We thank the Clinical Microbiology Service staff of the Memorial Sloan-Kettering Cancer Center for help in collecting clinical specimens. This study was supported in part by a research agreement between MSKCC and Alere Scarborough (SK2013-0262).

道德守则:使用遗留临床标本被批准，并遵循纪念斯隆 - 凯特琳癌症中心伦理审查委员会的指导方针。 1.在运行分析注:甲型和乙型流感检测已被批准用于鼻咽拭子标本和存储在病毒的传输介质鼻咽拭子。拭子被包括在试剂盒中，并应被用于最佳性能。然而，人造丝，泡沫，植绒拭子或聚酯鼻拭子也可用于收集鼻拭子样品。 <ol><li>收集鼻拭子样本，将拭子插入具有最明显的排水或者说是最拥挤的鼻孔。轻轻旋转，推拭子插入鼻孔，直至遇到阻力，并从鼻孔慢慢取出之前将针对鼻壁几次。存放拭子和装有3毫升病毒的传输介质（VTM）的小瓶运输他们。 注意:仔细注意appropr莱特标本采集必须被视为标本采集不足可能会产生错误的结果。 注意:拭子标本采集后尽早进行测试。然而，它们可在室温下保存长达2小时或在2-8℃下进行长达24小时的冷冻如果测试不是立即可用。新鲜采集的标本是理想的最佳测试性能。不当的样品处理，储存和/或运输可能产生错误的结果。 注:作为预防措施，执行此测试人员应该把所有的标本都当成潜在通过处理标本时注意以下事项普遍预防感染。 </li><li>将所有样品至室温测试之前。 注:所有的测试部件仅用于单次使用并且不应当被用来执行多个测试。不要混用不同批号试剂盒成分。不要使用试剂盒过去的到期日期。 </li><li>把蓝色样品接收器ROO测试前m温度。橙色测试碱可以无需加热到室温进行试验。 </li><li>留在箔包裹所有试验片直到即将使用前。通过按下仪器侧面的电源键打开仪器。输入用户ID，然后按"确定"。 </li></ol> 2.运行测试<ol><li>开始测试过程中，触摸"运行测试"在仪器屏幕上。通过屏幕上的键盘或条形码扫描仪，然后按Enter病人ID"确定"。打开盖子，然后插入橙色试验基地到橙色试验基地持有者。 </li><li>确认正确的测试显示屏幕，按下"确定"。将蓝色样品接收器插入蓝色样品接收机持有者。 注:在仪器放置，因为这将防止到达正确的工作温度下的洗脱缓冲液并可能影响考试成绩之前，不要打开示例接收器。 </li&gt; <li>等待样品接收器预热。当仪器提示，去掉铝箔封口，然后将病人拭子被测试到样品接收器。 注:当删除箔，将两个手指放在样品接收器的边缘，以确保它留在地方。 </li><li>大力混在液体中的棉签，持续10秒，按压棉签头靠在一侧的样品接收器，你混吧，帮助逐出拭子标本。按"确定"继续。如果存储在病毒的传输介质测试拭子，涡VTM，持续10秒，然后加入200μl的样品接收器。 </li><li>按白转移盒插入蓝色的样品接收器。继续按在样品接收，直到橙色指示灯上升。 </li><li>抬起，然后转送盒连接到测试基地。观察橙色指示灯下降，一旦盒传输连接正确。盖上盖子，不开，直到"测试完成"屏幕上显示的消息。 </li></ol> 3.质量控制<ol><li>触摸"运行QC测试"在主屏幕上。选择QC测试中运行。确认测试类型通过触摸"OK"，然后按照屏幕上的提示完成测试，用于测试的QC样品不一致。 </li></ol> 4.结果解释<ol><li>试运行结束后，观察与解释流感A，B，未知亚型的出现在屏幕上显示的测试结果。对于无效结果重复测试。 </li></ol> 5.保养和清洁<ol><li>清洁仪器和周围长凳用70％乙醇或10％的漂白剂溶液每日区域。喷在湿润的无绒布擦拭清洗液。不要在仪器上直接倒入解决方案。 </li></ol>

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The authors have nothing to disclose.

流感病毒是发病率和死亡率的显著世界范围的原因。流感的快速和准确的诊断是主要键期间呼吸季节管理流感爆发中的一个。其他基于抗原的测试是快速和容易执行;然而，它们具有低的灵敏度13。另一方面，传统的分子测试具有改善的敏感性，但需要更多的有经验的化验师来执行，而且更昂贵。在这项研究中和协议中描述的流感A和B测定是流感A和B的CLIA-放弃，快速的分子测试的?...

流感病毒感染导致发病率和死亡率，每年1，2，3的显著量。单纯性流感的特点是宪法和呼吸道症状，如发热，肌痛，头痛和非生产性咳嗽4,5。老年人，幼儿，免疫功能低下患者，和患者的潜在合并症处于严重的并发症，如肺炎，心肌炎，中枢神经系统疾病，甚至导致死亡6,7风险较高。 流感感染是抗病毒治疗的症状出现后48小时内及时服用能减少疾病的严重程度和长度从8其他呼吸道病毒唯一的。流感的快速鉴定也已示出减少使用的不必要的抗生素9,10。另外，住院患者感染流感病毒必须放置在适当的感染控制预防措施隔离室。然而，所造成的非流感病毒呼吸道疾病可能难以从流感临床区分。出于这个原因，对于流感快速准确的诊断测试是用于临床患者管理非常重要。 数测定法可用于检测和流感病毒鉴定。快速流感抗原检测试验（RIDTs）被广泛应用于临床实践中作为点护理测试，因为它们易于使用，并提供在15至30分钟11，12的结果;然而，他们的敏感性有很大的不同取决于制造商和人口（10-80％）进行测试，并且流感类型和subtyPE 13，14，15。直接荧光测定（的DFA）提供超过RIDTs优异灵敏度，但处理时间大于（〜3小时），并且必须由熟练的技术人员16，17来完成。病毒培养一直是流感的诊断的金标准，并已改善了两个RIDTs和DFA的18灵敏度。然而，流感病毒培养可以从2-14ð任何地方来完成，减少在帮助病人管理19其效用。最后，核酸扩增试验（NAAT）已经取代培养技术在流感诊断新的金标准。 NAAT被认为具有在几个小时检测流感的最大灵敏度。然而，NAAT是最昂贵的测定和需要专门的设备和技术来执行5 <s了&gt;，20，21，22，23，24，25。 此处所描述的流感A和B的测定法是第一CLIA-放弃分子快速流感测试是容易获得的。此法的工作原理是采用使用特定流感引物，其次是目标检测与分子信标探针等温扩增核酸内切酶泥炭截短扩增反应（NEAR）。该测定区分流感A选自B，需要2分钟来设置和处理一个样品，并且总共需要15分钟来完成。 这里，我们目前的流感A＆B试验的协议。此外，我们还提供了一个样本数据集比较流感A对已归档的鼻咽拭子（NPS）的性能和B检测标本保存在病毒的传输介质（VTM）到其他呼吸道病原体面板检测。

<table><tbody><tr><td>Alere i Instrument</td><td>Alere</td><td>NAT-000 (Global), NAT-024 (US)</td><td></td></tr><tr><td>Alere i Influenza A &amp;amp; B 24 Test Kit</td><td>Alere</td><td>425-000 (Global), 425-024 (US)</td><td></td></tr><tr><td>Alere i Barcode Scanner</td><td>Alere</td><td>EQ001001</td><td></td></tr><tr><td>Alere Universal Printer</td><td>Alere</td><td>55115 (Global), alereiprinter&amp;nbsp;(US)</td><td></td></tr><tr><td>200 &amp;micro;l precision pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;l disposable pipette tips</td><td></td><td></td><td></td></tr><tr><td>Viral transport medium</td><td>Remel</td><td>M4-RT</td><td></td></tr></tbody></table>

rapid molecular detection and differentiation of influenza viruses a and b

流感是在人中由流感病毒引起A和B的传染性呼吸系统疾病，并导致每年发病率和死亡率的一个显著量。的甲型和乙型流感检测是第一个可用的CLIA-放弃分子快速流感测试。流感A和B的测试工作通过采用等温扩增与流感特异性引物，接着目标检测的分子信标探针。这里，A型流感的性能和B测定冷冻，归档鼻咽拭子（NPS）试样储存在病毒运输介质（VTM）进行比较，以呼吸面板测定。 流感A和B测定的性能通过的结果相比较，以呼吸面板参考方法进行评价。总甲型流感病毒的敏感性为67.5％（95％CI（CI），56.6-78.5），特异性为86.9％（CI，71.0-100）。对于乙型流感病毒的检测，其敏感性和特异性分别为90.2％（CI，68.5-100）和98.8％（CI，68.5-100）表示。 该系统具有的显著缩短测试时间比其他任何现有的分子检测及简单，无吸管程序运行的优势一个完全集成的，封闭的，小尺寸的系统。总体而言，流行性感冒A和B测定本研究评估具有作为一个点 - - 关心快速流感诊断测试的可能性。

在这项研究中，归档NPS标本采自2013年NPS标本在3毫升住院患者提交2012年12月15日和3月1日之间的流感爆发期间与纪念斯隆 - 凯特琳癌症中心（MSKCC）流感样症状的收集VTM，并与检测呼吸道病毒（RP），其中包括流行性感冒A，A-1，A-3，和B小组在研究期间的分子检测常规的临床实践的一部分进行测试，3675 ​​NPS标本提交到MSKCC临床实验室进行检测。这些标本，45，425，37，77的药...

Watch this Scientific Journal Video about Rapid Molecular Detection and Differentiation of Influenza Viruses A and B at JoVE.com

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

我们描述了一个快速，分子为基础的甲型和乙型流感检测。流感检测采用等温扩增与流感特异性引物，其次是目标检测与分子信标探针检测15分钟内每一个目标。的甲型和乙型流感检测是用户友好的，并要求最少的动手时间来执行。

快速分子检测和流感病毒A和B的分化

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

rapid-molecular-detection-differentiation-influenza-viruses

Research

JoVE Journal

Immunology and Infection

15.7K 次观看.  Memorial Sloan Kettering Cancer Center. 我们描述了一个快速，分子为基础的甲型和乙型流感检测。流感检测采用等温扩增与流感特异性引物，其次是目标检测与分子信标探针检测15分钟内每一个目标。的甲型和乙型流感检测是用户友好的，并要求最少的动手时间来执行。 

视频: Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Generation of Recombinant Influenza Virus from Plasmid DNA

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

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

质粒DNA重组流感病毒的一代

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

科研

免疫与感染

ampliPHOX Colorimetric Detection on a DNA Microarray for Influenza

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5 For instance, many examples of the ability to type and subtype influenza virus have been demonstrated.6-11 The identification and subtyping of influenza on DNA microarrays has applications in both public health and the clinic for early detection, rapid intervention, and minimizing the impact of an influenza pandemic. Traditional fluorescence is currently the most commonly used microarray detection method. However, as microarray technology progresses towards clinical use,1 replacing expensive instrumentation with low cost detection technology exhibiting similar performance characteristics to fluorescence will make microarray assays more attractive and cost-effective.
The ampliPHOX colorimetric detection technology is intended for research applications, and has a limit of detection within one order of magnitude of traditional fluorescence11, with a main advantage being an approximate ten-fold lower instrument cost compared to the confocal microarray scanners required for fluorescence microarray detection. Another advantage is the compact size of the instrument which allows for portability and flexibility, unlike traditional fluorescence instruments. Because the polymerization technology is not as inherently linear as fluorescence detection, however, it is best suited for lower density microarray applications in which a yes/no answer for the presence of a certain sequence is desired, such as for pathogen detection arrays. Currently the maximum spot density compatible with ampliPHOX detection is &tilde;1800 spots/array. Because of the spot density limitations, higher density microarrays are not suitable for ampliPHOX detection.
Here, we present ampliPHOX colorimetric detection technology as a method of signal amplification on a low density microarray developed for the detection and characterization of influenza viruses (FluChip). Although this protocol uses the FluChip (a DNA microarray) as one specific application of ampliPHOX detection, any microarray incorporating biotinylated target can be labeled and detected in a similar manner. The microarray design and biotinylation of the target to be captured are the responsibility of the user. Once the biotinylated target has been captured on the array, ampliPHOX detection can be performed by first tagging the array with a streptavidin-label conjugate (ampliTAG). Upon light exposure using the ampliPHOX Reader instrument, polymerization of a monomer solution (ampliPHY) occurs only in regions containing ampliTAG-labeled targets. The polymer formed can be subsequently stained with a non-toxic solution to improve visual contrast, followed by imaging and analysis using a simple software package (ampliVIEW). The entire FluChip assay from un-extracted sample to result can be performed in about 6 hours, and the ampliPHOX detection steps described above can be completed in about 30 min.

ampliPHOX colorimetric detection technology is presented as an inexpensive alternative to fluorescence detection for microarrays. Based on photopolymerization, ampliPHOX produces solid polymer spots visible to the naked eye in just a few minutes. Results are then imaged and automatically interpreted with a simple yet powerful software package.

ampliPHOX比色法检测为流感的DNA芯片

DNA microarrays have emerged as a powerful tool for pathogen detection.1-5  For instance, many examples of the ability to type and subtype influenza virus ...

Quantitative Analyses of all Influenza Type A Viral Hemagglutinins and Neuraminidases using Universal Antibodies in Simple Slot Blot Assays

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle and the induction of protective immune responses1,2. As the main target for neutralizing antibodies, HA is currently used as the influenza vaccine potency marker and is measured by single radial immunodiffusion (SRID)3. However, the dependence of SRID on the availability of the corresponding subtype-specific antisera causes a minimum of 2-3 months delay for the release of every new vaccine. Moreover, despite evidence that NA also induces protective immunity4, the amount of NA in influenza vaccines is not yet standardized due to a lack of appropriate reagents or analytical method5. Thus, simple alternative methods capable of quantifying HA and NA antigens are desirable for rapid release and better quality control of influenza vaccines.
Universally conserved regions in all available influenza A HA and NA sequences were identified by bioinformatics analyses6-7. One sequence (designated as Uni-1) was identified in the only universally conserved epitope of HA, the fusion peptide6, while two conserved sequences were identified in neuraminidases, one close to the enzymatic active site (designated as HCA-2) and the other close to the N-terminus (designated as HCA-3)7. Peptides with these amino acid sequences were synthesized and used to immunize rabbits for the production of antibodies. The antibody against the Uni-1 epitope of HA was able to bind to 13 subtypes of influenza A HA (H1-H13) while the antibodies against the HCA-2 and HCA-3 regions of NA were capable of binding all 9 NA subtypes. All antibodies showed remarkable specificity against the viral sequences as evidenced by the observation that no cross-reactivity to allantoic proteins was detected. These universal antibodies were then used to develop slot blot assays to quantify HA and NA in influenza A vaccines without the need for specific antisera7,8. Vaccine samples were applied onto a PVDF membrane using a slot blot apparatus along with reference standards diluted to various concentrations. For the detection of HA, samples and standard were first diluted in Tris-buffered saline (TBS) containing 4M urea while for the measurement of NA they were diluted in TBS containing 0.01% Zwittergent as these conditions significantly improved the detection sensitivity. Following the detection of the HA and NA antigens by immunoblotting with their respective universal antibodies, signal intensities were quantified by densitometry. Amounts of HA and NA in the vaccines were then calculated using a standard curve established with the signal intensities of the various concentrations of the references used. 
Given that these antibodies bind to universal epitopes in HA or NA, interested investigators could use them as research tools in immunoassays other than the slot blot only.

A simple slot blot method was developed for the quantification of influenza viral hemagglutinin and neuraminidase using universal antibodies targeting their most conserved sequences identified through bioinformatics analyses. This innovative approach may provide a useful alternative to quantitative determination of all viral hemagglutinin and neuraminidase.

所有流感的定量分析类型使用通用抗体的一种病毒Hemagglutinins和Neuraminidases，在简单的狭缝杂交分析

Hemagglutinin (HA) and neuraminidase (NA) are two surface proteins of influenza viruses which are known to play important roles in the viral life cycle ...

Rapid Diagnosis of Avian Influenza Virus in Wild Birds: Use of a Portable rRT-PCR and Freeze-dried Reagents in the Field

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory flyways. Sampling of wild birds for avian influenza virus (AIV) is often conducted in remote regions, but results are often delayed because of the need to transport samples to a laboratory equipped for molecular testing. Real-time reverse transcriptase polymerase chain reaction (rRT-PCR) is a molecular technique that offers one of the most accurate and sensitive methods for diagnosis of AIV. The previously strict lab protocols needed for rRT-PCR are now being adapted for the field. Development of freeze-dried (lyophilized) reagents that do not require cold chain, with sensitivity at the level of wet reagents has brought on-site remote testing to a practical goal.
Here we present a method for the rapid diagnosis of AIV in wild birds using an rRT-PCR unit (Ruggedized Advanced Pathogen Identification Device or RAPID, Idaho Technologies, Salt Lake City, UT) that employs lyophilized reagents (Influenza A Target 1 Taqman; ASAY-ASY-0109, Idaho Technologies). The reagents contain all of the necessary components for testing at appropriate concentrations in a single tube: primers, probes, enzymes, buffers and internal positive controls, eliminating errors associated with improper storage or handling of wet reagents. The portable unit performs a screen for Influenza A by targeting the matrix gene and yields results in 2-3 hours. Genetic subtyping is also possible with H5 and H7 primer sets that target the hemagglutinin gene.
The system is suitable for use on cloacal and oropharyngeal samples collected from wild birds, as demonstrated here on the migratory shorebird species, the western sandpiper (Calidrus mauri) captured in Northern California. Animal handling followed protocols approved by the Animal Care and Use Committee of the U.S. Geological Survey Western Ecological Research Center and permits of the U.S. Geological Survey Bird Banding Laboratory. The primary advantage of this technique is to expedite diagnosis of wild birds, increasing the chances of containing an outbreak in a remote location. On-site diagnosis would also prove useful for identifying and studying infected individuals in wild populations. The opportunity to collect information on host biology (immunological and physiological response to infection) and spatial ecology (migratory performance of infected birds) will provide insights into the extent to which wild birds can act as vectors for AIV over long distances.

This study describes diagnosis of avian influenza in wild birds using a portable rRT-PCR system. The method takes advantage of freeze-dried reagents to screen wild birds in a non-laboratory setting, typical of an outbreak scenario. Use of molecular tools provides accurate and sensitive alternatives for rapid diagnosis.

在野生鸟类禽流感病毒快速诊断：使用便携式的RRT - PCR和在外地的冻干试剂

Wild birds have been implicated in the spread of highly pathogenic avian influenza (HPAI) of the H5N1 subtype, prompting surveillance along migratory ...

High-throughput Detection Method for Influenza Virus

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, precise diagnosis of the infecting strain and rapid high-throughput screening of vast numbers of clinical samples is paramount to control the spread of pandemic infections. Current clinical diagnoses of influenza infections are based on serologic testing, polymerase chain reaction, direct specimen immunofluorescence and cell culture 1,2.
Here, we report the development of a novel diagnostic technique used to detect live influenza viruses. We used the mouse-adapted human A/PR/8/34 (PR8, H1N1) virus 3 to test the efficacy of this technique using MDCK cells 4. MDCK cells (104 or 5 x 103 per well) were cultured in 96- or 384-well plates, infected with PR8 and viral proteins were detected using anti-M2 followed by an IR dye-conjugated secondary antibody. M2 5 and hemagglutinin 1 are two major marker proteins used in many different diagnostic assays. Employing IR-dye-conjugated secondary antibodies minimized the autofluorescence associated with other fluorescent dyes. The use of anti-M2 antibody allowed us to use the antigen-specific fluorescence intensity as a direct metric of viral quantity. To enumerate the fluorescence intensity, we used the LI-COR Odyssey-based IR scanner. This system uses two channel laser-based IR detections to identify fluorophores and differentiate them from background noise. The first channel excites at 680 nm and emits at 700 nm to help quantify the background. The second channel detects fluorophores that excite at 780 nm and emit at 800 nm. Scanning of PR8-infected MDCK cells in the IR scanner indicated a viral titer-dependent bright fluorescence. A positive correlation of fluorescence intensity to virus titer starting from 102-105 PFU could be consistently observed. Minimal but detectable positivity consistently seen with 102-103 PFU PR8 viral titers demonstrated the high sensitivity of the near-IR dyes. The signal-to-noise ratio was determined by comparing the mock-infected or isotype antibody-treated MDCK cells.
Using the fluorescence intensities from 96- or 384-well plate formats, we constructed standard titration curves. In these calculations, the first variable is the viral titer while the second variable is the fluorescence intensity. Therefore, we used the exponential distribution to generate a curve-fit to determine the polynomial relationship between the viral titers and fluorescence intensities. Collectively, we conclude that IR dye-based protein detection system can help diagnose infecting viral strains and precisely enumerate the titer of the infecting pathogens.

This method describes the use of Infrared dye based imaging system for detection of H1N1 in bronchioalveolar lavage (BAL) fluid of infected mice at a high sensitivity. This methodology can be performed in a 96- or 384-well plate, requires &lt;10 &mu;l volume of test material and has the potential for concurrent screening of multiple pathogens.

流感病毒的高通量检测方法

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

微流控芯片的制备及方法检测甲型流感

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

生物工程

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

流感病毒在小鼠感染模型中的研究

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Use of an Influenza Antigen Microarray to Measure the Breadth of Serum Antibodies Across Virus Subtypes

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge to the development of universal influenza vaccines is high antigenic diversity resulting from antigenic drift. Overcoming this challenge requires novel research tools to measure the breadth of serum antibodies directed against many virus strains across different antigenic subtypes. Here, we present a protocol for analyzing the breadth of serum antibodies against diverse influenza virus strains using a protein microarray of influenza antigens.
This influenza antigen microarray is constructed by printing purified hemagglutinin and neuraminidase antigens onto a nitrocellulose-coated membrane using a microarray printer. Human sera are incubated on the microarray to bind antibodies against the influenza antigens. Quantum-dot-conjugated secondary antibodies are used to simultaneously detect IgG and IgA antibodies binding to each antigen on the microarray. Quantitative antibody binding is measured as fluorescence intensity using a portable imager. Representative results are shown to demonstrate assay reproducibility in measuring subtype-specific and cross-reactive influenza antibodies in human sera.
Compared to traditional methods such as ELISA, the influenza antigen microarray provides a high throughput multiplexed approach capable of testing hundreds of sera for multiple antibody isotypes against hundreds of antigens in a short time frame, and thus has applications in sero-surveillance and vaccine development. A limitation is the inability to distinguish binding antibodies from neutralizing antibodies.

We present a protocol for using a protein microarray constructed by printing influenza antigens onto nitrocellulose-coated slides to simultaneously probe serum for multiple antibody isotypes against over 250 antigens from different virus strains, thus allowing measurement of the breadth of serum antibodies across virus subtypes.

使用流感抗原微阵列测量病毒亚型血清抗体的广度

The influenza virus remains a significant cause of mortality worldwide due to the limited effectiveness of currently available vaccines. A key challenge ...

用于呼吸道病毒检测的多重 RT-qPCR 检测

Development of Multiplex Real-Time RT-qPCR Assays for the Detection of SARS-CoV-2, Influenza A/B, and MERS-CoV

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general public&#39;s health. During influenza seasons, the spread of SARS-CoV-2 and other respiratory viruses may cause a population-wide burden of respiratory disease that is difficult to manage. For that, the respiratory viruses SARS-CoV-2, Influenza A, Influenza B, and Middle East respiratory syndrome (MERS-CoV) will need to be carefully watched over in the upcoming fall and winter seasons, particularly in the case of SARS-CoV-2, Influenza A, and Influenza B, which share similar epidemiological factors like susceptible populations, mode of transmission, and clinical syndromes. Without target-specific assays, it can be challenging to differentiate among cases of these viruses owing to their similarities. Accordingly, a sensitive and targeted multiplex assay that can easily differentiate between these viral targets will be useful for healthcare practitioners. In this study, we developed a real-time reverse transcriptase-PCR-based assay utilizing an in-house developed R3T one-step RT-qPCR kit for simultaneous detection of SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV. With as few as 10 copies of their synthetic RNAs, we can successfully identify SARS-CoV-2, Influenza A, Influenza B, and MERS-CoV targets simultaneously with 100% specificity. This assay is found to be accurate, reliable, simple, sensitive, and specific. The developed method can be used as an optimized SARS-CoV-2, Influenza A, Influenza B, and SARS-CoV-2, MERS-CoV diagnostic assay in hospitals, medical centers, and diagnostic laboratories as well as for research purposes.

作者聚焦:呼吸道病毒多重检测的进展 

We present two probe-based in-house one-step RT-qPCR kits for common respiratory viruses. The first assay is for SARS-CoV-2 (N), Influenza A (H1N1 and H3N2), and Influenza B. The second is for SARS-Cov-2 (N) and MERS (UpE and ORF1a). These assays can be successfully implemented in any specialized laboratory.

开发用于检测SARS-CoV-2、甲型/乙型流感和MERS-CoV的多重实时RT-qPCR检测

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes Coronavirus disease 2019 (COVID-19) is a serious threat to the general ...