מחקר זה מומן על ידי עמיתי הקרן Wellcome בכיר הוענק לאיאן גודפלו, וגם מארי קירי תוך אחוות האירופי (FP7 המועצה האירופית למחקר) הוענק ארמנדו אריאס. אנו רוצים להודות איו הונג צ&#39;ונג, ד&quot;ר רבקה וד&quot;ר רובי מייק סקינר שנתן לנו רשות להשתמש bioanalyser Agilent שלהם ולעזור בניהול דגימות רנ&quot;א.

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outbreaks+of+gastroenteritis+associated+with+noroviruses+on+cruise+ships--United+States.">Outbreaks of gastroenteritis associated with noroviruses on cruise ships--United States.</a> MMWR Morb. Mortal. Wkly. Rep. 51, 1112-1115 (2002).</li><li>Lopman, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+cost+of+nosocomial+gastroenteritis,+Avon,+England,+2002-2003.">Epidemiology and cost of nosocomial gastroenteritis, Avon, England, 2002-2003.</a> Emerg. Infect. Dis. 10, 2002-2003 (2004).</li><li>Duizer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+efforts+to+cultivate+noroviruses.">Laboratory efforts to cultivate noroviruses.</a> J. Gen. Virol. 85, 79-87 (2004).</li><li>Karst, S. M., Wobus, C. E., Lay, M., Davidson, J., Virgin, H. W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STAT1-dependent+innate+immunity+to+a+Norwalk-like+virus.">STAT1-dependent innate immunity to a Norwalk-like virus.</a> Science. 299, 1575-1575 (2003).</li><li>Wobus, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+Norovirus+in+cell+culture+reveals+a+tropism+for+dendritic+cells+and+macrophages.">Replication of Norovirus in cell culture reveals a tropism for dendritic cells and macrophages.</a> PLoS Biol. 2, e432 (2004).</li><li>Bok, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+norovirus+replication+by+morpholino+oligomers+targeting+the+5'-end+of+the+genome.">Inhibition of norovirus replication by morpholino oligomers targeting the 5'-end of the genome.</a> Virology. 380, 328-337 (2008).</li><li>Kim, Y., Thapa, M., Hua, D. H., Chang, K. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biodegradable+nanogels+for+oral+delivery+of+interferon+for+norovirus+infection.">Biodegradable nanogels for oral delivery of interferon for norovirus infection.</a> Antiviral Res. 89, 165-173 (2011).</li><li>McFadden, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Norovirus+Regulation+of+the+Innate+Immune+Response+and+Apoptosis+Occurs+via+the+Product+of+the+Alternative+Open+Reading+Frame+4.">Norovirus Regulation of the Innate Immune Response and Apoptosis Occurs via the Product of the Alternative Open Reading Frame 4.</a> PLoS Pathog. 7, e1002413 (2011).</li><li>Kormelink, R., van Poelwijk, F., Peters, D., Goldbach, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-viral+heterogeneous+sequences+at+the+5'+ends+of+tomato+spotted+wilt+virus+mRNAs.">Non-viral heterogeneous sequences at the 5' ends of tomato spotted wilt virus mRNAs.</a> J. Gen. Virol. 73, 2125-2128 (1992).</li><li>Shuman, S., Hurwitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+mRNA+capping+by+vaccinia+virus+guanylyltransferase:+characterization+of+an+enzyme--guanylate+intermediate.">Mechanism of mRNA capping by vaccinia virus guanylyltransferase: characterization of an enzyme--guanylate intermediate.</a> Proc. Natl. Acad. Sci. U.S.A. 78, 187-191 (1981).</li><li>Jang, S. K., Pestova, T. V., Hellen, C. U., Witherell, G. W., Wimmer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cap-independent+translation+of+picornavirus+RNAs:+structure+and+function+of+the+internal+ribosomal+entry+site.">Cap-independent translation of picornavirus RNAs: structure and function of the internal ribosomal entry site.</a> Enzyme. 44, 292-309 (1990).</li><li>Chaudhry, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Caliciviruses+differ+in+their+functional+requirements+for+eIF4F+components.">Caliciviruses differ in their functional requirements for eIF4F components.</a> J. Biol. Chem. 281, 25315-25325 (2006).</li><li>Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calicivirus+translation+initiation+requires+an+interaction+between+VPg+and+eIF+4.">Calicivirus translation initiation requires an interaction between VPg and eIF 4.</a> E. EMBO Rep. 6, 968-972 (2005).</li><li>Herbert, T. P., Brierley, I., Brown, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+protein+linked+to+the+genomic+and+subgenomic+mRNAs+of+feline+calicivirus+and+its+role+in+translation.">Identification of a protein linked to the genomic and subgenomic mRNAs of feline calicivirus and its role in translation.</a> J. Gen. Virol. 78 ( Pt5 ), 1033-1040 (1997).</li><li>Chaudhry, Y., Skinner, M. A., Goodfellow, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+genetically+defined+murine+norovirus+in+tissue+culture+by+using+a+fowlpox+virus+expressing+T7+RNA+polymerase.">Recovery of genetically defined murine norovirus in tissue culture by using a fowlpox virus expressing T7 RNA polymerase.</a> J. Gen. Virol. 88, 2091-20100 (2007).</li><li>Ward, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+infectious+murine+norovirus+using+pol+II-driven+expression+of+full-length+cDNA.">Recovery of infectious murine norovirus using pol II-driven expression of full-length cDNA.</a> Proc. Natl. Acad. Sci. U.S.A. 104, 11050-11055 (2007).</li><li>Yunus, M. A., Chung, L. M., Chaudhry, Y., Bailey, D., Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+optimized+RNA-based+murine+norovirus+reverse+genetics+system.">Development of an optimized RNA-based murine norovirus reverse genetics system.</a> J. Virol. Methods. 169, 112-118 (2010).</li><li>Arias, A., Perales, C., Escarmis, C., Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deletion+mutants+of+VPg+reveal+new+cytopathology+determinants+in+a+picornavirus.">Deletion mutants of VPg reveal new cytopathology determinants in a picornavirus.</a> PLoS One. 5, e10735 (2010).</li><li>Werf, S. v. a. n. d. e. r., Bradley, J., Wimmer, E., Studier, F. W., Dunn, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+infectious+poliovirus+RNA+by+purified+T7+RNA+polymerase.">Synthesis of infectious poliovirus RNA by purified T7 RNA polymerase.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 2330-2334 (1986).</li><li>Bailey, D., Thackray, L. B., Goodfellow, I. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+amino+acid+substitution+in+the+murine+norovirus+capsid+protein+is+sufficient+for+attenuation+in+vivo.">A single amino acid substitution in the murine norovirus capsid protein is sufficient for attenuation in vivo.</a> J. Virol. 82, 7725-7728 (2008).</li><li>Bailey, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+RNA+structures+present+at+the+3'+extremity+of+the+murine+norovirus+genome:+the+variable+polypyrimidine+tract+plays+a+role+in+viral+virulence.">Functional analysis of RNA structures present at the 3' extremity of the murine norovirus genome: the variable polypyrimidine tract plays a role in viral virulence.</a> J. Virol. 84, 2859-2870 (2010).</li></ol>

אין לנו מה למסור.

כאן אנו הדגימו שתי גישות שונות גנטית הפוכה המאפשרים התאוששות MNV מדבקות תרבית תאים. שתי הגישות יעילות לעקוף את הדרישה מוחלטת הצמדה קוולנטית של VPg עד הסוף &#39;5 של הגנום RNA ויראלי באמצעות הדור של כתרים תמלילי MNV המוכרים מכן על ידי הריבוזומים הסלולר. ב שעתוק במבחנה ואחריו enzymatically מכסת יעיל יותר ההתאוששות של MNV זיהומיות מאשר שעתוק של פלסמידים זיהומיות בתאים המבטאים T7 רנ&quot;א פולימרז, שבו תעתיקי ניתן כתרים על ידי אנזימים FPV פיקוק. וירוס של הסרט התאושש עם מערכות אלה גנטיקה הפוכה דומים אלה שהושגו על ידי transfection של VPg צמודות רנ&quot;א נגיפי מטוהרים מן בתרביות תאים נגועים 17 (איור 5). Transfection של כתרים MNV RNA לתוך תאים Raw264.7 מתירנית הופך titre וירוס רק 1-log נמוך ניסויים הקשורים transfection של RNA הכולל תאים נגועים המכילים ויראלי VPg צמוד RNA (איור 5). עובדה זו מעודדת בדיקות נוספות כדי לקבוע אם תוספת של מולקולת VPg עד הסוף &#39;5 של תמלילי שנוצר על ידי מערכות אלה עלול לגרום עלייה בתשואות וירוס אשר עשוי לחשוף היבטים פונקציונליים שבבסיס infectivity MNV בתאים הקשורים VPg. עם זאת, אנו רואים במערכת זו גנטיקה הפוכה כמו 1 היעילה ביותר להשוות אחרים וירוסים RNA להפוך מערכות גנטיקה המשמש כיום שבו במבחנה RNA עיבד מאפשר התאוששות של הסרט רק 10-100 נמוך יותר מאשר זיהומים אמיתיים עם 19 virions, 20. בסך הכל, את שיטות הנוכחי מהווה צעד משמעותי קדימה בתחום של ביולוגיה מולקולרית norovirus לספק לנו את הכלים לחקור את התפקידים תפקודית של חלבונים ונשמר מוטיבים רנ&quot;א ב הגנום norovirus. גישות אלה כבר בשילוב עם גבמודל עכבר urrent זמין הראו כי MNV התאושש cDNA זיהומיות יכול לגרום לזיהום קטלני של&gt; 80% STAT1-/ - העכברים בתוך פחות מ -10 ימים 4, 21. תוך שימוש במערכת זו אנו התאוששו קיימא מוטציות norovirus Murine של החלבון capsid של מערכת polypyrimidine מעורב מחייב גורמים מארחים שונים (PTB ו PCBP) המציגים פנוטיפים נחלש מעט ב 21 vivo, 22. בנוסף, הראו לאחרונה כי וירוסים חסרי יכולת לבטא את החלבון VF1 מ ORF4 ביעילות לשכפל תרבית תאים אבל שוב צמצמו ארסיות בעכברים לגבי WT MNV 8. מחקרים אלו מעודדים אותנו לעצב גרסאות נחלש noroviruses של אדם בהתבסס על מחקרים MNV אשר יכול להיחקר כמועמדים פוטנציאליים לחיסון.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </td></tr><tr><td> ליתיום כלוריד משקעים פתרון </td><td> Ambion </td><td> AM9480 </td></tr><tr><td> RNA פתרון האחסון </td><td> Ambion </td><td> AM7000 </td></tr><tr><td> MEGAscript T7 התסריט </td><td> Ambion </td><td> AM1333 </td></tr><tr><td> ScriptCap m7G תקרת מערכת </td><td> Epicentre ביוטכנולוגיות </td><td> SCCE0610 </td></tr><tr><td> Transfection ניאון מערכת </td><td> Invitrogen </td><td> MPK5000 </td></tr><tr><td> Transfection ניאון מערכת הערכה </td><td> Invitrogen </td><td> MPK1025 </td></tr><tr><td> Opti-MEM אני </td><td> Invitrogen </td><td> 31985070 </td></tr><tr><td> 2000 Lipofectamine transfection מגיב </td><td> Invitrogen </td><td> 11668-027 </td></tr><tr><td> Agilent RNA 600 ננו ערכת </td><td> Agilent </td><td> 5067-1511 </td></tr><tr><td> Agilent 2100 דוoanalyzer </td><td> Agilent </td><td> G2939AA </td></tr><tr><td> Illustra GFX PCR DNA וטיהור ג&#39;ל ערכת Band </td><td> GE Healthcare </td><td> 28-9034-70 </td></tr><tr><td> RiboMAX גדול Scale-RNA ייצור System-T7 </td><td> Promega </td><td> P1300 </td></tr></tbody></table>

reverse genetics mediated recovery of infectious murine norovirus

Noroviruses האדם אחראים לרוב המקרים של גסטרואנטריטיס האנושי (GE) ברחבי העולם, הם הבעיה חוזרת בסביבות שבהן בקשר הדוק אדם אל אדם לא ניתן להימנע מכך 1, 2. בשנים האחרונות עלייה בשכיחות של התפרצויות בבתי חולים דווחה, לגרום לשיבושים משמעותיים והיכולת המבצעית שלהם, כמו גם הפסדים כלכליים גדולים. זיהוי של גישות אנטי החדשים הייתה מוגבלת בשל חוסר היכולת של האדם להשלים noroviruses זיהום פרודוקטיבי תרבית תאים 3. הבידוד לאחרונה norovirus Murine (MNV), קשורה קשר הדוק norovirus האנושי 4 אך ניתן מופצות בתאים 5 פתחה אפיקים חדשים לחקר אלה פתוגנים 6, 7. תוצאות MNV שכפול בסינתזה של הגנומי חדש במובן החיובי subgenomic מולקולות רנ&quot;א, האחרון שבהם מתאים תי האחרוןrd של הגנום הויראלי (איור 1). MNV מכיל ארבע מסגרות שונות הקריאה פתוחים (ORFs), מתוכם ORF1 מעסיק את רוב הגנום מקודד שבע ללא חלבונים מבניים (ns1-7) שוחרר מבית מבשר polyprotein. ORF2 ו ORF3 נמצאים בתוך האזור RNA subgenomic ו לקודד את החלבונים קפסיד (VP1 ו VP2, בהתאמה) (איור 1). לאחרונה, זיהינו כי ORF4 נוסף חופפים ORF2 אבל בתוך מסגרת קריאה שונה הוא פונקציונלי מקודד עבור גורם ארסיות המיטוכונדריה מקומי (VF1) 8. שכפול וירוסים תחושת חיוביות רנ&quot;א, כולל noroviruses, מתרחש בציטופלסמה וכתוצאה מכך סינתזה של הגנום חדשים הסיר את המיכסה RNA. כדי לקדם את התרגום ויראלית, וירוסים לנצל אסטרטגיות שונות שמטרתן גיוס חלבון הסלולר סינתזה מכונות 9-11. מעניין לציין, תרגום norovirus הוא מונע על ידי VPg רב תכליתיים חלבונים פריימר ויראליקשור קוולנטית לקצה &#39;5 של שני RNAs גנומית subgenomic 12-14. זה מנגנון מתוחכם של התרגום עשוי להיות גורם מרכזי היעילות המוגבלת של התאוששות ויראלי ידי הפוכה הגישות המקובלות גנטיקה. כאן אנו מדווחים על שתי אסטרטגיות שונות המבוססות על הדור של Murine norovirus-1 (המכונה MNV בזאת) תעתיקים כתרים בסוף &quot;5. אחת השיטות כרוכה הן סינתזה במבחנה מכסת של רנ&quot;א נגיפי, ואילו הגישה השנייה כרוכה שעתוק של MNV cDNA בתאים המבטאים T7 רנ&quot;א פולימרז. הזמינות של מערכות אלה גנטיקה הפוכה לחקר MNV ומודל חיה קטנה סיפקה יכולת חסרת תקדים לנתח את התפקיד של רצפים ויראליים שכפול בפתוגנזה 15-17.

Watch this Scientific Journal Video about Reverse Genetics Mediated Recovery of Infectious Murine Norovirus at JoVE.com

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Noroviruses הם אחד הגורמים העיקריים של הקיבה והמעיים עדיין בטכניקות מולקולריות לאפיון שלהם עדיין חדש יחסית. כאן אנו מדווחים על שתי גישות שונות גנטיקה הפוכה להתאוששות יעילה של norovirus Murine (MNV), הנציג היחיד של סוג זה אשר ניתן מופצות על תרבית תאים.

שחזור הפוך גנטיקה מתווכת של norovirus Murine זיהומיות

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

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Immunology and Infection

16.5K Views.  Imperial College London. Noroviruses הם אחד הגורמים העיקריים של הקיבה והמעיים עדיין בטכניקות מולקולריות לאפיון שלהם עדיין חדש יחסית. כאן אנו מדווחים על שתי גישות שונות גנטיקה הפוכה להתאוששות יעילה של norovirus Murine (MNV), הנציג היחיד של סוג זה אשר ניתן מופצות על תרבית תאים.

Video: Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Using Reverse Genetics to Manipulate the NSs Gene of the Rift Valley Fever Virus MP-12 Strain to Improve Vaccine Safety and Efficacy

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal malformation in ruminants1, has been classified as a HHS/USDA overlap select agent and a risk group 3 pathogen. It belongs to the genus Phlebovirus in the family Bunyaviridae and is one of the most virulent members of this family. Several reverse genetics systems for the RVFV MP-12 vaccine strain2,3 as well as wild-type RVFV strains 4-6, including ZH548 and ZH501, have been developed since 2006. The MP-12 strain (which is a risk group 2 pathogen and a non-select agent) is highly attenuated by several mutations in its M- and L-segments, but still carries virulent S-segment RNA3, which encodes a functional virulence factor, NSs. The rMP12-C13type (C13type) carrying 69% in-frame deletion of NSs ORF lacks all the known NSs functions, while it replicates as efficient as does MP-12 in VeroE6 cells lacking type-I IFN. NSs induces a shut-off of host transcription including interferon (IFN)-beta mRNA7,8 and promotes degradation of double-stranded RNA-dependent protein kinase (PKR) at the post-translational level.9,10 IFN-beta is transcriptionally upregulated by interferon regulatory factor 3 (IRF-3), NF-kB and activator protein-1 (AP-1), and the binding of IFN-beta to IFN-alpha/beta receptor (IFNAR) stimulates the transcription of IFN-alpha genes or other interferon stimulated genes (ISGs)11, which induces host antiviral activities, whereas host transcription suppression including IFN-beta gene by NSs prevents the gene upregulations of those ISGs in response to viral replication although IRF-3, NF-kB and activator protein-1 (AP-1) can be activated by RVFV7. . Thus, NSs is an excellent target to further attenuate MP-12, and to enhance host innate immune responses by abolishing the IFN-beta suppression function. Here, we describe a protocol for generating a recombinant MP-12 encoding mutated NSs, and provide an example of a screening method to identify NSs mutants lacking the function to suppress IFN-beta mRNA synthesis. In addition to its essential role in innate immunity, type-I IFN is important for the maturation of dendritic cells and the induction of an adaptive immune response12-14. Thus, NSs mutants inducing type-I IFN are further attenuated, but at the same time are more efficient at stimulating host immune responses than wild-type MP-12, which makes them ideal candidates for vaccination approaches.

The reverse genetics system for the Rift Valley fever virus MP-12 vaccine strain is a useful tool for creating additional MP-12 mutants with increased attenuation and immunogenicity. We describe the protocol to generate and characterize NSs mutant strains.

באמצעות גנטיקה הפוכה כדי לתפעל את ג&#39;ין NSS של וירוס קדחת עמק השבר MP-12 מסננים כדי לשפר את בטיחות חיסון והיעילות

Rift Valley fever virus (RVFV), which causes hemorrhagic fever, neurological disorders or blindness in humans, and a high rate abortion and fetal ...

Plaque Assay for Murine Norovirus

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are observed during MNV-1 infection of murine dendritic cells or macrophages 1. This property of MNV-1 can be used to quantify the number of infectious particles in a given sample by performing a plaque assay 1. The plaque assay relies on the ability of MNV-1 to lyse cells and to form holes in a confluent cell monolayer, which are called plaques 3.
Multiple techniques can be used to detect viral infections in tissue culture, harvested tissue, clinical, and environmental samples, but not all measure the number of infectious particles (e.g. qRT-PCR). One way to quantify infectious viral particles is to perform a plaque assay 3, which will be described in detail below. A variation on the MNV plaque assay is the fluorescent focus assay, where MNV antigen is immunostained in cell monolayers 4. This assay can be faster, since viral antigen expression precedes plaque formation. It is also useful for titrating viruses unable to form plaques. However, the fluorescent focus assay requires additional resources beyond those of the plaque assay, such as antibodies and a microscope to count focus-forming units. Infectious MNV can also be quantified by determining the 50% Tissue Culture Infective Dose (TCID50) 3. This assay measures the amount of virus required to produce CPE in 50% of inoculated tissue culture cells by endpoint titration 5. However, its limit of detection is higher compared to a plaque assay 4.
In this article, we describe a plaque assay protocol that can be used to effectively determine the number of infectious MNV particles present in biological or environmental samples 1, 4, 6. This method is based on the preparation of 10-fold serial dilutions of MNV-containing samples, which are used to inoculate a monolayer of permissive cells (RAW 264.7 murine macrophage cells). Virus is allowed to attach to the cell monolayer for a given period of time and then aspirated before covering cells with a mixture of agarose and cell culture media. The agar enables the spread of viral progeny to neighboring cells while limiting spread to distantly located cells. Consequently, infected cells are lysed and form holes in the monolayer known as plaques. Upon sufficient spread of virus, plaques become visible following staining of cells with dyes, like neutral red, methylene blue, or crystal violet. At low dilutions, each plaque originates from one infectious viral particle and its progeny, which spread to neighboring cells. Thus, counting the number of plaques allows one to calculate plaque-forming units (PFU) present in the undiluted sample 3.

Here we describe a method to quantify infectious particles of murine norovirus (MNV), which is the only norovirus that efficiently replicates in cell culture. The plaque assay takes advantage of MNV&#x2019;s tropism for murine macrophages and can be adapted for use with biological or environmental samples containing MNV.

Assay רובד לnorovirus Murine

Murine norovirus (MNV) is the only member of the Norovirus genus that efficiently grows in tissue culture 1, 2. Cell lysis and cytopathic effect (CPE) are ...

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer's Patches

Peyer's patches (PPs) are integral components of the gut-associated lymphoid tissues (GALT) and play a central role in intestinal immunosurveillance and homeostasis. Particulate antigens and microbes in the intestinal lumen are continuously sampled by PP M cells in the follicle-associated epithelium (FAE) and transported to an underlying network of dendritic cells (DCs), macrophages, and lymphocytes. In this article, we describe protocols in which murine PPs are (i) dissociated into single cell suspensions and subjected to flow cytometry and (ii) prepared for cryosectioning and immunostaining. For flow cytometry, PPs are mechanically dissociated and then filtered through 70 &mu;m membranes to generate single cell suspensions free of epithelial cells and large debris. Starting with 20-25 PPs (from four mice), this quick and reproducible method yields a population of &gt;2.5 x 106 cells with &gt;90% cell viability. For cryosectioning, freshly isolated PPs are immersed in Optimal Cutting Temperature (OCT) medium, snap-frozen in liquid nitrogen, and then sectioned using a cryomicrotome. Tissue sections (5-12 &mu;m) are air-dried, fixed with acetone or methanol, and then subjected to immunolabeling.

Isolating And Immunostaining Lymphocytes and Dendritic Cells from Murine Peyer&#039;s Patches

There is an increasing interest in understanding the immunological functions of specific subpopulations of cells in Peyer's patches (PPs), the primary inductive sites of gut-associated lymphoid tissues. Here we outline parallel protocols for preparing PP single cell preparations for flow cytometric analysis and PP cryosections for immunostaining.

וגם בידוד Immunostaining ימפוציטים ותאי דנדריטים מהתיקונים של Peyer Murine

Peyer's  patches (PPs) are integral components of the gut-associated lymphoid tissues  (GALT) and play a central role in intestinal immunosurveillance and ...

VIGS-Mediated Forward Genetics Screening for Identification of Genes Involved in Nonhost Resistance

Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important for developing durable disease-resistant plants against wide range of pathogens. Virus-induced gene silencing (VIGS)-based forward genetics screening is a useful approach for identification of plant defense genes imparting nonhost resistance. Tobacco rattle virus (TRV)-based VIGS vector is the most efficient VIGS vector to date and has been efficiently used to silence endogenous target genes in Nicotiana benthamiana.

	In this manuscript, we demonstrate a forward genetics screening approach for silencing of individual clones from a cDNA library in N. benthamiana and assessing the response of gene silenced plants for compromised nonhost resistance against nonhost pathogens, Pseudomonas syringae pv. tomato T1, P. syringae pv. glycinea, and X. campestris pv. vesicatoria. These bacterial pathogens are engineered to express GFPuv protein and their green fluorescing colonies can be seen by naked eye under UV light in the nonhost pathogen inoculated plants if the silenced target gene is involved in imparting nonhost resistance. This facilitates reliable and faster identification of gene silenced plants susceptible to nonhost pathogens. Further, promising candidate gene information can be known by sequencing the plant gene insert in TRV vector. Here we demonstrate the high throughput capability of VIGS-mediated forward genetics to identify genes involved in nonhost resistance. Approximately, 100 cDNAs can be individually silenced in about two to three weeks and their relevance in nonhost resistance against several nonhost bacterial pathogens can be studied in a week thereafter. In this manuscript, we enumerate the detailed steps involved in this screening. VIGS-mediated forward genetics screening approach can be extended not only to identifying genes involved in nonhost resistance but also to studying genes imparting several biotic and abiotic stress tolerances in various plant species.

Virus-induced gene silencing is an useful tool for identifying genes involved in nonhost resistance of plants. We demonstrate the use of bacterial pathogens expressing GFPuv in identifying gene silenced plants susceptible to nonhost pathogens. This approach is easy, fast and facilitates large scale screening and similar protocol can be applied to studying various other plant-microbe interactions.

הקרנת גנטיקה קדימה VIGS בתיווך לזיהוי גני המעורבים בהתנגדות Nonhost


	Nonhost disease resistance of plants against bacterial pathogens is controlled by complex defense pathways. Understanding this mechanism is important ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Organoids כמודל למחלות זיהומיות: התרבות של האדם וOrganoids הקיבה Murine וMicroinjection של הליקובקטר פילורי

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Isolation and Flow Cytometric Characterization of Murine Small Intestinal Lymphocytes

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both microbial and dietary. Given an increasing understanding that these luminal antigens help shape the immune response and that education of immune cells within the intestine is critical for a number of systemic diseases, there has been increased interest in characterizing the intestinal immune system. However, many published protocols are arduous and time-consuming. We present here a simplified protocol for the isolation of lymphocytes from the small-intestinal lamina propria, intraepithelial layer, and Peyer&#39;s patches that is rapid, reproducible, and does not require laborious Percoll gradients. Although the protocol focuses on the small intestine, it is also suitable for analysis of the colon. Moreover, we highlight some aspects that may need additional optimization depending on the specific scientific question. This approach results in the isolation of large numbers of viable lymphocytes that can subsequently be used for flow cytometric analysis or alternate means of characterization.

There is growing interest in the quantitative characterization of intestinal lymphocytes owing to increasing recognition that these cells play a critical role in a variety of intestinal and systemic diseases. In this protocol, we describe how to isolate single cell populations from different small-intestinal compartments for subsequent flow cytometric characterization.

אפיון בידוד תזרים Cytometric של Murine קטנים מעיים לימפוציטים

The intestines &#8212; which contain the largest number of immune cells of any organ in the body &#8212; are constantly exposed to foreign antigens, both ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

שיטת הדגימה ספוגית לצורך זיהוי של norovirus האדם על משטחים

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...

A Method to Assess Fc-mediated Effector Functions Induced by Influenza Hemagglutinin Specific Antibodies

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and Fc-regions. Here, we describe how to measure the activation of Fc effector functions by monoclonal antibodies targeting the influenza virus hemagglutinin with the use of a genetically engineered Jurkat cell line expressing an activating type 1 Fc-Fc&#947;R. Using this method, the contribution of specific Fc-Fc&#947;R interactions conferred by immunoglobulins can be determined using an in vitro assay.

We describe a method to measure the activation of Fc-mediated effector functions by antibodies that target the influenza virus hemagglutinin. This assay can also be adapted to assess the ability of monoclonal antibodies or polyclonal sera targeting other viral surface glycoproteins to induce Fc-mediated immunity.

שיטה להערכת פונקציות אפקטור בתיווך Fc המושרה על ידי שפעת Hemagglutinin נוגדנים ספציפיים

Antibodies play a crucial role in coupling the innate and adaptive immune responses against viral pathogens through their antigen binding domains and ...

Isolating Lymphocytes from the Mouse Small Intestinal Immune System

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to dietary antigens and commensal bacteria while mounting effective immune responses to enteropathogenic microbes. In addition, it has become clear that local intestinal immunity has a profound impact on distant and systemic immunity. Therefore, it is important to study how an intestinal immune response is induced and what the immunologic outcome of the response is. Here, a detailed protocol is described for the isolation of lymphocytes from small intestine inductive sites like the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes and effector sites like the lamina propria and the intestinal epithelium. This technique ensures isolation of a large numbers of lymphocytes from small intestinal tissues with optimal purity and viability and minimal cross compartmental contamination within acceptable time constraints. The technical capability to isolate lymphocytes and other immune cells from intestinal tissues enables the understanding of immune responses to gastrointestinal infections, cancers, and inflammatory diseases.

Here we describe a detailed protocol for the isolation of lymphocytes from the inductive sites including the gut-associated lymphoid tissue Peyer&#39;s patches and the draining mesenteric lymph nodes, and the effector sites including the lamina propria and the intestinal epithelium of the small intestinal immune system.

לימפוציטים בידוד מן העכבר קטן המעי המערכת החיסונית

The intestinal immune system plays an essential role in maintaining the barrier function of the gastrointestinal tract by generating tolerant responses to ...

Preparation of Murine Submandibular Salivary Gland for Upright Intravital Microscopy

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, including cell biology, oncology, dentistry, and immunology. The SMG is an exocrine gland comprised of secretory epithelial cells, myofibroblasts, endothelial cells, nerves, and extracellular matrix. Dynamic cellular processes in the rat and mouse SMG have previously been imaged, mostly using inverted multi-photon microscope systems. Here, we describe a straightforward protocol for the surgical preparation and stabilization of the murine SMG in anesthetized mice for in vivo imaging with upright multi-photon microscope systems. We present representative intravital image sets of endogenous and adoptively transferred fluorescent cells, including the labeling of blood vessels or salivary ducts and second harmonic generation to visualize fibrillar collagen. In sum, our protocol allows for surgical preparation of mouse salivary glands in upright microscopy systems, which are commonly used for intravital imaging in the field of immunology.

We describe a protocol to surgically expose and stabilize the murine submandibular salivary gland for intravital imaging using upright intravital microscopy. This protocol is easily adaptable to other exocrine glands of the head and neck region of mice and other small rodents.

הכנה של בלוטת הרוק Submandibular מאתר מיקרוסקופ Intravital זקופה

The submandibular salivary gland (SMG) is one of the three major salivary glands, and is of interest for many different fields of biological research, ...