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

Forskning på influenzavirus i LM-S laboratorium er delvist finansieret af The New York Influenza Center of Excellence (NYICE), et medlem af NIAID Centers of Excellence for Influenza forskning og overvågning (CEIRS). Vi takker Wendy Bates for hendes støtte i korrektioner af manuskriptet.

alle animalske protokoller er beskrevet her blev godkendt af det institutionelle Animal Care og brug udvalg (IACUC) og institutionelle biosikkerhed Udvalget (IBC) på University of Rochester School of Medicine og tandpleje, og i overensstemmelse med anbefalinger i vejledningen for pleje og anvendelse af forsøgsdyr af den nationale forskning Rådet 22. Faciliteter og programmer af Vivarium og Division of laboratorium animalsk Medicine School of Medicine og tandpleje er akkrediteret af AAALAC International og i overensst...

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Musemodel af IAV er almindeligt brugt til i vivo studier af IAV patogenese, immunogenicitet og beskyttelse effektivitet. Den lille størrelse af mus gør dem nemme at manipulere og gemme i forhold til andre dyremodeller såsom fritter eller marsvin. Derudover lethed i form af dyr koster, boliger og reproduktion tillader deres brug i prækliniske vaccination tests hvor stort antal dyr, der er brug for. Især da mus har været brugt i flere forskning discipliner, adskillige molekylære og Immunologi murine reagens...

IAVs er kappebærende virus klassificeret i Orthomyxoviridae familien3. De indeholder otte enkeltstrenget RNA molekyler med negativ polaritet3. Hos mennesker forårsage IAVs årstidens epidemier og lejlighedsvis pandemier af vigtig konsekvens når romanen vira er indført i den menneskelige befolkning4. Derudover overføres sæsonbetonede IAVs højt og hurtigt mellem mennesker producerer en forhøjet økonomiske tab på verdensplan hvert år2,5. IAV symptomer er hoste, tilstoppet næse, feber, utilpashed, hovedpine, anoreksi og myalgi, men virus kan også producere en mere alvorlig sygdom i immunkompromitterede patienter6. I virkeligheden, beregner World Health Organization (WHO), at sæsonbestemt influenzavirus forårsager 300.000-500.000 dødsfald på verdensplan hvert år1. Der er kun to klasser af narkotika i øjeblikket godkendt af Food and Drug Administration (FDA) for influenza profylakse og behandling hos mennesker: neuraminidasetypen (NA) hæmmere (fx, oseltamivir) og blokkere M2 ion kanal (f.eks., amantadin); fremkomsten af resistente virus varianter er imidlertid en stigende bekymring. Vaccination, derfor, stadig den bedste medicinske mulighed for at beskytte mennesker mod IAVs infektioner. Til dato har tre typer af influenza vacciner licenseret af FDA til human brug er tilgængelige: rekombinant viral hemagglutinin (HA) protein vacciner, inaktiverede vacciner mod influenza (&amp; Videoside) og live-svækkede influenza vacciner (LAIV)5, 7. de tre vacciner er designet til at fremkalde adaptive immunrespons mod viral HA protein, det store mål af neutraliserende antistoffer mod IAVs.
En valideret musemodel at studere IAV infektion i vivo
Dyremodeller har været brugt til at studere, blandt andre, IAV patogenese8,9,10,11, viral faktorer, der bidrager til sygdom12 og/eller viral transmission13 ,14, og til at teste effekten af nye vacciner og antivirale lægemidler9,10,15. Mus (Mus musculus) er den mest omfattende brugte dyremodel for IAV forskning af flere grunde: 1) immunsystemet er evolutionært svarende til at præsentere i mennesker; 2) lave omkostninger, herunder animalske køb, bolig og reproduktion; 3) lille størrelse nemt manipulere og gemme; 4) minimal vært variation at opnå homogen svar og resultater; 5) et stort kendskab til mus biologi, herunder genomet sekvens; 6) mange tilgængelige Molekylærbiologi og/eller immunologi reagenser; 7) tilgængelige knock out (KO) mus at studere bidrag af en given vært protein på viral infektion; og 8) flere mus stammer, der kan udnyttes til at evaluere det genetiske grundlag for infektioner.
Der er flere mus-stammer, der er i øjeblikket tilgængelig at studere IAV i vivo. Alder, immunstatus, sex, genetiske baggrund og mus stamme samt ruter af infektion, dosis og viral stammer alle påvirke resultatet af IAV infektion i mus. De mest almindelige mus stammer anvendes i IAV forskning er C57BL/6, BALB/C og mere for nylig, DBA.2 mus, da de er mere modtagelige for IAV sygdom end de to tidligere stammer16,17,18, 19 , 20. vigtigere, immunresponset også kan være anderledes afhængigt af mus stamme18,19,20. Det er således meget vigtigt at inddrive alle de tilgængelige oplysninger om mus og IAV stamme til at vælge den bedste løsning for forsøget skal gennemføres.
Selv om mus er en god dyremodel af infektion i vivo undersøgelser med IAV, har de flere begrænsninger, der skal overvejes i den eksperimentelle design. For eksempel, er en stor begrænsning ved at bruge mus i vivo undersøgelser, at IAVs ikke overfører blandt mus. Således, for transmission studier, mere accepteret dyremodeller (fx, fritter eller marsvin) er anvendte16,17,21. Derudover er der adskillige forskelle mellem manifestationer af IAV i mus og mennesker. I modsætning til mennesker udvikler mus ikke feber efter IAV infektion; omvendt præsenterer de med hypotermi16,17. I mus, er IAV replikering koncentreret i de nedre luftveje (lungerne) i stedet for de øvre luftveje. Således er virulens af IAV i mus ikke altid korreleret til den, set hos mennesker. Helt, fordi fordelene, der opvejer de begrænsede ulemper, mus repræsenterer det første dyr model anvendes til at evaluere influenza viral patogenese, immunogenicitet og beskyttende effekt i undersøgelser af vaccine og antivirale. Desuden ville det ikke være etisk acceptabelt at udføre undersøgelser med IAV bruger store dyremodeller uden foregående beviser i en lille dyremodel af IAV infektion. I dette manuskript, vi beskriver, hvordan at inficere mus intranasalt (i.n.) med IAV, hvordan man kan overvåge sværhedsgraden og fremskridt af viral infektion og hvordan man udfører eksperimenter skal vurdere humorale immunrespons og beskyttelse effektivitet.

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			<td height="40" style="height:40px;width:295px;">Madin-Darby Canine Kidney (MDCK) epithelial cells</td>
			<td style="width:119px;">ATCC</td>
			<td style="width:123px;">CCL-34</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Six- to eight-week-old female C57BL/6 mice</td>
			<td style="width:119px;">National Cancer Institute (NCI)</td>
			<td>01XBE</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Turckey red blod cells</td>
			<td style="width:119px;">Biolink Inc</td>
			<td style="width:123px;"></td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td style="width:119px;">Corning Cellgro</td>
			<td style="width:123px;">15-013-CV</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:119px;">Seradigm</td>
			<td style="width:123px;">1500-050</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Penicillin/Streptomycin/L-Glutamine (PSG) 100X</td>
			<td style="width:119px;">Corning</td>
			<td style="width:123px;">30-009-CI</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Penicillin/Streptomycin (PS) 100X</td>
			<td style="width:119px;">Corning</td>
			<td style="width:123px;">30-00-CI</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Bovin Albumin solution (BA)</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:123px;">A7409</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Bovin Serum Albumin (BSA)</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:123px;">A9647</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin</td>
			<td style="width:119px;">Sigma-Aldrich</td>
			<td style="width:123px;">T8802</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Neutral Buffered Formalin 10%</td>
			<td style="width:119px;">EMD</td>
			<td style="width:123px;">65346-85</td>
			<td style="width:121px;">Store at RT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Triton X-100</td>
			<td style="width:119px;">J.T.Baker</td>
			<td style="width:123px;">X198-07</td>
			<td style="width:121px;">Store at RT</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Monoclonal Antibody anti-NP Influenza A Virus HB-65</td>
			<td style="width:119px;">ATTC</td>
			<td style="width:123px;">H16-L10-4R5</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Polyclonal rabbit anti-mouse immunoglobulins/FITC</td>
			<td style="width:119px;">Dako</td>
			<td style="width:123px;">F0261</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:295px;">ECL Anti-mouse IgG, Horseradish Peroxidase linked whole antibody</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:123px;">LNA931V/AG</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">TMB substrate set</td>
			<td style="width:119px;">BioLegend</td>
			<td style="width:123px;">421101</td>
			<td style="width:121px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Vmax Kinetic plate reader</td>
			<td style="width:119px;">Molecular Devices</td>
			<td style="width:123px;"></td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Dounce Tissue Grinders</td>
			<td style="width:119px;">Thomas Scientific</td>
			<td style="width:123px;">7722-7</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Receptor destroying enzyme, RDE (II)</td>
			<td style="width:119px;">Denka Seiken Co.</td>
			<td style="width:123px;">370013</td>
			<td style="width:121px;">Store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Crystal Violet</td>
			<td style="width:119px;">Fisher Scienctific</td>
			<td style="width:123px;">C581-100</td>
			<td style="width:121px;">Store at RT</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">96-well Cell Culture Plate</td>
			<td style="width:119px;">Greiner Bio-one</td>
			<td style="width:123px;">655-180</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Cell Culture dishes 100mm</td>
			<td style="width:119px;">Greiner Bio-one</td>
			<td style="width:123px;">664-160</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Nunc MicroWell 96-Well Microplates</td>
			<td style="width:119px;">Thermo Fisher Scienctific</td>
			<td style="width:123px;">269620</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:295px;">Nunc 96-Well Polystyrene Conical Bottom MicroWell Plates</td>
			<td style="width:119px;">Thermo Fisher Scienctific</td>
			<td style="width:123px;">249570</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Puralub Vet Ointment</td>
			<td style="width:119px;">Dechra</td>
			<td style="width:123px;">9N-76855</td>
			<td style="width:121px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:295px;">Fluorescent microscope</td>
			<td style="width:119px;">Olympus</td>
			<td>Olympus IX81</td>
			<td style="width:121px;"></td>
		</tr>
	</tbody>
</table>

influenza a virus studies in a mouse model of infection

Influenzavirus forårsager over 500.000 dødsfald på verdensplan1 og er forbundet med en årlig omkostning på 12-14 milliarder dollars i USA alene overvejer direkte medicinsk og indlæggelse udgifter og arbejde fravær2. Dyremodeller er afgørende i Influenza A virus (IAV) undersøgelser at vurdere viral patogenese, vært-patogen interaktioner, immunrespons, og effekten af nuværende og/eller roman vaccine tilgange og antivirale lægemidler. Mus er en fordelagtig lille dyremodel, fordi deres immunsystem er evolutionært magen til, findes i mennesker, de er tilgængelige fra kommercielle leverandører som genetisk identiske emner, der er flere stammer, der kan udnyttes til at evaluere det genetiske grundlag for infektioner, og de er forholdsvis billig og let at manipulere. For at sammenfatte IAV infektion hos mennesker via luftvejene, bedøvede mus først før intranasal podning med smitsomme IAVs under ordentlig biosikkerhed indeslutning. Efter infektion bestemmes patogenesen af IAVs overvågning dagligt sygelighed (krop vægttab) og dødelighed (overlevelse). Viral patogenese kan derudover også evalueres ved at vurdere virus replikering i øvre (næseslimhinden) eller lavere (lungerne) luftveje af inficerede mus. Humorale svar på IAV infektion hurtigt kan vurderes af non-invasiv blødning og påvisning af sekundær antistof assays sigter mod at påvise forekomst af total eller neutraliserende antistoffer. Her beskriver vi de almindelige metoder bruges til at inficere mus intranasalt (i.n) med IAV og evaluere patogenese, humorale immunrespons og beskyttelse virkning.

Karakterisering af viral patogenese i mus
Patogenesen af IAV er relateret til sygelighed og dødelighed forårsaget af sin infektion. Disse to parametre kan evalueres i mus let: IAV sygelighed er forbundet med kroppen vægttab i inficerede mus og procentdelen af overlevelse vil angive dødelighed (figur 1). Kropsvægt og overlevelse i IAV-inficerede mus er normalt overvåges dagligt i mindst to uger ef...

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

Influenza A Virus Studies in a Mouse Model of Infection

Influenza A virus (IAVs) er vigtige menneskelige respiratorisk patogener. At forstå IAVs patogenicitet og Udfør præklinisk afprøvning af nye vaccine tilgange er dyremodeller efterligne menneskelige fysiologi påkrævet. Her, beskriver vi teknikker til vurdering af IAV patogenese, humorale svar og vaccine virkning ved hjælp af en musemodel af infektion.

Influenza A Virus undersøgelser i en musemodel af infektion

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

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 ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza virusformering i befrugtede hønseæg

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous ...

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

Co-immunoudfældning af musen Mx1 Protein med influenza A-virus nukleoprotein

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

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Nasal Wipes for influenza A-virus Detection og Isolering fra svin

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

Measuring Attachment and Internalization of Influenza A Virus in A549 Cells by Flow Cytometry

Måling Attachment og internalisering af influenza A-virus i A549-celler ved flowcytometri

Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrofil Isolering og analyse for at fastslå deres rolle i lymfom Cell Følsomhed til terapeutiske midler

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

En miniature Glycan Microarray Assay til Vurdering Aviditet og specificitet influenza A-virus hæmagglutininer

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

In vitro Demontering af influenza A-virus capsider ved gradientcentrifugering

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

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

Imaging celle interaktion i trakeal slimhinde under Influenza virusinfektion ved hjælp af to-foton Intravital mikroskopi

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

En luciferase-fluorescerende reporter influenza virus til levende billeddannelse og kvantificering af viral infektion

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