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?...

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、<sup class="xr...

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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28.2K 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 ...

Co-immunoprecipitation of the Mouse Mx1 Protein with the Influenza A Virus Nucleoprotein

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

免疫共沉淀鼠标MX1蛋白与流感病毒核蛋白

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions ...

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 ...

流感病毒在小鼠感染模型中的研究

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