这篇文章的发表，是由来自美国田纳西州大学的研究办公室授予。作者感谢上一代的学生和研究人员一直在努力改进这些程序。研究是由美国国家科学基金会（NSF - 0851113，NSF - 0825405和NSF - 0550485）的赠款支持。

1。超滤海水生成“病毒”水（2001年威廉与Poorvin） <ol><li>约20L的海水/ lakewater是为在无菌条件下尽可能收集。 </li><li>水是连续预过滤通过142毫米直径的聚碳酸酯0.8微米的过滤器，保持社会分析，可在-20 ° C。更大的孔径过滤器可用于在此之前收获颇丰系统的步骤。 </li><li>为了获得Amicon的M12系统（Millipore公司）30 kDa的截止螺旋墨盒是用来排除所有的病毒，即使是小RNA病毒的超滤水。 </li><li>样品处理和集中〜25％的速度〜15-16千帕的背压。 </li><li>余下的〜500毫升水样本将包含集中的病毒社区（可用于其他实验保存），而其余19.5无病毒水L将用于病毒性生产实验。 </li><li> Amicon的M12系统使用的每一天后，一定要清洗干净，以防止损坏滤芯膜。 </li><li>如果你与海水的工作，至少Milli - Q水6L冲洗膜与0.1N的NaOH洗30-45分钟的解决方案遵循。 </li><li>再次冲洗至少Milli - Q水6L硒鼓。 </li><li>完成后，使用M12系统，螺旋墨盒应存放在一个0.05MH 3 PO 4溶液，在4 ° C 。 </li></ol> 2。病毒病毒生产还原法（威廉等人 2002年） <ol><li>高达500毫升的海水/ lakewater样品获得主机和病毒放置在一个sterifilter单元和0.2微米的标称孔径低蛋白结合过滤器（例如，Durapore）置于其上。 </li><li>该样本是轻轻真空压力&lt;200毫米汞柱，而不断resuspending样品用无菌移液管，抑制细菌细胞的过滤器上集中。 </li><li>三卷超滤液慢慢地被添加到的菌悬液，免费病毒样本数量显着减少。 </li><li>细菌分数稀释回无病毒水至500 ml，并分为三重复每次150毫升，并明确250毫升聚碳酸酯瓶。 </li></ol> 3。切向流过滤（TFF）方法对病毒的生产（Weinbauer等 。2002年， 迪尔等人，2005年） TFF病毒还原法的替代方法。 <ol><li>如上所述，约500毫升的天然样品收集。 </li><li>这个范例是集中使用0.2微米孔径名义切向流过滤系统。 </li><li>当细菌分数减少到大约10-15毫升，超滤，无病毒加水以上。 </li><li>复制瓶培养在原位条件，使用环境试验箱。 <ol><li>使用筛选净蓝有色丙烯酸或丙烯酸减少光照强度，光照水平改变表面状况。 </li><li>环境表面温度通常是通过使用一个流动海水甲板孵化器。 </li></ol></li><li>细菌和病毒的丰度估计的样品到离心管中添加的2.0-2.5％无菌戊二醛最终浓度在0时刻。这些样品立即用液氮速冻和储存冷冻，直到处理。 <ol><li>如果液氮不可用，可能是准备和显微镜幻灯片立即处理（参见下面的步骤） </li></ol></li><li>子样本是由上面介绍的方法收集至少10小时，每隔2.5小时。 <ol><li>此时水可收集定量PCR分析。 5毫升的样品可能被添加到一个离心管，没有立即闪光冷冻在液氮中的固定液代理。 </li></ol></li></ol> 4。病毒生产显微镜（贵族和富尔曼1998年， 温家宝等人，2004年） <ol><li>将0.02微米的过滤显微镜冷冻样品应在冰上解冻。 </li><li>准备用无菌水稀释原液1:10原液的SYBR GREEN。接下来，从股票的解决方案，准备加入1ml至39毫升无菌水原液一个可行的解决方案。 50％甘油，50％的磷酸盐缓冲盐溶液（PBS，0.05 mol娜2 HPO 4，0.85％氯化钠，pH值7.5）和2.5％的对苯二胺的新鲜原液也应准备开始之前。 50％glycerol/50％PBS液保持在4 ° C和P - phenylenediamINE股票在-20 ° C黑暗环境中，直到开始。右键过滤前添加苯二胺50％glycerol/50％的PBS作为抗淬灭的解决方案中使用的终浓度为0.1％。 </li><li>将25毫米0.02微米，0.45微米MicronStep，纤维素支持过滤器的顶部Anodisc过滤。固定样本850μL加入到20的pKa Anodisc和真空顶部，直至完全干燥。将100μL的SYBR GREEN工作无菌培养皿中的解决方案，并与真空，小心取出过滤塔和SYBR GREEN Anodisc。样品在室温下孵育20分钟，在黑暗中。小心取出从SYBR绿色解决方案的过滤器和灯芯与一个Kimwipe过滤器的背面，清除所有残留的染料。如果需要，返回过滤塔，并通过800μL0.02 -微米的过滤水或无菌媒体，通过过滤器，冲洗掉多余的污渍。 </li><li>小滴在显微镜幻灯片添加抗淬灭的解决方案，并放置顶盖上滑。取出盖玻片，干燥过滤器，并添加与抗淬灭溶液，在显微镜幻灯片湿。再次，添加少量的抗淬灭的解决方案，以盖玻片，慢慢地放置在过滤器顶部，一定要排除任何可能形成的泡沫。 </li><li>立即冻结在-20 ° C的幻灯片，直到需要（这些应该在几个月内使用，以防止褪色和降低病毒计数） </li><li>病毒枚举使用一个全蓝色的过滤器设置（λ防爆= 450至490纳米，λEM抑制滤波器在λ= 510纳米= 510 nm处）的荧光显微镜（徕卡DMRXA显微镜）在我们的例子。每个滤波器将有至少20个领域的计算视图，确保量化每个字段电网总的病毒，以确保整个滤膜甚至病毒的分布。 </li><li>平均的病毒再次发生率从三个独立的复制，然后计算和确定一个标准偏差是从生产速度。 </li></ol> 5。代表性的成果研究人员所收集的原始数据，需要最少的数学处理，生成病毒丰度的再次发生率。设置这项研究的主要数据是病毒丰度的再次发生率从孵化的子样本。这些结果形成独立的回归，每个样本的病毒丰度随时间。每个样本的个体孵化作为一个治疗，所以完成三个复制，孵化，研究者可以计算税率，以及估计的变化（如标准偏差）（参见图1。） 这个过程中需要注意的是，减少病毒丰度不变在宿主细胞样品中，携带病毒的负担减少。为了弥补这个损失，从源（未过滤海水或湖水）和T = 0的孵化样品的细菌数量的枚举是必要的。这些信息可以用来核算失去细胞的百分比：假设减少病毒丰度的过程中是没有选择性的微生物群落的任何成员或反对，这个因素可以被用来估计在病毒的原位生产速度。 <img src="/files/ftp_upload/2196/2196fig1.jpg" alt="图1" /> 图1。生产类病毒颗粒超过10小时的潜伏期 ，在使用epifluorescence显微镜原位条件。样品收集过程中浮游植物绽放2008年9月在新西兰海岸。 <img src="/files/ftp_upload/2196/2196fig2.jpg" alt="图2" /> 图2。这一过程始于生成无病毒水超滤水样化验病毒生产的工作流程过程的示意图。。这是使用超滤系统完成。在平行水样收集来自同一个站点，并通过一个过滤器的免费病毒而保留的微生物群落（含感染和非感染细胞的混合物）。这个社区是悬浮于病毒的自由水和就地条件下培养。然后监测病毒的再次发生率在未来10小时内，以确定病毒的生产率。

<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>

一个病毒如何影响海洋微生物群落的重要组成部分的理解是，以确定该病毒粒子产生率。鉴于，在大多数系统中的丰度（或多或少）静态（威廉净重1999年，Weinbauer 2004年），以及病毒被删除或呈现非传染性迅速在水生系统（ 威廉等，1998），产率必须相对迅速，以取代失去颗粒。 估计死亡率病毒引起的微生物群落，需要多少病毒产生的每一次病毒裂解单元格（“突发大小”）的知识。天然样品中的病毒突发流量大小可以相差很大。脉冲大小，可直接通过透射电子显微镜（例如，Weinbauer Peduzzi 1994年）确定的，但往往超出这是一个给定的实验室的能力或不总是可行的。他们不能凭经验确定的情况下，24％病毒裂解事件的文学价值，可用于海洋生态系统和淡水生态系统的 34（帕拉达等。2006年）。如果这个数字除以病毒的生产速度，其结果是每量由每天的病毒破坏的细胞丰度。裂解价值的微生物可以分为由细菌病毒引起系统问题的死亡率造成的丰的常备现货：现有的估计范围从百分之几到几乎整个人口，往往依赖于系统问题的其他因素（威廉 - 马特森2008）。为了确定总死亡率的百分比，这个数字往往是乘以两个（假设50％的细胞去复制和50％的细胞失去了，Weinbauer 2004年）。 由于养分和微量元素的生物利用度（如氮，磷，铁），可以限制初级生产力的速度，如碳通量，通过水生生态系统，驱动的微生物病毒的死亡率在这个过程中作用的认识已成为海洋地球化学的兴趣。现在存在几个估计表明病毒释放一个重要的营养元素浓度每天的基础上，以水柱（ 罗等人，2008年， 希金斯等人，2009年），这些元素的微生物群落迅速吸收（Poorvin等人，2004年，Mioni 等人，2005年）。营养每单元（表示“配额”）的金额破坏细胞的数量乘以养分通量率的环境，才能确定。这些信息可以提供一个关键组成部分，我们如何微生物食物链的整个水生生态系统功能的理解。 涉及持续发展：通过了一系列的研究小组目前正在努力适应上述策略来枚举特定社区内的病毒，因此，以确定如何具体的生物体是由病毒活性的影响。要做到这一点的研究人员使用定量聚合酶链反应（qPCR的），估计在平行丰富的特定病毒的团体或家庭总病毒界的估计。结果，然后直接应用提供病毒的死亡率，营养的营业额，为特定的浮游生物群体等的估计。这种强大的新方法将在未来几年中，研究人员更深入地挖掘到与病毒生态相关的进程，并首次量化特定的病毒主机超越实验室系统的制约社区之间的相互作用。

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		<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

病毒是无孔不入的海洋和淡水系统的组成部分，是已知的微生物死亡率显着的代理商。发展的定量估计，这一过程是至关重要的，我们可以开发更好的模型的微生物群落结构和功能以及推进我们了解病毒如何工作，以改变水生生物地球化学循环。减少病毒技术可以让研究人员估计在病毒颗粒的释放速率从特有的微生物群落。总之，大量的自由（外）病毒是减少样品中，而维持在附近的环境浓度的微生物群落。没有免费的病毒和病毒再次出现在样品通过已被感染的社会成员的裂解epifluorescence显微镜可以通过量化，或在特定的病毒的情况下，定量率的微生物群落，然后孵育PCR扩增。然后，这些比率可以被用来估计微生物死亡，由于病毒介导的细胞溶解率。

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

Estimating Virus Production Rates in Aquatic Systems

估计周转率在海洋和淡水系统的病毒可以通过减少和再陷技术。这些数据使研究人员推断病毒介导的水生生态系统中的微生物死亡率。

估计在水生系统的病毒产率

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 次观看.  University of Tennessee. 估计周转率在海洋和淡水系统的病毒可以通过减少和再陷技术。这些数据使研究人员推断病毒介导的水生生态系统中的微生物死亡率。

视频: 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.

可视登革病毒通过的Alexa Fluor标签

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

科研

免疫与感染

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).

重组细小病毒与腺病毒派生系统的帮助下高效生产

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.

在活体成像系统（IVIS）的神经侵入性脑炎病毒的检测

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.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

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.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

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).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

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.

猪全血表达编码和非编码 rna 类的鉴定

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.

功能金报告系统测量特定分枝杆菌误调速率

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.

体外ELISA测试评估狂犬病疫苗效力

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.

内生菌与人类肠道微生物群使用体外浴发酵系统的相互作用分析

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