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: Observações gerais: executar todos os procedimentos que envolvem a manipulação do ovo em condições estéreis, e uma técnica estéril deve ser usado em conformidade. Pré-limpa todos os equipamentos com etanol 70% antes do uso. Em geral, a inoculação e colheita vírus influenza deve ser realizado em um laboratório BSL-2. No entanto, se esse protocolo é usado para propagar muito mais patogênico vírus da gripe (por exemplo, cepas pandêmicas e pré-pandemia, estirpes que representam um perigo para as aves de capoeira e, cepas altamente patogênicas da gripe aviária, o H1N1 estirpe 1918 gado), níveis de biossegurança mais elevadas e em alguns casos, as precauções de biosegurança e autorizações são necessários. O Comitê de Biossegurança Institucional local deve aprovar todas as pesquisas envolvendo o vírus da gripe. 1. Preparação de ovos para a inoculação <ol><li> Obter ovos de aves (SPF) isentos de agentes patogénicos recém-fertilizado de soro de um fornecedor adequado. Tipicamente, os ovos de galinha são utilizados. Os ovos devem ser nósed imediatamente após a chegada. </li><li> Após ovos lugar chegada em uma incubadora de ovos umidificado com uma turner ovo automática para rodar ovos regularmente. Se um turner ovo automática não está disponível, rodar manualmente ovos pelo menos 2-3 vezes por dia (certifique-se de usar luvas e manter tudo tão estéril quanto possível). </li><li> Incubar os ovos a 37 ° C e 55-60% de humidade durante 10-11 dias. </li></ol> 2. Egg Candling <ol><li> Use um candler ovo para examinar o ovo contra a luz em um quarto escuro para verificar os ovos para a infertilidade por candling após cerca de 7 ou 8 dias de incubação. <ol><li> Limpe a candler ovo com etanol 70%. Mudar de luvas frequentemente e desinfectar com etanol a 70% para minimizar o risco de contaminação por agentes patogénicos. </li><li> Retire os ovos da incubadora e colocá-los em caixas de ovos vazias. </li><li> Desligue as luzes na sala. </li><li> Segurar a extremidade mais larga de cada ovo, um de cada vez contra a candler. </li><li> Observar o ovo para determine se é fértil ou estéril. NOTA: os vasos sanguíneos finos que conduzem a um embrião em forma de feijão deve ser claramente visível. Ovos não fertilizados aparece como uma mancha de sangue pequena, com uma gema de ovo visível. </li></ol></li><li> Descartar os ovos que são não fertilizado, e devolver os ovos viáveis ​​para a incubadora. Não deixar ovos fora da incubadora durante mais de 30 min. Se o status de um ovo não pode ser confirmada, marcá-lo e observá-lo mais tarde. </li></ol> 3. Preparação de Vírus Inoculante <ol><li> Diluir stock de virus da gripe e cerca de marco 10-abril 10 pfu / ml em PBS estéril contendo 10 mM de HEPES (pH 7,4). Passo crítico: Diluir a reserva de vírus imediatamente antes da utilização e manter o vírus diluído em gelo durante todo o tempo. </li></ol> 4. Influenza inoculação do vírus através da Route alantóica <ol><li> No dia da inoculação com o vírus (dia 10 ou 11), os ovos novamente vela e eliminar quaisquer embriões mortos. Distinguir embriões vivos porolhando para o movimento no interior do ovo. </li><li> Remova três ovos da incubadora, e organizar os ovos em um suporte em separado com o saco de ar para cima. </li><li> Marque o espaço aéreo dos ovos com um marcador permanente. </li><li> Lava-se a casca de ovo, acima do espaço de ar com etanol a 70%. </li><li> Coloque os ovos com o espaço-se ar em uma caixa de ovos e em uma capa de biossegurança (BSL-2). </li><li> Use uma agulha de 18G estéril para perfurar um pequeno buraco no shell sobre o saco de ar de cada ovo. Tenha cuidado para não inserir a agulha muito profundamente para evitar esfaquear o embrião ou gema. </li><li> Elaborar o vírus influenza diluído em uma estéril seringa de 1 ml e anexar uma agulha 22G. </li><li> Cuidadosamente avançar a seringa com a agulha com um ângulo de na cavidade alantóide de 45 °. </li><li> Injectar 0,2 mL de vírus da gripe diluiu-se. </li><li> Repetir a injecção com os outros dois ovos, e, em seguida, descartar a seringa e agulha. </li><li> Selar o buraco com uma gota de cola de uma pistola de cola (ou cera de parafina derretida). Be cuidado que a cola não vaza dentro do ovo. </li><li> Coloque os ovos de volta para a incubadora de ovos com o espaço aéreo apontou para cima. </li><li> Repita os passos 4,2-4,12 até que todos os ovos foram inoculados. </li></ol> 5. Tempo de Incubação <ol><li> Incubar os ovos infectados sem virar a 37 ° C e ~ 60% de humidade. </li><li> Escolha tempo de incubação óptimo, dependendo do tipo de vírus da gripe. Incubar a influenza A por 48 h; incubar gripe B e C durante 72 h. </li></ol> 6. Vírus da Influenza Colheita <ol><li> Após o período de incubação, refrigerar os ovos a 4 ° C durante um mínimo de 4 h (ou S / N) para matar o embrião e a constrição dos vasos sanguíneos para reduzir o risco de contaminação do fluido alantóico infectado com sangue. Fazer isso para evitar que as células vermelhas do sangue de absorção do vírus, o que reduz o rendimento. Alternativamente, os ovos frio durante 30 min a -20 ° C; no entanto, isso aumenta o risco de contaminação por sangue. </li><li> Depois que os ovos tenham sido suficientemente refrigerados, transferir os ovos para uma capa de biossegurança. Coloque o ovo em um suporte firme com o saco de ar voltada para cima. Limpe a superfície do ovo com etanol 70%. </li><li> Use uma tesoura esterilizada para abrir a casca de ovo por cima do saco de ar. Retire a casca ao redor do saco de ar, tendo o cuidado para não destruir a membrana corioalantóide. </li><li> Abrir a membrana corioalantóica com fórceps estéreis rombas. Gentilmente afastar o embrião e do saco vitelino com uma pequena espátula ou colher. Tome cuidado para não romper a gema. </li><li> Utilizando uma pipeta de Eppendorf de 1 mL, cuidadosamente recolher o fluido alantóico. Se necessário, incline o ovo um pouco para o lado para coletar o máximo possível de líquidos. </li><li> Combina-se o fluido alantóico a partir de todos os ovos em 50 ml de um tubo cónico de plástico. Manter tubos em gelo em todos os momentos durante a colheita de vírus. Um ovo rendimentos de 5 - 10 ml de um fluido de cor ligeiramente amarelada. </li><li> Girar o fluido alantóide que contém o vírus a 1.000 xg durante 10 min a 46; C para sedimentar os detritos. Transferir o fluido límpido para um novo tubo de 50 ml mantido em gelo. Descarte ovos em um recipiente de resíduos BSL-2. </li></ol> 7. Armazenamento <ol><li> Aliquotar imediatamente o líquido alantóico clarificado (por exemplo, 50, 100 ou 500 ul aliquotas) e encaixe por congelação em azoto líquido. Transferir stocks de vírus congeladas a -80 ° C congelador para armazenamento a longo prazo. </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 provoca uma grande carga global de doenças, eo trabalho continua para a compreensão da patogênese da lesão pulmonar 7. Para facilitar a investigação nesta doença mortal, vários métodos têm sido desenvolvidos para propagar o vírus da gripe 6. Aqui, descrevemos uma técnica para produzir vírus influenza em ovos de galinha. A vantagem deste método é que é altamente reprodutível e resulta em grandes quantidades de estoques de vírus de título elevado gripe, o que é muitas ve...

Influenza A continua a ser uma grande ameaça para a saúde humana. É uma doença respiratória potencialmente devastador com uma grande carga global causando até 500.000 mortes no mundo anualmente 1. Os vírus da gripe estão na família Orthomyxoviridae e levar de cadeia simples RNA 8 negativas-sense em seu genoma 1,2. A mutabilidade alta (isto é, &quot;deriva&quot; antigénico) do genoma viral impede a imunidade a longo prazo. Além disso, a gripe é cada vez mais resistentes aos medicamentos anti-virais 3. A pandemia de gripe H1N1 2009 em destaque todas as questões desafiadoras associados à doença influenza (estirpe pandémica, a resistência anti-viral, a produção de vacinas em atraso). A formulação da vacina é determinado anualmente pela Organização Mundial de Saúde para as cepas de influenza provavelmente patogênicos (um para cada H1N1, H3N2 e influenza B) 4. Uma vez que este método é baseado na estirpe da gripe previus, estirpes patogénicas são ocasionalmente incorretamente identificado e eficácia da vacina cai drasticamente. Além disso, o surgimento de novas cepas de influenza pode contornar esses programas preventivos que causam doenças pandêmicas 2. A criação de uma vacina contra a gripe universal tem sido difícil 5. Portanto, a pesquisa deve continuar na compreensão da patogênese da lesão pulmonar. Para pesquisas de laboratório instalação, vários métodos foram desenvolvidos para o isolamento viral e propagação 6. Os vírus influenza humanos pode ser amplificado em uma variedade de substratos de células de mamíferos. No entanto, gerando vírus de título elevado em grandes quantidades são atingidas mais facilmente em ovos embrionados. O protocolo seguinte descreve uma técnica para a propagação de vírus influenza A e armazenamento de stocks de vírus 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

A infecção por influenza está associada com cerca de 36.000 mortes e mais de 200 mil internações por ano nos Estados Unidos. O surgimento contínuo de novas estirpes do vírus da influenza, devido a mutação e de re-sortido complica o controlo do vírus e necessita o desenvolvimento permanente de novas drogas e vacinas. O estudo baseado em laboratório da gripe requer um método confiável e de custo eficaz para a propagação do vírus. Aqui, um protocolo global prestada para influenza A propagação do vírus em ovos de galinhas férteis, que consistentemente produz stocks virais título elevado. Em breve, isentos de agentes patogénicos (SPF) de ovos de galinha fertilizados de soro são incubadas a 37 ° C e 55-60% de humidade durante 10-11 dias. Durante este período, o desenvolvimento do embrião pode ser facilmente monitorizado utilizando um candler ovo. A inoculação do virus é realizada por injecção de estoque de vírus na cavidade alantóide usando uma agulha. Após 2 dias de incubação a 37 ° C, os ovos sãorefrigeradas durante pelo menos 4 horas a 4 ° C. A casca de ovo de cima do saco de ar e a membrana corioalantóica são então cuidadosamente aberto e o fluido alantóide que contém o vírus é colhido. O fluido é limpa de detritos por centrifugação, aliquotado e transferido para -80 ° C para armazenamento a longo prazo. A grande quantidade (5-10 ml de fluido por ovo contendo vírus) e título de alta vírus quais geralmente são obtidas com este protocolo fez com que o uso de ovos para a preparação de vírus nosso método favorável, em particular, para estudos in vitro que necessitem de grandes quantidades de vírus em que são necessárias doses elevadas da mesma reserva de vírus.

Vários métodos têm sido desenvolvidos para titular o vírus da gripe. Uma consideração na escolha de um método adequado é que alguns determinar partículas totais, independentemente da viabilidade (por exemplo, o ensaio de hemaglutinina), enquanto outros são baseados em infectividade, que avaliará virions viáveis ​​(por exemplo, teste de placas) 6. Aqui, o título virai de líquido alantóico recolhidas a partir de ovos de galinha inoculados com um vírus da gripe adaptada-rato...

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.

Propagação do vírus Influenza em ovos de galinha embrionados

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 visualizações.  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.

Vídeo: 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.

Geração de recombinante do vírus Influenza de DNA plasmidial

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

Imunologia e Infecção

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.

Usando imagens Bioluminescent para Investigar sinergismo entre Streptococcus pneumoniae E Vírus da Influenza A em Ratos infantil

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.

Resgate de Vírus da Doença de Newcastle recombinante 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.

Produção de Vírus da Doença de Newcastle Recombinante de Alta Titer a partir de fluido allantóico

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

Pesquisa do câncer

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.

Usando embrião de frango como ferramenta poderosa na avaliação de cardiotoxi cidades do desenvolvimento

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

Biologia do Desenvolvimento

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.

Transfecção única célula em embriões de galinha

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

Biologia

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.

Em Ovo Injeção intravascular em embriões de frango

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.

Usando modelos de Zebrafish de infecções por vírus gripe humana A para Screen antivirais e Caracterizar imune do hospedeiro respostas de 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.

Produção de partículas pseudodigitadas de gripe infecciosa de alto titer com glicoproteínas envelope de vírus H5N1 e H7N9 altamente patogênicos

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.

Nucleofection e In Vivo Propagação de Parasitas de Eimeria de Frango

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 ...