Queremos agradecer Scullion M. e R. Crisp de Idaho Technologies para o suporte técnico eo USGS ocidental Centro de Pesquisa Ecológica de financiamento (S. Schwarzbach) e assistência (K. Spragens, T. Graham). Esta pesquisa foi realizada sob os auspícios do Centro para Inovação Tecnológica - Instituto de Defesa e Segurança Interna (www.idhs.org), em apoio ao Departamento de Defesa e Laboratório de Pesquisas da Força Aérea. Manuseio dos animais seguiu protocolos aprovados pelo Comitê Animal Care e Uso do Geological Survey Research Centro Oeste dos EUA e permite Ecológica do Pássaro Serviço Geológico dos EUA Banding Laboratory. Qualquer uso do comércio, produto ou empresa nomes nesta publicação é apenas para fins descritivos e não implica o endosso pelo governo dos EUA.

1. Captura de aves selvagens usando redes de neblina <ol><li> Para a captura de shorebird, configurar redes de neblina em um sítio ativo de forrageamento como um pântano, costa, ou lama plana. </li><li> Deslize linha de tresmalho loops de uma extremidade da rede de neblina em torno do pólo e inserir pole verticalmente em lama. </li><li> Estende a net, insira segundo pólo através de loops de tresmalho na outra extremidade da rede de neblina e verticalmente inserir pole em lama, certificando-se que as linhas de tresmalho são ensinados. </li><li> Uma vez que um pássaro é capturado, extrair o pássaro da rede e voltar para a estação de bandas. </li></ol> 2. Cloacal técnica swab <ol><li> Coletar swabs cloacais imediatamente após a captura </li><li> Localize a cloaca e use os dedos para empurrar para trás as penas ao redor </li><li> Desembalar um Dacron ou estéril com ponta de poliéster algodão, caule primeira extremidade, e umedecer a ponta com estéril meios de transporte viral para maior facilidade na raspagem. Depois de umedecimento, a cabeça inteira do swab é gentilmente inserido na cloaca. Swab circunferência no interior da cloaca, lentamente girando o cotonete e retire o swab de cloaca. </li><li> Inserindo a ponta swab diretamente no frasco da amostra viral meios de transporte; girar o eixo do swab entre o polegar eo indicador, enquanto a mecha é na mídia. Ter um assistente concluir o procedimento puxando a ponta do cotonete de volta do fundo do frasco ~ 1 cm e corte o eixo da haste com um par de tesouras para a ponta swab permanece no frasco e no eixo não interfere com fechamento da tampa. Frasco tampa. Desinfectar a tesoura com álcool entre os eixos de corte cotonete. </li><li> Coloque os frascos em uma caixa contendo um ou outro freezer gelo seco ou bolsas de gelo. </li></ol> 3. A técnica swab de orofaringe <ol start="1"><li> Abra cuidadosamente o bico do pássaro para que a fenda coanal é visível </li><li> Desembrulhar um cotonete limpo do pacote, caule primeira extremidade, e umedecer a ponta com meios de transporte viral para maior facilidade na swabbing </li><li> Sem tocar na ponta de quaisquer outras superfícies, insira-o na boca e swab da abertura coanal no céu da boca e continuar em direção a região da orofaringe ou da garganta </li><li> Coloque a ponta da cotonete diretamente no frasco de meio de transporte viral. Girar o eixo do swab entre o polegar eo indicador, enquanto a mecha é na mídia. Como acima, ter um assistente puxar a ponta da haste de volta do fundo do frasco ~ 1 cm e corte o eixo da haste com um par de tesouras para a ponta swab permanece no frasco e no eixo não interfere com tampa encerramento. Frasco tampa. Desinfectar a tesoura com álcool entre os eixos de corte cotonete. </li><li> Coloque os frascos em uma caixa freezer contendo gelo seco ou bolsas de gelo. </li></ol> 4. Manuseio de aves e de liberação <ol start="1"><li> Retorno das aves para o local de captura e liberação em tempo hábil (&lt;20 minutos). Manipuladores devem estar cientes dos princípios de bem-estar animal e estar alerta para sinais de sofrimento durante a amostragem de aves (abrasões ou seja, a captura de miopatia e hipo ou hipertermia). Um kit básico de primeiros socorros deve ser incluído na lista de equipamento. </li></ol> 5. Instalações de teste <ol start="1"><li> Amostras podem ser testadas em qualquer espaço fechado, como um reboque de pesquisa ou tenda, com uma fonte de alimentação (tensão regulada gerador de energia) para executar a unidade de PCR e computador ligado. </li></ol> 6. A extração de RNA <ol start="1"><li> Extração de RNA foi realizada com o kit RNeasy Mini com pequenas modificações para as instruções do fabricante seguintes Spackman et al 1. </li><li> Médio Vortex o cotonete para 3-5 s e transferência de 350 mL para um tubo de microcentrífuga de 2 ml. Nota: Mantenha amostra restante no gelo em caso PCR amostras positivas precisam ser submetida a novo rastreio ou teste H5 precisa ser executado. </li><li> Adicionar 350 mL de tampão RLT (com β-me) e vortex por 5 s. </li><li> Adicionar 350 uL de RNA de etanol de grau de 70%. </li><li> Centrifugar a 5.000 xg por 5 min. </li><li> Adicionar 600 mL do sobrenadante para uma coluna de spin colocado em um tubo de coleta de 2 ml. Manter a amostra restante em rack tubo de ensaio. </li><li> Centrifugar durante 20 s em 10.000 xg e descarte por escoamento. </li><li> Repita os passos 17-18 até que todas as amostras foi passada através da coluna spin. </li><li> Colocar a coluna girar em um tubo de coleta limpa 2 ml. </li><li> Adicionar 700 mL de tampão RW1 para a coluna de spin e centrifugar 25 s em 10.000 x g. Descartar o escoamento. </li><li> Adicionar 500 mL tampão RPE para a coluna girar e centrifugar por 25 segundos a 10.000 x g. Descartar o escoamento. </li><li> Repita o passo 22 para um total de duas lavagens com tampão RPE. </li><li> Centrifugar a coluna girar por mais 2 minutos a 14.000 x g. Descartar tubo de coleta. </li><li> Coloque a coluna girar em um tubo de microcentrífuga de 1,5 ml e adicionar 50μl de água livre de nuclease. Incubar a temperatura ambiente por 60 s. </li><li> Eluir RNA girando por 60 s em 10.000 xgfou 60 s. Lugar purificado amostra no gelo. </li></ol> 7. Preparando-se controles negativos <ol start="1"><li> Reagentes liofilizados foram preparados para serem utilizados no ensaio de PCR de acordo com instruções do Idaho Technologies Kit de Detecção de Freeze-Dried Reagente para TaqMan Sondas Influenza A Meta 1 (Asay-ASY-0109). </li><li> Centrífuga frasco de reagente controle negativo (vermelho) para 3 s para garantir pelotas são na parte inferior. </li><li> Retire a tampa, visualmente fazer pellets certeza estão na parte inferior. </li><li> Adicionar 20 mL de Tampão de Reconstituição 2 X e 20 mL de água grau reagente. </li><li> Recap e vortex por 5 s na velocidade máxima. </li><li> Centrifugar por 3 s para levar líquidos para o fundo do frasco. </li><li> Verificar visualmente o pellet foi reidratado, se não repita os passos 31-32. </li><li> Pipetar 19 mL da mistura hidratada em um tubo capilar LightCycler, repita o passo pipeta capilar em um segundo para obter uma segunda via. </li></ol> 8. Preparação de amostras de teste <ol start="1"><li> Centrifugar o frasco reagente desconhecido (azul) durante 3 segundos para garantir pelotas são na parte inferior. </li><li> Retire a tampa, visualmente fazer pellets certeza estão na parte inferior. </li><li> Adicionar 40 mL de RNA purificado. </li><li> Recap e vortex por 5 s na velocidade máxima. </li><li> Centrifugar por 3 s para levar líquidos para o fundo do frasco. </li><li> Verificar visualmente o pellet foi reidratado, se não repita os passos 38-39. </li><li> Pipetar 19 mL da mistura hidratada em um tubo capilar LightCycler, repita o passo pipeta capilar em um segundo para obter uma segunda via. </li></ol> 9. Preparar controles positivos <ol start="1"><li> Centrifugar o frasco de reagente de controlo positivo (verde) para 3 s para garantir pelotas são na parte inferior. </li><li> Retire a tampa, visualmente fazer pellets certeza estão na parte inferior. </li><li> Adicionar 20 mL de Tampão de Reconstituição 2 X e 20 mL de água grau reagente. </li><li> Recap e vortex por 5 s na velocidade máxima. </li><li> Centrifugar por 3 s para levar líquidos para o fundo do frasco. </li><li> Verificar visualmente o pellet foi reidratado, se não repita os passos 45-46. </li><li> Pipetar 19 mL da mistura hidratada em um tubo capilar LightCycler, repita o passo pipeta capilar em um segundo para obter uma segunda via. </li></ol> 10. Centrifugação os tubos capilares <ol start="1"><li> Depois de todos os tubos capilares para o lote foram preparadas carregá-los no mini-centrífuga equipados com adaptadores capilar. </li><li> Centrífuga na configuração mais baixa, segurando o botão de pulso para baixo por 1 s. Isso transfere líquido para o fundo, em preparação para o teste. </li></ol> 11. Operação do RAPID <ol start="1"><li> Criar um novo arquivo. Controle de rótulo positivo, controle negativo e amostras (pelo seu número de ID). </li><li> Programa 1: Segure RNA Preparação da Amostra <ol><li> Ciclos = 1 </li><li> Modo de análise = None </li><li> Temperatura target = 40 </li><li> Tempo de incubação = 30 min </li><li> Taxa de transição de temperatura = 20 ° C / s (padrão) </li><li> Alvo de temperatura secundário = 0 (default) </li><li> Tamanho passo = 0 (default) </li><li> Atraso passo = 0 (default) </li><li> Modo de aquisição = Nenhum (padrão) </li></ol></li><li> Programa 2: RNA Desnaturação <ol><li> Ciclos = 1 </li><li> Modo de análise = None </li><li> Temperatura target = 94 </li><li> Tempo de incubação = 120s </li><li> Taxa de transição de temperatura = 20 ° C / s (padrão) </li><li> Alvo de temperatura secundário = 0 (default) </li><li> Tamanho passo = 0 (default) </li><li> Atraso passo = 0 (default) </li><li> Modo de aquisição = Nenhum (padrão) </li></ol></li><li> Programa 3: Ampliação RNA <ol><li> Ciclos = 45 </li><li> Modo de Análise de Análise de Auto = </li><li> Temperatura-alvo, Segmento 1 = 94; Segmento 2 = 60 </li><li> Tempo de incubação, Segmento 1 = 0 s, Segmento 2 = 20 s </li><li> Taxa de temperatura de transição, Seg 1 &amp; 2 = 20 ° C / s (padrão) </li><li> Alvo de temperatura secundário, Seg 1 &amp; 2 = 0 (default) </li><li> Tamanho passo, Seg 1 &amp; 2 = 0 (default) </li><li> Atraso passo, Seg 1 &amp; 2 = 0 (default) </li><li> Modo de aquisição, Segmento 1 = None; Segmento 2 = single </li></ol></li><li> Parâmetros de fluorescência <ol><li> Modos de Exibição: canal de fluorescência 2 (Ch2 / 1) Ganhos: <ol><li> Ch 1 (F1) = 1 </li><li> Ch 2 (F2) = 8 </li><li> Ch 3 (F3) = 1 </li></ol></li><li> Para exibir os dados em tempo real de fluorescência para cada amostra, seleção da amostra de interesse e clique no botão &#39;Gráfico Show&#39; na parte inferior da tela </li></ol></li><li> Para cada capilar, os resultados são os seguintes: <ul><li> # - Identifica a posição do carrossel carrossel </li><li> Nome da amostra - fornece o nome da amostra </li><li> Controle - indentifies o tipo de amostra para cada amostra </li><li> Pontuação - calculada utilizando a relação entre a fluorescência da amostra e do nível de fluorescência de fundo na amostra </li><li> Resultado - exibe o resultado global para a amostra </li></ul></li></ol> 12. Resultados representativos: Presente: Uma chamada de vermelha &quot;presente&quot; para um teste / amostra desconhecida indica que o alvo foi identificado em que a amostra e os controles foram bem sucedidos. Não detectado: Uma chamada de verde &quot;não foi detectado&quot; por um teste / amostra desconhecida indica que o alvo não foi identificado em que a amostra de teste e os controles foram bem sucedidos. Por favor, repito: A &#39;repetir, por favor&#39; indica que o controlo positivo ou negativo falhou. Um exemplo de uma análise bem sucedida pelo RAPID 7200 é mostrado na Figura 4. O controle positivo é amplificada e gera fluorescência detectável (eixo y) em aproximadamente 25 ciclos (x-eixo). Um limite de ciclo (CT)&gt; 35 é o cut-off acordado para laboratórios de referência mais AIV. Portanto, os controles positivos para este método produziu um sinal fluorescente dentro do número de ciclos aceita (0-35) para a detecção de AIV. Em contraste, o controle negativo não gera um sinal fluorescente, mesmo após 45 ciclos. Da mesma forma, os doze amostras coletadas de maçaricos ocidental não gerar um sinal fluorescente que indica que as aves foram negativos para AIV. Testes de acompanhamento de todas as amostras positivas em um laboratório tradicional é incentivado para garantir que nenhum falso positivo são erroneamente produzido. <img src="files/ftp_upload/2829/2829fig1.jpg" alt="Figura 1" /> Figura 1. Capturar Shorebird usando redes de neblina. <img src="files/ftp_upload/2829/2829fig2.jpg" alt="Figura 2" /> Figura 2. Coleta de amostras de cloaca e orofaringe de pelo maçaricos. <img src="files/ftp_upload/2829/2829fig3.jpg" alt="Figura 3" /> Figura 3. Programação do RAPID 7200 para a amplificação de RNA. <img src="files/ftp_upload/2829/2829fig4.jpg" alt="Figura 4" /> Figura 4. Fluorogram gerado pelo RAPID 7200 portátil rRT-PCR mostrando como controles positivos e negativos devem aparecer em um ensaio bem-sucedido. <a href="http://www.jove.com/files/ftp_upload/2829/2829fig4large.jpg">Clique aqui para ver a imagem de tamanho completo</a>

<ol>
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Não há conflitos de interesse declarados.

O método de diagnóstico rápido aqui apresentados facilita os testes de tempo eficiente e preciso de amostras de aves selvagens para a vigilância de AIV. Os requisitos espécime muito menos rigorosas de armazenamento portátil de rRT-PCR são adequadas para situações remotas, onde a manutenção de uma cadeia de frio pode ser impraticável se carregadores de nitrogênio líquido ou gelo seco não está disponível. Além disso, descobrimos que a análise da amostra com os reagentes liofilizados foi suficientemente ...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número Catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Coluna de spin RNeasy mini- </td><td> Qiagen </td><td> 74106 </td><td> Incluído no Kit Mini RNeasy </td></tr><tr><td> Tubos de coleta (1,5 e 2 mL) </td><td> Qiagen </td><td> 74106 </td><td> Incluído no Kit Mini RNeasy </td></tr><tr><td> Tampão RLT </td><td> Qiagen </td><td> 74106 </td><td> Incluído no Kit Mini RNeasy </td></tr><tr><td> Tampão RW1 </td><td> Qiagen </td><td> 74106 </td><td> Incluído no Kit Mini RNeasy </td></tr><tr><td> Tampão RPE </td><td> Qiagen </td><td> 74106 </td><td> Incluído no Kit Mini RNeasy </td></tr><tr><td> RNase água </td><td> Qiagen </td><td> 74106 </td><td> Incluído no Kit Mini RNeasy </td></tr><tr><td> 14,3 M de solução de β-mercaptoetanol </td><td> Fisher Scientific </td><td> BP176100 </td><td></td></tr><tr><td> Etanol 100% </td><td> Fisher Scientific </td><td> NC9602322 </td><td></td></tr><tr><td> Vortex Genie 2, 120V </td><td> Scientific Industries </td><td> SI-0236 </td><td></td></tr><tr><td> Taqman Influenza A Meta 1 (Probe Hidrólise) </td><td> Idaho Technologies </td><td> Asay-ASY-0109 </td><td></td></tr><tr><td> LightCycler 20ml tubos capilares </td><td> Roche Applied Science </td><td> 04929292001 </td><td></td></tr><tr><td> Micro-centrífuga com rotor para tubos de 2 mL </td><td> Idaho Technologies </td><td></td><td> Incluído no kit RAPID </td></tr><tr><td> Dispositivo de Identificação Ruggedized avançada de patógenos (RAPID) 7200 </td><td> Idaho Technologies </td><td></td><td> Incluído no kit RAPID </td></tr><tr><td> Pentium laptop com Windows XP Professional </td><td> Idaho Technologies </td><td></td><td> Incluído no kit RAPID </td></tr><tr><td> LightCycler software de Análise de Dados </td><td> Idaho Technologies </td><td></td><td> Incluído no kit RAPID </td></tr></tbody></table>

rapid diagnosis of avian influenza virus in wild birds: use of a portable rrt-pcr and freeze-dried reagents in the field

Aves selvagens têm sido implicados na disseminação da Gripe aviária altamente patogénica (GAAP) do subtipo H5N1, o que levou a vigilância ao longo de rotas migratórias. Amostragem de aves selvagens para o vírus da gripe aviária (AIV) é muitas vezes realizado em regiões remotas, mas os resultados são muitas vezes adiada devido à necessidade de transportar amostras para um laboratório equipado para o teste molecular. Em tempo real da transcriptase reversa reação em cadeia da polimerase (PCR rRT-) é uma técnica molecular que oferece um dos métodos mais precisos e sensíveis para o diagnóstico de AIV. Os protocolos de laboratório anteriormente estritamente necessário para rRT-PCR estão sendo adaptados para o campo. Desenvolvimento de reagentes liofilizados (liofilizado) que não exigem cadeia de frio, com sensibilidade ao nível dos reagentes molhada trouxe o teste no local remoto a um objetivo prático. Aqui apresentamos um método para o diagnóstico rápido de AIV em aves selvagens usando uma unidade rRT-PCR (Dispositivo de Identificação Ruggedized avançada de patógenos ou RAPID, Idaho Technologies, Salt Lake City, UT), que emprega reagentes liofilizados (Influenza A Meta 1 Taqman; Asay ASY-0109-, Idaho Technologies). Os reagentes contêm todos os componentes necessários para o teste em concentrações adequadas, em um único tubo: primers, sondas, enzimas, amortecedores e controles positivos internos, eliminando erros associados com o armazenamento inadequado ou manuseio de reagentes molhado. A unidade portátil executa uma tela para Influenza A, visando o gene matriz e produz resultados em 2-3 horas. Subtipagem genética também é possível com primer H5 e H7 conjuntos que visam o gene hemaglutinina. O sistema é adequado para uso coletadas de aves selvagens, como demonstrado aqui sobre as espécies migratórias shorebird, o maçarico ocidental (Calidrus mauri) capturado no norte da Califórnia. Em amostras de cloaca e orofaringe Manuseio dos animais seguiu protocolos aprovados pelo Comitê Animal Care e Uso do Geological Survey Research Centro Oeste dos EUA e permite Ecológica do Pássaro Serviço Geológico dos EUA Banding Laboratory. A principal vantagem desta técnica é para agilizar o diagnóstico das aves selvagens, aumentando as chances de conter um surto em um local remoto. No local, o diagnóstico também ser útil para identificar e estudar os indivíduos infectados em populações selvagens. A oportunidade para coletar informações sobre a biologia do hospedeiro (resposta imunológica à infecção e fisiológicas) e ecologia espacial (performance migratória de aves infectadas) fornecerá insights sobre a medida em que as aves selvagens podem atuar como vetores para AIV a longas distâncias.

Watch this Scientific Journal Video about Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field at JoVE.com

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Este estudo descreve o diagnóstico de influenza aviária em aves selvagens usando um sistema rRT-PCR portátil. O método aproveita liofilizadas reagentes para a tela aves selvagens em um ambiente não-laboratorial, típico de um cenário de surto. Uso de ferramentas moleculares oferece alternativas precisas e sensíveis para o diagnóstico rápido.

Diagnóstico rápido de vírus da gripe aviária em aves selvagens: o uso de um Portable rRT-PCR e liofilizados Reagentes no Campo

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

rapid-diagnosis-avian-influenza-virus-wild-birds-use-portable-rrt-pcr

Research

JoVE Journal

Immunology and Infection

15.6K visualizações.  USGS Western Ecological Research Center. Este estudo descreve o diagnóstico de influenza aviária em aves selvagens usando um sistema rRT-PCR portátil. O método aproveita liofilizadas reagentes para a tela aves selvagens em um ambiente não-laboratorial, típico de um cenário de surto. Uso de ferramentas moleculares oferece alternativas precisas e sensíveis para o diagnóstico rápido.

Vídeo: Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

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 ...

Imunologia e Infecção

Avian Influenza Surveillance with FTA  Cards: Field Methods, Biosafety, and Transportation Issues Solved

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral subtypes2. Human pandemics of flu originally stem from AIVs3. Many fatal human cases during the H5N1 outbreaks in recent years were reported. Lately, a new AIV related strain swept through the human population, causing the 'swine flu epidemic'4. Although human trading and transportation activity seems to be responsible for the spread of highly pathogenic strains5, dispersal can also partly be attributed to wild birds6, 7. However, the actual reservoir of all AIV strains is wild birds.
In reaction to this and in face of severe commercial losses in the poultry industry, large surveillance programs have been implemented globally to collect information on the ecology of AIVs, and to install early warning systems to detect certain highly pathogenic strains8-12. Traditional virological methods require viruses to be intact and cultivated before analysis. This necessitates strict cold chains with deep freezers and heavy biosafety procedures to be in place during transport. Long-term surveillance is therefore usually restricted to a few field stations close to well equipped laboratories. Remote areas cannot be sampled unless logistically cumbersome procedures are implemented. These problems have been recognised13, 14 and the use of alternative storage and transport strategies investigated (alcohols or guanidine)15-17. Recently, Kraus et al.18 introduced a method to collect, store and transport AIV samples, based on a special filter paper. FTA cards19 preserve RNA on a dry storage basis20 and render pathogens inactive upon contact21. This study showed that FTA cards can be used to detect AIV RNA in reverse-transcription PCR and that the resulting cDNA could be sequenced and virus genes and determined.
In the study of Kraus et al.18 a laboratory isolate of AIV was used, and samples were handled individually. In the extension presented here, faecal samples from wild birds from the duck trap at the Ottenby Bird Observatory (SE Sweden) were tested directly to illustrate the usefulness of the methods under field conditions. Catching of ducks and sample collection by cloacal swabs is demonstrated. The current protocol includes up-scaling of the work flow from single tube handling to a 96-well design. Although less sensitive than the traditional methods, the method of FTA cards provides an excellent supplement to large surveillance schemes. It allows collection and analysis of samples from anywhere in the world, without the need to maintaining a cool chain or safety regulations with respect to shipping of hazardous reagents, such as alcohol or guanidine.

A method to preserve, detect and sequence RNA from Avian Influenza Viruses was validated and extended using natural faecal samples from birds. This technique removes the necessity of maintaining a cool chain and handling of infectious viruses and can be applied in a 96-well high-throughput setup.

Vigilância da gripe aviária com FTA Cards: Métodos de campo, de Biossegurança, e questões de transporte Resolvido

Avian Influenza Viruses (AIVs) infect many mammals, including humans1. These AIVs are diverse in their natural hosts, harboring almost all possible viral ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

Microondas Assistida Diagnóstico Rápido de Doenças vírus de planta por Microscopia Eletrônica de Transmissão

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

Co-imunoprecipitação da proteína do rato Mx1 com a nucleoproteína do vírus da gripe A

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

Nasal Wipes para Influenza A detecção de vírus e Isolamento de suínos

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Isolamento de neutrófilos e análise para determinar seu papel na linfoma sensibilidade das células a agentes terapêuticos

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

In Vitro A desmontagem do vírus influenza A capsï¿½eos por centrifugação em gradiente

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Avaliação da eficácia e toxicidade de RNAs Segmentação HIV-1 Produção para uso em Gene ou Quimioterapia

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.