部分支持这项工作是由西班牙政府"部教育Ÿ Ciencia"（项目AGL2008-05275-C01/ALI），由欧盟研究框架7 VIROBATHE资助项目（合同号513648），VIROCLIME（合同号243923 ）和水的加泰罗尼亚机构，通讯社Catalana艾瓜（ACA），Departament去控制我Millora dels Ecosistemes游泳。在研究开发，玛塔Rusiñol是加泰罗尼亚政府"AGAUR"（FI - DGR）的研究员。

1。病毒颗粒的浓度水样中<ol><li>水样的采集和空调<ol><li>收集最低为2 10％的样本大​​号平底塑料容器和过程控制作为一个额外的样品复制。这最后的样本将会被添加与已知的病毒颗粒量，并作为控制使用。 注意:建议有特殊独立的材料加标样品（瓶，管件，等）。 </li><li>检查校准conductimeter，如果有必要重新调整。准备一个额外的塑料10升容器中使用以前调整到足够的导电性（见下文1.6）的自来水阴性对照。 </li><li>加标样品:样品添加量控制病毒的标准（每10升水的基因组约10份）。混合搅拌避免喷溅和气溶胶。阳性对照可包括在腺病毒小号少见株UCH作为HAdV - 35或噬菌体MS2等。 </li><li>如果样品呈现高量的悬浮物（沙或其他材料），让泥沙为15分钟。转移到一个新的容器中的水。 </li><li>水样的pH值调整为3.5（± 0.1）1 N盐酸。病毒的浓度，这一步很重要，所以一定要确保pH值已适当调整。清水彻底剧烈搅拌混合，同时加入的HCl。 （注:如果pH值超过3.5添加1 M氢氧化钠）。 </li><li>调整的电导率。如果样品电导率1500μS/ cm或更高，这​​一步是没有必要的。如果电导率超过1500μS/厘米形成的絮凝物质（絮状物）不能保证较低，所以调整到1500μS/ cm的电导率此外人工海盐（Sigma公司）。大力搅拌，同时加入海盐混合。 </li><li>之前和之后，空调以及一个记录样品的pH值S的盐酸量使用。电导率也应调整pH值后记录。始终消毒用新鲜的盐酸溶液的pH计和conductimeter电极，脱氯10％的钠tiosulphate溶液，最后用蒸馏水冲洗。 </li></ol></li><li>絮凝前1％的脱脂奶粉的制备系统（PSM） <ol><li>检查校准pH计和conductimeter，如果有必要重新调整。 </li><li>准备在1升人工海水溶解10克脱脂奶粉（DIFCO）絮凝预脱脂奶粉溶液（1％的PSM，W / V）（33.3克的人工海盐溶解在1 L脱氯自来水和高压灭菌）和仔细调节pH至3.5 1N盐酸。絮体应该是可见的的。准备24小时的解决方案之前，使用或储存于4 ° C脱氯使用1毫升的10％，每100毫升水tiosulphate溶液。 </li></ol></li><li>目前在水样病毒颗粒的絮凝<ol><li>添加1％的PSM 100毫升到10 L的水样。 </li><li>搅拌8-10 h的样本，以使病毒吸附到的絮体。使用定时关机搅拌后8-10小时。 </li><li>停止搅拌，让絮体沉积物的比重为8-10小时</li></ol></li><li>收集和重新溶解的絮体。离心<ol><li>使用蠕动泵和塑料管连接到一个塑料吸管去除上清。应加标样品上清收集到一个瓶子，根据内部程序和消毒。在所有情况下照顾不收集沉淀。 </li><li>收集成离心瓶（约500毫升）的絮体的泥沙。 </li><li>平衡此外PSM的pH值3.5盆。 </li><li>离心机在高速离心机的盆，在30分钟8000 XG 4 ° C。只要停止离心机，小心地取出离心机离心机盆。 </li><li>很克ently倒出上清液。按照传染性物质的适当措施。 </li><li>添加7毫升磷酸盐缓冲液溶解沉淀在每个离心瓶。 </li><li>絮体已被解散后，测量和添加磷酸盐缓冲液总体积达到10毫升。 </li><li>均质涡旋病毒集中和分配到10毫升干净的微管，应冻结在-80℃直至需要在进一步的分析。 </li></ol></li></ol> 2。核酸提取<ol><li>执行与QIAamp病毒RNA试剂盒制造商的说明的核酸提取。该套件使一个自动化平台的使用（如Qiacube，QIAGEN）。 </li></ol> 3。人类腺病毒（HAdV），JC多瘤病毒（JCPyV），猪腺病毒（反对家庭暴力伙伴关系）和牛多瘤病毒定量PCR（BPyV） <ol><li>定量样品中的基因组副本<ol><li>准备使用TaqMan环境PCR扩增预混试剂2X（Applied Biosystems公司）的qPCR在一个干净的独立的区域组合。反应在96孔光学反应板与光学胶盖覆盖的地方。表1描述了主结构的浓度，引物和探针。 </li><li>一旦组合已经做好准备，分装15μl，包括控制（见3.2）。将一个目标后，除了反应总体积25μl（15μl混合+加入10μl样品或标准）。 </li><li>添加核酸提取的样品在一个单独的区域（10μL）。运行直接和十倍稀释重复每个样品纯化水。盖井样品含有粘合剂盖的一部分，继续以下步骤盖的其他部分。 </li><li>从10 0 10 6一式三份GC/10μl添加的DNA标准悬浮液（10μL）的稀释和使用专为斯坦一个微量准的DNA。明智的做法是添加标准配备销毁质粒DNA，每次使用后清洁微量的紫外线辐射面积。含标准的预切胶盖，盖井。 注意:要准备用于在基因组复制的量化标准悬浮液，应克隆到质粒和线性目标DNA区域。在下面的地址，你会发现对如何建立标准曲线在qPCR的质粒DNA模板的详细过程: <a href="http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf">http://www.appliedbiosystems.com/support/tutorials/pdf/quant_pcr.pdf</a> </li><li>执行到一个适当的系统选择适当的参数（考虑使用粘合剂的封面和总量在每口井等）的qPCR。以下激活AmpliTaq黄金10分钟在95℃，40个扩增循环执行如下:15在95 ° C小号和HAdV，JCPyV BPyV，15日95章第1分钟在60 ° C ° C，20岁，在55 ° C和为反对家庭暴力伙伴关系在60 20岁°彗星。 </li><li>一旦反应完成后，用于存储数据，并在用户的设备手册所述的结果。 DNA的金额将被定义为纠正稀释倍数后，需要时获得的数据的中位数。 </li></ol></li><li>控制<ol><li>使用阳性和阴性对照。检测必须包括一个以上的非模板对照（NTC）证明混合不产生荧光。明智的做法是执行积极的过程控制，以评估潜在的酶抑制由于抑制剂目前在研究的样本。 </li><li>两个不同稀释度的标准DNA和过程控制的qPCR实验记录结果。使用结果的灵敏度和检测效率相关的质量控制（QC）计划，以备控制图。 </li></ol></li><li>确认结果<ol><li>积极的结果可能会进一步证实，采用巢式PCR扩增和核苷酸测序，生产株的核苷酸序列的额外数据检测1,5,6,7,9,12。 </li></ol></li></ol>所述的程序之后，人类和动物病毒检测和洗浴水，海水和河水2,3量化。作为一个代表性的例子，提出高水平的硝酸盐地区地下水样品进行了评估，以确定污染的来源。十公升水样收集了来自4个不同井在西班牙东北部地区的农村。五复制被收集在每个福祉用于过程控制的人类腺病毒2号种子的复制。根据图1所代表的协议处理样品。四个复制分析研究的4个地点之一，表明反对家庭暴力伙伴关系的积极结果（平均值7.74 × 2 GC / L），这将是相关的采样点周围地区的猪泥浆和支持粪猪污染的地下水中硝酸盐（见表2）的来源。 4。代表性的成果: <img src="/files/ftp_upload/2820/2820fig1.jpg" alt="图1" /> 图1:在水中病毒的检测和量化的过程。 <img src="/files/ftp_upload/2820/2820table1.jpg" alt="表1" /> qPCR实验的引物和探针的浓度见表 1 。。 <img src="/files/ftp_upload/2820/2820table3.jpg" alt="表3" /> 表2:动物和人类腺病毒和地下水样品中的多瘤病毒的检测和量化。 N多的复制分析 积极％的比例复制 （ - ）非检测

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两项专利申请量在2009年，以保护知识产权的协议，为反对家庭暴力伙伴关系和BPyV量化。

<html>描述的步骤将履行为日常的环境和公众健康实验室的拟合方法的条件是:重现性好，质量可靠，简单和成本效益。该协议是简单的，但它必须严格遵守。低电导率样品中没有添加人工海水盐的要求的浓度将大大降低病毒的复苏将的情况下，如果搅拌时间对絮凝显着降低（例如少于5小时）。 目前应用的MST工具一般都是基于分子生物学技术。由不同的群体开发的研究显示出，目前使用的参数（即细菌基因）没有具体需要。因此，使用这些参数的组合已建议作为最佳逼近的粪便污染水体的起源8,11的有效跟踪。 jove_content"&gt;近年来，在许多情况下会产生临床症状的情况下持续感染所选择的DNA病毒的研究，又出现了一个源跟踪粪便污染环境的方法。定量PCR技术和浓度的方法这些病毒的浓度和非常有价值的数据的风​​险评估研究和整治行动的发展提供可靠的值，这些技术也高度敏感和特异的的，这是一个用于跟踪污染源的绝对要求，此外，他们将代大型数据库，建议为微生物源追踪工具在不同地理区域的特定病毒的传播和排泄的定量表征是一个非常有用的工具。 描述的过程允许多个样品的分析，同时在约48小时，没有劳动密集型的要求第一个高效低成本的浓度方法的可用性支持定量微生物源追踪研究和地下水中污染物的来源识别的成本效益分析HAdV和JCPyV，反对家庭暴力伙伴关系和BPyV在水中的适用性。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>高速离心机（8000 XG） </td><td> Berckman库尔特</td><td>阿凡提J - 20XP </td><td></td></tr><tr><td> pH计，温度计和conductimeter </td><td> Afora </td><td> LPPC3003 </td><td></td></tr><tr><td>塑料管长100-200厘米</td><td> Deltalab </td><td> 350059 </td><td></td></tr><tr><td>无菌毕业于一次性移液器</td><td> Labclinics </td><td> PN10E1 </td><td></td></tr><tr><td> 1.5和10-15毫升（Eppendorf公司，猎鹰等）的无菌塑料管</td><td> Afora </td><td> KA298/00 </td><td></td></tr><tr><td>离心机壶（500毫升） </td><td> Fisher Scientific则</td><td> SE5753512 </td><td></td></tr><tr><td>磁力搅拌器和磁铁（每样本） </td><td> Fisher Scientific则</td><td> 10510 </td><td></td></tr><tr><td>平底的玻璃或塑料容器，允许使用磁力搅拌器</td><td> Deltalab </td><td> 191642 </td><td></td></tr><tr><td>一个蠕动泵取出上清液（或水喷射真空泵） </td><td>沃森的马洛</td><td> 323E / D转换</td><td></td></tr><tr><td>定时关机搅拌后8-10小时</td><td> Deltalab </td><td> 900400 </td><td></td></tr><tr><td>盐酸（1N和0.1N） </td><td> Panreac </td><td> 141020.1611 </td><td></td></tr><tr><td>氢氧化钠（1N） </td><td> Panreac </td><td> 131687.1211 </td><td></td></tr><tr><td>人工海水的海盐</td><td>西格玛</td><td> S9883 </td><td></td></tr><tr><td>脱脂牛奶（SM） </td><td> DIFCO </td><td> 232100 </td><td></td></tr><tr><td>磷酸盐缓冲液pH值75 </td><td></td><td></td><td> 1:2 V / V无菌娜2 HPO 4 0,2中号的NaH 2 PO 4 0.2，在pH 7.5中号</td></tr><tr><td>硫代</td><td> Panreac </td><td> 121879.1209 </td><td>请在水中10％的解决方案</td></tr><tr><td> QIAamp病毒RNA试剂盒</td><td> Qiagen公司</td><td> 52904 </td><td></td></tr><tr><td> 96孔光学反应板（500个单位） </td><td>应用生物系统公司</td><td> 43426659 </td><td></td></tr><tr><td>光学粘合剂覆盖（100个单位） </td><td>应用生物系统公司</td><td> 4311971 </td><td></td></tr><tr><td> TaqMan探针环境PCR扩增预混试剂2X </td><td>应用生物系统公司</td><td> 4396838 </td><td></td></tr></tbody></table>

cost-effective method for microbial source tracking using specific human and animal viruses

微生物污染的环境，代表着一个重大的健康风险。古典细菌的粪便指标显示有显著的局限性，病毒更耐许多失活过程和标准的粪便指标不告知的污染源。成本效益的方法为水和分子检测病毒浓度的发展，有利于病毒的适用性作为粪便污染的指标，并作为微生物源追踪（MST）工具。腺病毒，多瘤病毒是DNA病毒感染具体包括人类在内的脊椎动物物种，并坚持在粪便和/或研究所有地区的尿液排出体外。在以往的研究中，我们建议人类腺病毒（HAdV）和JC多瘤病毒定量PCR（qPCR）（JCPyV）作为人类粪便污染指数的量化。最近，我们已经制定了具体量化的POR qPCR实验电影的腺病毒（反对家庭暴力伙伴关系）和牛多瘤病毒（BPyV）每1-10试管基因组副本的敏感动物粪便污染的标记。在这项研究中，我们目前所应遵循的程序，以确定在使用这些工具的水样本的污染来源。作为代表性的结果为例，提出高水平的硝酸盐在地下水的病毒分析显示。 低或中度污染水域的病毒检测需要从几升的水至少到一个更小的体积，一个过程，通常包括两个系列的浓缩步骤的病毒浓度。这有些繁琐的程序，观察病毒回收率的变异显著妨碍同时处理大量的水样。 为了消除由两个步骤的程序所造成的瓶颈，我们已经申请了一个一步到位的协议，在以前的studieS和适用于水基质的多样性。该程序包括:10升的水样酸化，絮凝脱脂奶，重力沉淀絮凝材料，沉淀，离心收集，在磷酸盐缓冲液10毫升沉淀再悬浮。病毒集中用于提取病毒核酸和具体利益的腺病毒和多瘤病毒是由qPCR的量化。使用这种低成本的浓度的方法，可同时分析大量样品。 该程序已应用于洗浴水，海水和河水的分析，在这项研究中，我们目前的地下水样品分析的结果。这种高通量的定量方法是可靠的，简单，成本效益。

Watch this Scientific Journal Video about Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses at JoVE.com

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

这项研究描述为粪/尿污染或使用人/猪/牛的DNA病毒，腺病毒和提出的MST工具多瘤病毒，具体量化的qPCR在水中的硝酸盐污染的来源识别成本效益的方法。

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 ...

cost-effective-method-for-microbial-source-tracking-using-specific

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Statistics

Immunology and Infection

Unit 1

Control Charts

SCIENTISTS IN ACTION

12.1K 次观看.  University of Barcelona. 这项研究描述为粪/尿污染或使用人/猪/牛的DNA病毒，腺病毒和提出的MST工具多瘤病毒，具体量化的qPCR在水中的硝酸盐污染的来源识别成本效益的方法。

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

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

科研

免疫与感染

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing &gt; 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Anti-retroviral therapy to treat HIV/AIDS is monitored in South Africa on a large scale. Flow cytometry is combined for haematology (CD45), immunology (CD4) and viral-load linked CD38 assay. Recorded at NHLS-SA laboratories, Johannesburg, these modern methods are cost-efficient with heightened local internal quality control, serving as role-models for resource-limited diagnostics.

艾滋病毒/艾滋病的综合成本有效的实验室监测:非洲的角色模型

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

用荧光素酶在小鼠细菌性感染的形象

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

S. aureus is a pathogenic bacterium that requires iron to carry out vital metabolic functions and cause disease. The most abundant reservoir of iron inside the human host is heme, which is the cofactor of hemoglobin. To acquire iron from hemoglobin, S. aureus utilizes an elaborate system known as the iron-regulated surface determinant (Isd) system1. Components of the Isd system first bind host hemoglobin, then extract and import heme, and finally liberate iron from heme in the bacterial cytoplasm2,3. This pathway has been dissected through numerous in vitro studies4-9. Further, the contribution of the Isd system to infection has been repeatedly demonstrated in mouse models8,10-14. Establishing the contribution of the Isd system to hemoglobin-derived iron acquisition and growth has proven to be more challenging. Growth assays using hemoglobin as a sole iron source are complicated by the instability of commercially available hemoglobin, contaminating free iron in the growth medium, and toxicity associated with iron chelators. Here we present a method that overcomes these limitations. High quality hemoglobin is prepared from fresh blood and is stored in liquid nitrogen. Purified hemoglobin is supplemented into iron-deplete medium mimicking the iron-poor environment encountered by pathogens inside the vertebrate host. By starving S. aureus of free iron and supplementing with a minimally manipulated form of hemoglobin we induce growth in a manner that is entirely dependent on the ability to bind hemoglobin, extract heme, pass heme through the bacterial cell envelope and degrade heme in the cytoplasm. This assay will be useful for researchers seeking to elucidate the mechanisms of hemoglobin-/heme-derived iron acquisition in S. aureus and possibly other bacterial pathogens.

Staphylococcus aureus Growth using Human Hemoglobin as an Iron Source

Here we describe a growth assay for Staphylococcus aureus using hemoglobin as the sole source of available nutrient iron. This assay establishes the role of bacterial factors involved in hemoglobin-derived iron acquisition.

金黄色葡萄球菌生长使用人血红蛋白作为铁源

S. aureus is a pathogenic bacterium that requires iron to  carry out vital metabolic functions and cause disease. The most abundant  reservoir of iron ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

细菌人工染色体:对于正链RNA病毒研究的功能基因组学工具

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Measuring Influenza Neutralizing Antibody Responses to A(H3N2) Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for influenza infections. Microneutralization (MN) assays are often used to measure neutralizing antibody responses in human sera after influenza vaccination or infection. Madine Darby Canine Kidney (MDCK) cells are the commonly used cell substrate for MN assays. However, currently circulating 3C.2a and 3C.3a A(H3N2) influenza viruses have acquired altered receptor binding specificity. The MDCK-SIAT1 cell line with increased &#945;-2,6 sialic galactose moieties on the surface has proven to provide improved infectivity and more faithful replications than conventional MDCK cells for these contemporary A(H3N2) viruses. Here, we describe a MN assay using MDCK-SIAT1 cells that has been optimized to quantify neutralizing antibody titers to these contemporary A(H3N2) viruses. In this protocol, heat inactivated sera containing neutralizing antibodies are first serially diluted, then incubated with 100 TCID50/well of influenza A(H3N2) viruses to allow antibodies in the sera to bind to the viruses. MDCK-SIAT1 cells are then added to the virus-antibody mixture, and incubated for 18 - 20 h at 37 &#176;C, 5% CO2 to allow A(H3N2) viruses to infect MDCK-SIAT1 cells. After overnight incubation, plates are fixed and the amount of virus in each well is quantified by an enzyme-linked immunosorbent assay (ELISA) using anti-influenza A nucleoprotein (NP) monoclonal antibodies. Neutralizing antibody titer is defined as the reciprocal of the highest serum dilution that provides &#8805;50% inhibition of virus infectivity.

Measuring Influenza Neutralizing Antibody Responses to AH3N2 Viruses in Human Sera by Microneutralization Assays Using MDCK-SIAT1 Cells

Influenza neutralizing antibodies correlate with protection of influenza infections. Microneutralization assays measure neutralizing antibodies in human sera and are often used for influenza human serology. We describe a microneutralization assay using MDCK-SIAT1 cells to measure neutralizing antibody titers to contemporary 3C.2a and 3C.3a A(H3N2) viruses following influenza vaccination or infection.

用 MDCK-SIAT1 细胞 Microneutralization 法测定人血清中中和 (H3N2) 病毒的流感中和抗体反应

Neutralizing antibodies against hemagglutinin (HA) of influenza viruses are considered the main immune mechanism that correlates with protection for ...

VirWaTest, A Point-of-Use Method for the Detection of Viruses in Water Samples

Viruses excreted by humans and animals may contaminate water sources and pose a risk to human health when this water is used for drinking, food irrigation, washing, etc. The classical fecal bacteria indicator does not always check for the presence of viral pathogens so the detection of viral pathogens and viral indicators is relevant in order to adopt measures of risk mitigation, especially in humanitarian scenarios and in areas where water-borne viral outbreaks are frequent.
At present, several commercial tests allowing the quantification of fecal indicator bacteria (FIB) are available for testing at the point of use. However, such commercial tests are not available for the detection of viruses. The detection of viruses in environmental water samples requires concentrating several liters into smaller volumes. Moreover, once concentrated, the detection of viruses relies on methods such as nucleic acid extraction and molecular detection (e.g., polymerase chain reaction [PCR]-based assays) of the viral genomes.
The method described here allows the concentration of viruses from 10 L water samples, as well as the extraction of viral nucleic acids at the point of use, with simple and portable equipment. This allows the testing of water samples at the point of use for several viruses and is useful in humanitarian scenarios, as well as at any context where an equipped laboratory is not available. Alternatively, the method allows concentrating viruses present in water samples and the shipping of the concentrate to a laboratory at room temperature for further analysis.

Here we present VirWaTest, which is a simple, affordable and portable method for the concentration and detection of viruses from water samples at the point of use.

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

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