Este trabalho foi financiado pelo Fundo de Ciência austríaco (FWF, P20619 e P22988 a BZ).

Microondas preparação da amostra assistida <ol><li> Antes do início da preparação da amostra é necessário para programar o processador de tecido automático microondas por microscopia electrónica (EM Leica AMW; Leica Microsystems, Viena, Áustria), com os seguintes protocolos (Tabela 1) para a preparação de amostras de microondas assistida por TEM: <img src="/files/ftp_upload/2950/2950table1.jpg" alt="Tabela 1" /> Tabela 1. Protocolo para a preparação da amostra, utilizando a irradiação de microondas. As diferentes colunas mostrar (da esquerda para a direita): <ol><li> Número frasco (frasco n °.) Representa a ordem na qual o frasco é colocado no carrossel do processador. </li><li> Etapa do processo real. </li><li> Reagentes nos frascos. </li><li> Duração da etapa actual. </li><li> Temperatura máxima que é alcançada no frasco antes da irradiação de microondas é desligado. </li><li> Configuração de irradiação de microondas: Contínuo = incrementos rápida de temperaturaase, mantendo a temperatura desejada; Inclinação = aumento de temperatura suave, temperatura final alcançado no final; Pulsada = rápido aumento da temperatura de alimentação, desligado até a temperatura caiu 5 ° C, o poder voltou a atingir a temperatura. </li><li> Potência máxima da irradiação por microondas. </li></ol></li><li> Recém preparar as soluções para as diferentes etapas descritas no protocolo de preparação da amostra (por Agar resina epoxi 100 ver 1.7), enchê-los nos frascos designados de acordo com o protocolo programado (Tabela 1), carregar os frascos no carrossel, em seguida, inserir o carrossel para o processador de tecido micro-ondas, e, finalmente, carregar o primeiro frasco para dentro da câmara mono-modo. </li><li> Cortar secções de folhas pequenas (1mm 2) a partir de Nicotiana tabacum infectadas com vírus do mosaico do tabaco (TMV) com uma lâmina de barbear sobre uma placa de cera de modelagem de uma gota de glutaraldeído a 3% (Agar Scientific Ltd., Stansted, Inglaterra) em 60mM Sørensen tampão fosfato (pH 7,2) à temperatura ambienteratura. </li><li> Transferir as seções com pinças finas imediatamente para as cestas designados com uma largura de malha de cerca de 200 um. Empilhar as cestas em cima uns dos outros e inseri-los na câmara de mono-modo. Deve ser tomado cuidado de que as amostras estão constantemente coberta com a solução fixadora durante o carregamento e empilhamento dos cestos de modo que eles não secam. </li><li> Comece o programado anteriormente microondas protocolo de preparação assistida amostra para desidratação, fixação e infiltração. </li><li> Enquanto a preparação da amostra é realizada automaticamente pelo processador de tecidos microondas continuar com coloração negativa com o material vegetal remanescente, conforme descrito na seção 3 (coloração negativa). </li><li> Agar de fresco preparar resina epóxi 100 através da mistura dos seguintes componentes, conforme descrito: Agar 24g enchimento 100, 16g de anidrido dodecenil succínico e 10 g de anidrido metil nádico (para todos os componentes ver Agar Scientific Ltd., Stansted, Inglaterra) num copo de plástico de calor, -a40 ° C e misture bem. Adicionar 1,2 g de benzil dimetilamina e misture bem. Encha Agar resina epoxi 100 para a polimerização designado forma imediatamente antes do protocolo de preparação da amostra chega ao fim (por exemplo, durante o passo 22 na tabela 1). </li><li> Após o protocolo está terminada (após o passo 22 na tabela 1) libertar os cestos empilhados que contêm as amostras a partir da câmara de infiltrados mono-modo para dentro do frasco passado do carrossel. Retire o carrossel do dispositivo de microondas, desempilhar as cestas e carregá-los usando uma pinça fina para as formas de polimerização designados. Deve ser tomado cuidado de que as amostras são sempre cobertas com Agar de resina epoxi 100 durante desempilhar e carregamento de modo a que eles não secam. </li><li> Empilhar as formas de polimerização em cima uns dos outros. Deve ser tomado cuidado de que as amostras são sempre cobertas com Agar de resina epoxi 100 durante o carregamento e empilhamento de modo a que eles não secam. </li><li> Remover os frascos previamente utilizadas a partir do carrossel de microondas tissue processador, carregá-lo com as cestas empilhadas e inserir o carrossel para o processador de tecidos de microondas. </li><li> Iniciar o protocolo de polimerização previamente programado (tabela 1). </li><li> Enquanto polimerização é realizada automaticamente pelo processador de tecidos microondas, examinar grades negativamente manchadas com um microscópio eletrônico de transmissão [por exemplo Philips CM10 TEM, FEI (antiga Philips), Eindhoven, Holanda] e realizar análise de imagem, como descrito na seção 3 e 4 ( coloração negativa e a análise de imagem). </li><li> Depois de terminar o protocolo é remover as formas de polimerização a partir da câmara de mono-modo, desempilhar as formas de polimerização e remover os blocos de polimerizados que contêm as amostras. Eles já estão prontos para ser seccionadas em micrótomo. </li></ol> 2. Desbastar e cortar <ol><li> Insira um ou mais blocos em porta-amostras separadas para ultrafino seccionamento com a amostra no topo degola cerca de 1cm para fora do suporte. </li><li>Apare o bloco com um aparador de amostra para TEM (por exemplo, Leica Reichert Ultratrim; Leica Microsystems), de modo que um rosto bloco de max. 1 mm de comprimento e 200 um de largura, que contém o material da folha, tanto quanto possível, é alcançado (o tamanho do bloco cara pode ter de ser ajustada para o tamanho da faca de diamante). </li><li> Secção do bloco com um ultramicrótomo (por exemplo, Reichert Ultracut Leica S; Leica Microsystems), usando uma faca de diamante num ângulo de faca de 45 ° (por exemplo, Ultra Diatome 45, Gröpl, Tulln, Áustria). Espessura de corte deve ser ajustada para cerca de 70 a 90 nm e a velocidade de corte deve ser cerca de 1 mm / s. Pegue várias seções com um revestimento formvar cobre ou níquel (Agar Scientific Ltd.) 200 grade de malha quadrada. </li><li> Post-mancha as secções sobre a grade com citrato de chumbo (Agar Scientific Ltd., 1,1 g de citrato de chumbo dissolvido em 42 mL de água duplamente destilada e 8 mL de NaOH 1N) durante 5 minutos numa placa de Petri parcialmente cheios com NaOH para criar um CO 2-livre meio ambiente e para 15 miNUTES com acetato de uranilo a 1% (Agar Scientific Ltd.) dissolvida em água destilada à temperatura ambiente. Lave as grades com água destilada durante 1 minuto, entre cada passo de pós-coloração. Secar as grades de uma caixa de grade. </li><li> Examine as seções com um microscópio eletrônico de transmissão (por exemplo Philips CM10 TEM, FEI, Eindhoven, Holanda). </li></ol> 3. Coloração negativa <ol><li> Colheita cerca de 100mg de TMV-infectado material foliar e preparar seiva em bruto por homogeneização do material durante 2 minutos com uma lâmina de barbear sobre uma lâmina de microscópio em 100 ul de 60 mM Søfrensen tampão fosfato (pH 7,2). </li><li> Transferir 20 uL de homogenato resultante para o primeiro poço de uma lâmina de microscópio revestidas de teflon com 4 ou mais poços (em alternativa, também é possível transferir o homogeneizado em um pedaço de Parafilm). </li><li> Colocar uma grade formvar revestida na parte superior do homogenato com o lado formvar (Agar Scientific Ltd.) revestida voltada para a gota e INCUbate por 5 minutos. </li><li> Lava-se a grelha de 2 vezes, durante 2 minutos cada, colocando a grade na parte superior de duas gotas de 200 ul de tampão fosfato 60 mM Sørensen (pH 7,2). </li><li> Incubar a grelha durante 1 minuto, com um preparado de fresco de 2% de ácido fosfotúngstico (Agar Scientific Ltd.) em solução 60 mM de fosfato de Sorensen (pH 6,5). </li><li> Retire a grelha e deixe-a secar ao ar numa caixa de grelha. </li><li> Examine a grade com um microscópio eletrônico de transmissão (por exemplo Philips CM10 TEM, FEI, Eindhoven, Holanda). Tomar, pelo menos, 10 imagens escolhidas aleatoriamente virions coradas negativamente (com pelo menos 10 ou mais viriões deve ser visível em cada imagem), com o microscópio electrónico de transmissão com uma ampliação de 21000X primária ou superior. Cuidados devem ser tomados para que todas as imagens têm a mesma ampliação. </li></ol> 4. A análise de imagem <ol><li> Medir o comprimento e largura de pelo menos 100 escolhidos aleatoriamente partículas de vírus individuais sobre as micrografias tiradasas amostras coradas negativamente usando qualquer software de computador de análise [Cell por exemplo, D (Olympus, Vida e Ciência dos Materiais Europe GmbH, Hamburgo, Alemanha) com a ferramenta de análise de partículas ou Optimas 6.5.1 (Media Cybernetics Inc., Bethesda, Maryland, EUA)]. </li><li> Calcular médias e desvios padrão, a fim de atingir um comprimento médio e a largura das partículas de vírus visualizados por métodos de coloração negativa e TEM. </li><li> Comparar o comprimento e as características ultra-estruturais (obtido em 2.5) com tamanhos de vírus e de alterações ultra-estruturais induzidas por doenças de vírus conhecidos na literatura, a fim de identificar claramente as doenças virais. </li></ol> 5. Os resultados representativos: Depois de microondas assistidas de preparação de amostra típica TMV induzidas alterações ultra-estruturais, tais como grandes áreas contendo viriões alinhadas em forma paralela pode ser observado com o microscópio electrónico de transmissão no citosol de fumante infectadoum células tabacum (Fig. 1A). Além disso, na seiva em bruto de TMV-infected deixa partículas TMV pode ser observada como flexuosas, em forma de bastonete estruturas após coloração negativa (Fig. 1B). A análise de imagem de 100 partículas de vírus revelou um tamanho médio de 280 nm para o TMV em comprimento e em largura 17nm (Fig. 2). A ultra-estrutura, em células infectadas com TMV e do tamanho dos viriões observados neste estudo revelaram-se de acordo com TMV induzidas propriedades ultra-estruturais do tabaco e o intervalo de tamanho de partículas de TMV previamente relatados na literatura 9-15. <img src="/files/ftp_upload/2950/2950fig1.jpg" alt="Figura 1" /> Figura 1. Micrografias electrónicas de transmissão de células infectadas com TMV folha e viriões. A) A imagem mostra a ultra-estrutura das células infectadas TMV mesofilo de folhas de Nicotiana tabacum após microondas preparação assistida planta amostra. Note-se a grande área de viriões alinhadas paralelas que se acumularam no citosol.C = cloroplasto com amido (St), M = mitocôndria, N = núcleo, V = vacúolo, Bar = 2 um. B) A imagem mostra vírions que foram detectados pela coloração negativa na seiva de folhas infectadas. <img src="/files/ftp_upload/2950/2950fig2.jpg" alt="Figura 2" /> Figura 2. Tamanho relativo dos virions. Distribuição relativa de comprimento e largura de TMV-partículas após coloração negativa a seiva das folhas infectadas como apareceram no microscópio eletrônico. A média dos valores (média de comprimento / largura de ± desvio padrão) foram calculados a partir de 100 virions.

<ol>
	<li>Bozzola, J. J., Russell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Electron Microscopy. , (1999).</li><li>Kuo, J., Kuo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+plant+tissues+for+ultrastructural+study.">Processing plant tissues for ultrastructural study.</a> Methods in Molecular Biology, Electron Microscopy, Methods and Protocols. 369, 47-65 (2007).</li><li>Schroeder, J. A., Gelderblom, H. R., Hauroeder, B., Schmetz, C., Milios, J., Hofstaedter, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-assisted+tissue+processing+for+sameday+EM-diagnosis+of+potential+bioterrorism+and+clinical+samples.">Microwave-assisted tissue processing for sameday EM-diagnosis of potential bioterrorism and clinical samples.</a> Micron. 37, 577-590 (2006).</li><li>Webster, P., Kuo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave-assisted+processing+and+embedding+for+transmission+electron+microscopy.">Microwave-assisted processing and embedding for transmission electron microscopy.</a> Methods in Molecular Biology, Electron Microscopy, Methods and Protocols. 369, 47-65 (2007).</li><li>Zechmann, B., Zellnig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+assisted+rapid+plant+sample+preparation+for+transmission+electron+microscopy.">Microwave assisted rapid plant sample preparation for transmission electron microscopy.</a> J. Microsc. 233, 258-268 (2009).</li><li>Ler&#237;a, F., Marco, R., Medina, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+antigenic+preservation+of+plant+samples+by+microwave-enhanced+fixation,+using+dedicated+hardware,+minimizing+heat-related+effects.">Structural and antigenic preservation of plant samples by microwave-enhanced fixation, using dedicated hardware, minimizing heat-related effects.</a> Micr. Res. Techniq. 65, 86-100 (2004).</li><li>Giberson, R. T., Austin, R. L., Charlesworth, J., Adamson, G., Herrera, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+and+digital+imaging+technology+reduce+turnaround+times+for+diagnostic+electron+microscopy.">Microwave and digital imaging technology reduce turnaround times for diagnostic electron microscopy.</a> Ultrastruct. Pathol. 27, 187-196 (2003).</li><li>Cavusoglu, I., Minbay, F. Z., Temel, S. G., Noyan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+polymerisation+with+microwave+irradiation+for+transmission+electron+microscopy.">Rapid polymerisation with microwave irradiation for transmission electron microscopy.</a> Eur. J. Morph. 39, 313-317 (2001).</li><li>Ermolina, I., Morgana, H., Greena, N. G., Milnerb, J. J., Feldmanc, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dielectric+spectroscopy+of+tobacco+mosaic+virus.">Dielectric spectroscopy of tobacco mosaic virus.</a> Biochim. Biophys. Acta. 1622, 57-63 (2003).</li><li>Maeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+atomic+force+microscopy+study+for+the+assembly+structures+of+tobacco+mosaic+virus+and+their+size+evaluation.">An atomic force microscopy study for the assembly structures of tobacco mosaic virus and their size evaluation.</a> Langmuir. 13, 4150-4161 (1997).</li><li>Milne, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplication+of+tobacco+mosaic+virus+in+tobacco+leaf+palisade+cells.">Multiplication of tobacco mosaic virus in tobacco leaf palisade cells.</a> Virology. 28, 527-532 (1966).</li><li>Reunov, A. V., Gnutova, I. V., Lapshina, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+tobacco+mosaic+virus+strains+on+the+ultrastructure+of+tobacco+leaf+parenchymal+cells.">Effect of tobacco mosaic virus strains on the ultrastructure of tobacco leaf parenchymal cells.</a> Biol. Bull. 33, 409-415 (2006).</li><li>Sachse, C., Chen, J. Z., Coureux, P. D., Stroupe, M. E., F&#228;ndrich, M., Grigorieff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+electron+microscopy+of+helical+specimens:+a+fresh+look+at+tobacco+mosaic+virus.">High-resolution electron microscopy of helical specimens: a fresh look at tobacco mosaic virus.</a> J. Mol. Biol. 371, 812-835 (2007).</li><li>Smith, M. L., Lindbo, J. A., Dillard-Telm, S., Brosio, P. M., Lasnik, A. B., McCormick, A. A., Nguyen, L. V., Palmer, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modified+tobacco+mosaic+virus+particles+as+scaffolds+for+display+of+protein+antigens+for+vaccine+applications.">Modified tobacco mosaic virus particles as scaffolds for display of protein antigens for vaccine applications.</a> Virology. 348, 475-488 (2006).</li><li>Zechmann, B., Zellnig, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+TEM+diagnosis+of+plant+virus+diseases.">Rapid TEM diagnosis of plant virus diseases.</a> J. Virol. Meth. 162, 163-169 (2009).</li><li>Giberson, R. T., Demaree, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microwave+processing+techniques+for+electron+microscopy:+a+four-hour+protocol.">Microwave processing techniques for electron microscopy: a four-hour protocol.</a> Meth. Molec. Biol. 117, 145-158 (1999).</li></ol>

Não há conflitos de interesse declarados.

Microondas preparação de amostras assistida planta para TEM tenha sido comprovada a fornecer rápida e confiável de dados ultra-estruturais dentro de algumas horas 3,4,16. A preservação estrutural fina e membranas de organelos alcançadas com o método utilizado no presente estudo foi semelhante para amostras convencionais e cryofixed 5,15, com a vantagem de uma redução drástica na fixação da amostra e incorporação de tempo de 3 dias ou mais, para cerca de 2 horas. Isto representa o protocolo mais rápido para a preparação da amostra TEM actualmente disponíveis na literatura. O método descrito neste estudo combinado de microondas a preparação de amostras para TEM assistida planta com métodos de coloração negativa o que permitiu uma identificação clara e rápida de TMV induzidas alterações ultra-estruturais e do agente viral em si. Alterações ultra-estruturais TMV induzidas poderiam ser investigados após o corte, de corte e de pós-coloração dentro de cerca de 4 horas após o início da fixação na transmissão electron microscópio. Usando o modo de preparação de forma totalmente automática espécime libertou o pesquisador para conduzir coloração negativa no intercalar de modo a determinar o tamanho e largura do que o agente viral. Assim, podemos concluir que este método permite um diagnóstico claro e rápido de doenças de plantas de vírus em cerca de metade de um dia que é de grande importância para uso futuro na agricultura e científica experimentos em planta fitopatologia. Como este método também pode ser utilizado para o diagnóstico rápido de doenças de animais e humanos, tem um grande potencial para aplicação futura em patologia médica e veterinária.

<table border="1"><tbody><tr><td> Nome do reagente / equipamento </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Glutaraldeído </td><td> Agar Scientific Ltd. </td><td> R1312 </td><td></td></tr><tr><td> Tetróxido de ósmio </td><td> Agar Scientific Ltd. </td><td> R1022 </td><td></td></tr><tr><td> Resina Agar 100 </td><td> Agar Scientific Ltd. </td><td> R1043 </td><td></td></tr><tr><td> Anidrido succínico dodecenilo </td><td> Agar Scientific Ltd. </td><td> R1051 </td><td></td></tr><tr><td> Metil anidrido nádico </td><td> Agar Scientific Ltd. </td><td> R1081 </td><td></td></tr><tr><td> Benzílico dimetilamina </td><td> Agar Scientific Ltd. </td><td> R1060 </td><td></td></tr><tr><td> Citrato de chumbo </td><td> Agar Scientific Ltd. </td><td> R1210 </td><td></td></tr><tr><td> Acetato de uranilo </td><td> Agar Scientific Ltd. </td><td> R1260A </td><td></td></tr><tr><td> Ácido fosfotúngstico </td><td> Agar Scientific Ltd. </td><td> R1213 </td><td></td></tr><tr><td> Formvar </td><td> Agar Scientific Ltd. </td><td> R1202 </td><td></td></tr><tr><td colspan="4"></td></tr><tr><td> Leica EM AMW </td><td> Leica Microsystems </td><td></td><td></td></tr><tr><td> Leica (Reichert) Ultratrim </td><td> Leica Microsystems </td><td></td><td> Novo modelo está disponível </td></tr><tr><td> Leica (Reichert) Ultracut S </td><td> Leica Microsystems </td><td></td><td> Novo modelo está disponível </td></tr><tr><td> Diatome Ultra 45 </td><td> Gröpl </td><td></td><td></td></tr><tr><td> Philips CM10 TEM </td><td> FEI (antiga Philips) </td><td> &lt;/ Td&gt; <td> Novo modelo está disponível </td></tr><tr><td> Célula D </td><td> Olympus Inc. </td><td></td><td></td></tr><tr><td> Óptimas 6.5.1 </td><td> Media Cybernetics Inc. </td><td></td><td> Nova versão estiver disponível </td></tr></tbody></table>

microwave assisted rapid diagnosis of plant virus diseases by transmission electron microscopy

Investigações de alterações ultra-estruturais induzidas por vírus são muitas vezes necessários para identificar claramente doenças virais nas plantas. Com a preparação da amostra convencional para microscopia eletrônica de transmissão (TEM) tais investigações podem demorar vários dias 1,2 e portanto não são adequados para o diagnóstico rápido de doenças de vírus de plantas. Fixação de microondas pode ser utilizada para reduzir drasticamente o tempo de preparação de amostras para TEM investigações com resultados semelhantes, como observado após ultraestruturais convencionalmente preparação de amostras 3-5. Diversos dispositivos de micro-ondas personalizados feitos estão actualmente disponíveis, que pode ser usada para a fixação de sucesso e de incorporação de amostras biológicas para investigações TEM 5-8. Neste estudo mostramos em Vírus do Mosaico do Tabaco (TMV) infectadas plantas de Nicotiana tabacum, que é possível diagnosticar alterações ultra-estruturais em folhas de cerca de meio dia, utilizando micro-ondas a preparação da amostra assistida por TEM. Optou-se por realizar este estudo, com um dispositivo de micro-ondas comercialmente disponível, uma vez que a preparação da amostra efectua quase totalmente automática 5 em contraste com os outros dispositivos disponíveis em vários passos têm ainda de ser realizadas manualmente, 6-8 e são, portanto, mais tempo de trabalho e consumo. Como a preparação da amostra é realizada de forma totalmente automática coloração negativa de partículas virais na seiva das restantes folhas infectadas com TMV e do exame seguinte da ultra-estrutura e tamanho pode ser realizada durante a fixação e a incorporação.

Watch this Scientific Journal Video about Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy at JoVE.com

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

Este estudo descreve um método que permite o diagnóstico rápido e preciso de doenças de plantas de vírus em cerca de meio dia, utilizando uma combinação de micro-ondas preparação assistida planta amostra para microscopia electrónica de transmissão e métodos de coloração negativa.

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 ...

microwave-assisted-rapid-diagnosis-plant-virus-diseases-transmission

Research

JoVE Journal

Immunology and Infection

13.4K visualizações.  University of Graz. Este estudo descreve um método que permite o diagnóstico rápido e preciso de doenças de plantas de vírus em cerca de meio dia, utilizando uma combinação de micro-ondas preparação assistida planta amostra para microscopia electrónica de transmissão e métodos de coloração negativa.

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

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

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

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 ...

Imunologia e Infecção

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing &le;50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.

We describe a method to process and screen field-collected mosquitoes for a diversity of viruses by Vero cell culture assay. By employing this technique, we have detected 9 different viruses from 4 taxonomic families in mosquitoes collected in Connecticut.

Detecção de vírus infecciosos de Campo mosquitos coletados por Vero Ensaio Cultura de Células

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

Estrutura do HIV-1 Assembléias Cápside por Cryo-Microscopia eletrônica e reconstrução iterativa helicoidal espaço-Real

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials 9, vaccines 14, 15, electronic materials, chemical tools, and molecular electronic containers4, 5, 10, 11, 16, 18, 12.
In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups 8. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications 8. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed 13. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus 3, Carnation mottle virus 12, Cowpea chlorotic mottle virus 6, Tobacco mosaic virus 17, Turnip yellow mosaic virus 1, and MRFV 13.

Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Análise da acessibilidade de solvente de resíduos de cisteína em<em&gt; Milho rayado fino vírus</em&gt; Partículas semelhantes a vírus produzidas em<em&gt; Nicotiana benthamiana</em&gt; Plantas e cross-linking de peptídeos para VLP

Mimicking  and exploiting virus properties and physicochemical and physical  characteristics holds promise to provide solutions to some of  the world's ...

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2. H. pylori is the primary cause of gastric cancer, the second leading cause of cancer-related deaths worldwide3. One virulence factor that has been associated with increased risk of gastric disease is the Cag-pathogenicity island, a 40-kb region within the chromosome of H. pylori that encodes a type IV secretion system and the cognate effector molecule, CagA4,5. The Cag-T4SS is responsible for translocating CagA and peptidoglycan into host epithelial cells5,6. The activity of the Cag-T4SS results in numerous changes in host cell biology including upregulation of cytokine expression, activation of proinflammatory pathways, cytoskeletal remodeling, and induction of oncogenic cell-signaling networks5-8. The Cag-T4SS is a macromolecular machine comprised of sub-assembly components spanning the inner and outer membrane and extending outward from the cell into the extracellular space. The extracellular portion of the Cag-T4SS is referred to as the &#8220;pilus&#8221;5. Numerous studies have demonstrated that the Cag-T4SS pili are formed at the host-pathogen interface9,10. However, the environmental features that regulate the biogenesis of this important organelle remain largely obscure. Recently, we reported that conditions of low iron availability increased the Cag-T4SS activity and pilus biogenesis. Here we present an optimized protocol to grow H. pylori in varying conditions of iron availability prior to co-culture with human gastric epithelial cells. Further, we present the comprehensive protocol for visualization of the hyper-piliated phenotype exhibited in iron restricted conditions by high resolution scanning electron microscopy analyses.

High Resolution Electron Microscopy of the Helicobacter pylori Cag Type IV Secretion System Pili Produced in Varying Conditions of Iron Availability

Here we describe a method to visualize the oncogenic bacterial organelle known as the Cag Type IV Secretion System (Cag-T4SS). We find that the Cag-T4SS is differentially produced on the surface of H. pylori in response to varying conditions of iron availability.

Alta resolução de Microscopia Eletrônica da<em&gt; Helicobacter pylori</em&gt; Cag Tipo IV Secreção Sistema Pili Produzido em diferentes condições de Ferro Disponibilidade

Helicobacter pylori is a helical-shaped, gram negative bacterium that colonizes the human gastric niche of half of the human population1,2.  H. pylori is ...

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or specific treatments for the disease caused by these viruses, and work with infectious Ebola viruses is restricted to biosafety level 4 laboratories, significantly limiting the research on these viruses. Lifecycle modeling systems model the virus lifecycle under biosafety level 2 conditions; however, until recently such systems have been limited to either individual aspects of the virus lifecycle, or a single infectious cycle. Tetracistronic minigenomes, which consist of Ebola virus non-coding regions, a reporter gene, and three Ebola virus genes involved in morphogenesis, budding, and entry (VP40, GP1,2, and VP24), can be used to produce replication and transcription-competent virus-like particles (trVLPs) containing these minigenomes. These trVLPs can continuously infect cells expressing the Ebola virus proteins responsible for genome replication and transcription, allowing us to safely model multiple infectious cycles under biosafety level 2 conditions. Importantly, the viral components of this systems are solely derived from Ebola virus and not from other viruses (as is, for example, the case in systems using pseudotyped viruses), and VP40, GP1,2 and VP24 are not overexpressed in this system, making it ideally suited for studying morphogenesis, budding and entry, although other aspects of the virus lifecycle such as genome replication and transcription can also be modeled with this system. Therefore, the tetracistronic trVLP assay represents the most comprehensive lifecycle modeling system available for Ebola viruses, and has tremendous potential for use in investigating the biology of Ebola viruses in future. Here, we provide detailed information on the use of this system, as well as on expected results.

Work with infectious Ebola viruses is restricted to biosafety level 4 laboratories. Tetracistronic minigenome-containing replication and transcription-competent virus like particles (trVLPs) represent a lifecycle modeling system that allows us to safely model multiple infectious cycles under biosafety level 2 conditions, relying exclusively on Ebola virus components.

Modelando o ciclo de vida do vírus Ebola Sob Nível de Biossegurança 2 condições com partículas semelhantes a vírus contendo Tetracistronic minigenomas

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and resilience of Bacillus subtilis biofilms. First, methods are presented that select for small molecule inhibitors with biofilm-specific targets in order to separate the effect of the small molecule inhibitors on planktonic growth from their effect on biofilm formation. Next, we focus on how inoculation conditions affect the sensitivity of multicellular, floating B. subtilis cultures to small molecule inhibitors. The results suggest that discrepancies in the reported effects of such inhibitors such as D-amino acids are due to inconsistent pre-culture conditions. Furthermore, a recently developed protocol is described for evaluating the contribution of small molecule treatments towards biofilm resistance to antibacterial substances. Lastly, scanning electron microscopy (SEM) techniques are presented to analyze the three-dimensional spatial arrangement of cells and their surrounding extracellular matrix in a B. subtilis biofilm. SEM facilitates insight into the three-dimensional biofilm architecture and the matrix texture. A combination of the methods described here can greatly assist the study of biofilm development in the presence and absence of biofilm inhibitors, and shed light on the mechanism of action of these inhibitors.

Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors

This study presents the development of reproducible methodologies to study biofilm inhibitors and their effects on Bacillus subtilis multicellularity.

Metodologias para o estudo<em&gt; B. subtilis</em&gt; Os biofilmes como um modelo para a caracterização de pequena molécula biofilme Inibidores

This work assesses different methodologies to study the impact of small molecule biofilm inhibitors, such as D-amino acids, on the development and ...

Protocols for Investigating the Host-tissue Distribution, Transmission-mode, and Effect on the Host Fitness of a Densovirus in the Cotton Bollworm

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, Helicoverpa armigera densovirus (HaDV2), in a lepidopteran species, the cotton bollworm, Helicoverpa armigera (Hubner). Here, we describe the protocols that are currently used to study the effect of HaDV2 on its host. First, we establish a HaDV2-free cotton bollworm colony from a single breeding pair. Then, we orally inoculate some neonate larval offspring with HaDV2-containing filtered liquid to produce two colonies with the same genetic background: one HaDV2-infected, the other uninfected. A protocol to compare life table parameters (e.g., larval, pupal, and adult periods and fecundity) between the HaDV2-infected and -uninfected individuals is also presented, as are the protocols for determining the host-tissue distribution and transmission efficiency of HaDV2. These protocols would also be suitable for investigating the effects of other orally transmitted viruses on their insect hosts, lepidopteran hosts in particular.

Here, we present a protocol to investigate the host-tissue distribution, transmission mode, and effect on the host fitness of a densovirus within a lepidopteran species, the cotton bollworm. This protocol can also be used for studying the interaction between other orally-transmitted viruses and their insect hosts.

Protocolos para investigar a distribuição no tecido hospedeiro, o modo de transmissão e o efeito na aptidão do hospedeiro de um Densovírus na lagarta do algodoeiro

Many novel viruses have been discovered in animal hosts using next-generation sequencing technologies. Previously, we reported a mutualistic virus, ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

Extração rápida, segura e simples Manual cabeceira de ácidos nucleicos para a detecção de vírus em amostras de sangue total

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Antimicrobial Synergy Testing by the Inkjet Printer-assisted Automated Checkerboard Array and the Manual Time-kill Method

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR bacteria are increasingly becoming a necessity. One such approach is combination therapy, in which two or more antibiotics are used together to treat an infection against which one or both of the drugs may be ineffective alone. When two drugs, in combination, exert a greater than additive effect, they are considered synergistic. In vitro investigation of synergistic activity is an important first step in evaluating the possible efficacy of drug combinations. Two main in vitro synergy testing methods have been developed: the checkerboard array and the time-kill study. In this paper, we present an automated checkerboard array method that makes use of inkjet printing technology to increase the efficiency and accuracy of this technique, as well as a standard manual time-kill synergy method. The automated checkerboard array can serve as a high-throughput screening assay, while the manual time-kill study provides additional, complementary data on synergistic activity and killing.
The checkerboard array is a modification of standard minimum inhibitory concentration (MIC) testing, in which bacteria are incubated with antibiotics at different concentration combinations and evaluated for growth inhibition after overnight incubation. Manual performance of the checkerboard array requires a laborious and error-prone series of calculations and dilutions. In the automated method presented here, the calculation and dispensing of required antibiotic stock solution volumes are automated through the use of inkjet printer technology. In the time-kill synergy assay, bacteria are incubated with the antibiotics of interest, both together and individually, and sampled at intervals over the course of 24 h for quantitative culture. The results can determine whether a combination is synergistic and whether it is bactericidal, and provide data on inhibition and killing of bacteria over time.

Antimicrobial synergy testing is used to evaluate the effect of two or more antibiotics used in combination and is typically performed by one of two methods: the checkerboard array or the time-kill assay. Here, we present an automated, inkjet printer-assisted checkerboard array synergy technique and a classic time-kill synergy study.

Antimicrobiana sinergia testes pela impressora jato de tinta-assistida matriz Checkerboard automatizado e o método Manual de tempo-kill

As rates of multidrug-resistant (MDR) pathogens continue to rise, outpacing the development of new antimicrobials, novel approaches to treatment of MDR ...