A publicação deste artigo foi apoiado por uma bolsa do Instituto de Pesquisa da Universidade de Tennessee. Os autores agradecem a geração anterior de estudantes e investigadores que têm trabalhado para aperfeiçoar esses procedimentos. Pesquisa foi suportada por concessões do National Science Foundation (NSF-0851113, 0825405 e NSF-NSF-0550485).

1. Ultrafiltração da água do mar para gerar &quot;Virus-free&quot; de água (Wilhelm &amp; Poorvin 2001) <ol><li> Cerca de 20L de água do mar / lakewater é coletado como assepticamente possível. </li><li> A água é serialmente através prefiltered 142 mm de diâmetro em policarbonato 0,8 mM filtros que podem ser mantidos a -20 ° C para análise da comunidade. Filtros de maior tamanho dos poros pode ser usado antes de este passo para sistemas muito produtivos. </li><li> Para obter água de um ultrafiltrado M12 Amicon sistema (Millipore) de 30 kDa cartucho espiral de corte é usada para excluir todos os vírus, mesmo pequenos vírus RNA. </li><li> As amostras são processadas e concentradas em ~ 25% a velocidade com ~ 15-16 kPa de contrapressão. </li><li> A amostra restante de aproximadamente 500 mL de água irá conter uma comunidade vírus concentrado (que pode ser guardado para outras experiências), enquanto os restantes 19,5 L de água livre de vírus será utilizado para ensaios de produção viral. </li><li> Após cada dia de uso, o M12 Amicon sistema devem ser limpos para evitar danos à membrana do filtro. </li><li> Se você estiver trabalhando com água do mar, lavar a membrana com, pelo menos, 6L de água Milli-Q seguida por uma lavagem com solução de NaOH 0,1 N por 30-45 minutos. </li><li> Lavar novamente o cartucho com, pelo menos, 6L de água Milli-Q. </li><li> Quando terminar de usar o sistema M12, o cartucho espiral deve ser armazenado em um 3 PO 4 0.05MH solução a 4 ° C. </li></ol> 2. Método de Redução do vírus para produção Viral (Wilhelm et al. 2002) <ol><li> Até 500 mL da amostra da água do mar / lakewater com o anfitrião e os vírus são obtidas e colocadas em uma unidade sterifilter com 0,2 mícrons nominal dos poros do tamanho de baixa ligação às proteínas do filtro (por exemplo, Durapore) colocados sobre ele. </li><li> A amostra é suavemente pressionado a vácuo em &lt;200 mmHg, enquanto continuamente ressuspender a amostra com uma pipeta de transferência estéril para inibir as células bacterianas de se concentrar no filtro. </li><li> Lentamente, três volumes de ultrafiltrado são adicionados à suspensão bacteriana para reduzir significativamente o número de vírus livre na amostra. </li><li> A fracção bacteriana é diluído de volta para 500 mL com água livre de vírus e dividido em três repetições de 150 mL cada e são colocados em garrafas de 250ml claro policarbonato. </li></ol> 3. Filtração tangencial Fluxo Método (TFF) para a Produção Viral (Weinbauer et al. 2002, Winget et al. 2005) TFF representa uma abordagem alternativa para o método de redução viral. <ol><li> Aproximadamente 500 mL de amostra natural é coletado como descrito acima. </li><li> Esta amostra é concentrada através de um 0,2 mícrons nominal dos poros do tamanho de sistema de filtração de fluxo tangencial. </li><li> Quando a fração de bactérias é reduzida a cerca de 10-15 mL, ultrafiltrado, a água livre de vírus é adicionada e distribuída como acima. </li><li> As garrafas são incubadas a replicar em condições in situ, usando câmaras ambientais. <ol><li> Níveis de luz são alteradas para as condições da superfície, usando de cor azul de acrílico transparente com acrílico ou screening net para diminuir a intensidade da luz. </li><li> Temperaturas da superfície do ambiente são muitas vezes obtidos através de um fluxo de água do mar baralho incubadora. </li></ol></li><li> Amostras para estimativas de abundância bacteriana e viral são tomadas no tempo 0, com uma concentração final de 2,0-2,5% glutaraldeído estéril adicionado em criotubos. Estas amostras são imediatamente flash congelado com nitrogênio líquido e armazenadas congeladas até ser transformado. <ol><li> Se nitrogênio líquido não estiver disponível, lâminas de microscopia podem ser preparados e transformados imediatamente (ver procedimento abaixo) </li></ol></li><li> Subamostras são coletadas a cada 2,5 horas para pelo menos 10 horas pelo método descrito acima. <ol><li> Neste momento a água pode ser recolhida para análise de PCR quantitativo. Até 5 mL da amostra pode ser adicionado a uma cryovial com nenhum agente fixador com congelamento de flash imediata em nitrogênio líquido. </li></ol></li></ol> 4. Microscopia de Produção viral (Noble &amp; Fuhrman 1998, Wen et al. 2004) <ol><li> Amostras congeladas a ser 0,02 mM filtrada para microscopia devem ser descongeladas no gelo. </li><li> Prepare uma solução estoque de SYBR Green diluindo a 1:10 solução estoque com água estéril. Em seguida, a partir da solução estoque, preparar uma solução de trabalho, adicionando 1 ml da solução estoque para 39 mL de água estéril. A glicerol a 50%, 50% de fosfato tamponado soluções salinas (PBS, 0,05 M Na 2 HPO 4, 0,85% NaCl, pH 7,5) e solução estoque fresco de 2,5% de p-fenilenodiamina também deve estar preparado antes de começar. Manter os 50% glycerol/50% de solução PBS a 4 ° C e os p-phenylenediamine estoque a -20 ° C no escuro até de partida. Direito antes de adicionar a filtragem p-fenilenodiamina ao PBS 50% glycerol/50% para uma concentração final de 0,1% para ser utilizado como a solução Antifade. </li><li> Coloque um de 25 mm 0,02 mícrons filtro Anodisc em cima de um MicronStep 0,45 mícrons, filtro backing celulósico. Adicionar 850 mL da amostra fixa para o topo da Anodisc e vácuo de 20 pKa até que esteja completamente seco. ML colocar 100 de SYBR solução Verde trabalhando para uma placa de Petri estéril e, com o vácuo ainda sobre, remova cuidadosamente a Anodisc a partir da torre do filtro e colocar on the Green SYBR. Incubar as amostras no escuro à temperatura ambiente por 20 minutos. Remova cuidadosamente o filtro da solução Verde SYBR e pavio parte de trás do filtro com um Kimwipe para remover todas as corante residual. Se desejar, o retorno do filtro para a torre e passar até 800 mL de 0,02 mícrons água filtrada ou meios estéreis através do filtro para enxaguar mancha excesso. </li><li> Adicionar uma pequena gota de solução Antifade a uma lâmina de microscópio e coloque uma lamínula por cima. Remover a lamínula e adicionar o filtro seco para a lâmina de microscópio molhado com a solução Antifade. Mais uma vez, adicionar uma pequena quantidade de solução Antifade à lamínula e lentamente coloque-o em cima do filtro, certificando-se de se livrar de todas as bolhas que podem se formar. </li><li> Congelar imediatamente os slides a -20 ° C até ser necessário (estes devem ser usados ​​dentro de alguns meses para evitar desbotamento e reduziu a contagem de vírus) </li><li> Os vírus são enumerados usando microscopia de fluorescência (no nosso caso um microscópio Leica DMRXA) com um conjunto azul gama de filtros (λ Ex = 450-490 nm, λ Em = 510 nm com um filtro de supressão em λ = 510 nm). Cada filtro terá pelo menos 20 campos de vista contadas, certificando-se de quantificar vírus total de cada grade de campo para assegurar uma distribuição de vírus através da membrana do filtro. </li><li> Taxas médias de recorrência do vírus da independente três repetições são então calculados e um desvio padrão é determinada a partir das taxas de produção. </li></ol> 5. Resultados representante Os dados brutos colhidos pela pesquisadora requer processamento matemático mínimo para gerar taxas de recorrência da abundância de vírus. O conjunto de dados primários resultantes deste estudo é a taxa de recorrência da abundância de vírus no subamostras das incubações. Estes resultados do formulário regressões independentes de vírus tempo vs abundância para cada uma das amostras. Para cada amostra, o incubações individuais atuam como um tratamento, por isso, completar três replicate-incubações o pesquisador pode calcular as taxas, bem como uma estimativa de variação (por exemplo, o desvio padrão) (ver Figura 1). Uma ressalva deste processo é que a redução na abundância invariável vírus leva a uma redução nas células hospedeiras na amostra que estão carregando o fardo de vírus. Para compensar esta perda, a enumeração de abundância de bactérias, tanto a fonte (a água do mar não filtrada ou água do lago) e T = amostra de incubação 0 são necessárias. Esta informação pode ser usada para explicar o percentual de células perdidas: supondo que o processo de redução da abundância de vírus não é seletiva a favor ou contra qualquer membro da comunidade microbiana, este fator pode então ser usada para estimar a taxa de produção em situ de vírus . <img src="/files/ftp_upload/2196/2196fig1.jpg" alt="Figura 1" /> Figura 1. A produção de amostras de vírus como partículas ao longo de um 10 horas de incubação em condições in situ por microscopia de epifluorescência. Foram coletados durante um bloom de fitoplâncton na costa da Nova Zelândia em setembro de 2008. <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="Figura 2" /> Figura 2. Diagrama esquemático do processo de fluxo de trabalho para ensaio de produção de vírus. O processo começa com a ultrafiltração de água da amostra para gerar vírus sem água. Este é preenchido por um sistema de ultrafiltração. Em amostras de água paralelas são recolhidos a partir do mesmo local e os vírus livres passados ​​por um filtro, enquanto a comunidade microbiana (contendo uma mistura de células infectadas e não infectadas) é mantida. Esta comunidade é então ressuspendido em água livre de vírus e incubadas sob condições in situ. A taxa de recorrência do vírus é então monitorada para os próximos 10 horas para determinar as taxas de produção de vírus.

<ol>
	<li>Higgins, J. L., Kudo, I., Nishioka, J., Tsuda, A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+response+of+the+virus+community+to+a+mesoscale+iron+fertilization+in+the+sub-Arctic+Pacific+Ocean.">The response of the virus community to a mesoscale iron fertilization in the sub-Arctic Pacific Ocean.</a> Deep-Sea Res II. 56, 2788-2795 (2009).</li><li>Mioni, C. E., Poorvin, L., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+and+siderophore-mediated+transfer+of+available+Fe+between+heterotrophic+bacteria:+characterization+using+a+Fe-specific+bioreporter.">Virus and siderophore-mediated transfer of available Fe between heterotrophic bacteria: characterization using a Fe-specific bioreporter.</a> Aquat Microb Ecol. 41, 233-245 (2005).</li><li>Noble, R. T., Fuhrman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+SYBR+Green+I+for+rapid+epifluorescence+counts+of+marine+viruses+and+bacteria.">Use of SYBR Green I for rapid epifluorescence counts of marine viruses and bacteria.</a> Aquat Microb Ecol. 14, 113-118 (1998).</li><li>Parada, V., Herndl, G., Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+burst+size+of+heterotrophic+prokaryotes+in+aquatic+systems.">Viral burst size of heterotrophic prokaryotes in aquatic systems.</a> J Mar Biol Assoc U K. 86, 613-621 (2006).</li><li>Poorvin, L., Rinta-Kanto, J. M., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+release+of+Fe+and+its+bioavailability+to+marine+plankton.">Viral release of Fe and its bioavailability to marine plankton.</a> Limnol Oceanogr. 49, 1734-1741 .</li><li>Rowe, J. M., Saxton, M. A., Cottrell, M. T., DeBruyn, J. M., Berg, G. M., Kirchman, D. L., Hutchins, D. A., Wilhelm, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constraints+on+virus+production+in+the+Sargasso+Sea+and+North+Atlantic.">Constraints on virus production in the Sargasso Sea and North Atlantic.</a> Aquat Microb Ecol. 52, 233-244 (2008).</li><li>Weinbauer, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+of+prokaryotic+viruses.">Ecology of prokaryotic viruses.</a> FEMS Microbiol Rev. 28, 127-181 (2004).</li><li>Weinbauer, M. G., Peduzzi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency,+size+and+distribution+of+bacteriophages+in+different+marine+morphotypes.">Frequency, size and distribution of bacteriophages in different marine morphotypes.</a> Mar Ecol Prog Ser. 108, 11-20 (1994).</li><li>Weinbauer, M. G., Winter, C., H&amp;ouml, f. l. e., G, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconsidering+transmission+electron+microscopy+based+estimates+of+viral+infection+of+bacterioplankton+using+conversion+factors+derived+from+natural+communities.">Reconsidering transmission electron microscopy based estimates of viral infection of bacterioplankton using conversion factors derived from natural communities.</a> Aquat Microb Ecol. 27, 103-110 (2002).</li><li>Wen, K., Ortmann, A. C., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+estimation+of+viral+abundance+by+epifluorescence+microscopy.">Accurate estimation of viral abundance by epifluorescence microscopy.</a> Appl Environ Microbiol. 70, 3862-3867 (2004).</li><li>Wilhelm, S. W., Brigden, S. M., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dilution+technique+for+the+direct+measurement+of+viral+production:+a+comparison+in+stratified+and+tidally+mixed+coastal+waters.">A dilution technique for the direct measurement of viral production: a comparison in stratified and tidally mixed coastal waters.</a> Microb Ecol. 43, 168-173 (2002).</li><li>Wilhelm, S. W., Matteson, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Freshwater+and+marine+virioplankton:+a+brief+overview+of+commonalities+and+differences.">Freshwater and marine virioplankton: a brief overview of commonalities and differences.</a> Freshw Biol. 53, 1076-1089 (2008).</li><li>Wilhelm, S. W., Poorvin, L., Paul, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Quantification of algal viruses in marine samples. , 53-66 (2001).</li><li>Wilhelm, S. W., Suttle, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viruses+and+nutrient+cycles+in+the+sea.">Viruses and nutrient cycles in the sea.</a> Bioscience. 49, 781-788 (1999).</li><li>Wilhelm, S. W., Weinbauer, M. G., Suttle, C. A., Jeffrey, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+sunlight+in+the+removal+and+repair+of+viruses+in+the+sea.">The role of sunlight in the removal and repair of viruses in the sea.</a> Limnol Oceanogr. 43, 586-592 (1998).</li><li>Winget, D. M., Williamson, K. E., Helton, R. R., Wommack, 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=Tangential+flow+diafiltration:+an+improved+technique+for+estimation+of+virioplankton+production.">Tangential flow diafiltration: an improved technique for estimation of virioplankton production.</a> Aquat Microb Ecol. 41, 221-232 (2005).</li></ol>

Não há conflitos de interesse declarados.

Um componente crítico na compreensão de como os vírus influência comunidades microbianas marinhos é determinar a taxa em que partículas de vírus são produzidos. Dado que a abundância são (mais ou menos) estática na maioria dos sistemas (Wilhelm &amp; Suttle 1999, Weinbauer 2004), e que os vírus são removidos ou tornados não-infecciosa rapidamente em sistemas aquáticos (Wilhelm et al. 1998), então as taxas de produção deve ser relativa rápida para substituir partículas perdido. Estimar o vírus causas de mortalidade para a comunidade microbiana requer um conhecimento de quantos vírus são produzidos cada vez que um vírus lise de uma célula (o &quot;tamanho da rajada&quot;). Tamanhos vírus estourou em amostras naturais pode variar muito. Tamanhos explosão pode ser determinada diretamente por microscopia eletrônica de transmissão (por exemplo, Weinbauer &amp; Peduzzi, 1994), mas este é muitas vezes além das capacidades de um dado laboratório ou não sempre prático. Em situações onde eles não podem ser empiricamente determinadas, valores de literatura de 24 vírus por evento lítico pode ser usado para sistemas marinhos e 34 para sistemas de água doce (Parada et al. 2006). Se a taxa de produção de vírus é dividido por este número, o resultado é a abundância de células por volume destruída por vírus em uma base diária. Os micróbios lisadas valor pode ser dividido pelo estoque de pé de abundância de bactérias, resultando na mortalidade induzida por vírus para o sistema em questão: faixa estimativas existentes a partir de alguns por cento para quase toda a população e muitas vezes são dependentes de outros fatores no sistema em questão (Wilhelm &amp; Matteson 2008). Para determinar a porcentagem de mortalidade total este número é muitas vezes multiplicada por dois (trabalhando a partir do pressuposto de que 50% das células passam a se reproduzir e 50% das células são perdidas, Weinbauer 2004). Dado que a biodisponibilidade de nutrientes e elementos traço (por exemplo, N, P, Fe) pode limitar a taxa de produtividade primária e, como tal fluxo de carbono, através de sistemas aquáticos, e compreensão do papel do vírus-driven mortalidade microbiana nesse processo tornou-se de interesse para geoquímicos marinhos. Várias estimativas sugerem que existem agora os vírus lançar um concentrações significativas de elementos nutrientes de volta para a coluna de água diariamente (Rowe et al. 2008, Higgins et al. 2009) e que estes elementos são rapidamente assimilados pela comunidade microbiana (Poorvin et al. 2004, Mioni et al. 2005). A taxa de fluxo de nutrientes para o meio ambiente pode ser calculado multiplicando o número de células destruídas pela quantidade de nutrientes por célula (denotado &quot;quotas&quot;). Esta informação pode fornecer um componente crítico para a nossa compreensão de como teias alimentares microbianas em função de sistemas aquáticos. Desenvolvimentos em curso: Os esforços atuais por uma série de grupos de pesquisa envolvem a adaptação da estratégia acima para enumerar vírus específicos dentro da comunidade e, como tal, para determinar como organismos específicos são influenciados pela atividade de vírus. Para fazer isso os pesquisadores utilizam a reação em cadeia da polimerase quantitativa (qPCR) para estimar a abundância de grupos específicos vírus ou famílias em paralelo com as estimativas da comunidade virus total. Os resultados são então aplicados diretamente para fornecer estimativas de mortalidade do vírus, de nutrientes, etc para grupos específicos de plâncton. Esta abordagem nova e poderosa permitirá que os pesquisadores nos próximos anos para cavar muito mais profundamente nos processos associados com a ecologia do vírus e, pela primeira vez, para quantificar as interações específicas do vírus-hospedeiro comunidades além das limitações dos sistemas de laboratório.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>2.5% p-phenylenediamine</td>
<td>&nbsp;</td>
<td>Acros</td>
<td>130575000</td>
<td>Stock for Antifade</td>
</tr>
<tr>
<td>Amicon Proflux M12 system</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>N/A</td>
<td>Any ultrafiltration device may be used for this step</td>
</tr>
<tr>
<td>SYBR Green I nucleic acid gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>S-7563</td>
<td>&nbsp;</td>
<tr>
<td>Helicon S10 30kDa Filter</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>CDUF010LT</td>
<td>&nbsp;</td>
<tr>
<td>Pelicon XL filters 0.22 &mu;m</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>PXGVPPC50</td>
<td>&nbsp;</td>
<tr>
<td>GE 0.2 &mu;m PCTE membrane filters (47mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-732-35</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Labscale Tangential Flow Filtration System</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>XX42LSS11</td>
<td>Other TFF systems may be used for this step</td>
</tr>
<tr>
<td>0.45 &mu;m Micronstep, Cellulosic, white plain filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>E04WP02500</td>
<td>&nbsp;</td>
<tr>
<td>GE Whatman 0.02 &mu;m Anodisc filters (25mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>68-09-6002</td>
<td>&nbsp;</td>
<tr>
<td>Leica DMRXA microscope</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Any epifluorescence microscope with a blue filter set may be used</td>
</tr>
<tr>
<td>20L polycarbonate carboys</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP229-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS (0.05 M Na2HPO4, 0.85% NaCl, pH 7.5)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP329-500, S640-500</td>
<td>&nbsp;</td>
<tr>
<td>50% Glutaraldehyde </td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>G151-1</td>
<td>&nbsp;</td>
<tr>
<td>Corning 2 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-71</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td>Corning 5 mL Cryovials-External Thread polypropylene</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>09-761-74</td>
<td>Any cryovials may be used</td>
</tr>
<tr>
<td> 85% H3PO4</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A242-212</td>
<td>Spiral Membrane storage</td>
</tr>
<tr>
<td>NaOH pellets</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>S318-3</td>
<td>M12 cleaning</td>
</tr>
<tr>
<td>Graduated Cylinders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Isopore 0.8-&mu;m pore-size membrane filter (142mm)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>ATTP14250</td>
<td>&nbsp;</td>
<tr>
<td>Millipore Stainless Steel Pressure Filter Holder (142mm)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>YY30 090 00</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

estimating virus production rates in aquatic systems

Os vírus são componentes generalizada dos sistemas marinhos e de água doce e são conhecidos por serem agentes significativa da mortalidade microbiana. Desenvolver estimativas quantitativas deste processo é fundamental como podemos então desenvolver melhores modelos de estrutura da comunidade microbiana e função, bem como o avanço na compreensão do funcionamento dos vírus para alterar aquáticos ciclos biogeoquímicos. A técnica de redução de vírus permite que os pesquisadores para estimar a taxa em que partículas de vírus são liberados da comunidade microbiana endêmica. Em breve, a abundância de livre (extracelular) Os vírus é reduzida em uma amostra enquanto a comunidade microbiana é mantida a concentração ambiente próximo. A comunidade microbiana é, então, incubadas na ausência de vírus livres ea taxa em que os vírus reaparecer na amostra (através da lise de membros já infectados da comunidade) podem ser quantificados por microscopia de epifluorescência, ou, no caso de vírus específicos, quantitativos PCR. Essas taxas podem ser usados ​​para estimar a taxa de mortalidade microbiana devido ao vírus mediada por lise celular.

Watch this Scientific Journal Video about Estimating Virus Production Rates in Aquatic Systems at JoVE.com

Estimating Virus Production Rates in Aquatic Systems

A taxa de rotatividade de vírus em sistemas marinhos e de água doce pode ser estimado por uma redução e técnica recorrência. Os dados permitem aos pesquisadores inferir as taxas de mortalidade do vírus mediada microbiana em sistemas aquáticos.

Estimar as taxas de produção de vírus em Sistemas Aquáticos

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

estimating-virus-production-rates-in-aquatic-systems

Research

JoVE Journal

Immunology and Infection

12.5K visualizações.  University of Tennessee. A taxa de rotatividade de vírus em sistemas marinhos e de água doce pode ser estimado por uma redução e técnica recorrência. Os dados permitem aos pesquisadores inferir as taxas de mortalidade do vírus mediada microbiana em sistemas aquáticos.

Vídeo: Estimating Virus Production Rates in Aquatic Systems

Visualizing Dengue Virus through Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that influence this interaction could lead to improved understanding of disease pathogenesis and thus influence vaccine or therapeutic design. Hence, the development 
of methods to probe this interaction would be useful. Recent advancements in fluorophores development1-3 and imaging technology4 can be exploited to 
improve our current knowledge on dengue pathogenesis and thus pave the way to reduce the millions of dengue infections occurring annually.

The enveloped dengue virus has an external scaffold consisting of 90 envelope glycoprotein (E) dimers protecting the nucleocapsid shell, 
which contains a single positive strand RNA genome5. The identical protein subunits on the virus surface can thus be labeled with an amine reactive dye and visualized 
through immunofluorescent microscopy. Here, we present a simple method of labeling of dengue virus with Alexa Fluor succinimidyl ester dye dissolved directly in a sodium 
bicarbonate buffer that yielded highly viable virus after labeling. There is no standardized procedure for the labeling of live virus and existing manufacturer&#x2019;s protocol 
for protein labeling usually requires the reconstitution of dye in dimethyl sulfoxide. The presence of dimethyl sulfoxide, even in minute quantities, can block 
productive infection of virus and also induce cell cytotoxicity6. The exclusion of the use of dimethyl sulfoxide in this protocol thus reduced this possibility. 
Alexa Fluor dyes have superior photostability and are less pH-sensitive than the common dyes, such as fluorescein and rhodamine2, making them ideal for 
studies on cellular uptake and endosomal transport of the virus. The conjugation of Alexa Fluor dye did not affect the recognition of labeled dengue 
virus by virus-specific antibody and its putative receptors in host cells7. This method could have useful applications in virological studies.

Taking advantage of the advancements in fluorophore development and imaging technology, a simple method of Alexa Fluor labeling of dengue virus was devised to visualize the early interactions between virus and cell.

Visualizando Vírus da Dengue por meio Alexa Fluor Labeling

The early events in the interaction between virus and cell can have profound influence on the outcome of infection. Determining the factors 
that ...

Imunologia e Infecção

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Produção Parvovirus eficiente recombinante com a ajuda de Adenovirus sistemas derivados

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

In Vivo Imaging Systems (IVIS) Detection of a Neuro-Invasive Encephalitic Virus

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by Russel and Burch in Hume's 3Rs (replacement, reduction, refinement), the utilization of animal models in scientific research is under constant pressure to identify new methodologies to reduce animal usage while improving scientific accuracy and speed. A major challenge to Hume's principals however, is how to ensure the studies are statistically accurate while reducing animal disease morbidity and overall numbers. Vaccine efficacy studies currently require a large number of animals in order to be considered statistically significant and often result in high morbidity and mortality endpoints for identification of immune protection. We utilized in vivo imaging systems (IVIS) in conjunction with a firefly bioluminescent enzyme to progressively track the invasion of the central nervous system (CNS) by an encephalitic virus in a murine model. Typically, the disease progresses relatively slowly, however virus replication is rapid, especially within the CNS, and can lead to an often, lethal outcome. Following intranasal infection of the mice with TC83-Luc, an attenuated Venezuelan equine encephalitis virus strain modified to expresses a luciferase gene; we are able to visualize virus replication within the brain at least three days before the development of clinical disease symptoms. Utilizing CNS invasion as a key encephalitic disease development endpoint we are able to quickly identify therapeutic and vaccine protection against TC83-Luc infection before clinical symptoms develop. With IVIS technology we are able to demonstrate the rapid and accurate testing of drug therapeutics and vaccines while reducing animal numbers and morbidity.

In Vivo Imaging Systems IVIS Detection of a Neuro-Invasive Encephalitic Virus

Utilizing luciferase and in vivo imaging systems (IVIS) as a novel means to identify disease endpoints before clinical developments occur. IVIS has allowed us to visualize in real time the invasion of encephalitic viruses over multiple days, providing a more accurate disease model for future study. It has also allowed us to identify the potential protective features of antivirals and vaccines faster than currently utilized animal models. The capability to utilize individual animals over multiple time points ensures reduced animal requirements, costs, and overall morbidity to the animals utilized ensuring a more humane and more scientific means of disease study.

In Vivo Imaging Systems Detecção (IVIS) de um vírus de Neuro-invasiva encefalítico

Modern advancements in imaging technology encourage further development and refinement in the way viral research is accomplished. Initially proposed by ...

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

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

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

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

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

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

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

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

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

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

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 ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identificação de codificação e Classes de RNA não-codificante expressa em suínos, sangue total

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

Medição de taxas de mistranslation micobacterianas específicas com sistemas de repórter de ganho de função

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

Teste in vitro ELISA para avaliar a potência da vacina antirrábica

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Described here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems.

Análise de interações entre endobióticos e microbiota intestinal humana usando sistemas de fermentação de banho in vitro

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, ...