación por patógenos. </li><li> Retire los huevos de la incubadora y colocarlos en cajas de huevos vacías. </li><li> Apague las luces en la habitación. </li><li> Mantenga el extremo grande de cada huevo de una en una en contra de la candler. </li><li> Observe que el huevo se determine si es fértil o estéril. NOTA: los vasos sanguíneos finos que conducen a un embrión en forma de frijol debe ser claramente visible. Los huevos no fertilizados aparecerá como una pequeña mancha de sangre con una yema de huevo visible. </li></ol></li><li> Deseche los huevos que son sin fertilizar, y devolver los huevos viables a la incubadora. No deje los huevos fuera de la incubadora durante más de 30 min. Si el estado de un huevo no se puede confirmar, marcarlo, y observar más tarde. </li></ol> 3. Preparación de inóculo del virus <ol><li> Diluir virus de la gripe de stock a alrededor de 03 10 hasta 04 10 pfu / ml en PBS estéril que contiene HEPES 10 mM (pH 7,4). Paso crítico: Diluir el stock de virus inmediatamente antes de su uso y mantener el virus diluido en hielo todo el tiempo. </li></ol> 4. Influenza inoculación del virus a través de la Ruta alantoideo <ol><li> En el día de la inoculación del virus (día 10 o 11), Vela los huevos de nuevo y eliminar cualquier embriones muertos. Distinguir embriones vivos poren busca de movimiento dentro del huevo. </li><li> Retire los tres huevos de la incubadora, y organizar los huevos en un soporte independiente con la bolsa de aire hacia arriba. </li><li> Marca el espacio de aire de los huevos con un marcador permanente. </li><li> Lavar la cáscara del huevo por encima del espacio de aire con etanol al 70%. </li><li> Poner los huevos con el espacio hasta el aire en un cartón de huevos y en una campana de bioseguridad (BSL-2). </li><li> Use una aguja 18G estéril para perforar un pequeño orificio en la cubierta sobre el saco de aire de cada huevo. Tenga cuidado de no insertar la aguja demasiado profundamente para evitar apuñalando al embrión o yema. </li><li> Elaborar el virus de la influenza diluida en una jeringa de 1 ml estéril y conecte una aguja 22G. </li><li> Avanzar con cuidado la jeringa con la aguja en un ángulo de 45 ° en la cavidad alantoidea. </li><li> Inyectar 0,2 ml de virus de la gripe diluida. </li><li> Repetir la inyección con los otros dos huevos, y luego desechar la jeringa y la aguja. </li><li> Sellar el agujero con una gota de pegamento de una pistola de pegamento (o cera de parafina fundida). Be cuidado de que el pegamento no se filtre en el interior del huevo. </li><li> Coloque los huevos de nuevo en la incubadora de huevo con el espacio de aire apuntando hacia arriba. </li><li> Repita los pasos 04.02 a 04.12, hasta todos los huevos han sido inoculados. </li></ol> 5. Tiempo de incubación <ol><li> Incubar los huevos infectados sin girar a 37 ° C y ~ 60% de humedad. </li><li> Elige tiempo óptimo de incubación dependiendo del tipo de virus de la gripe. Incubar la gripe A durante 48 h; incubar la influenza B y C durante 72 h. </li></ol> 6. Influenza Virus Cosecha <ol><li> Después del período de incubación, los huevos enfriar a 4 ° C durante un mínimo de 4 horas (o O / N) para matar el embrión y constreñir los vasos sanguíneos para reducir el riesgo de contaminar el fluido alantoideo infectado con sangre. Hacer esto para evitar que las células rojas de la sangre de la absorción del virus, lo que reduce el rendimiento. Alternativamente, los huevos de enfriamiento durante 30 minutos a -20 ° C; sin embargo, esto aumenta el riesgo de contaminación de la sangre. </li><li> Después de que los huevos han sido suficientemente refrigerado, transferir los huevos a una campana de bioseguridad. Coloca el huevo en un soporte firme con la bolsa de aire hacia arriba. Limpie la superficie del huevo con etanol al 70%. </li><li> Con unas tijeras estériles para abrir la cáscara de los huevos encima de la bolsa de aire. Retire la cáscara alrededor del saco de aire con cuidado de no destruir la membrana corioalantoidea. </li><li> Abra la membrana corioalantoidea con unas pinzas romas estériles. Mover suavemente a un lado el embrión y el saco vitelino con una pequeña espátula o una cuchara. Tenga cuidado de no romper la yema. </li><li> Utilizando una pipeta Eppendorf de 1 ml, recoger cuidadosamente el fluido alantoideo. Si es necesario, incline el huevo un poco hacia un lado para recoger la mayor cantidad de líquido posible. </li><li> Combinar el fluido alantoideo de todos los huevos en un tubo cónico de plástico de 50 ml. Mantenga los tubos en hielo en todo momento durante la cosecha de virus. Los rendimientos de un huevo de 5 - 10 ml de un líquido ligeramente amarillento. </li><li> Girar el fluido alantoideo que contiene el virus a 1.000 xg durante 10 min a 46; C para sedimentar los restos. Transferir el líquido claro en un nuevo tubo de 50 ml se mantuvo en hielo. Deseche los huevos en un recipiente de residuos BSL-2. </li></ol> 7. Almacenamiento <ol><li> Inmediatamente la alícuota aclarado alantoideo fluido (por ejemplo, 50, 100 o 500 alícuotas ul) y complemento de congelación en nitrógeno líquido. Transferencia de existencias congeladas virus a -80 ° C congelador para el almacenamiento a largo plazo. </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.

La gripe causa una gran carga mundial de la enfermedad, y se sigue trabajando en la comprensión de la patogénesis de la lesión pulmonar 7. Para la investigación facilitado en esta enfermedad mortal, varios se han desarrollado métodos para propagar el virus de la gripe 6. Aquí se describe una técnica para producir virus de la gripe en huevos de gallina. La ventaja de este método es que es altamente reproducible y resulta en grandes cantidades de reservas de virus de la gripe de alta titulaci...

La gripe A sigue siendo una amenaza importante para la salud humana. Es una enfermedad respiratoria potencialmente devastadora con una gran carga mundial que causa más de 500.000 muertes en el mundo cada año 1. Los virus de influenza son de la familia Orthomyxoviridae y llevan 8 de sentido negativo de ARN de cadena sencilla en su genoma 1,2. La alta mutabilidad (es decir, &quot;deriva&quot; antigénica) del genoma viral impide inmunidad a largo plazo. Por otra parte, la gripe es cada vez más resistente a los medicamentos antivirales 3. La pandemia de gripe H1N1 2009 ponía de relieve todas las cuestiones difíciles asociados con la enfermedad de la gripe (cepa pandémica, la resistencia antiviral, la producción de vacunas retardada). La formulación de la vacuna es determinada anualmente por la Organización Mundial de la Salud para las cepas de gripe más probable patógenos (uno cada uno para H1N1, H3N2 y la influenza B) 4. Debido a que este método se basa en la cepa de gripe previstas, las cepas patógenas son ocasionalmente incorrectamente identificados y eficacia de la vacuna se reduce drásticamente. Por otra parte, la aparición de nuevas cepas puede eludir estos programas preventivos que causan enfermedad pandémica 2. La creación de una vacuna contra la gripe universal ha sido difícil de alcanzar 5. Por lo tanto, la investigación debe continuar en la comprensión de la patogénesis de la lesión pulmonar. Para la investigación de laboratorio instalación, varios métodos se han desarrollado para el aislamiento viral y la propagación 6. Virus de influenza humana pueden ser amplificados en una variedad de substratos de células de mamíferos. Sin embargo, la generación de virus de alta titulación en grandes cantidades se logran mejor en huevos embrionados. El siguiente protocolo describe una técnica para la gripe A la propagación de virus y el almacenamiento de reservas de virus existentes.

<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

La infección por influenza se asocia con alrededor de 36,000 muertes y más de 200,000 hospitalizaciones cada año en los Estados Unidos. La aparición continua de nuevas cepas de virus de la gripe debido a la mutación y re-surtido complica el control del virus y requiere el desarrollo permanente de nuevos fármacos y vacunas. El estudio de laboratorio de influenza requiere de un método fiable y rentable para la propagación del virus. Aquí, se proporciona un protocolo completo para la gripe A la propagación de virus en huevos de pollo fértiles, que produce consistentemente stocks virales de alto título. En resumen, (SPF) huevos de gallina fertilizados libres de patógenos de suero se incuban a 37 ° C y 55-60% de humedad durante 10 - 11 días. Durante este período, el desarrollo del embrión se puede controlar fácilmente con un candler huevo. La inoculación del virus se lleva a cabo mediante la inyección de stock de virus en la cavidad alantoidea usando una aguja. Después de 2 días de incubación a 37 ° C, los huevos sonrefrigerada durante al menos 4 horas a 4 ° C. La cáscara de huevo por encima de la bolsa de aire y la membrana corioalantoidea continuación, se abrió cuidadosamente, y el fluido alantoideo que contiene el virus se recoge. El líquido se elimina de los desechos por centrifugación, se dividió en alícuotas y se transfiere a -80 ° C para almacenamiento a largo plazo. La gran cantidad (5-10 ml de virus que contiene fluido por huevo) y de alto título de virus que se consigue normalmente con este protocolo ha hecho que el uso de huevos para la preparación de virus nuestro método favorable, en particular para los estudios in vitro que requieren grandes cantidades de virus en los que se necesitan altas dosis de la misma stock de virus.

Múltiples métodos se han desarrollado para el título del virus de la influenza. Una consideración al momento de elegir un método apropiado es que algunos determinar partículas totales, independientemente de la viabilidad (por ejemplo, ensayo de hemaglutinina), mientras que otros se basan en la infectividad, que evaluará viriones viables (por ejemplo, la placa de ensayo) 6. Aquí, el título viral de fluido alantoideo recogido a partir de huevos de pollo inoculados con un virus de la g...

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.

Propagación del virus de la influenza en embrionados de pollo Huevos

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

43.1K vistas.  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.

Video: 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.

Generación de recombinantes del virus de influenza a partir del ADN plásmido

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

Investigación

Inmunología e Infección

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.

Utilizando imágenes bioluminiscentes encargado de investigar la sinergia entre Streptococcus pneumoniae Y el Virus de la Influenza en ratones lactantes

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.

Rescate del virus de la enfermedad de Newcastle recombinante a partir de 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.

Producción de virus de la enfermedad de Newcastle recombinante de alto título a partir de líquido alantoico

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

Investigación sobre el cáncer

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.

Uso del embrión de pollo como una herramienta poderosa en la evaluación de cardiotoxicidades del desarrollo

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

Biología del Desarrollo

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.

Transfección de células individuales en embriones de pollo

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

Biología

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.

En Ovo Inyección intravascular en embriones de pollo

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.

El uso de modelos de pez cebra de Infecciones por Virus de la Influenza Humana A a la pantalla medicamentos antivirales y caracterizar inmune del huésped respuestas de las células

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.

Producción de partículas pseudotipadas de influenza infecciosa de alto valor con glicoproteínas envolventes de virus H5N1 altamente patógenos y aviares H7N9

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.

Nucleofección y Propagación In Vivo de Parásitos de La Eimeria de Pollo

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 ...