This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

以下数据收集中使用的所有的猪都在美国俄亥俄州立大学机构动物护理和使用委员会保护（动物使用的协议号2009A0134-R1）。 1.准备病毒传输介质和样品收集瓶<ol><li>加37个克脑心浸液的900毫升纯化水中，并用搅拌棒和磁力搅拌器充分混合，同时加热至70℃以完全溶解粉末。 </li><li>高压灭菌的肉汤在121℃下进行15分钟。酷板凳上的顶部，直至汤汁达到室温。冷藏于4℃过夜如果需要的话。 </li><li>溶解，通过用磁力搅拌棒，6.02克青霉素G钠盐和10g硫酸链霉素到50ml的纯净水中，在室温下混合。添加额外的纯化水，使总体积为100毫升。过滤通过0.22μm聚醚砜膜的溶液到无菌瓶。 </li><li>无菌添加过滤青霉素和链霉素溶液到冷却的脑心浸液肉汤调匀（搅拌棒或用手涡旋烧瓶）以使病毒运输介质。 </li><li>无菌测试病毒运输介质的pH;如果需要的话，用盐酸或氢氧化钠将pH调节至7.4±0.2。 </li><li>通过测试的A型流感病毒基质蛋白RRT-PCR检测，所描述的张某和哈蒙执行病毒传输介质的质量保证测试。22 </li><li>分配将5ml病毒运输介质的到无菌的高密度聚乙烯瓶用8ml容量。标签小瓶在现场使用。 </li><li>存储准备使用的小瓶中标准的冷冻箱在-20℃下直到样品收集。 </li></ol> 2.收集鼻湿巾从猪<ol><li>解冻小瓶样品采集前夕适当数量的;一小瓶将每头猪的采样是必要的。解冻在室温温度大约需要30-45分钟。保持解冻瓶清凉冰袋直至使用。 </li><li>唐适当的个人防护装备的情况所决定（ 如工作服，靴套，口罩，耳塞等 ）。 </li><li>进入动物区。如果需要，限制了猪的小区域，但也没有限制。不要吵醒休息的动物。 </li><li>穿上一双一次性检查手套。请注意不要触摸猪，人，或无生命的物体污染的手套。 </li><li> （在2×2英寸）从包装用戴着手套的手，除去无菌5.08厘米×5.08厘米棉花纱布垫。无菌纱布垫应尽可能使用。独立包装纱布垫比那些包裹着每个套餐二纱布垫使用起来更加方便。 </li><li>持棉花纱布垫用一只手指尖暴露尽可能多的垫尽可能进行采样。 </li><li>擦拭整个猪的口鼻部的纱布垫，以如果可能的话格外小心，输入外部鼻孔。收集可见鼻腔分泌物（约1毫升）使用相同的纱布垫。 </li><li>其自身使用相同的手，折叠纱布垫，以方便放置于含有病毒运输介质中的小瓶中。 </li><li>使用另一只手，除去从药瓶的盖和将折叠的纱布垫入小瓶中。重新盖上小瓶并摇动小瓶以确保纱布垫和病毒运输介质的混合。 </li><li>在适当的容器中取出手套和地点。更改每头猪的手套。 </li><li>验证小瓶标识符。记录猪识别号码，年龄，性别，临床症状，并认为必要的研究者其它注释。 </li><li>采集后立即冷却该样品。考虑冻结防止多个冻融循环之前使样品的等分试样。一旦收集后可能，将样品在干冰上运送到实验室。存放小瓶于-80°C，直到试验开始。 </li></ol>检测流感3.病毒核酸<ol><li>解冻在37℃的干燥珠浴样品5分钟，然后完成解冻置于工作台顶部，将样品在室温下20-30分钟。同时加入几个小瓶至珠会导致浴加热;应谨慎行事，以不过热的小瓶。 </li><li>取出100μl病毒传输介质的RNA提取。 <ol><li>使用高通量（96孔板格式）磁珠平台，用于从大量样本中提取RNA。31 </li></ol></li><li>使用实时逆转录聚合酶链反应检测的快速检测甲型流感病毒。31 </li></ol> 4.隔离流感从鼻湿巾病毒<ol><li>如第3.1节上述解冻的样本</li><li>治疗解冻样品硫酸庆大霉素（1000微克/毫升），两性霉素B（22.5微克/毫升），和硫酸卡那霉素（325微克/毫升）。 </li><li>接种入的Madin-Darby犬肾（MDCK）的上皮细胞如前所述。32 </li></ol>

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</ol>

The authors have nothing to disclose.

收集使用聚酯嘴鼻拭子样品的猪在开展IAV监控已经证明是有益的;然而，使用鼻拭子过程的阻碍，由于所需使用圈套克制的监测工作。鼻湿巾代表目前的猪采样技术的细化，旨在样品采集过程中减少压力对人和猪。而该方法已被开发用于和验证展猪设置，它可以很容易地应用于其它情形宰前检测IAV的猪是必要的（ 即商业猪，活的动物中的市场，生物医学研究等 ） <p class="jove_content"...

<html> A型流感病毒（IAV）引起的呼吸系统疾病在许多物种，包括家禽，猪和人类。由于分段IAV基因组快速病毒进化的重配可发生新IAV株频频出现。猪是可以作为从多个宿主物种重配IAVs的混合容器中的物质。1目前IAV三个主要亚型间的北美猪（H1N1，H1N2，H3N2）通常循环，但来自人多IAV引入有导致大量IAV多样性的亚型内。IAVs感染猪2快速发展自1998年以来，当含有来自人类，禽流感和猪的病毒基因片段一个三重配IAV 3成为普遍在美国猪群中已可见一斑。4，内部基因从三重配IAV段保持IAVs目前感染猪中非常普遍。5 "&gt;在世界范围内，IAV是显著造成呼吸系统疾病的猪其典型的临床症状包括发烧，厌食，嗜睡，咳嗽，呼吸困难，打喷嚏，流鼻涕和体重不增。IAV可以是特别昂贵的母猪养殖场，其中生殖失败是由于IAV引起发热和弱仔猪已被记录在案，6,7在美国，IAV是常用的商业猪群和广泛的抗原和基因组多样性与持续发展之间的IAV感染猪检测阻碍了该控制病毒8-11 关于大流行IAV株来自重配导致猪分别实现在2009年时包含从三重配的北美猪血统和欧亚禽样猪的血统基因片段猪谱系IAV引起了世界范围内大流行的出现公共健康问题在人类12大流行性流感病毒（A型（H1N1）pdm09）具有自重配地方性猪IAV株13,14和一些新的重配株已传回人类。15重配事件和新IAV株可能引起大流行的出现，使循环IAV病毒在猪势在必行的主动监测的频率，特别是在猪的人机界面。 猪 - 人接口是IAV的双向种间传播的重要。人到猪的商业化养猪生产产生的传输是负责大量当前存在于猪群IAV多样性。农产品展销会是在美国人民和猪的comingling最大的设置和已知位点IAV的动物传播。15-21在2012年，一个变种H3N2 IAV爆发期间，案件93％的受访出席农业博览会在未来的日子发病前15基因组分析21展猪感染IAV从展猪病毒分离株与人分离株相比，证实人畜共患传输。常常不显示疾病，21-23指示需要直接诊断测试的临床症状。 对明显的病猪采样本身并不能成功地识别IAV流行的猪，不能依赖于识别IAV新毒株出现的猪之一。主动监测为检测IAV的猪，并评估其对两个猪和公共卫生威胁新兴菌株的绝对必要的。大多数IAV侦察活动是自愿的，因此需要最低限度的破坏性的方法。为IAV感染猪三大生前样本收集程序是:鼻拭子，唾液，鼻腔擦拭。对于个别采样猪检测IAV名单合成纤维插入当前建议拭子插入鼻孔的首选方法收集鼻分泌物和上皮细胞。24,25由于猪可能尽量避免此过程中，一组训练有素的人员必须手动或使用圈套根据动物的大小抑制猪26的限制处理是费力的人员，和紧张的猪。此外，展览猪经常参与多个比赛在一个公平的这样增加的压力对比赛的动物的感受可以让车主耐监测工作。 随着IAV检测的概率感染牛群从80-100％的IAV，口服液体已经成为一种流行的替代鼻拭子在猪群体的分子检测IAV的。27,28此外，口服液体可以提供更广泛的窗口IAV检测不是遵循最初的感染鼻拭子。然而，从唾液IAV隔离一直是个问题，只有50％的病毒分离的尝试导致IAV恢复。29 代替期间在猪IAV监视鼻拭子的使用鼻腔擦拭物克服了上述限制。鼻腔擦拭物不需要使用限制圈套的，可以在不强调过程的动物或证人来执行。最少的技术培训是需要收集鼻腔擦拭，这使得非兽医专业人士，包括猪的业主，收集监测样本。鼻腔擦拭物以前相比鼻拭子对甲病毒30和对于这种非侵入性采样的方法的详细的协议下面描述的检测和流感的隔离。

<table><tbody><tr><td>BBL Brain Heart Infusion</td><td>Becton, Dickinson and Company</td><td>211059</td><td></td></tr><tr><td>Penicillin G Sodium Salt</td><td>MP Biomedicals, LLC</td><td>021194537</td><td>1500 u/mg</td></tr><tr><td>Streptomycin Sulfate</td><td>AMRESCO LLC</td><td>0382</td><td></td></tr><tr><td>Gentamicin Solution</td><td>Mediatech, Inc.</td><td>30-005-CR</td><td>50 mg/ml</td></tr><tr><td>Amphotericin B Solution</td><td>Fisher</td><td>S 24 25</td><td>250 ug/ml</td></tr><tr><td>Kanamycin Sulfate</td><td>Teknova</td><td>K2105</td><td>5000 ug/ml</td></tr><tr><td>TPP Rapid Filtermax System</td><td>TPP Techno Plastic Products AG&amp;nbsp;</td><td>99150</td><td></td></tr><tr><td>Nalgen Diagnostic Bottles</td><td>Thermo Scientific</td><td>342002-9025</td><td>HDPE with white PP closure</td></tr><tr><td>Dermacea Gauze Sponge, 8 ply</td><td>Covidien</td><td>441211</td><td>5.08 cm &amp;times; 5.08 cm (2 in. &amp;times; 2 in)&amp;nbsp;</td></tr><tr><td>Nitrile Exam Gloves</td><td>Saftey Choice</td><td>19-170-010 (A-D)</td><td></td></tr></tbody></table>

nasal wipes for influenza a virus detection and isolation from swine

在监测猪流感A型病毒对人类和动物的健康至关重要，因为A型流感病毒迅速演变的猪群和新品种不断涌现。猪能够被感染甲型流感病毒的使得它们的出现和维持的新的流感病毒株重要主机多样谱系。抽样猪在不同的设置，如商业猪场，农产品集贸市场和活禽交易市场是非常重要的，提供当前流行IAV株的全面视图。目前的黄金标准宰前抽样技术（鼻拭子的，即集合）是劳动密集型的，因为它需要猪的物理限制。鼻抹布擦涉及在尽量减少猪的口鼻部一块布来节制的动物。鼻腔擦拭过程简单进行，并不需要专业的兽医或动物处理人员培训。 W¯¯往往微不足道略高于鼻拭子不太敏感，病毒检测和​​隔离率足以使鼻湿巾抽检个别猪在低应力抽样方法都需要一个可行的选择。该诉讼协议概述了需要收集一个可行鼻的步骤从个体猪擦拭。

成功使用这种方法产生RRT-PCR结果是，伴随着RNA提取和RRT-PCR过程中使用的内部控制，显示样本不含有PCR抑制剂从任何环境碎片捡起取样过程。样品在接种后，分离病毒井应不受样品可见环境碎屑。有与PCR通常产生更高IAV阳性率较病毒分离因为PCR检测病毒核酸，不一定可行病毒的理解RRT-PCR结果和病毒分离结果之间吻合。 结果表明，鼻抹布是一个有用的替代鼻拭子，这对于IAV目?...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

鼻湿巾从猪流感病毒检测和​​隔离

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and ...

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.4K 次观看.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

视频: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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 ...

科研

免疫与感染

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, ...

Detection of Viruses from Bioaerosols Using Anion Exchange Resin

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid impingement-based air sampling devices for efficacious concentration of negatively-charged viruses from bioaerosols. Thus, the resin serves as an additional concentration step in the bioaerosol sampling workflow. Nucleic acid extraction of the viral particles is then performed directly from the anion exchange resin, with the resulting sample suitable for molecular analyses. Further, this protocol describes a custom-built bioaerosol chamber capable of generating virus-laden bioaerosols under a variety of environmental conditions and allowing for continuous monitoring of environmental variables such as temperature, humidity, wind speed, and aerosol mass concentration. The main advantage of using this protocol is increased sensitivity of viral detection, as assessed via direct comparison to an unmodified conventional liquid impinger. Other advantages include the potential to concentrate diverse negatively-charged viruses, the low cost of anion exchange resin (~$0.14 per sample), and ease of use. Disadvantages include the inability of this protocol to assess infectivity of resin-adsorbed viral particles, and potentially the need for the optimization of the liquid sampling buffer used within the impinger.

An anion exchange resin-based method, adapted to liquid impingement-based bioaerosol sampling of viruses is demonstrated. When coupled with downstream molecular detection, the method allows for facile and sensitive detection of viruses from bioaerosols.

用阴离子交换树脂检测 Bioaerosols 病毒

This protocol demonstrates a customized bioaerosol sampling method for viruses. In this system, anion exchange resin is coupled with liquid ...

环境科学

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

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 ...

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 ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

中和流感病毒单克隆抗体逃逸变种的产生

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...

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 ...

Imaging Cell Interaction in Tracheal Mucosa During Influenza Virus Infection Using Two-photon Intravital Microscopy

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. Two-photon intravital microscopy (2P-IVM) allows the observation of cell interactions in deep tissue in living animals, while minimizing the photobleaching generated during image acquisition. To date, different models for 2P-IVM of lymphoid and non-lymphoid organs have been described. However, imaging of respiratory organs remains a challenge due to the movement associated with the breathing cycle of the animal.
Here, we describe a protocol to visualize in vivo immune cell interactions in the trachea of mice infected with influenza virus using 2P-IVM. To this purpose, we developed a custom imaging platform, which included the surgical exposure and intubation of the trachea, followed by the acquisition of dynamic images of neutrophils and dendritic cells (DC) in the mucosal epithelium. Additionally, we detailed the steps needed to perform influenza intranasal infection and flow cytometric analysis of immune cells in the trachea. Finally, we analyzed neutrophil and DC motility as well as their interactions during the course of a movie. This protocol allows for the generation of stable and bright 4D images necessary for the assessment of cell-cell interactions in the trachea.

In this study, we present a protocol to perform two-photon intravital imaging and cell interaction analysis in the murine tracheal mucosa after infection with influenza virus. This protocol will be relevant for researchers studying immune cell dynamics during respiratory infections.

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

The analysis of cell-cell or cell-pathogen interaction in vivo is an important tool to understand the dynamics of the immune response to infection. ...

An Optimized Hemagglutination Inhibition (HI) Assay to Quantify Influenza-specific Antibody Titers

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody production pre- and post-vaccination is required to understand vaccine-induced immunity. This article describes a reliable point-by-point protocol to determine influenza-specific antibody titers. The first protocol describes a method to specify the antigen amounts required for hemagglutination, which standardizes the concentrations for subsequent usage in the second protocol (hemagglutination assay, HA assay). The second protocol describes the quantification of influenza-specific antibody titers against different viral strains by using a serial dilution of human serum or cell culture supernatants (hemagglutination inhibition assay, HI assay).
As an applied example, we show the antibody response of a healthy cohort, which received a trivalent inactivated influenza vaccine. Additionally, the cross-reactivity between the different influenza viruses is shown and methods to minimize cross-reactivity by using different types of animal red blood cells (RBCs) are explained. The discussion highlights advantages and disadvantages of the presented assays and how the determination of influenza-specific antibody titers can improve the understanding of vaccine-related immunity.

An Optimized Hemagglutination Inhibition HI Assay to Quantify Influenza-specific Antibody Titers

The presented protocols describe how to perform a hemagglutination inhibition assay to quantify influenza-specific antibody titers from serum samples of influenza vaccine recipients. The first assay determines optimal viral antigen concentrations by hemagglutination. The second assay quantifies influenza-specific antibody titers by hemagglutination inhibition.

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

Antibody titers are commonly used as surrogate markers for serological protection against influenza and other pathogens. Detailed knowledge of antibody ...

鼻湿巾从猪流感病毒检测和隔离

摘要

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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

流感病毒的高通量检测方法

用阴离子交换树脂检测 Bioaerosols 病毒

快速分子检测和流感病毒A和B的分化

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

流感病毒在小鼠感染模型中的研究

中和流感病毒单克隆抗体逃逸变种的产生

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

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

鼻湿巾从猪流感病毒检测和​​隔离

摘要

探索更多视频

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

流感病毒的高通量检测方法

用阴离子交换树脂检测 Bioaerosols 病毒

快速分子检测和流感病毒A和B的分化

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

流感病毒在小鼠感染模型中的研究

中和流感病毒单克隆抗体逃逸变种的产生

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

双光子活体显微镜感病毒感染时气管黏膜的影像细胞相互作用

一种优化的血凝抑制 (HI) 测定流感特异抗体滴

鼻湿巾从猪流感病毒检测和隔离