Os autores agradecem as contribuições de Greg Smith, Pomeranz Lisa, Matt Lyman, Eldridge Marlies, Staniszewska Halina Goraczniak, e os membros do laboratório Enquist in fine-tuning protocolo isso.

1. Dia primeiro: Infecção viral e Preparação de Buffers <ol><li> Preparar pratos 50-10 (15 cm de diâmetro) de células de cultura de tecidos para a infecção, por exemplo, PK-15 células por vírus pseudo-raiva (PRV) ou células Vero para o vírus herpes simplex (HSV). </li><li> Quando as células são 95 - confluentes 100%, infectá-los em um (MOI) de 10/05. Para fazer isso, inocular cada placa usando o estoque de vírus em um volume total de 4 ml por placa, em seguida, incubar as placas por 1 hora a 37 ° C. Placas de rocha suavemente a cada 15 minutos para garantir que a monocamada de células permanece totalmente revestida pela inóculo do vírus. Enquanto isso, aqueça o meio para a próxima etapa. </li><li> Após uma hora de infecção, aspirado inóculo viral das placas, adicione 15 ml de meio quente, e incubar a 37 ° C por 12-20 horas em uma incubadora umidificada. </li><li> Prepare buffers LCM (ver Tabela 1) e rock durante a noite a 4 ° C para homogeneização da mistura. Prepare TNE para a ressuspensão de DNA viral (ver Tabela 2). </li></ol> 2. Segundo dia, Fase 1: Lise Celular e Ultracentrifugação <ol><li> Confirmar visualmente que a infecção de células causou efeito citopático uniforme (CPE). Permitir a infecção progredindo até que todas as células têm arredondado para cima, mas ainda não decolou no prato. </li><li> Raspe as células infectadas no meio, e combinar as células e médio em 50 ml tubos cônicos. O número de tubos cônicos necessários para esta etapa vai depender do número de pratos utilizados para a infecção no Passo 1.2 acima, por exemplo, um tubo de 50 ml cônica pode ser usado para pellet um máximo de 3 pratos de 15 ml de meio de cada Opcional:. Lave o placas com 10 ml de PBS e combinam com as células e médias empresas. </li><li> Células centrífuga e médio por 10 minutos a 2000 centifugal força relativa (RCF) em temperatura ambiente, em seguida, aspire o sobrenadante e ressuspender cada pellet em 10 ml de PBS. Se o pellet de uma infecção viral foi espalhada em vários tubos cônicos na Etapa 2.2, estes podem ser recombinados em um tubo cônico nesta etapa. Repetir a centrifugação e aspiração Opcional:. Este pellet podem ser armazenadas a -20 ° C e do protocolo continuou em um momento posterior. </li></ol> 3. Segundo dia, Fase 2: Ultracentrifugação Mantenha o pellet celular e buffers LCM no gelo sempre que possível durante as etapas seguintes. <ol><li> Adicionar β-mercaptoetanol para 0% de glicerol - buffer de LCM. Começar a resfriar a ultracentrífuga (para o passo 3.9), definindo-a 4 ° C e ligar o vácuo. </li><li> Ressuspender o pellet celular em 5 ml de glicerol 0% - tampão LCM até que não é mais clumpy. Se necessário, o tubo de vórtice para quebrar qualquer célula não dissolvido pellet. </li><li> Extrato, adicionando 1,5 ml Freon (1,1,2-tricloro-1 ,2,2-trifluoroetano) e vórtex vigorosamente. Imediatamente centrifugar por 10 min a 2000 rcf a uma temperatura de 4 ° C. Coletar a camada superior com uma ponteira de grande furo (evitando a interface) e transferir para um novo tubo Opcional:. Se o pellet é um sólido gelatinoso, você pode rapidamente despeje a camada superior para um tubo novo. </li><li> Repetir a extracção Freon e centrifugar novamente. </li><li> Coletar a camada superior (~ 5 ml) e transferir para um novo tubo polyallomer ultracentrífuga. Adicionar β-mercaptoetanol para o glicerol dois restantes - buffers LCM. </li><li> Prepare um gradiente no tubo de ensaio polyallomer por underlaying o extrato Freon (camada superior) com 3 ml de glicerol 5% - LCM (camada intermediária), e depois underlaying novamente com 2,5 ml de glicerol 45% - LCM (camada inferior). Use uma pipeta fina para evitar o transbordamento do conteúdo do tubo. </li><li> Se necessário, prepare um tubo de equilíbrio equivalente usando as mesmas proporções de buffers LCM. </li><li> Transferência de tubos em baldes ultracentrífuga. Equilíbrio baldes para dentro de 0,1 g do outro, suavemente adicionando glicerol 0% extra - buffer de LCM para a camada superior (~ 5-10 mL de cada vez). </li><li> Usando uma ultracentrífuga frio com um rotor horizontal balde ou balançando, spin amostras equilibrada durante 1 hora a 77.000 rcf (por exemplo, 25.000 rpm para um rotor SW41Ti) a uma temperatura de 4 ° C. </li><li> Durante ultracentrifugação, fazer um gancho para capturar DNA de vidro flutuante na etapa de precipitação de etanol (4.6). Segure ambas as extremidades de uma pipeta Pasteur de vidro e suspender a parte do meio sobre uma chama. Quando uma seção de vidro é quase derretido, retire com cuidado para esticar o copo, dobre-o suavemente para criar um gancho bem anguladas (&lt;90 graus), e puxe bruscamente para selar o fim. Se a ponta do anzol de vidro está aberto, segure a ponta sobre a chama para que o vidro derrete o suficiente para a fechar. </li><li> Após a ultracentrifugação, você deve ver uma fina opaco pellet com uma mancha escura no meio. Aspirar cuidadosamente o líquido do tubo, incluindo drippings ao longo dos lados, mas evitando a pelota no fundo do tubo. </li><li> Adicionar 0,5 ml TNE em temperatura ambiente. </li><li> Deixe o pellet sentar-se por pelo menos 10 minutos para permitir a hidratação do sedimento. Optional: O pellet também pode ressuspender em TNE durante a noite, se for mantida a 4 ° C. </li></ol> 4. Terceiro Dia: &quot;Ghost&quot; Precipitação DNA <ol><li> Quebrar o pellet por pipetagem com uma ponteira de pequeno calibre (por exemplo, um p200 ponta da pipeta). Não se preocupe com corte nesta etapa, porque o DNA viral ainda está contida em capsídeos neste momento. Adicionar os reagentes a seguir ao tubo contendo o pellet viral, à temperatura ambiente, para destruir capsídeos: <ul><li> 4,25 ml TNE </li><li> 0,25 ml SDS 10% </li><li> 2 mg de proteinase K </li></ul> Agite levemente para misturar estes reagentes. Deve haver um ligeiro aumento na viscosidade. </li></ol> Cuidado com corte a partir de agora, por exemplo, uso de grande calibre pipetas e não vortex o material viral <ol start="2"><li> Extrato de proteínas virais, adicionando 5 ml de fenol-clorofórmio e imediatamente começar a inverter ou vórtex para manter uma emulsão. Misture por 10 segundos, em seguida, centrifugar a emulsão de 5 minutos a 2000 rcf à temperatura ambiente. Coletar a camada superior com uma ponteira de grande diâmetro, evitando a interface, e transferir para um novo tubo Opcional:. Use fase de bloqueio de gel (PLG) tubos nesta etapa para evitar a contaminação a partir da interface. </li><li> Repita a extração fenol-clorofórmio e centrifugar novamente. </li><li> Após a extração do fenol-clorofórmio segundo, coletar a camada superior em um vidro de 30 ml tubo Corex e descontrair o tubo a -20 ° C por 10-15 minutos, tomando cuidado para que não congela. </li><li> Adicionar 10 ml de etanol gelado, cobrir o tubo (por exemplo, que se estende um pedaço de Parafilm M sobre a parte superior), e inverta para misturar imediatamente. Assistir a um DNA &quot;fantasma&quot; para aparecer, que consiste de um precipitado ropy fina. Inverter o tubo suavemente para misturar mais e fazer com que o DNA sticky precipita a se agrupar. </li><li> Use um gancho de vidro para capturar o fantasma DNA (s). Cuidadosamente blot o DNA para remover o excesso de líquido, em seguida, coloque o gancho (ponta para baixo) em um tubo de 1,5 ml e deixar secar. Adicionar ,25-,5 ml de TE para dissolver o fantasma DNA. Permitir a ressuspensão de DNA para continuar por pelo menos uma hora. DNA armazenar a 4 ° C. Opcional: Para maximizar o rendimento de DNA, romper o gancho de vidro dentro do tubo de 1,5 ml, de modo que a ressuspensão fora os fragmentos de vidro podem continuar ao longo do tempo. </li></ol> 5. Resultados representante Com uma alta suficientemente MOI, as células devem atingir CPE total dentro ~ infecção pós 18 horas (hpi) para as estirpes do tipo selvagem PRV, e ~ hpi 24 para tipo selvagem HSV-1 amostras (Figura 1). Uma vez que o pellet celular infectado é colhido, os passos do protocolo restante pode ser completa em um dia inteiro ou realizadas durante dois dias (Figura 2). Ao preparar anzóis de vidro para capturar o fantasma DNA, é importante fazer o ângulo de gancho ser inferior a 90 graus, para que mais tarde pode caber na base de um tubo de 1,5 ml (Figura 3). Vedação a ponta deste gancho impede a perda de DNA no interior da pipeta. Fantasmas DNA formam durante a etapa de precipitação e vai aparecer como o branco, threads ropy (Figura 4). Estes agregados e ficar uns aos outros e / ou o gancho de vidro usado para capturar o DNA. Assim, o gancho mesmo vidro pode ser usado para juntar vários segmentos de DNA a partir da solução de precipitação. <table border="1"><tbody><tr><td> Escolha coluna com base no volume desejado: </td><td> 10 ml </td><td> 15 ml </td><td> 20 ml </td><td> 40 ml </td></tr><tr><td> KCL 1M </td><td> 1,3 ml </td><td> 1,9 ml </td><td> 2,5 ml </td><td> 5 ml </td></tr><tr><td> 1M Tris (pH 7,4) </td><td> 300 ml </td><td> 450 mL </td><td> 600 mL </td><td> 1,2 ml </td></tr><tr><td> 1M MgCl 2 </td><td> 50 ul </td><td> 75 mL </td><td> 100 l </td><td> 200 mL </td></tr><tr><td> EDTA 0,5 </td><td> 10 ml </td><td> 15 mL </td><td> 20 l </td><td> 40 mL </td></tr><tr><td> NP40/IGEPAL </td><td> 50 ul </td><td> 75 mL </td><td> 100 l </td><td> 200 mL </td></tr><tr><td> β-mercaptoetanol (adicione imediatamente antes da utilização) </td><td> 4,3 mL </td><td> 6,5 mL </td><td> 8,6 mL </td><td> 17,2 mL </td></tr><tr><td colspan="5"></td></tr><tr><td colspan="5"> Use proporções nas pesquisas de glicerol por cento desejado (0, 5 ou 45%) </td></tr><tr><td> 0% de glicerol </td><td> Nenhum </td><td> Nenhum </td><td> Nenhum </td><td> Nenhum </td></tr><tr><td> água </td><td> 8,3 ml </td><td> 12,5 ml </td><td> 16,7 ml </td><td> 33,4 ml </td></tr><tr><td> Glicerol 5% </td><td> 0,5 ml </td><td> 0,8 ml </td><td> 1,0 ml </td><td> 2,0 ml </td></tr><tr><td> água </td><td> 7,8 ml </td><td> 11,7 ml </td><td> 15,7 ml </td><td> 31,4 ml </td></tr><tr><td> 45% glycerol </td><td> 4,5 ml </td><td> 6,8 ml </td><td> 9,0 ml </td><td> 18,0 ml </td></tr><tr><td> água </td><td> 3,8 ml </td><td> 5,7 ml </td><td> 7,7 ml </td><td> 15,4 ml </td></tr></tbody></table> Tabela 1. Receitas para a preparação de buffers LCM <table border="1"><tbody><tr><td> Para 50 ml </td></tr><tr><td> 5 ml NaCl 1M </td></tr><tr><td> 2,5 ml 1M Tris, pH 7.5 </td></tr><tr><td> 1 ml de EDTA 0,5 </td></tr><tr><td> 41,5 ml H 2 O </td></tr></tbody></table> Tabela 2. Preparação de TNE (0,1 M NaCl, 50 mM Tris, pH 7,5, 10 mM EDTA) <table border="1"><tbody><tr><td> Para 1 volume L </td></tr><tr><td> 0,2 g KCl </td></tr><tr><td> 0,2 g KH 2 PO 4 </td></tr><tr><td> 8 g de NaCl </td></tr><tr><td> 1.15g Na 2 HPO 4 </td></tr><tr><td> H 2 O para 1L </td></tr></tbody></table> Tabela 3. Preparação de PBS (2,7 mM KCl, 1,5 mM KH 2 PO 4; 137 mM NaCl, 8,1 mM Na 2 HPO 4, pH 7,0) <img alt="Figura 1" src="/files/ftp_upload/3151/3151fig1.jpg" /> Figura 1. Células PK15 (A) antes da infecção, (B) no meio de infecção, e (C) no efeito citopático (CPE), depois de uma multiplicidade de infecção. <img alt="Figura 2" src="/files/ftp_upload/3151/3151fig2.jpg" /> Figura 2. Visão geral do protocolo. <img alt="Figura 3" src="/files/ftp_upload/3151/3151fig3.jpg" /> Figura 3. Três exemplos representativos de vidro ganchos utilizados para recolher os fantasmas DNA viral. <img alt="Figura 4" src="/files/ftp_upload/3151/3151fig4.jpg" /> Figura 4. Exemplo representativo de uma bem-formados e abundantes de DNA &quot;fantasma&quot;.

<ol>
	<li>Szpara, M. L., Parsons, L., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+variability+in+clinical+and+laboratory+isolates+of+herpes+simplex+virus+1+reveals+new+mutations.">Sequence variability in clinical and laboratory isolates of herpes simplex virus 1 reveals new mutations.</a> J Virol. 84, 5303-5313 (2010).</li><li>Banfield, B. W., Kaufman, J. D., Randall, J. A., Pickard, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+pseudorabies+virus+strains+expressing+red+fluorescent+proteins:+new+tools+for+multisynaptic+labeling+applications.">Development of pseudorabies virus strains expressing red fluorescent proteins: new tools for multisynaptic labeling applications.</a> J Virol. 77, 10106-10112 (2003).</li><li>Kobiler, O., Lipman, Y., Therkelsen, K., Daubechies, I., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpesviruses+carrying+a+Brainbow+cassette+reveal+replication+and+expression+of+limited+numbers+of+incoming+genomes.">Herpesviruses carrying a Brainbow cassette reveal replication and expression of limited numbers of incoming genomes.</a> Nat Commun. 1, 146-146 (2010).</li><li>Enquist, L. W., Madden, M. J., Schiop-Stanley, P., Vande Woude, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+of+herpes+simplex+type+1+DNA+fragments+in+a+bacteriophage+lambda+vector.">Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.</a> Science. 203, 541-544 (1979).</li><li>Smith, G. A., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+and+transposon+mutagenesis+in+Escherichia+coli+of+a+full-length+infectious+clone+of+pseudorabies+virus,+an+alphaherpesvirus.">Construction and transposon mutagenesis in Escherichia coli of a full-length infectious clone of pseudorabies virus, an alphaherpesvirus.</a> J Virol. 73, 6405-6414 (1999).</li><li>Flint, S. J., Enquist, L. W., Racaniello, V. R., Skalka, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Principles of virology. , (2008).</li><li>Pomeranz, L. E., Reynolds, A. E., Hengartner, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+pseudorabies+virus:+impact+on+neurovirology+and+veterinary+medicine.">Molecular biology of pseudorabies virus: impact on neurovirology and veterinary medicine.</a> Microbiol Mol Biol Rev. 69, 462-500 (2005).</li><li>Roizman, B., Pellett, P. E., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Fields Virology. , 2381-2397 (2001).</li><li>Mendez, I. I., Hermann, L. L., Hazelton, P. R., Coombs, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+analysis+of+freon+substitutes+in+the+purification+of+reovirus+and+calicivirus.">A comparative analysis of freon substitutes in the purification of reovirus and calicivirus.</a> J Virol Methods. 90, 59-67 (2000).</li><li>Gharabaghi, F., Aymard, M., Trotemann, P., Gerdil, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+simplified+micromethod+for+subtyping+varicella-zoster+virus.">A rapid and simplified micromethod for subtyping varicella-zoster virus.</a> J Med Virol. 31, 129-134 (1990).</li><li>Granstedt, A. E., Szpara, M. L., Kuhn, B., Wang, S. S., Enquist, L. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+monitoring+of+in+vivo+neural+activity+using+a+circuit-tracing+pseudorabies+virus.">Fluorescence-based monitoring of in vivo neural activity using a circuit-tracing pseudorabies virus.</a> PLoS One. 4, e6923-e6923 (2009).</li></ol>

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Partes deste protocolo foram originalmente desenvolvidos para o isolamento do DNA viral no BSL4 condições, mas adapta-se igualmente bem a não-BSL condições. 4 Nós geralmente usam este protocolo para isolar o DNA do PRV alfa-herpesvírus e HSV-1, que têm genomas de DNA fechado em um capsídeo protéico e cercado por um envelope lipídico. 7,8 No entanto, é provável diretamente adaptável a outros vírus DNA de grande porte, incluindo beta-e gama-herpesvírus e adenovírus. Extrações semel...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Freon (1,1,2-tricloro-1 ,2,2-trifluoroetano) </td><td> Pescador </td><td> T178-4 </td><td> Verifique com sua instituição, para obter orientações sobre o descarte adequado de resíduos contendo Freon, ou ver Mendez et al. de alternativas Freon potencial 9. </td></tr><tr><td> Bloqueio fase de tubos de gel, capacidade de 15ml Pesada </td><td> 5 PRIME </td><td> 2302850 </td><td> Opcional </td></tr><tr><td> Ultracentrífuga tubos Polyallomer </td><td> Beckman * </td><td> 331372 * </td><td> * Selecione tubos apropriados para o seu próprio ultracentrífuga, que são incluídas apenas como exemplo </td></tr><tr><td> NP-40 / IGEPAL </td><td> Sigma </td><td> I-3021 </td><td></td></tr><tr><td> PK-15 células </td><td> ATCC </td><td> CCL-33 </td><td></td></tr><tr><td> Células Vero </td><td> ATCC </td><td> CCL-81 </td><td></td></tr><tr><td> PBS </td><td> HyClone </td><td> SH30028.03 </td><td></td></tr></tbody></table>

preparation of viral dna from nucleocapsids

Vírus são parasitas obrigatórios do interior celular e, assim, o estudo do seu DNA requer material isolante viral longe de contaminantes da célula hospedeira e DNA. Várias aplicações a jusante exigem grandes quantidades de DNA viral puro, que é fornecido por este protocolo. Estas aplicações incluem o seqüenciamento do genoma viral, onde a remoção do DNA do hospedeiro é fundamental para otimizar a saída de dados para seqüências virais, ea produção de novas linhagens recombinantes viral, onde co-transfecção de plasmídeos purificados e DNA viral linear facilita a recombinação. 1,2, 3 Este procedimento utiliza uma combinação de extrações e densidade baseado centrifugação para isolar DNA purificado herpesvirus linear nucleocapsídeo das células infectadas. 4,5 As etapas de purificação inicial visam isolar purificada capsídeos virais, que contêm e protegem o DNA viral durante a extração e as etapas de centrifugação que removem as proteínas celulares e DNA. Lise de nucleocapsids então libera DNA viral, e duas finais fenol-clorofórmio passos eliminar as proteínas restantes. O DNA final capturada a partir de solução é altamente concentrada e pura, com uma média de 260/280 OD de 1,90. Dependendo da quantidade de células infectadas utilizado, os rendimentos da faixa DNA viral 150-800 mg ou mais. A pureza deste DNA torna-se estável durante armazenamento a longo prazo em 4C. Este DNA é, portanto, ideal para high-throughput seqüenciamento de alta fidelidade reações PCR e transfections. Antes de iniciar o protocolo, é importante saber o número médio de células por prato (por exemplo, uma média de 8 x 10 6 células PK-15 em um prato centímetros confluentes 15), eo título do estoque viral a ser usado ( por exemplo, 1 x 10 8 unidades formadoras de placas por ml). Estes são necessários para calcular a multiplicidade adequada de infecção (MOI) para o protocolo. 6 Por exemplo, para infectar um prato de 15 cm de PK-15 células com o estoque acima viral, em um MOI de 5, você usaria 400 mL de ações virais e diluir com 3,6 ml de meio (volume de inoculação total de 4 ml para uma placa de 15 cm). Várias preparações de DNA viral podem ser preparados ao mesmo tempo. O número de preparações simultâneas é limitado apenas pelo número de tubos de posse do rotor ultracentrífuga (um por vírus; veja o passo 3.9). Aqui nós descrevemos o procedimento de como se está sendo feito por um vírus.

Watch this Scientific Journal Video about Preparation of Viral DNA from Nucleocapsids at JoVE.com

Preparation of Viral DNA from Nucleocapsids

Descrevemos o processo de isolamento de DNA de alta pureza herpesvirus nucleocapsídeo das células infectadas. O DNA final capturada a partir de solução é de alta concentração e pureza, tornando-a ideal para high-throughput seqüenciamento de alta fidelidade reações PCR e transfections para produzir novos recombinantes viral.

Preparação de DNA viral a partir nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

preparation-of-viral-dna-from-nucleocapsids

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JoVE Journal

Immunology and Infection

20.9K visualizações.  Princeton University. Descrevemos o processo de isolamento de DNA de alta pureza herpesvirus nucleocapsídeo das células infectadas. O DNA final capturada a partir de solução é de alta concentração e pureza, tornando-a ideal para high-throughput seqüenciamento de alta fidelidade reações PCR e transfections para produzir novos recombinantes viral.

Vídeo: Preparation of Viral DNA from Nucleocapsids

Generation of Recombinant Influenza Virus from Plasmid DNA

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

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

Geração de recombinante do vírus Influenza de DNA plasmidial

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

Imunologia e Infecção

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

Usando um ensaio Microarray Pan-Viral (Virochip) para Tela de amostras clínicas para patógenos virais

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Isolamento de variantes de vírus de RNA Fidelity e Caracterização de frequência de mutação de vírus

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Development of Cell-type specific anti-HIV gp120 aptamers for siRNA delivery

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering (si)RNAs to inhibit the expression of complementary RNA transcripts is being exploited as a new class of therapeutics for a variety of diseases including HIV. Many previous reports have shown that novel RNAi-based anti-HIV/AIDS therapeutic strategies have considerable promise; however, a key obstacle to the successful therapeutic application and clinical translation of siRNAs is efficient delivery. Particularly, considering the safety and efficacy of RNAi-based therapeutics, it is highly desirable to develop a targeted intracellular siRNA delivery approach to specific cell populations or tissues. The HIV-1 gp120 protein, a glycoprotein envelope on the surface of HIV-1, plays an important role in viral entry into CD4 cells. The interaction of gp120 and CD4 that triggers HIV-1 entry and initiates cell fusion has been validated as a clinically relevant anti-viral strategy for drug discovery. 
Herein, we firstly discuss the selection and identification of 2'-F modified anti-HIV gp120 RNA aptamers. Using a conventional nitrocellulose filter SELEX method, several new aptamers with nanomolar affinity were isolated from a 50 random nt RNA library. In order to successfully obtain bound species with higher affinity, the selection stringency is carefully controlled by adjusting the conditions. The selected aptamers can specifically bind and be rapidly internalized into cells expressing the HIV-1 envelope protein. Additionally, the aptamers alone can neutralize HIV-1 infectivity. Based upon the best aptamer A-1, we also create a novel dual inhibitory function anti-gp120 aptamer-siRNA chimera in which both the aptamer and the siRNA portions have potent anti-HIV activities. Further, we utilize the gp120 aptamer-siRNA chimeras for cell-type specific delivery of the siRNA into HIV-1 infected cells. This dual function chimera shows considerable potential for combining various nucleic acid therapeutic agents (aptamer and siRNA) in suppressing HIV-1 infection, making the aptamer-siRNA chimeras attractive therapeutic candidates for patients failing highly active antiretroviral therapy (HAART).

Several 2&#x2019;-Fluoro RNA aptamers against HIV-1Ba-L gp120 with nanomole affinity are isolated from a RNA library by in vitro SELEX procedure. A new dual inhibitory function anti-gp120 aptamer-siRNA chimera is created and shows considerable promise for systemic anti-HIV therapy.

Desenvolvimento de células de tipo específico anti-HIV gp120 aptâmeros para entrega siRNA

The global epidemic of infection by HIV has created an urgent need for new classes of antiretroviral agents. The potent ability of small interfering ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Separação de Single-stranded DNA, Double-stranded DNA e RNA de uma Comunidade Ambiental Viral Usando Cromatografia hidroxiapatita

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs1,2,3,4,5. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential6,7,8. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens.
We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km2 vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 &mu;l of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 &mu;l was retained while the other 10 &mu;l was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol9,10. The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction9.
Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality11, a PCR for identification of Anopheles gambiae sibling species10 and a nested PCR for typing of Plasmodium falciparum infection12. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min' processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain.

A Simple Chelex Protocol for DNA Extraction from Anopheles spp.

A rapid and affordable way to extract quality malaria parasite and vector DNA from mosquito specimens is described. Capitalizing on chelating properties of Chelex resin, the simple method enables genotyping of malaria parasites in mosquito mid-gut and salivary gland phases, as well as molecular identification of the Anopheles sibling species by PCR.

Um Protocolo de Chelex simples para extração de DNA de<em&gt; Anopheles</em&gt; Spp.

Endemic countries are increasingly adopting molecular tools for  efficient typing, identification and surveillance against malaria parasites and  vector ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

Estabelecimento de um<em&gt; In vitro</em&gt; Sistema para estudar o comportamento intracelular de<em&gt; Candida glabrata</em&gt; Em humanos THP-1 macrófagos

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Calculando a média de Viral glicoproteína do envelope Spikes de Electron Cryotomography reconstruções com Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

Isolamento de Frações Sub-nuclear Viral Replication Compartimento enriquecidas de Adenovirus-infectados células normais Humanos

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

O Desenvolvimento da liofilizado Loop-mediadas isotérmicas de amplificação Reagentes para a detecção de<em&gt; Burnetii Coxiella</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...