Name: influenza a virus studies in a mouse model of infection
Uploaded: 2017-09-07T13:00:00.000+00:00
Duration: 10 min 44 s
Description: Watch this Scientific Journal Video about Influenza A Virus Studies in a Mouse Model of Infection at JoVE.com

流感病毒在 LM 实验室的研究部分由纽约流感卓越中心 (NYICE) 提供, 这是 NIAID 流感研究和监测卓越中心 (CEIRS) 的成员。我们感谢温迪. 贝茨对手稿的修正表示支持。

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作者没有什么可透露的。

IAV 的小鼠模型广泛应用于体内的 IAV 病机、免疫原性和保护功效的研究。与其他动物模型 (如雪貂或豚鼠) 相比, 小老鼠可以使它们易于操作和储存。此外, 在动物成本、住房和生殖方面的便利使它们能够用于需要大量动物的前体免疫试验。值得注意的是, 由于小鼠已被用于多个研究学科, 一些分子和免疫学鼠试剂可以方便用于 IAV 研究。此外, 最小的寄主变异性和存在的免疫系统, 在进化上类似于目前在人类, 使他们一个强大的小动物模型的 IAV 研究。最后, 研究宿主病毒蛋白相互作用及其对病毒病机的贡献目前是可行的, 以获得高鼠。然而, 除了使用小鼠进行 IAV 研究的几个优点之外, 它们对于 IAV 传输研究是无用的。因此, 雪貂或豚鼠是更适合研究 IAV 传播的动物模型。感染 IAV 的小鼠的临床症状通常出现在 2-3 天感染, 虽然这可能会因老鼠的年龄、免疫状况、性别、遗传背景和应变而有所不同;以及使用的病毒株和剂量。症状包括厌食、嗜睡、蜷缩、毛绒、体重损失和死亡16,17。
在这篇手稿中, 我们描述了如何评估感染. 的小鼠病毒病机, 通过监测体重损失 (发病率), 生存率 (死亡率) 和评估病毒复制在上 (鼻黏膜) 和下 (肺) 的 IAV。呼吸道 (图 1和 图 2)。IAV 的发病机制是一个非常重要的参数, 不仅在新的 IAV 菌株的情况下, 而且在安全实施 LAIV 疫苗候选 (图 3)。季节性 IAVs 和 IAVs 感染其他哺乳动物 (如马、狗或猪) 通常不会在小鼠体内产生疾病症状11,27。在这种情况下, 对感染小鼠的肺部或鼻腔黏膜中病毒滴的分析是评价 IAV 菌株病毒病机的主要参数。一旦肺部和鼻腔粘膜被收集, 它们均质化, 并通过斑块法、免疫荧光法、组织培养感染剂量 50 (TCID50) 或其他经验证的方法来分析这些器官中存在的病毒的数量,8 ,29。我们建议使用间接免疫荧光法评估病毒滴, 因为斑块检测需要较高数量的 MDCK 细胞 (6-井板格式), 并且, 像 TCID50, 需要更多的时间 (3-4 天) 来计算病毒滴。然而, 要进行间接免疫荧光分析, 有必要有: 1) 抗 IAV 蛋白的初级特异抗体 (建议使用抗体对 IAV 核, NP, 因为它是最丰富的病毒蛋白产生病毒感染期间);2) 二次抗体共轭荧光;和 3) 荧光显微镜对荧光阳性感染细胞计数。
由于小鼠的免疫系统在进化上类似于人类的免疫系统16 , 而且由于小鼠可以诱发对 IAV 感染的强大和保护性体液反应, IAVs (或候选疫苗) 的免疫原性可以很容易通过测定总 (ELISA;图 4)和中和 (海,图 5) 或网络 (数字 6) 抗体应答。为了分析免疫后的体液反应, 可以采用不同的方法, 如逆行眶穿刺, 尾夹, 大隐静脉穿刺或下颌下腺穿刺。我们选择了下颌下腺穿刺技术26 , 因为该手术不需要使用麻醉, 并允许我们获得可接受的血液免疫研究量;与逆行眶穿刺相反, 在那里小鼠应麻醉, 并与尾夹或隐静脉穿刺, 在那里的血容量恢复通常是低的。
在这篇手稿中, 我们描述了如何通过 ELISA 使用病毒感染细胞提取物来评估抗体对总病毒蛋白的存在。由于 IAV 感染诱导一种保护性免疫, 主要是通过对病毒 ha (主要抗原病毒靶) 的抗体进行介导, 因此强烈建议用重组纯化 ha 对 ha 的特异抗体进行评估。蛋白质10。为了分析中和抗体的存在, 海和网络方法都接受, 但都有利弊。海是快速 (2-3 h) 并且由疾病和控制中心 (CDC) 批准评估存在 IAV 中和抗体30。网络是更费时 (3-4 天), 但更具体, 因为它只评估抗体, 可以中和病毒感染。值得注意的是, 网络已被证明可以检测到茎-反应性 HA 中和抗体31, 以及 NA 中和抗体32。网络的另一个优点是检测灵敏度, 因为相对于需要大约 104-105 FFU 的口检测, 需要较少的 IAV (200 FFU)。此外, 由于难以解释结果33、34、35, 因此, 海试检测可能会引起对禽 IAVs 的中和抗体的检出问题。此外, 网络不需要使用红细胞来鉴别流感中和抗体。
最后, 我们描述了评估在开发新的 IAV 疫苗 (图 3): 1) 安全方面必须考虑的三主要方面的实验程序: 疫苗必须是安全的, 而不是产生疾病;2) 免疫原性: 该疫苗必须免疫, 同时诱发体液和细胞保护反应;3) 保护功效: 该疫苗必须通过相应的和/或异源病毒来诱导保护免受挑战。小鼠模型是一个很好的选择, 第一次筛选新的 IAV 疫苗在体内, 因为它可以评估不同的疫苗接种计划和条件, 包括使用各种佐剂, 抗原/疫苗剂量, 管理和广泛的保护应对与同源或异源 IAVs 菌株的挑战16,17。然而, 在进行人体临床试验之前, 疫苗候选者至少应在第二个体内模型中进行测试。

IAVs 被封装在Orthomyxoviridae系列3中的病毒分类。他们包含八单 RNA 分子与阴性极性3。在人类中, IAVs 导致季节性流行病和偶然大流行的重要后果, 当新的病毒在人类人口中引入4。此外, 季节性 IAVs 是高度和迅速传播之间的人产生了高的经济损失世界各地每年2,5。IAV 症状包括咳嗽、鼻塞、发热、不适、头痛、厌食和肌痛, 但病毒也会在免疫缺陷患者中产生更严重的疾病6。事实上, 世界卫生组织 (WHO) 计算, 季节性流感病毒每年造成30万至50万人死亡, 在世界各地1。目前只有两类药物被食品和药物管理局 (FDA) 批准用于人类的流感预防和治疗: 神经氨酸酶 (NA) 抑制剂 (例如, 奥司他韦) 和阻断剂的 M2 离子通道 (例如,金刚烷胺);然而, 耐药性病毒变种的出现日益引起人们的关注。因此, 疫苗接种仍然是保护人类免受 IAVs 感染的最佳医疗选择。迄今为止, 有三种类型的流感疫苗是由 FDA 授权供人使用的: 重组病毒血 (HA) 蛋白疫苗、灭活流感疫苗 (IIV) 和活减流感疫苗 (LAIV)5,7. 三种疫苗的设计目的是诱导对病毒 HA 蛋白的适应性免疫反应, 这是中和抗体对抗 IAVs 的主要目标。
一种验证的小鼠模型, 用于研究 IAV 感染在体内
动物模型被用于研究, 除其他以外, IAV 发病机制8,9,10,11, 导致疾病的病毒因素12和/或病毒传输13 ,14, 并测试新疫苗或抗病毒药物的功效9,10,15。老鼠 (小家鼠) 是最广泛使用的动物模型为 IAV 研究有几个原因: 1) 免疫系统在进化上类似于人类的存在;2) 低成本, 包括动物购买、住房和繁殖;3) 体积小, 易于操作和贮存;4) 最小主机的变异性, 以获得均匀的反应和结果;5) 大鼠生物学知识, 包括基因组序列;6) 许多可用的分子生物学和/或免疫学试剂;7) 可用的敲出 (KO) 小鼠研究某一宿主蛋白对病毒感染的贡献;和, 8) 多小鼠菌株, 可以利用, 以评估遗传基础的感染。
目前有几个老鼠菌株可用于研究 IAV在体内。年龄、免疫状况、性别、遗传背景和小鼠品系以及感染途径、剂量和病毒株均影响小鼠 IAV 感染的结果。IAV 研究中使用的最常见的小鼠菌株是 C57BL/6、balb/c/C 和最近的 DBA. 2 只小鼠, 因为它们比两个前菌株更容易 IAV 疾病16,17,18,19,20. 重要的是, 免疫应答也可以根据鼠标的应变情况而有所不同18,19,20。因此, 恢复所有关于鼠标和 IAV 应变的可用信息是非常重要的, 以选择最佳的实验方法进行。
虽然鼠标是一个良好的动物模型感染的在体内研究与 IAV, 他们有几个限制, 需要考虑的实验设计。例如, 使用小鼠进行体内研究的一个主要限制是, IAVs 不在小鼠之间传播。因而, 为传输研究, 更加被接受的动物模型 (例如, 雪貂或试验品) 使用16,17,21。此外, IAV 在小鼠和人类中的表现也存在着一些差异。与人类不同, 老鼠在 IAV 感染时不会发热;相反, 他们目前与低温16,17。在小鼠中, IAV 的复制集中在下呼吸道 (肺部) 而不是上层气管。因此, IAV 在小鼠中的毒力并不总是与人类所看到的相关联。总之, 由于优势大于有限的劣势, 鼠标代表了第一个动物模型, 用于评估流感病毒的发病机制, 免疫原性和保护功效的疫苗和抗病毒研究。此外, 在 IAV 感染的小动物模型中, 使用大型动物模型进行 IAV 研究, 并没有以前的证据, 在道德上是不能接受的。在这篇手稿中, 我们描述了如何感染小鼠鼻 (.) 与 IAV, 如何监测病毒感染的严重性和进展, 以及如何进行的实验, 以评估体液免疫反应和保护功效。

<table><tbody><tr><td>Madin-Darby Canine Kidney (MDCK) epithelial cells</td><td>ATCC</td><td>CCL-34</td><td></td></tr><tr><td>Six- to eight-week-old female C57BL/6 mice</td><td>National Cancer Institute (NCI)</td><td>01XBE</td><td></td></tr><tr><td>Turckey red blod cells</td><td>Biolink Inc</td><td></td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Corning Cellgro</td><td>15-013-CV</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1500-050</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Penicillin/Streptomycin/L-Glutamine (PSG) 100x</td><td>Corning</td><td>30-009-CI</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Penicillin/Streptomycin (PS) 100x</td><td>Corning</td><td>30-00-CI</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Bovin Albumin solution (BA)</td><td>Sigma-Aldrich</td><td>A7409</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Bovin Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A9647</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin</td><td>Sigma-Aldrich</td><td>T8802</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Neutral Buffered Formalin 10%</td><td>EMD</td><td>65346-85</td><td>Store at RT</td></tr><tr><td>Triton X-100</td><td>J.T.Baker</td><td>X198-07</td><td>Store at RT</td></tr><tr><td>Monoclonal Antibody anti-NP Influenza A Virus HB-65</td><td>ATTC</td><td>H16-L10-4R5</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Polyclonal rabbit anti-mouse immunoglobulins/FITC</td><td>Dako</td><td>F0261</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody</td><td>GE Healthcare</td><td>LNA931V/AG</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>TMB substrate set</td><td>BioLegend</td><td>421101</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Vmax Kinetic plate reader</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>Dounce Tissue Grinders</td><td>Thomas Scientific</td><td>7722-7</td><td></td></tr><tr><td>Receptor destroying enzyme, RDE (II)</td><td>Denka Seiken Co.</td><td>370013</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Crystal Violet</td><td>Fisher Scienctific</td><td>C581-100</td><td>Store at RT</td></tr><tr><td>96-well Cell Culture Plate</td><td>Greiner Bio-one</td><td>655-180</td><td></td></tr><tr><td>Cell Culture dishes 100 mm</td><td>Greiner Bio-one</td><td>664-160</td><td></td></tr><tr><td>Nunc MicroWell 96-Well Microplates</td><td>Thermo Fisher Scienctific</td><td>269620</td><td></td></tr><tr><td>Nunc 96-Well Polystyrene Conical Bottom MicroWell Plates</td><td>Thermo Fisher Scienctific</td><td>249570</td><td></td></tr><tr><td>Puralub Vet Ointment</td><td>Dechra</td><td>9N-76855</td><td></td></tr><tr><td>Fluorescent microscope</td><td>Olympus</td><td>Olympus IX81</td><td></td></tr></tbody></table>

influenza a virus studies in a mouse model of infection

流感病毒在全球范围内造成超过50万人死亡1 , 并与每年的费用 12-140亿 USD 在美国单独考虑直接医疗和住院费用和工作缺勤的2。动物模型是关键的流感 A 病毒 (IAV) 研究评估病毒发病机制, 宿主-病原体相互作用, 免疫反应, 以及目前和/或新的疫苗方法的功效, 以及抗病毒药物。小鼠是一种有利的小动物模型, 因为它们的免疫系统在进化上与人类相似, 它们可从商业供应商那里获得, 作为基因上相同的学科, 有多种菌株可以利用来评估感染的遗传基础, 它们相对便宜, 易于操作。为了总结 IAV 感染的人通过呼吸道, 小鼠首先被麻醉之前, 鼻腔接种与传染性 IAVs 在适当的生物安全控制下。感染后, IAVs 的发病机制是通过每日监测发病率 (体重损失) 和死亡率 (存活率) 来确定的。此外, 病毒病机还可以评估病毒复制在上 (鼻黏膜) 或较低 (肺) 呼吸道感染小鼠。对 IAV 感染的体液反应可以通过无创性出血和二级抗体检测方法快速评估, 目的是检测总或中和抗体的存在。在这里, 我们描述了常用的方法感染小鼠鼻 (i. n) 与 IAV 和评估发病机制, 体液免疫反应和保护功效。

小鼠病毒病机的特征
IAV 的发病机制与其感染引起的发病率和死亡率有关。这两个参数可以很容易地在小鼠中进行评估: IAV 发病率与受感染小鼠的体重下降有关, 存活率的百分比将表明死亡率 (图 1)。IAV 感染小鼠的体重和生存率通常在感染8、9、10、29后每天监测至少两周。获得的生存数据可以确定使用簧片和明奇25方法计算的 MLD50 。IAV 发病机制的另一个指标是与下 (肺) 和上 (鼻黏膜) 呼吸道的病毒复制有关 (图 1)。这些器官的病毒滴度所获得的信息对应于发病率和死亡率, 并一起提供了一个很好的迹象, 病毒病机。据了解, IAV 复制在老鼠肺部峰值之间的天2和4感染 (私家侦探), 所以我们建议恢复这些器官在天2和4私家侦探
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55898/55898fig1.jpg"> 
图 1: 小鼠 IAV 病机的特征:小鼠的 IAV 病机与其发病率 (体重下降) 和死亡率 (% 存活率) 有关, 也与 IAV 在上 (鼻黏膜) 和下 (肺) 呼吸道中复制的能力有关。简要地, 在天0小鼠称重和麻醉腹腔与 22, 2-tribromoethanol (未), 在他们被传染的鼻与 IAV 之前。从每天1天到 14, 小鼠每日体重评估体重损失 (发病率) 和% 生存率 (死亡率) 计算鼠标致死剂量 50 (MLD50)。在天2和天4感染, 老鼠肺和鼻黏膜被恢复并且均匀性评估病毒复制。小鼠实验的特点 IAV 发病机制使用 PR8 体重在 BSL-2 条件下进行。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55898/55898fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在图 2中, PR8 体重机制评估中获得的数据表示。四组6至8周老 C57BL/6 小鼠 (n = 11) 被感染. 与指示剂量 (10, 102, 103和 104 FFU/鼠标) 的 PR8 的重量。每组5只小鼠的体重和存活率由14天的私家侦探 (图 2A-B) 进行测量。所有的小鼠免疫 104 FFU PR8 体重迅速减轻 (图 2A), 他们都死在天5和6私家侦探 (图 2B)。用 103 FFU 免疫小鼠 PR8 体重在以后的时间里丢失了身体重量 (图 2A), 并且他们全部死了由天7和8私家侦探 (图 2B)。所有的小鼠接种了 102 FFU PR8 体重减轻 (图 2A), 但其中只有2人死于9私家侦探 (图 2B)。最后, 接种了 10 FFU PR8 的小鼠并没有减轻体重 (图 2A), 并且它们都存活了感染 (图 2B)。在本实验中, 用簧片和明奇方法计算出的 PR8 MLD50 25是 1.5 x 102 FFU (图 2C)。
评估感染. 的小鼠上、下呼吸道的 PR8 复制量 (10, 102、103和 104 FFU/鼠标), 肺部和鼻腔粘膜在天2和4私家侦探 (n = 3) 中恢复。在肺均质性后, 用免疫荧光法 (图 2D) 对该器官中的病毒数量进行分析。在不同小鼠的肺中检测到的病毒滴度与用于免疫的剂量有关: 在2和4私家侦探中, 104 FFU 免疫的小鼠肺部检测出较高的病毒滴, 而低病毒滴被检测到 用 10 FFU PR8 免疫小鼠的肺。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55898/55898fig2.jpg"> 
图 2: 在病毒病机评估中获得的数据的表示:分析 PR8 的发病率和死亡率, 6 至8周老的雌性 C57BL/6 小鼠 (n = 5) 被感染., 其荧光形成单位 (FFU) 的数量显示为 PR8, 然后每日监测2周 (A) 体重减轻 (发病率)和 (B) 生存 (死亡率)。数据代表单个小鼠所确定结果的平均值和标准偏差 (n = 5)。(C) 2 周以上的生存率计算值用于计算 MLD50 , 使用簧片和明奇方法25。(D) 评估病毒复制, 6 至8周老的雌性 C57BL/6 小鼠 (n = 6) 被感染., 其数量为 FFU。病毒复制在肺部或鼻腔粘膜感染的小鼠通常评估在2天和4私家侦探使用 immunofocus 化验。病毒滴被记录为 FFU/毫升。数据代表每只老鼠获得的结果的手段和 SDs。虚线被包括在内, 以表明检测的限制 (200 FFU/毫升) 的化验。在 BSL-2 条件下, 利用 PR8 进行小鼠和组织培养试验, 以评估 IAV 发病率、死亡率和病毒滴。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55898/55898fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
免疫接种后体液反应的评价
IAV 感染引起的体液反应对控制病毒感染起着至关重要的作用。因此, 分析 IAV 感染或新的 IAV 疫苗引起的体液反应是非常重要的 (图 3)。在免疫接种后14天的小鼠模型中, 通过 ELISA (图 4) 分析了抗体对总病毒蛋白的存在, 以及以病毒 HA 蛋白为靶的中和抗体的存在, 这通常是分析了常见的血清学方法, 如海化验 (<stro></stro>ng 类 = "xfig" > 图 5) 和/或网络 (图 6)。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55898/55898fig3.jpg"> 
图 3: LAIV 的安全性、免疫原性和保护功效的不同步骤的方案:在开发新的 LAIV 时, IAV 感染的小鼠模型通常用于测试安全性、免疫原性和保护功效。在该方案中, 对候选 LAIV 的安全性 (A)、免疫原性 (B) 和保护效能 (C) 进行了实验。(a) 为了评估 LAIV 的安全性, 必须对图 1中描述的相同参数 (发病率、死亡率和病毒复制在上下呼吸道中) 进行检测。为了评估免疫原性, 小鼠免疫接种后14天, 他们被下颌下腺穿刺出血。通过酶联免疫吸附试验 (ELISA) 和用血凝抑制试验 (海) 和/或病毒评价中和抗体的存在来分析体液反应, 以评估 IAV 蛋白的总抗体microneutralization 测定 (网络)。(C) 为了评估保护效果, 在接种疫苗15天后, 小鼠受到 IAV 的挑战. 通过对小鼠的发病率、死亡率和病毒滴的测量来评估保护功效, 如图1所述.在 BSL-2 条件下, 对 LAIV 候选者的安全性、免疫原性和保护效果进行小鼠实验。其他 BSL 的西装或遏制条件可能需要根据 IAV 应变所使用的挑战接种小鼠。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55898/55898fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
评估 PR8 LAIV 的免疫原性三组6至8周老 C57BL/6 小鼠 (n = 5) 接种了不同剂量 (102, 103, 和 104 FFU/鼠标) 的 PR8 LAIV 或模拟疫苗接种 1x PBS 作为一个负控制。接种后十四天, 小鼠血清由下颌下腺出血26 (图 3) 采集。用1:200 稀释 PR8-infected MDCK 细胞提取物 (图 4) 对总病毒蛋白的抗体反应进行了 ELISA 评价。每个血清分别进行分析。结果表明, PR8 LAIV 高剂量 (104 FFU) 免疫小鼠血清对总 PR8 蛋白的抗体滴高于接种低剂量 (102 FFU) 的小鼠血清。血清控制 (模拟感染小鼠) 没有表现出对 PR8 抗原的反应。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55898/55898fig4.jpg"> 
图 4: 酶联免疫吸附试验 (ELISA) 的示意图表示法评估体液抗体应答:受感染小鼠的 PR8-specific 抗体水平是由酶联免疫吸附法测定的 96-好聚苯乙烯板涂层的细胞提取物从模拟 (控制) 或 PR8-infected MDCK 细胞。简单地说, 在14天的私家侦探 (图 3) 中, 用 PR8 LAIV 免疫的小鼠通过下颌下腺穿刺和血清进行了出血。每个血清通过 ELISA 对总 PR8 蛋白的 IgG 抗体单独评估。图形数据表示从单个小鼠血清 (n = 6) 获得的结果的方法和标准偏差。光学密度。ELISA 测定是在 BSL-2 的柜子里进行的。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55898/55898fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了分析在小鼠血清中中和抗体的存在, 使用海化验, 2 倍稀释血清 (以前灭活在56° c) 每组接种不同剂量 (102, 103, 104FFU) PR8 LAIV 在 RT (图 5) 中孵育了4口 PR8 重量为1小时。然后, 0.5% 的火鸡红细胞被添加到 PR8-sera 混合物中。图 5的上部是可能的海结果的示意性表示: 当血清中含有中和抗体时, 在井底观察到红细胞沉淀, 因为与病毒 HA 蛋白结合的抗体阻止红细胞的血凝另一方面, 在缺乏中和抗体的情况下, 观察了红细胞的血凝。然后, 在最后一井缺乏血凝的情况下, 将海滴度计算为血清稀释的倒数。海的结果显示, 在图 5的下部表明, PR8 LAIV (104 FFU) 接种的小鼠的血清中和抗体的效价较高, 而从小鼠的血清免疫低剂量 (102 FFU) 对 PR8 的中和抗体的效价最低,.在血清控制 (模拟感染小鼠) 中, 没有中和抗体存在。
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55898/55898fig5.jpg"> 
图 5: 血凝抑制 (海) 试验:通过海化验可以很容易地评价感染小鼠 PR8 的中和抗体水平。简单地说, 2 倍序列稀释 (开始稀释 1:10) 的血清从免疫小鼠的 PR8 LAIV 的指示剂量是混合 (1:1) 与4口 PR8 重量为 1 h 在 RT 在一个96井 V 底板。1 h 潜伏期后, 0.5% 红细胞被添加到病毒-血清混合物 30-60 分钟冰。海滴是确定的最后井, 其中红细胞形成一个红色按钮和血凝不发生 (顶部)。在 BSL-2 柜中进行了 PR8 重量的海化验。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55898/55898fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
在图 6中, 用网络表示了在评估小鼠血清中和抗体中所获得的数据。200 FFU PR8 混合2倍稀释血清 (以前 inactivated 在56° c) 从接种了不同剂量的小鼠 (102, 103, 和 104 FFU) 的 PR8 LAIV 在 RT 的1小时。每一个血清样本进行三份评估。病毒-血清混合物被用来感染 MDCK 细胞的单分子膜在一个96井板。图 6的上部是可能的网络结果的示意性表示: 血清中和抗体的缺乏使 CPE 的结果在大约 3-4 天的私家侦探相反, 当中和抗体存在 (完整的细胞单层), 他们绑定到病毒 HA 和防止病毒感染, 并没有 CPE。CPE 的存在通常是通过染色被感染的细胞与结晶紫, 和病毒中和滴被计算为最后的稀释的倒数, 在其中完全阻断感染。网络的结果在图 6的下部表示。高剂量 PR8 LAIV (104 FFU) 免疫小鼠的血清中和抗体的效价最大, 而免疫小鼠或 PR8 LAIV (102 FFU) 的血清中和抗体的效价最低,与海化验所得的结果相似。模拟感染小鼠血清中不存在中和抗体。
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/55898/55898fig6.jpg"> 
图 6: 病毒 Microneutralization 检测 (网络):另一种方法, 网络测定可以评估 PR8 中和抗体的存在。简单地说, 接种了 PR8 LAIV 的小鼠的血清是连续2倍稀释 (开始稀释 1:5) 在 96-井板和混合1:1 与 200 FFU PR8 重量为 1 h 在 RT。1小时后, 病毒-血清混合物被用来感染96个 MDCK 细胞板。感染细胞孵育 3-4 天, 直到完全病变的效果。96井板然后被染色与0.1% 水晶紫罗兰色。网络滴是确定的最高稀释保留一个汇合的细胞单层。网络 PR8 在 BSL-2 条件下进行。<a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/55898/55898fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
小鼠 IAV 疫苗保护效果评价
当一个新的 LAIV 的开发, 它的安全性, 免疫原性和保护功效必须在体内测试, 而鼠标模型通常是第一个动物模型, 选择分析这些参数。图 3是一种在小鼠中描述新 LAIV 所需的不同步骤的方案。为了评估 LAIV 的安全性 (图 3A), 小鼠被感染. 使用类似的协议的实验过程中描述的发病机制部分 (4 节), 和体重和生存监测将提供有关信息LAIV 的发病率和死亡率 (图 2A-2B), 包括 MLD50。另外, 在接种用不同的疫苗药量时, LAIV 的复制在下 (肺) 和在上部 (鼻黏膜) 呼吸道应该被评估8,9,10, 11,29 (图 2D)。为了测试免疫原性, 接种 LAIV 疫苗的小鼠的血清在挑战前收集 PR8 (同源挑战), 使用与免疫原性部分所描述的类似的协议 (5 节) (图 3B)。血清中的总和中和抗体的存在可以通过 ELISA 和海和/或网络分别检测。
最后, 为了测试保护功效, LAIV 免疫小鼠受到 PR8 的挑战, 其保护功效的特点是监测发病率、死亡率和在肺部存在的挑战 PR8, 如上文4节所述 (图 3C)8,9,10,11。如果 LAIV 诱导保护抗 PR8 的挑战, 小鼠不会失去体重, 他们将生存的致命挑战与 IAV 重量。此外, IAV 病毒滴在肺部将不会被检测到, 或将大大减少与模拟 (PBS) 疫苗接种小鼠8,9,10,11。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>组织培养介质及解决方案</td>
			<td>组成</td>
			<td>存储</td>
			<td>使用</td>
			<td>评论</td>
		</tr>
		<tr>
			<td>组织培养介质:Dulbecco 氏改良的鹰培养基 (DMEM), 10% 胎牛血清 (FBS), 1% 青霉素-链霉素-l-谷氨酰胺 (DMEM 10% FBS 1%)</td>
			<td>445毫升 DMEM、50毫升 FBS 和5毫升 100x 1% 青霉素 (100 单位/毫升)-链霉素 (100 µg/毫升)-l-谷氨酰胺 (2 mM)</td>
			<td>贮存在4° c</td>
			<td>该培养基用于维持 Madin-达比犬肾脏 (MDCK) 上皮细胞</td>
			<td></td>
		</tr>
		<tr>
			<td>后感染介质: DMEM 0.3% 牛白蛋白 (ba)、1% 例 (DMEM 0.3% ba 1%)</td>
			<td>491毫升 DMEM, 4.2 毫升 35% BA 和5毫升的100x。</td>
			<td>贮存在4° c</td>
			<td>此介质用于在感染甲型流感病毒后维持 MDCK 细胞 (IAV)</td>
			<td></td>
		</tr>
		<tr>
			<td>10x 磷酸盐缓冲盐水 (10x PBS)</td>
			<td>80克氯化钠, 2 克氯化钾, 11.5 克 Na2HPO4. 7H2O, 2 g 的下一2PO4。添加 ddH2O 最多 1 l 调整 pH 值到7。3</td>
			<td>室温贮存 (RT)</td>
			<td></td>
			<td>灭菌釜</td>
		</tr>
		<tr>
			<td>1x PBS</td>
			<td>用 ddH2O 稀释 10x PBS 1:10</td>
			<td>存储在 RT</td>
			<td></td>
			<td>灭菌釜</td>
		</tr>
		<tr>
			<td>感染媒体: 1x pbs, 0.3% BA, 1% 青霉素-链霉素 (ps) (pbs/BA/ps)</td>
			<td>487毫升 1x PBS 无菌, 4.2 毫升 35% BA 和5毫升 100x 1% 青霉素 (100 单位/毫升)-链霉素 (100 µg/毫升) (PS)</td>
			<td>贮存在4° c</td>
			<td>此介质用于 IAV 感染</td>
			<td></td>
		</tr>
		<tr>
			<td>固定/性溶液: 4% 甲醛, 0.5% 海卫 X-100 稀释在 1x PBS</td>
			<td>400毫升中性缓冲福尔马林 10%, 5 毫升的海卫 X-100 和595毫升的 1x PBS</td>
			<t></t></tr></tbody></table>d > 存储在 RT
			<td>本解用于病毒滴定实验中的 MDCK 细胞的固定和 permeabilize</td>
			<td>在通风柜中准备解决方案, 防止接触甲醛</td>
		
		<tr>
			<td>阻止解决方案: 1x PBS 中的2.5% 牛血清白蛋白 (BSA)</td>
			<td>2.5 克 BSA 97.5 毫升 1x PBS</td>
			<td>贮存在4° c</td>
			<td>本解决方案被用作免疫荧光检测和 ELISAs 的阻断溶液。</td>
			<td>用0.2 µm 过滤器进行过滤消毒。</td>
		</tr>
		<tr>
			<td>抗体稀释液 (1% BSA 在 1x PBS)</td>
			<td>1克 BSA 99 毫升 1x PBS</td>
			<td>贮存在4° c</td>
			<td>本解决方案用于免疫荧光分析和 ELISAs 中的主次抗体稀释</td>
			<td>0.2 µm 过滤器过滤消毒</td>
		</tr>
		<tr>
			<td>0.1% 水晶紫罗兰色溶液</td>
			<td>1克结晶紫在400毫升甲醇。添加600毫升的 ddH2O</td>
			<td>存储在 RT</td>
			<td>本解决方案用于修复和染色 MDCK 细胞在病毒中和化验</td>
			<td></td>
		</tr>
		<tr>
			<td>Tosylsulfonyl phenylalanyl 氯甲基酮 (TPCK)-治疗胰蛋白酶</td>
			<td>在 ddH2O 中准备1毫克/毫升的1,000x 库存解决方案</td>
			<td>贮存在-20 ° c</td>
			<td>TPCK-胰蛋白酶在 IAV 感染中增加</td>
			<td>制作100µl 等分</td>
		</tr>
		<tr>
			<td>帕缓冲区</td>
			<td>10毫米三氯 (pH 8.0), 1 毫米 EDTA, 1% 海卫 X-100, 0.1% 钠胆酸, 0.1% SDS, 140 毫米氯化钠</td>
			<td>贮存在4° c</td>
			<td>此解决方案用于制作细胞提取物</td>
			<td></td>
		</tr>
	

表 1: 组织培养介质和解决方案。

Watch this Scientific Journal Video about Influenza A Virus Studies in a Mouse Model of Infection at JoVE.com

小鼠感染模型中的甲型流感病毒研究免疫学和感染|视频

Influenza A Virus Studies in a Mouse Model of Infection

甲型流感病毒 (IAVs) 是人类重要的呼吸道病原体。为了了解 IAVs 的致病性和对新疫苗方法进行临床前测试, 需要模仿人体生理学的动物模型。在这里, 我们描述的技术, 以评估 IAV 病机, 体液反应和疫苗的效果使用小鼠感染模型。

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 ...

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SCIENTISTS IN ACTION

28.1K 次观看.  University of Rochester School of Medicine and Dentistry. 甲型流感病毒 (IAVs) 是人类重要的呼吸道病原体。为了了解 IAVs 的致病性和对新疫苗方法进行临床前测试, 需要模仿人体生理学的动物模型。在这里, 我们描述的技术, 以评估 IAV 病机, 体液反应和疫苗的效果使用小鼠感染模型。

视频: Influenza A Virus Studies in a Mouse Model of Infection

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

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, ...

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 ...

Using Zebrafish Models of Human Influenza A Virus Infections to Screen Antiviral Drugs and Characterize Host Immune Cell Responses

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare burden. The principle strategy to curtail infections is yearly vaccination. In individuals who have contracted influenza, antiviral drugs can mitigate symptoms. There is a clear and unmet need to develop alternative strategies to combat influenza. Several animal models have been created to model host-influenza interactions. Here, protocols for generating zebrafish models for systemic and localized human influenza A virus (IAV) infection are described. Using a systemic IAV infection model, small molecules with potential antiviral activity can be screened. As a proof-of-principle, a protocol that demonstrates the efficacy of the antiviral drug Zanamivir in IAV-infected zebrafish is described. It shows how disease phenotypes can be quantified to score the relative efficacy of potential antivirals in IAV-infected zebrafish. In recent years, there has been increased appreciation for the critical role neutrophils play in the human host response to influenza infection. The zebrafish has proven to be an indispensable model for the study of neutrophil biology, with direct impacts on human medicine. A protocol to generate a localized IAV infection in the Tg(mpx:mCherry) zebrafish line to study neutrophil biology in the context of a localized viral infection is described. Neutrophil recruitment to localized infection sites provides an additional quantifiable phenotype for assessing experimental manipulations that may have therapeutic applications. Both zebrafish protocols described faithfully recapitulate aspects of human IAV infection. The zebrafish model possesses numerous inherent advantages, including high fecundity, optical clarity, amenability to drug screening, and availability of transgenic lines, including those in which immune cells such as neutrophils are labeled with fluorescent proteins. The protocols detailed here exploit these advantages and have the potential to reveal critical insights into host-IAV interactions that may ultimately translate into the clinic.

Systemic and localized zebrafish infection models for human influenza A virus are demonstrated. Using a systemic infection model, zebrafish can be used to screen antiviral drugs. Using a localized infection model, zebrafish can be used to characterize host immune cell responses.

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

Each year, seasonal influenza outbreaks profoundly affect societies worldwide. In spite of global efforts, influenza remains an intractable healthcare ...

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. ...

A Murine Model of Dengue Virus-induced Acute Viral Encephalitis-like Disease

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is characterized by lethal complications due to plasma leakage, ascites, pleural effusion, respiratory distress, severe bleeding, and organ impairment. A few cases of DENV infection present neurological manifestations; however, studies have not explored DENV-induced neuropathogenesis further. In this study, we present a protocol to use an immunocompetent outbred ICR (Institute of Cancer Research) mouse for investigating the induction of central nervous system (CNS) infection with DENV, followed by the progression of acute viral encephalitis-like disease.

Here, we present a protocol for creating an immunocompetent ICR (Institute of Cancer Research) murine model of central nervous system infection to display the development of neuropathy. Monitoring acute viral encephalitic disorders by identical disease scores could be performed for showing dengue virus-induced neuropathy in vivo.

登革热病毒诱发急性病毒性脑炎样疾病的鼠害模型

Dengue virus (DENV), an arthropod-borne virus transmitted by mosquitoes, may cause the severe disease known as dengue hemorrhagic fever, which is ...

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Evaluation of T Follicular Helper Cells and Germinal Center Response During Influenza A Virus Infection in Mice

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and generation of high-affinity antibodies. In influenza virus infection, robust Tfh and GC B cell responses are induced to facilitate effective virus eradication, which confers a qualified mouse model for Tfh-associated study. In this paper, we described protocols in detection of basic Tfh-associated immune response during influenza virus infection in mice. These protocols include: intranasal inoculation of influenza virus; flow cytometry staining and analysis of polyclonal and antigen-specific Tfh cells, GC B cells and plasma cells; immunofluorescence detection of GCs; enzyme-linked immunosorbent assay (ELISA) of influenza virus-specific antibody in serum. These assays basically quantify the differentiation and function of Tfh cells in influenza virus infection, thus providing help for studies in elucidating differentiation mechanism and manipulation strategy.

This paper describes protocols of evaluating Tfh and GC B response in mouse model of influenza virus infection.

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

T Follicular Helper (Tfh) cells are an independent CD4+&#160;T cell subset specialized in providing help for germinal center (GC) development and ...

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic pathogen upon influenza A virus (IAV) infection. Advanced age enhances host susceptibility to secondary pneumococcal pneumonia and is associated with worsened disease outcomes. The host factors driving those processes are not well defined, in part due to a lack of animal models that reproduce the transition from asymptomatic colonization to severe clinical disease. 
This paper describes a novel mouse model that recreates the transition of pneumococci from asymptomatic carriage to disease upon viral infection. In this model, mice are first intranasally inoculated with biofilm-grown pneumococci to establish asymptomatic carriage, followed by IAV infection of both the nasopharynx and lungs. This results in bacterial dissemination to the lungs, pulmonary inflammation, and obvious signs of illness that can progress to lethality. The degree of disease is dependent on the bacterial strain and host factors. 
Importantly, this model reproduces the susceptibility of aging, because compared to young mice, old mice display more severe clinical illness and succumb to disease more frequently. By separating carriage and disease into distinct steps and providing the opportunity to analyze the genetic variants of both the pathogen and the host, this S. pneumoniae/IAV co-infection model permits the detailed examination of the interactions of an important pathobiont with the host at different phases of disease progression. This model can also serve as an important tool for identifying potential therapeutic targets against secondary pneumococcal pneumonia in susceptible hosts.

A Mouse Model for the Transition of Streptococcus pneumoniae from Colonizer to Pathogen upon Viral Co-Infection Recapitulates Age-Exacerbated Illness

This paper describes a novel mouse model for the transition of pneumococcus from an asymptomatic colonizer to a disease-causing pathogen during viral infection. This model can be readily adapted to study polymicrobial and host-pathogen interactions during the different phases of disease progression and across various hosts.

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

Streptococcus pneumoniae (pneumococcus) is an asymptomatic colonizer of the nasopharynx in most individuals but can progress to a pulmonary and systemic ...

A Luciferase-Fluorescent Reporter Influenza Virus to Track Viral Infections

Source: Chiem, K., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/59890">A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection.</a> J. Vis. Exp. (150), (2019).
This video demonstrates a method to track viral infections in mouse models using a recombinant luciferase and fluorescence protein-expressing bi-reporter Influenza A virus. This method enables both in vivo and ex vivo studies, enabling researchers to gain insights into viral dynamics and the effects of interventions.

一种用于追踪病毒感染的荧光素酶荧光报告性流感病毒

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. In ...

流感病毒在小鼠感染模型中的研究

摘要

探索更多视频

此视频中的章节

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

流感病毒的高通量检测方法

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

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

登革热病毒诱发急性病毒性脑炎样疾病的鼠害模型

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

一种用于追踪病毒感染的荧光素酶荧光报告性流感病毒

流感病毒在小鼠感染模型中的研究

摘要

探索更多视频

此视频中的章节

利用生物发光成像之间调查的协同作用肺炎链球菌 A型流感病毒的婴儿小鼠

流感病毒的高通量检测方法

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

使用人类流感病毒感染的斑马鱼模型到屏幕抗病毒药物和表征宿主免疫细胞反应

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

登革热病毒诱发急性病毒性脑炎样疾病的鼠害模型

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

小鼠感染甲型流感病毒期间T卵泡帮助细胞和胚芽中心反应的评价

病毒合并感染后 肺炎链球菌 从定植者转变为病原体的小鼠模型概括了年龄加重的疾病

一种用于追踪病毒感染的荧光素酶荧光报告性流感病毒

病毒合并感染后肺炎链球菌从定植者转变为病原体的小鼠模型概括了年龄加重的疾病