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

LM S 研究室でインフルエンザ ウイルスに関する研究が部分的に、ニューヨーク インフルエンザ センターの卓越性 (NYICE)、インフルエンザ研究と監視 (CEIRS) のための卓越性の NIAID センターのメンバーによって資金を供給します。ウェンディ ・ ベイツは、原稿の修正に彼女のサポートを感謝いたします。

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IAV マウス モデル広く IAV 病態、免疫原性と保護の有効性のvivo の研究のため。マウスのサイズが小さいため簡単に操作して、フェレット、モルモットなど他の動物モデルと比較して保存します。また、動物がコストの面で使いやすさ、ハウジングおよび再生に使用できる多数の動物が必要となる予防接種前臨床テストで。とりわけ、マウスが使用されているので複数の研究分野は、い?...

IAVs は、オルトミクソ ウイルス科の家族3に分類されるエンベロープ ウイルスです。負の極性3と 8 の単一座礁させた RNA の分子が含まれます。ヒトでは、IAVs は、新規ウイルスは人間の人口4で導入されたときに、季節性のエピデミックおよび重要な結果の時折パンデミックを引き起こします。また、季節 IAVs は、生産の世界的な高い経済損失毎年2,5人間高と急速に送信されます。IAV 症状咳、鼻づまり、発熱、倦怠感、頭痛、食欲不振、筋肉痛、しかし、ウイルス免疫不全患者6のより深刻な病気を引き起こすことができます。実際には、世界保健機関 (WHO) は、季節性インフルエンザのウイルスは、毎年1300,000-500,000 死亡、世界を引き起こすことを計算します。現在のインフルエンザ予防とヒトの治療食品医薬品局 (FDA) によって承認された医薬品の唯一の 2 つのクラスがある: ノイラミニダーゼ (NA) 阻害剤 (例えばオセルタミビル) と (は例えば、M2 イオン チャネルのブロッカーアマンタジン);ただし、薬剤耐性ウイルスの亜種の出現は、関心の高まりです。予防接種は、したがって、IAVs 感染症から人間を守る最良の医療の選択肢になります。までに、インフルエンザの 3 つのタイプ人間の使用のために FDA によって認可されるワクチンが利用可能な: 組換えウイルス赤血球凝集素 (HA) 蛋白ワクチン、不活化インフルエンザ ワクチン (IIV) とライブ弱毒インフルエンザ ワクチン (LAIV)5,7. 3 つのワクチンは、ウイルスの HA 蛋白質、IAVs に対する中和抗体の主要なターゲットに対する適応免疫応答を誘導するために設計されています。
IAV 感染研究生体内で検証済みのマウス ・ モデル
要因を検討し、とりわけ、IAV 病態8,9,10,11, ウイルス病12および/またはウイルス伝送13 に貢献する動物モデルを使用されています。 ,14,9,10,15を薬新しいワクチンや抗ウイルス剤の有効性をテスト。マウス (ハツカネズミ) は、いくつかの理由 IAV 研究の最も広く使用された動物モデル: 1) 免疫系は進化的に似たような人間の存在を2) 低コストなど動物の購入、住宅再生;簡単に操作および格納; 3) 小型サイズ均一な反応と結果を取得する 4) 最小ホスト変動5) マウス生物学、ゲノム シーケンスを含む大規模な知識6) 多くの利用可能な分子生物学や免疫学の試薬;7) 使用可能なノックアウト (KO) マウス ウイルス感染の特定のホスト蛋白質の貢献を研究するには8) 感染症の遺伝的な根拠を評価するために利用できる複数のマウス系統。
体内IAV 勉強する現在利用可能ないくつかのマウスの系統があります。感染症, 線量, ウイルス株は、すべてのルートと同様、年齢、免疫状態、セックス、遺伝的背景とマウスひずみ IAV 感染マウスの結果に影響します。彼らは元系統16,17,18,より IAV 病気にかかりやすいので、IAV 研究で使用される最も一般的なマウス系統は BALB/C および、もっと最近、DBA.2 マウス C57BL/619,20します。 重要なことは、免疫反応も異なることがありますマウスひずみ18,19,20によって。したがって、マウスの IAV 緊張が行われる実験に最適なオプションを選択するすべての入手可能な情報を回復する非常に重要です。
マウスは、IAV と研究生体内で感染症の動物モデルは、実験的なデザインで考慮する必要があるいくつかの制限がある.例えば、マウスを用いた生体内での研究の主な制限は、IAVs をマウスの間で送信しないことです。したがって、伝送の研究より受け入れられる動物モデル (例えばフェレット、モルモット) が使用される16,17,21。さらに、マウスで経年の症状と人間のいくつかの違いがあります。人間と違って、マウスを開発しない IAV 感染時に発熱逆に、彼らは低体温症16,17を提示します。マウス、IAV レプリケーションは上気道ではなく下気道 (肺) に集中しています。したがって、IAV のマウスに対する病原性は人間に見られるように常に相関しません。完全に、メリットが限られている不利な点を上回る、のでマウスはインフルエンザ ウイルスの病原性、免疫原性およびワクチンと抗ウイルス剤の研究で予防効果を評価するために使用する最初の動物モデルを表します。さらに、それは IAV IAV 感染の小さな動物モデルで以前証拠なし大動物モデルを使用して、研究を実施する倫理的に許容できません。本稿で述べるマウスの鼻腔内感染する方法 (i.n と) IAV、重大度とウイルス感染の進行状況を監視する方法および液性免疫応答と保護の有効性を評価するために必要な実験を実施する方法。

<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

インフルエンザ ウイルスは引き起こす以上 500,000 の死世界1と単独で直接医療と入院費と仕事欠勤2を考慮した米国の 120 億ドルの年間コストに関連付けられています。動物モデルはウイルスの病原性、宿主-病原体相互作用、免疫反応を評価する A 型インフルエンザ ウイルス (経年) 研究で重要な現在および/または新規ワクチンの効果は抗ウイルス薬だけでなく、アプローチします。彼らの免疫システムは進化的人間に似ているので、マウスが有利な小さな動物モデル、彼らが遺伝的に同一科目として商用ベンダーから利用できる、複数の系統を評価するために悪用される可能性があります。感染症の遺伝的な根拠、比較的安価で操作が容易です。IAV 感染気道を介して人間を要約するには、マウスはバイオ セーフティの適切な抑制の下で伝染性の IAVs 鼻腔内接種前に麻酔最初。感染後 IAVs の病因は毎日 (体重) の罹患率と死亡率 (生存率) 率を監視することによって決定されます。さらに、ウイルスの病因は上 (鼻粘膜) または下 (肺) 感染マウスの気道でウイルスの複製を評価することによっても評価できます。IAV 感染体液性反応を非侵襲的な出血、二次抗体の検出によって急速に評価できる合計の存在を検出または中和抗体アッセイ向け。ここでは、方法について述べる、一般的なマウスの鼻腔内感染するために使用 (i.n) IAV とし、病因、液性免疫応答と保護の有効性を評価。

マウスにおけるウイルスの病因の評価
IAV の病因罹患率とその感染による死亡率に関連しています。これらの 2 つのパラメーターをマウスで簡単に評価できる: IAV 罹患率は感染マウスの体重減少と関連し、生存率は死亡率 (図 1) を示します。体重、IAV 感染マウスの生存率は通常感染8<s...

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

マウス感染モデルにおけるインフルエンザ A ウイルスの研究 |免疫学と感染症 |ビデオ

Influenza A Virus Studies in a Mouse Model of Infection

インフルエンザ A ウイルス (IAVs) は、重要な人間の呼吸器病原体です。IAVs の病原性を理解して新しいワクチン接種のアプローチの臨床テストを実行するには、人間の生理を模倣した動物モデルが必要です。ここでは、IAV 病因、体液性応答および感染症のマウスモデルを用いたワクチンの有効性を評価する手法について述べる。

感染症のマウスモデルでの A 型インフルエンザ ウイルス研究

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 ビュー.  University of Rochester School of Medicine and Dentistry. インフルエンザ A ウイルス (IAVs) は、重要な人間の呼吸器病原体です。IAVs の病原性を理解して新しいワクチン接種のアプローチの臨床テストを実行するには、人間の生理を模倣した動物モデルが必要です。ここでは、IAV 病因、体液性応答および感染症のマウスモデルを用いたワクチンの有効性を評価する手法について述べる。

ビデオ: 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.

の間に相乗効果を調査するために生物発光イメージングを使用して肺炎球菌幼児マウスで>とインフルエンザウイルス

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

免疫・感染学

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.

インフルエンザウイルスのためのハイスループット検出方法

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.

キメラマウスモデルにおける組換えインフルエンザワクチンの鼻腔内投与は、粘膜免疫を研究するために、

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.

インフルエンザAウイルス核を持つマウスMx1のタンパク質の共免疫沈降

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.

抗ウイルス薬をスクリーニングし、宿主の免疫細胞応答を特徴づけるために、ヒトインフルエンザAウイルス感染のゼブラフィッシュモデルを使用して、

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.

イメージング 2 光子生体顕微鏡を用いたインフルエンザ ウイルス感染症の中に気管粘膜の細胞相互作用

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.

デングウイルス誘発急性ウイルス脳炎様疾患のマウスモデル

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.

ウイルス感染のライブイメージングと定量化のためのルシフェラーゼ蛍光レポーターインフルエンザウイルス

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.

マウスにおけるインフルエンザAウイルス感染時のT濾胞ヘルパー細胞と胚葉センター応答の評価

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.

肺炎 連鎖 球菌のウイルス重感染によるコロニー形成者から病原体への移行に関するマウスモデルが加齢増悪疾患を再現

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

感染症のマウスモデルでの A 型インフルエンザウイルス研究

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感染症のマウスモデルでの A 型インフルエンザ ウイルス研究

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感染症のマウスモデルでの A 型インフルエンザウイルス研究