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

أبحاث على فيروس الإنفلونزا في مختبر LM-S تموله جزئيا "نيويورك الإنفلونزا مركز التميز" (نيس)، عضو نييد مراكز التميز "الإنفلونزا للبحث" والمراقبة (سيرس). ونحن نشكر بيتس يندى لها الدعم في تصويبات المخطوطة.

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نموذج الماوس عياف يستخدم على نطاق واسع في فيفو الدراسات فعالية عياف المرضية، الاستمناع والحماية. الحجم الصغير للفئران يجعلها سهلة لمعالجة وتخزين مقارنة بنماذج حيوانية أخرى مثل النموس أو خنازير غينيا. وعلاوة على ذلك، السهولة من حيث تكلفة الحيوان، السكن والإنجاب السماح باستخدامها في...

IAVs هي الفيروسات المغلفة مصنفة في أسرة اورثوميكسوفيريداى 3. أنها تحتوي على ثمانية جزيئات الحمض النووي الريبي المفرد-الذين تقطعت بهم السبل مع الأقطاب السالبة3. في البشر، وتسبب IAVs الأوبئة الموسمية والأوبئة العرضية تترتب عليها آثار هامة عندما يتم إدخال فيروسات جديدة في عدد السكان4. وعلاوة على ذلك، IAVs الموسمية العالية وسرعة تنتقل بين البشر تنتج خسارة اقتصادية مرتفعة في جميع أنحاء العالم كل سنة2،5. وتشمل الأعراض عياف السعال، احتقان الآنف، الحمى والشعور بالضيق، والصداع، وفقدان الشهية وألم عضلي، ولكن الفيروس يمكن أن تنتج أيضا مرض أكثر شدة في مرضى المناعة6. في الواقع، أن منظمة الصحة العالمية (WHO) يحسب أن فيروسات الإنفلونزا الموسمية تتسبب في 300,000-500,000 حالة وفاة في العالم كل سنة1. وهناك فقط فئتين من الأدوية المعتمدة حاليا للأغذية والدواء (FDA) للوقاية من الإنفلونزا ومعاملة البشر: مثبطات النورامينيداز (غ) (مثلاً، الأوسيلتاميفير) وسد القناة أيون M2 (مثلاً، أمانتادين)؛ ومع ذلك، ظهور متغيرات الفيروس المقاوم للأدوية تثير قلقا متزايداً. ولذلك، يظل التطعيم، خيار طبية أفضل لحماية البشر ضد الإصابات IAVs. وحتى الآن، ثلاثة أنواع من الإنفلونزا تتوفر لقاحات مرخصة من قبل إدارة الأغذية والعقاقير للاستخدام البشري: اللقاحات البروتين المؤتلف الفيروسية هيماغلوتينين (ها)، إبطال لقاحات الإنفلونزا (IIV)، ويعيش يخفف الإنفلونزا اللقاحات (لايف)5، 7-اللقاحات الثلاثة مصممة لحفز الاستجابة المناعية التكيفية ضد البروتين الفيروسي هكتار، هدفا رئيسيا لتحييد الأجسام المضادة ضد IAVs.
نموذج ماوس المصادق عليها لدراسة IAV العدوى في فيفو
وقد استخدمت نماذج حيوانية لدراسة، بين أمور أخرى، IAV المرضية8،،من910،11، العوامل الفيروسية التي تساهم في المرض12 و/أو انتقال العدوى الفيروسية13 ،14، والأدوية لاختبار فعالية اللقاحات الجديدة أو المضادة للفيروسات9،،من1015. الفئران (Mus musculus) هي نموذج الحيوانية المستخدمة على نطاق واسع معظم البحوث عياف لعدة أسباب: 1) المناعي يشبه تقحم إلى أن هناك حاليا في البشر؛ 2) منخفضة التكلفة، بما في ذلك شراء الحيوان والسكن والإنجاب؛ 3) صغيرة الحجم سهولة التعامل معها وتخزينها؛ 4) تقلب المضيف الحد الأدنى للحصول على استجابات متجانسة والنتائج؛ 5) على معرفة كبيرة بالبيولوجيا الفئران، بما في ذلك تسلسل الجينوم؛ 6) العديد من البيولوجيا الجزيئية المتاحة و/أو الكواشف علم المناعة؛ 7) متاح ضرب الفئران (كو) لدراسة مساهمة البروتين مضيف معين على العدوى الفيروسية؛ و، 8) سلالات الماوس المتعددة التي يمكن استغلالها لتقييم الأساس الوراثي للعدوى.
وهناك عدة سلالات الماوس المتوفرة حاليا لدراسة عياف في فيفو. العمر والحالة المناعية، والجنس، والسلالة الوراثية الخلفية والماوس، فضلا عن طرق العدوى والجرعة وسلالات فيروسية التأثير على نتيجة الإصابة إياف في الفئران. هي سلالات الماوس الأكثر شيوعاً المستخدمة في البحوث IAV C57BL/6، بالب/ج، وفي الآونة الأخيرة، الفئران DBA.2 نظراً لأنهم أكثر عرضه للأمراض إياف من السلالات السابقة هما16،،من1718، 19 , 20-الأهم من ذلك الاستجابة المناعية أيضا يمكن أن تختلف تبعاً للماوس السلالة18،،من1920. وهكذا، المهم جداً لاسترداد كافة المعلومات المتوفرة عن الماوس وسلالة إياف لاختيار أفضل خيار للتجربة التي ستجري.
على الرغم من أن الفأر نموذج حيوان جيد للعدوى في فيفو الدراسات مع عياف، لديهم العديد من القيود، التي تحتاج إلى النظر في التصميم التجريبي. على سبيل المثال، قيداً رئيسيا لاستخدام الفئران في فيفو الدراسات أن لا ترسل IAVs بين الفئران. وهكذا، لانتقال قبول الدراسات، أكثر النماذج الحيوانية (مثلاً، فرت أو خنازير غينيا) هي المستعملة16،،من1721. وبالإضافة إلى ذلك، هناك العديد من الاختلافات بين مظاهر عياف في الفئران والبشر. بخلاف البشر، لا تتطور الفئران الحمى عند عياف العدوى؛ على العكس من ذلك فإنها تعرض مع انخفاض درجة حرارة الجسم16،17. ويتركز في الفئران، عياف النسخ المتماثل في الجهاز التنفسي السفلي (الرئتين) بدلاً من الخطوط الجوية العليا. وهكذا، ضراوة عياف في الفئران لا يرتبط دائماً بأن ينظر في البشر. بالإجمال، نظراً للمزايا التي تفوق المساوئ محدودة، الماوس يمثل أول نموذج الحيوانات المستخدمة لتقييم أمراض الإنفلونزا الفيروسية، الاستمناع والنجاعة الوقائية في دراسات اللقاحات والأدوية المضادة. وعلاوة على ذلك، سيكون غير مقبولة أخلاقيا لإجراء دراسات مع عياف باستخدام نماذج حيوانية كبيرة دون الأدلة السابقة في نموذج حيوانات صغيرة للعدوى عياف. في هذه المخطوطة، ونحن تصف كيفية تصيب الفئران إينتراناسالي (i.n.) مع عياف وكيفية مراقبة بشدة والتقدم للعدوى الفيروسية وكيفية إجراء التجارب اللازمة لتقييم الاستجابات المناعية خلطيه وفعالية الحماية.

<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

فيروسات الإنفلونزا تتسبب في وفاة ما يزيد على 500,000 في العالم1 ، وترتبط بتكلفة سنوية تبلغ 12 بیلیون دولار في الولايات المتحدة وحدها النظر الطبية المباشرة، ونفقات العلاج في المستشفيات و التغيب عن العمل2. نماذج حيوانية تشكل عاملاً حاسما في فيروس الإنفلونزا أ الفيروس (عياف) دراسات لتقييم المرضية الفيروسية، والتفاعلات المضيف الممرض، والاستجابات المناعية، وفعالية اللقاحات الحالية و/أو رواية نهج فضلا عن الأدوية المضادة للفيروسات. الفئران نموذج حيوانات صغيرة مفيدة لأن نظامهم المناعي تقحم مشابهة لتلك الموجودة في البشر، فمتوفرة من البائعين التجاريين كمواضيع متطابقة وراثيا، وهناك سلالات متعددة يمكن استغلالها لتقييم الأساس الوراثي للإصابة، وغير مكلفة نسبيا وسهلة للتلاعب. أن الخص عياف الإصابة في البشر عبر الخطوط الجوية، أولاً يتم تخديره الفئران قبل التلقيح داخل الآنف مع IAVs المعدية تحت الاحتواء السلامة الأحيائية السليمة. بعد الإصابة، تتحدد الآلية المرضية ل IAVs الرصد اليومي الاعتلال (فقدان وزن الجسم) ومعدل الوفيات (البقاء على قيد الحياة). وبالإضافة إلى ذلك، يمكن أيضا تقييم المرضية الفيروسية بتقييم النسخ المتماثل الفيروس في الأعلى (الغشاء المخاطي للأنف) أو الجهاز التنفسي (الرئتين) أقل من الفئران المصابة. يمكن تقييم استجابات خلطيه عند الإصابة عياف سريعاً بالنزيف غير الغازية وكشف الأجسام المضادة الثانوية فحوصات تهدف إلى الكشف عن وجود مجموعة أو تحييد الأجسام المضادة. وهنا، يمكننا وصف الطرق الشائعة المستخدمة لتصيب الفئران إينتراناسالي (i.n) مع عياف وتقيم المرضية، والاستجابات المناعية خلطيه وفعالية الحماية.

وصف المرضية الفيروسية في الفئران
أمراض ال عياف تتصل المراضة والوفيات الناجمة عن الإصابة به. ويمكن تقييم هذه معلمتين من المعلمات في الفئران بسهولة: الاعتلال عياف يرتبط بفقدان وزن الجسم في الفئران المصابة وسوف تبين النسبة المئوية لبقاء معد?...

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

دراسات فيروس الأنفلونزا أ في نموذج الفأر للعدوى | علم المناعة والعدوى | فيديو

Influenza A Virus Studies in a Mouse Model of Infection

فيروسات الإنفلونزا A (IAVs) هي العوامل الممرضة التنفسية البشرية الهامة. لفهم الحالة المرضية التي تحدثها IAVs وإجراء الاختبار السريري نهج لقاح الرواية، نماذج حيوانية تقليد الفسيولوجيا البشرية مطلوبة. هنا، نحن وصف تقنيات تقييم IAV المرضية واستجابات خلطيه وفعالية اللقاحات باستخدام نموذج الفأر من العدوى.

فيروس الإنفلونزا أ الفيروس دراسات في نموذج ماوس للإصابة

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 Views.  University of Rochester School of Medicine and Dentistry. فيروسات الإنفلونزا A (IAVs) هي العوامل الممرضة التنفسية البشرية الهامة. لفهم الحالة المرضية التي تحدثها IAVs وإجراء الاختبار السريري نهج لقاح الرواية، نماذج حيوانية تقليد الفسيولوجيا البشرية مطلوبة. هنا، نحن وصف تقنيات تقييم IAV المرضية واستجابات خلطيه وفعالية اللق...

Video: 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.

باستخدام التصوير Bioluminescent بالتحقيق في التآزر بين العقدية الرئوية والأنفلونزا من الفيروسات في الفئران الرضع

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

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.

الإنتاجية العالية لطريقة الكشف عن فيروس الإنفلونزا

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.

إدارة الأنف المؤتلف لقاحات الأنفلونزا في همي ماوس نماذج لدراسة الأغشية المخاطية الحصانة

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.

شارك في مناعي من البروتين ماوس MX1 مع الأنفلونزا من الفيروسات البروتين النووي

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.

باستخدام نماذج الزرد من الإنسان الأنفلونزا فيروس العدوى الى الشاشة المضادة للفيروسات المخدرات وتوصيف المضيف المناعي الردود خلية

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.

تصوير تفاعل الخلية في الغشاء المخاطي الرغامى أثناء عدوى فيروس الإنفلونزا باستخدام الفحص المجهري إينترافيتال اثنين-فوتون

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.

نموذج مورين من مرض التهاب الدماغ الفيروسي الحاد الناجم عن فيروس حمى الضنك

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.

فيروس أنفلونزا مراسل لوسيفيراز الفلورسنت للتصوير الحي والقياس الكمي للعدوى الفيروسية

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.

تقييم T الخلايا المساعدة الجريب و استجابة مركز Germinal أثناء عدوى فيروس الأنفلونزا A في الفئران

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.

نموذج فأر لانتقال العقدية الرئوية من المستعمر إلى العامل الممرض عند العدوى الفيروسية المشتركة يلخص المرض المتفاقم حسب العمر

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

فيروس الإنفلونزا أ الفيروس دراسات في نموذج ماوس للإصابة

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