We would like to thank Dr. Nicholas J. Ashbolt and Dr. G. Shay Fout for their review of the manuscript.

1.准备玻璃器皿和过滤器外壳<ol style="margin-left: 40px;"><li>除非另有说明，所有消毒玻璃器皿，过滤器外壳和溶液在121℃下进行15分钟。为了保证无菌，覆盖所有开口或暴露的表面或者用铝箔或胶带固定前消毒纸。 </li><li>通过将过滤器壳（47毫米直径）以1 L侧臂Erlenmeyer烧瓶装配过滤装置。收集所需的过滤器:直径47 mm正电/负电盘式过滤器。 </li><li>制备1L的1.5％牛肉提取物（凝聚;产生的絮凝物颗粒时溶液pH值降低至3.5，或不絮凝;其中不聚集在pH 3.5），用0.05M甘氨酸，溶解15克牛肉提取物中的1升的去离子水并加入3.75克甘氨酸。 注:非絮凝牛肉提取物将需要叔胺浓度，然后加入硅藻土。 </li><li>加入洗脱添加剂钠polyphosphate牛肉提取物在0.1％的浓度。 </li><li>添加1M盐酸溶液，逐滴，以混合牛肉提取物，pH值溶液至所需的pH值。蒸压牛肉提取物在121℃15-30分钟。 </li><li>衡量0.1克精细，中型或大型粒子硅藻土（与唯一的非絮凝牛肉膏使用）的。 </li><li>通过附加兼容油管锥形侧臂烧瓶中，并于真空出口准备真空/抽吸。 </li></ol> 2.准备解决方案和病毒股票<ol style="margin-left: 40px;"><li>制备1升的1M HCl中。 </li><li>制备1升的1M NaOH处理。 </li><li>制备的1×PBS溶液（137 mM氯化钠，2.7 mM的氯化钾，1.47毫K 2 PO 4，和4.3毫摩尔的NaH 2 PO 4）。 <ol><li>调节pH的1×PBS的9.0使用1M的NaOH。 </li></ol></li><li>准备和稀释股票的病毒，以10 4 -10 5最可能数（MPN）毫升-1混合1毫升股票病毒的9毫升的PBS（pH值7），如先前所描述的8,13。储存在-80℃下在1ml等分试样。 <ol><li>消毒病毒的股票通过注射器过滤（0.22微米孔径），以消除潜在的污染物。 </li></ol></li><li>脱氯自来水<ol><li>措施1升用无菌无菌自来水量筒并倒入含有磁性搅拌棒的2升烧杯中。 </li><li>加入将0.7克的硫代硫酸钠并混合直到颗粒溶解。如果使用1M HCl或1M的NaOH溶液根据需要调整pH值的自来水至7-7.5。 </li><li>解冻的病毒刺突（1毫升）中，并添加穗总体积为1升的无菌脱氯自来水。混合10分钟。 </li><li>制备方法空白（无腺病毒脱氯自来水）与每个实验和过程中相同的方式加标样品。 </li></ol></li></ol> 3.制备微孔过滤装置<ol style="margin-left: 40px;"><li>删除无菌占地过滤器外壳。保证过滤器外壳上碗，下滤网之间适当的配合在过滤过程中，避免样品泄漏。 </li><li>从1升锥形瓶中取出无菌覆盖。确保过滤器单元的软木紧密地配合在锥形瓶中的顶部开口，创造了良好的密封。 </li><li>从较低的过滤网拆下顶部碗过滤单元，并将47毫米阳电滤盘正视屏幕上用无菌镊子。安装过滤器外壳碗底部的屏幕并锁定到位逆时针扭动。 </li></ol> 4.自来水过滤（主浓度） <ol style="margin-left: 40px;"><li>取100mL病毒的尖刺用无菌量筒自来水。 </li><li>倒入百毫升病毒飙升自来水到含有无论是47毫米折叠玻璃/纤维素过滤器或纳米氧化铝/玻璃纤维盘滤清器滤芯外壳。 </li><li>让水慢慢穿过过滤器。适用于温和真空去除剩余的残留物Ø从过滤面F的自来水。 </li><li>如果使用胰蛋白酶处理，加入5 ml 0.25％胰蛋白酶的过滤表面并孵育5分钟。 </li><li>从用50毫升无菌去离子水过滤表面清洗胰蛋白酶。 </li></ol>从过滤器5.病毒洗脱<ol style="margin-left: 40px;"><li>从2升Erlenmeyer烧瓶中除去过滤器壳体，并放置到250毫升侧臂烧瓶中。 </li><li>取100mL牛肉膏的使用量筒。慢慢倒入100毫升牛肉膏进入过滤器外壳。 </li><li>允许牛肉提取倒通过过滤器，捕获牛肉提取在一个小的（250毫升）的Erlenmeyer烧瓶中。运用真空过滤从通过残余的牛肉膏拉。 </li></ol> 6.硅藻土二级浓度<ol style="margin-left: 40px;"><li>传输瓶中含有洗脱病毒的轰动板。加灭菌磁力搅拌棒并将其放置在搅拌盘，搅拌创造轻微的漩涡。 </li><li>添加0.1克的硅藻​​土粉末至100毫升牛肉提取物，并允许硅藻土分散。将无菌的pH探头放入牛肉膏。 </li><li>添加逐滴1 M盐酸至牛肉膏实现pH值4.0。允许缓慢混合10分钟。 </li><li>放置使用的2L Erlenmeyer烧瓶47毫米预过滤到过滤器外壳（如前所述）。 </li><li>在预过滤倒入百毫升牛肉膏/硅藻土混合。让牛肉膏倒过。适用于轻微的真空过滤器，从通过剩余牛肉膏拉。 </li><li>将金属管夹上的过滤器外壳喷结束。附加的无菌15mL的聚丙烯管中收集到金属管夹。放置过滤器壳体回Erlenmeyer烧瓶。 </li><li>加入5毫升的1×PBS缓冲液，pH 9.0经硅藻土上收集的预过滤器。 </li><li>让PBS滴通过预过滤器。适用于轻微的真空通过残余的PBS拉成15个ml收集管。 </li></ol> 7.有机絮凝二级浓度<ol style="margin-left: 40px;"><li>传输含病毒洗脱的烧杯，以小的轰动板。加灭菌磁力搅拌棒至100毫升牛肉提取物和混合创造轻微的漩涡。 </li><li>将无菌的pH探头放入牛肉膏。 </li><li>添加1M的HCl滴至牛肉提取物和调节pH值至3.5。拌30分钟。 </li><li>倾洗脱液于250毫升的离心锥形管和离心机在2500×g离心15分钟，在4ºC。 </li><li>倒掉上清，注意不要不要破坏沉淀。重悬在5ml的1×PBS，pH值9的颗粒。 </li><li>离心该悬浮液在4000×g离心10分钟，在4ºC。倒掉上清弃沉淀。 </li><li>将pH调节至7-7.5，过滤消毒通过0.22微米注射器式滤器并冷冻在-80℃。 </li></ol> 8.病毒核酸提取<ol style="margin-left: 40px;"><li>提取根据制造商的说明，用适当的市售试剂盒的病毒核酸。 </li></ol> &lt;P类="j​​ove_title"&gt; 9。腺病毒4​​0/41定量PCR <ol style="margin-left: 40px;"><li>使用引物和探针，以及反应混合物和热循环曲线所公布8,13。 注:检测化验的估计上限是每反应约5 qPCR的单位。 </li><li>添加的PCR扩增混合物组分的浓度如下:10毫摩尔Tris（pH值8.3），50mM的氯化钾，4.5毫的MgCl 2，10毫的dNTPs，10μM引物和1μM的探针和0.5微升聚合酶以便在45最终卷微升可以加入到孔中的适当数量的96孔PCR反应板内。 </li><li>负载45微升的PCR扩增混合物中，96孔，快速反应的PCR板的每个相应井。 </li><li>加入5微升DNA提取样本的PCR板的合适的孔中。 </li><li>捂盘与耐热盖密封，旋转盘移动任何液滴PCR板孔底部。 </li><li>设置PCR扩增循环的FOLL流入的:95℃10分钟，随后为15秒的40个循环的95℃和60℃进行1分钟。 </li></ol> 10.数据分析<ol style="margin-left: 40px;"><li>为了确定病毒颗粒的最大可能数（MPN）625稀释范围，如前所述8,13:分析跨越1的样本的序列5倍稀释液的一式三份测量:5至1。 </li><li>为了确定病毒颗粒的最大可能数（MPN）15625稀释范围:腺病毒实验尖峰跨越1的串行5倍稀释液分析:5到1。 </li><li>使用的EPA MPN计算器（http://www.epa.gov/nerlcwww/online.html），调整计算器占稀释的类型（1:5或1:10），进行稀释的数量，以及数复制样品连续稀释。 </li><li>执行日志中的数据，随后归一体积的单位（ 例如 1毫升，100毫升， 等 10变换</EM&gt;）。除以存在于样品的病毒颗粒的数目计算回收率为给定的实验集中与存在于实验尖峰病毒颗粒的数目。 </li><li>评估不同实验变量的影响（ 例如，不同的硅藻土的类型，pH值范围，不同的滤波器类型和不同的次级浓缩技术）上的AdV回收率通过执行参数或非参数统计测试（ 例如 ，配对t检验，一个或两个往来港澳ANOVA），根据数据的分布和实验设计。 </li></ol>

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No competing financial interests. 
The views expressed in this article are those of the authors and do not necessarily reflect the views or policies of the U.S. Environmental Protection Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use.

阳电过滤器是集中在水的病毒有效;然而，这些过滤器可以在它们的结构和组合物，其可以反过来改变其效力不同。私了这个问题，衣壳的结构和收费病毒株需要浓缩技术进行定制，以确保最佳恢复15之间变化。通过对现有的浓度的技术（ 例如，正电性的过滤器，牛肉提取物洗脱），靶病毒更有效浓度可以达到16,17简单的修改。 研究迄今为止通常集中于?...

人类肠道病毒是水源性疾病1-3中重要病原体，但一般存在于被污染的环境水低的数字，使得它们的检测困难而不浓度。用于浓缩病毒程序通常包括一个过滤步骤中的过滤器的洗脱液中，接着过滤洗脱，和仲浓度。一个常见的过滤方法依赖于使用荷电膜，如正电的过滤器（最近在4,5审查）的。这些过滤器依赖于捕捉的病毒悬浮在水中使用的过滤表面（正电荷）和靶向的病毒​​颗粒之间的静电相互作用（带负电）。两个正电的过滤器是商业上可用的依赖于该技术中，玻璃/纤维素和纳米氧化铝/玻璃纤维过滤器。该玻璃/​​纤维素滤器成本高达10倍，该纳米氧化铝/玻璃纤维的，其限制使用的玻璃/ CE的llulose过滤器进行例行病毒监控。最近的研究已经得出结论差异是名义在从室温水中6,7肠道病毒的恢复这两个滤波器之间，证明使用一种更便宜的过滤器的替代。其他过滤选项，例如带负电和玻璃毛过滤器进行了研究，然而，他们要么需要源水（电负性过滤器）的预处理或没有市售的（玻璃毛过滤器）。的病毒浓度程序的发展主要集中在以提高病毒回收率从水优化初级浓缩技术（过滤器）。然而，次级浓度程序，其中降低洗脱液的体积通常为1升至毫升卷，也可对病毒回收率8显著影响。 的肠道病毒次要浓度通常依赖于絮凝剂如一些类型的牛肉提取物（有机floccuLATION）或脱脂奶絮凝9-12从过滤器表面除去病毒颗粒。最近，利用再加上另外硅藻土（细颗粒）的牛肉膏另一二次浓度过程已表明承诺回收腺病毒，肠道病毒，诺如病毒和8,13,14。根据相似的原理，以在该病毒颗粒中的有机絮凝方法的硅藻土的浓度的作品附加到并通过改变悬浮液的pH释放从粒子（絮凝物或硅藻土）。这两个次级浓缩技术之间的比较中尖刺腺病毒（ADV）类型40和41 8的恢复已被评估。该研究的结论是，两个次级浓缩技术人腺病毒的回收统计学相似。然而，有机絮凝方法需要30分钟。孵育在pH 3.5，而硅藻土技术需要一个较短的温育（10分钟），在pH 4.0。有机flocculat离子也需要使用昂贵的实验室设备（离心机）叔浓缩过程中收集的絮凝物颗粒，与此相反的硅藻土技术仅使用基本的实验室设备（真空过滤），以硅藻土颗粒从悬浮液中分离出来。 过滤器和二次洗脱技术的某些组合也可影响病毒的回收率。一项研究的结论是，初级（正电的过滤器）的某些组合和次级浓缩技术（硅藻土或有机絮凝）的 ​​腺病毒13的回收的显著影响。这些结果表明，优化使用这些技术时，需要以最佳地从给定的水基质回收靶病毒。优化是一个耗时的，艰苦的过程许多研究者积极避免，因为无数的变量进行评估（过滤器类型/品牌，pH值洗脱液，硅藻土/有机絮凝）。 对于这种Study，一个步骤，开发了使用加标人腺病毒株40和41可以推测，以确定病毒浓度的最佳条件，从水，由于每个病毒类型显示了独特的衣壳形态和特定的衣壳电荷浓度的协议可能需要对每个病毒进行优化目标，以达到最佳的病毒回收。本研究为腺病毒40和41浓度的方法:1）评估病毒回收率自来水使用阳电过滤盘之后2）建立有机絮凝法对硅藻土技术作为辅助的浓度评价，以及3）评估洗脱缓冲液为大专浓度。

<table><tbody><tr><td>Adenovirus 40 stock</td><td>ATCC</td><td>VR-931</td><td></td></tr><tr><td>Adenovirus 41 stock</td><td>ATCC</td><td>VR-930</td><td></td></tr><tr><td>Sodium Thiosulfate</td><td>Fluka Chemical Co.</td><td>72051</td><td></td></tr><tr><td>Celites #577</td><td>Fluka Chemical Co.</td><td>22142</td><td></td></tr><tr><td>NanoCeram 47 mm</td><td>Argonide</td><td>N/A</td><td></td></tr><tr><td>1MDS 47 mm</td><td>3M</td><td>6408502</td><td></td></tr><tr><td>AP-20 Prefilter 47 mm</td><td>Millipore Corp.</td><td>AP2004700</td><td></td></tr><tr><td>Glycine&amp;nbsp;</td><td>Sigma</td><td>50046-1KG</td><td></td></tr><tr><td>Sodium Polyphosphate</td><td>Acros Organics</td><td>390930010</td><td></td></tr><tr><td>Trypsin</td><td>Gibco</td><td>25200</td><td></td></tr><tr><td>PBS</td><td>Sigma</td><td>P5368</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher</td><td>A481-212</td><td></td></tr><tr><td>BBL Beef Extract</td><td>BD Biosciences</td><td>212303</td><td></td></tr><tr><td>Difco Beef Extract</td><td>BD Biosciences</td><td>211520</td><td></td></tr><tr><td>ABI 7900 Real-time PCR system</td><td>ABI</td><td>N/A</td><td></td></tr><tr><td>Stainless Steel Filter Housing</td><td>Millipore Corp.</td><td>XX2004720</td><td></td></tr><tr><td>Blood DNA Extraction Kit</td><td>Qiagen&amp;nbsp;</td><td>51104</td><td></td></tr><tr><td>EPA MPN Calculator</td><td></td><td></td><td>http://www.epa.gov/nerlcwww/online.html</td></tr></tbody></table>

a small volume procedure for viral concentration from water

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to identify optimal parameters for virus concentration. Viruses can be concentrated from water using filtration (electropositive, electronegative, glass wool or size exclusion), followed by secondary concentration with beef extract to release viruses from filter surfaces, and finally tertiary concentration resulting in a 5-30 ml volume virus concentrate. In order to identify optimal concentration procedures, two different electropositive filters were evaluated (a glass/cellulose filter [1MDS] and a nano-alumina/glass filter [NanoCeram]), as well as different secondary concentration techniques; the celite technique where three different celite particle sizes were evaluated (fine, medium and large) followed by comparing this technique with that of the established organic flocculation method. Various elution additives were also evaluated for their ability to enhance the release of adenovirus (AdV) particles from filter surfaces. Fine particle celite recovered similar levels of AdV40 and 41 to that of the established organic flocculation method when viral spikes were added during secondary concentration. The glass/cellulose filter recovered higher levels of both, AdV40 and 41, compared to that of a nano-alumina/glass fiber filter. Although not statistically significant, the addition of 0.1% sodium polyphosphate amended beef extract eluant recovered 10% more AdV particles compared to unamended beef extract.

<html>硅藻土选择三种不同类型的硅藻土进行之前表现最好的变体的选择进行测试。 Celites细到中等大小的粒子产生的最高腺病毒回收率。使用较大celites导致较低的回收率为AdV40和41（范围为32％-100％）（ 图1）。腺病毒4​​0的平均回收率为144％±52％（精细），115％±28％（中）和82％±53％（大）颗粒celites和AdV41，132％±39％（精细），83％±的25％（培养基）和50％±1...

Watch this Scientific Journal Video about A Small Volume Procedure for Viral Concentration from Water at JoVE.com

A Small Volume Procedure for Viral Concentration from Water

An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation.

小体积的方法从水中病毒浓度

Small-scale concentration of viruses (sample volumes 1-10 L, here simulated with spiked 100 ml water samples) is an efficient, cost-effective way to ...

a-small-volume-procedure-for-viral-concentration-from-water

Research

JoVE Journal

Environment

10.0K 次观看.  U.S. EPA. An approach was developed for identifying optimal viral concentration conditions for small volume water samples using spikes of human adenovirus. The techniques described here are used to identify concentration parameters for other viral targets, and applied to large-scale viral concentration experimentation. 

视频: A Small Volume Procedure for Viral Concentration from Water

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

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 ...

科研

免疫与感染

Unraveling the Unseen Players in the Ocean - A Field Guide to Water Chemistry and Marine Microbiology

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.

Here, we present a comprehensive protocol to assess the organic and inorganic nutrient availability and the abundance and structure of microbial and viral communities in remote marine environments.

揭开看不见的玩家在海洋 - 一个领域指南水化学和海洋微生物

Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling ...

环境科学

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. II. Total Culturable Virus Assay

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. The U.S Environmental Protection Agency&#8217;s (USEPA) Method 1615 was developed with the goal of providing such a standard for measuring Enterovirus and Norovirus in these waters. Virus is concentrated from water using an electropositive filter, eluted from the filter surface with beef extract, and then concentrated further using organic flocculation. Herein we present the protocol from Method 1615 for filter elution, secondary concentration, and measurement of total culturable viruses. A portion of the concentrated eluate from each sample is inoculated onto ten replicate flasks of Buffalo Green Monkey kidney cells. The number of flasks demonstrating cytopathic effects is used to quantify the most probable number (MPN) of infectious units per liter. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. Laboratories must meet defined performance standards. Method 1615 was evaluated by examining virus recovery from reagent-grade and ground waters seeded with Sabin poliovirus type 3. Mean poliovirus recoveries with the total culturable assay were 111% in reagent grade water and 58% in groundwaters.

Here we present a procedure to measure total culturable viruses using the Buffalo Green Monkey kidney cell line. The procedure provides a standardized tool for measuring the occurrence of infectious viruses in environmental and drinking waters.

EPA方法1615年测量和肠道病毒水中诺如病毒发生由文化部和RT-qPCR的的。 II。总可培养病毒试验

A standardized method is required when national studies on virus occurrence in environmental and drinking waters utilize multiple analytical laboratories. ...

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA方法1615测量肠道病毒和诺瓦克病毒发生水由文化和RT-qPCR的了。第三部分。病毒检测用RT-qPCR的

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

微生物源使用特定的人类和动物病毒的追踪成本效益的方法

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

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

Glass Wool Filters for Concentrating Waterborne Viruses and Agricultural Zoonotic Pathogens

The key first step in evaluating pathogen levels in suspected contaminated water is concentration. Concentration methods tend to be specific for a particular pathogen group, for example US Environmental Protection Agency Method 1623 for Giardia and Cryptosporidium1, which means multiple methods are required if the sampling program is targeting more than one pathogen group. Another drawback of current methods is the equipment can be complicated and expensive, for example the VIRADEL method with the 1MDS cartridge filter for concentrating viruses2. In this article we describe how to construct glass wool filters for concentrating waterborne pathogens. After filter elution, the concentrate is amenable to a second concentration step, such as centrifugation, followed by pathogen detection and enumeration by cultural or molecular methods. The filters have several advantages. Construction is easy and the filters can be built to any size for meeting specific sampling requirements. The filter parts are inexpensive, making it possible to collect a large number of samples without severely impacting a project budget. Large sample volumes (100s to 1,000s L) can be concentrated depending on the rate of clogging from sample turbidity. The filters are highly portable and with minimal equipment, such as a pump and flow meter, they can be implemented in the field for sampling finished drinking water, surface water, groundwater, and agricultural runoff. Lastly, glass wool filtration is effective for concentrating a variety of pathogen types so only one method is necessary. Here we report on filter effectiveness in concentrating waterborne human enterovirus, Salmonella enterica, Cryptosporidium parvum, and avian influenza virus.

Glass wool filters have been used to concentrate waterborne viruses by a number of research groups around the world. Here we show a simple approach for constructing glass wool filters and demonstrate the filters are also effective in concentrating waterborne viral, bacterial and protozoan pathogens.

为集中的的水性病毒和农业人畜共患病病原体的玻璃纤维过滤器

The key first step in evaluating pathogen levels in suspected contaminated water is concentration.  Concentration methods tend to be specific for a ...

Concentration of Virus Particles from Environmental Water and Wastewater Samples Using Skimmed Milk Flocculation and Ultrafiltration

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The recovery of microbial fractions, including viruses, bacteria, and microeukaryotes from wastewater and environmental water samples&#160;is one of the challenging steps in these approaches. In this study, we focused on the recovery efficiency of sequential ultrafiltration and skimmed milk flocculation (SMF) methods using Armored RNA as a test virus, which is also used as a control by some other studies. Prefiltration with 0.45 &#181;m and 0.2 &#181;m membrane disc filters were applied to eliminate solid particles before ultrafiltration to prevent the clogging of ultrafiltration devices. Test samples, processed with the sequential ultrafiltration method, were centrifuged at two different speeds. An increased speed resulted in lower recovery and positivity rates of Armored RNA.&#160;On the other hand, SMF resulted in relatively consistent recovery and positivity rates of Armored RNA. Additional tests conducted with environmental water samples demonstrated the utility of SMF to concentrate other microbial fractions. The partitioning of viruses into solid particles might have an impact on the overall recovery rates, considering the prefiltration step applied before the ultrafiltration of wastewater samples. SMF with prefiltration performed better when applied to environmental water samples&#160;due to lower solid concentrations in the samples and thus lower partitioning rates to solids. In the present study, the idea of using a sequential ultrafiltration method arose from the necessity to decrease the final volume of the viral concentrates during the COVID-19 pandemic, when the supply of the commonly used ultrafiltration devices was limited, and there was a need for the development of alternative viral concentration methods.

Virus concentration from environmental water and wastewater samples is a challenging task, carried out primarily for the identification and quantification of viruses. While several virus concentration methods have been developed and tested, we demonstrate here the effectiveness of ultrafiltration and skimmed milk flocculation for RNA viruses with different sample types.

使用脱脂牛奶絮凝和超滤法浓缩环境水和废水样品中的病毒颗粒

Water and wastewater-based epidemiology have emerged as alternative methods to monitor and predict the course of outbreaks in communities.&#160;The ...

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

慢病毒生产

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

生物学

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant viruses may be potentially pathogenic for many species of protists. They belong to the recently described order of Megavirales. The new lineage Faustovirus that has been isolated from sewage samples is distantly related to the mammalian pathogen African swine fever virus. This virus is also specific to its amoebal host, Vermamoeba vermiformis, a protist common in health care water systems. It is crucial to continue isolating new Faustovirus genotypes in order to enlarge its genotype collection and study its pan-genome. We developed new strategies for the isolation of additional strains by improving the use of antibiotic and antifungal combinations in order to avoid bacterial and fungal contaminations of the amoeba co-culture and favoring the virus multiplication. We also implemented a new starvation medium to maintain V. vermiformis in optimal conditions for viruses co-culture. Finally, we used flow cytometry rather than microscopic observation, which is time-consuming, to detect the cytopathogenic effect. We obtained two isolates from sewage samples, proving the efficiency of this method and thus widening the collection of Faustoviruses, to better understand their environment, host specificity and genetic content.

A Rapid Strategy for the Isolation of New Faustoviruses from Environmental Samples Using Vermamoeba vermiformis

We describe here the latest advances in viral isolation for the characterization of new genotypes of Faustovirus, a new asfarvirus-related lineage of giant viruses. This protocol can be applied to the high throughput isolation of viruses, especially giant viruses infecting amoeba.

一种快速战略新Faustoviruses从环境样品使用隔离<em&gt; Vermamoeba vermiformis</em

The isolation of giant viruses is of great interest in this new era of virology, especially since these giant viruses are related to protists. Giant ...