המחברים רוצה להודות לחברי בעבר ובהווה של אדולפו גרסיה-סאסטרה ופיטר Palese מעבדות לפיתוח טכניקות שפעת הפוך גנטיקה פלסמידים. מחקר AG-S מעבדות ממומנת בחלקה על ידי נכה, מרכז NIAID במימון מצוינות למחקר שפעת ומעקב (HHSN266200700010C) וכן על ידי מענקים NIAD R01AI046954, U01AI070469 ו P01AI058113. מחקר LM-S מעבדה ממומנת חלקית על ידי מענק NIAID RO1AI077719.

<ol>
	<li>Neumann, G., Watanabe, T., Ito, H., Watanabe, S., Goto, H., Gao, P., Hughes, M., Perez, D. R., Donis, R., Hoffmann, E., Hobom, G., Kawaoka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+influenza+A+viruses+entirely+from+cloned+cDNAs.">Generation of influenza A viruses entirely from cloned cDNAs.</a> Proc Natl Acad Sci U S A. 96, 9345-9350 (1999).</li><li>Fodor, E., Devenish, L., Engelhardt, O. G., Palese, P., Brownlee, G. G., Garcia-Sastre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rescue+of+influenza+A+virus+from+recombinant+DNA.">Rescue of influenza A virus from recombinant DNA.</a> J Virol. 73, 9679-9682 (1999).</li><li>Martinez-Sobrido, L., Garcia-Sastre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+influenza+virus+vectors.">Recombinant influenza virus vectors.</a> Future Virology. 2, 401-416 (2007).</li><li>Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthomyxoviridae.+The+viruses+and+their+replication.">Orthomyxoviridae. The viruses and their replication.</a> Fields Virology. , 1647-1689 (2006).</li><li>Schickli, J. H., Flandorfer, A., Nakaya, T., Martinez-Sobrido, L., Garcia-Sastre, A., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmid-only+rescue+of+influenza+A+virus+vaccine+candidates.">Plasmid-only rescue of influenza A virus vaccine candidates.</a> Philos Trans R Soc Lond B Biol Sci. 356, 1965-1973 (2001).</li><li>Quinlivan, M., Zamarin, D., Garcia-Sastre, A., Cullinane, A., Chambers, T., Palese, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attenuation+of+equine+influenza+viruses+through+truncations+of+the+NS1+protein.">Attenuation of equine influenza viruses through truncations of the NS1 protein.</a> J Virol. 79, 8431-8439 (2005).</li><li>Niwa, H., Yamamura, K., Miyazaki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+selection+for+high-expression+transfectants+with+a+novel+eukaryotic+vector.">Efficient selection for high-expression transfectants with a novel eukaryotic vector.</a> Gene. 108, 193-199 (1991).</li></ol>

הר סיני הספר לרפואה של בעל זכויות הקניין הרוחני בתחום של נגיפי שפעת רקומביננטי, AG ו-S הוא ממציא בקניין רוחני זה.

ההצלה של נגיפי שפעת רקומביננטי מ-DNA פלסמיד הוא תהליך פשוט וברור פעם פרוטוקול מבוצע באופן שגרתי במעבדה, אבל בהתחלה, דברים רבים יכולים להשתבש. זה הכרחי להיות הכנה טובה פלסמיד כדי ליצור את הנגיף. תחזוקה נכונה של שורות תאים (293T ו MDCK) הוא חיוני להצלת ויראלי מוצלח. באופן מסור?...

שורות תאים 293T (מספר קטלוג CRL-11268) ו MDCK (מספר קטלוג CCL-34) שורות תאים נשמרות 37 ° C חממה עם 5 CO 2% DMEM 10% FBS, PS 1%. תאים הם צורה זמין סוג התרבות האמריקאית אוסף (ATCC, 10801 אוניברסיטת שדרות, Manassas, VA. 20110-2209 ארה&quot;ב). עוף וביצים Embryonated Embryonated 10 יום בן ביצי תרנגולת ניתן לקבל צ&#39;ארלס ריבר מעבדות, העופות ספציפי פתוגן אגרת התשלום (SPAFAS) העופות מוצרים ושירותים. פרנקלין מלאי, 106 כביש 32, צפון פרנקלין, CT 06254 ארה&quot;ב. ביצים מודגרת ב 37 ° C הקודמת ואחרי זיהום ויראלי. לפני ואחרי זיהום ויראלי, בדק את הביצים כדי לקבוע את הכדאיות של העוברים. חשוב מאוד לחפש ביצים מת לפני ואחרי זיהום ויראלי. לפני זיהום ביצה מת ניתן הבחין בקלות על ידי בהעדר כלי הדם, כמו גם העדר ניידות העובר. כאשר בדק את, עוברים לגור לזוז. לאחר זיהום ויראלי ביצה מת (כנראה קשור זיהום בנגיף שפעת) יהיה הבחין בקלות על ידי הופעתו של ביצה רע כפי שהוא נראה על ידי נפח קטן של נוזל דם allantoic. אינפקטד, ביצים מושלכות בשקיות autoclavable כפול autoclaved הבאים נהלים סטנדרטיים. עוף אדום דם תאים (RBC) עוף RBC ניתן לרכוש חוות Truslow, 201 כביש בעמק, Chestertown, MD 21620. חנות ב 4 ° C. עבור מבחני HA, לשטוף 5 מ&quot;ל של RBC עוף עם 45 מ&quot;ל של PBS 1X בצינור צנטריפוגה 50 מ&quot;ל. צנטריפוגה במשך 5 דקות ב 1000 rpms, RT. מחק בזהירות את supernatant ולהשתמש דילול 1:1000 של RBC pelleted ב PBS 1X (הריכוז הסופי של RBC 0.5-1.0%). תרבות רקמות ונוזלים supernatants allantoic שניהם, תרבות supernatants רקמות ונוזלים allantoic ניתן לאחסן 4 ° C למשך תקופה קצרה של זמן. לאחר אישור הצלה וירוס, וירוסים supernatants תא או נוזל allantoic מאוחסנים ב -80 ° C. פלסמידים כל פלסמידים מוכנים באמצעות המלצות maxi פלסמיד ערכת הבא של היצרן. פלסמידים כל aliquot בריכוזים של 1 מיקרוגרם / מ&quot;ל ב DDH 2 O ו המאוחסן ב -20 ° C. עבור אחסון לטווח קצר, פלסמיד ניתן לשמור על 4 ° C. הריכוז של ה-DNA פלסמיד מטוהרים נקבעת על ידי לספקטרופוטומטריה ב 260 ננומטר, עם טוהר להיות מוערך באמצעות יחס 260:280 ננומטר. תכשירים עם 260:280 יחס 1.8-2.0 ננומטר נחשבים הופקע למטרות הצלה וירוס. בנוסף, ריכוז טוהר פלסמיד ויש אישר agarose עם ג&#39;ל כרומטוגרפיה. Ambisense pDZ פלסמידים (6) המכיל שמונה שפעת A/PR/8/34 גנים ויראליים (7) הן באיור 2. וירוסים הפרוטוקול המתואר להצלת שפעת A/PR/8/34 יכול להתבצע תחת biosafety רמה (מנהלת הליגה) 2 תנאים. חומר מזוהם, כולל תרבות supernatants רקמות ביצים embryonated, יש לעקר לפני סילוק. ההצלה של וירוס השפעת אחרים עשויים לדרוש תנאים מנהלת הליגה גבוהה יותר, ולכן, תנאים מיוחדים / מדידות הביטחון יהיה צורך אחר. תרבות רקמות התקשורת ופתרונות DMEM 10% FBS PS 1%: 445 מ&quot;ל מדיום שונה Dulbecco של הנשר (DMEM), 50 מ&quot;ל של סרום שור עוברית (FBS), ו - 5 מ&quot;ל של 100x פניצילין / סטרפטומיצין (PS). חנות ב 4 ° C. מדיה זה ישמש כדי לשמור על התאים 293T ו MDCK וכן transfections. DMEM BA 0.3% 1% נ.ב.: 495.7 מ&quot;ל של DMEM, 4.3 מ&quot;ל של 35% שור אלבומין (BA). חנות ב 4 ° C. רק לפני השימוש, מוסיפים טריפסין TPCK שטופלו לריכוז סופי של 1 מיקרוגרם / מ&quot;ל. זיהומיות התקשורת. פוספט 10X שנאגרו מלוחים (PBS): 80 גרם של NaCl, 2 גרם של KCl, 11.5 גרם של Na 2 HPO 4 0.7 H 2 O, 2 גרם של KH 2 PO 4. הוסף DDH 2 O עד 1 ליטר. התאם ל-pH 7.3. לעקר ידי החיטוי. אחסן בטמפרטורת החדר. 1X PBS: 10X PBS לדלל 1:10 עם DDH 2 O. לעקר ידי החיטוי ולאחסן בטמפרטורת החדר.

generation of recombinant influenza virus from plasmid dna

מאמצים על ידי מספר קבוצות מחקר שפעת כבר מרכזי פיתוח ושיפור של שפעת A גנטיקה הפוכה וירוס. הוקמה במקור בשנת 1999<sup&gt; 1,2</sup&gt; פלסמיד טכניקות מבוססות גנטית הפוכה לייצר וירוסים רקומביננטי חוללו מהפכה בתחום המחקר שפעת בגלל שאלות ספציפיות כבר ענה על ידי הנדסה גנטית, וירוסים זיהומיות שפעת רקומביננטי. מחקרים אלה כוללות שכפול הנגיף, פונקציה של חלבונים נגיפיים, התרומה של מוטציות ספציפיות חלבונים נגיפיים שכפול נגיפי ו / או בפתוגנזה ו, גם, וקטורים ויראליים באמצעות נגיפי שפעת רקומביננטי לבטא חלבונים זרים<sup&gt; 3</sup&gt;.

Watch this Scientific Journal Video about Generation of Recombinant Influenza Virus from Plasmid DNA at JoVE.com

Generation of Recombinant Influenza Virus from Plasmid DNA

הצלה של שפעת וירוסים מ-DNA פלסמיד היא טכניקה ניסויית בסיסי וחיוני המאפשר לחוקרים שפעת לייצר וירוסים רקומביננטי ללמוד היבטים מרובים הביולוגיה של נגיף השפעת, וכן לשמש וקטורים או חיסונים פוטנציאליים.

הדור של וירוס שפעת רקומביננטי מ-DNA פלסמיד

Efforts by a number of influenza research groups have been pivotal in the development and improvement of influenza A virus reverse genetics. Originally ...

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Research

JoVE Journal

Immunology and Infection

30.1K Views.  University of Rochester School of Medicine and Dentistry. הצלה של שפעת וירוסים מ-DNA פלסמיד היא טכניקה ניסויית בסיסי וחיוני המאפשר לחוקרים שפעת לייצר וירוסים רקומביננטי ללמוד היבטים מרובים הביולוגיה של נגיף השפעת, וכן לשמש וקטורים או חיסונים פוטנציאליים.

Video: Generation of Recombinant Influenza Virus from Plasmid DNA

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.

תפוקה גבוהה איתור שיטת וירוס השפעת

Influenza virus is a respiratory pathogen that causes a high degree of morbidity and mortality every year in multiple parts of the world. Therefore, ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

זיקה טיהור של נגיף השפעת תסביכים Ribonucleoprotein של הכרומטין של תאים נגועים

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

הצלה של וירוס רקומביננטי ניוקאסל מחלות מcDNA

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Expression of Functional Recombinant Hemagglutinin and Neuraminidase Proteins from the Novel H7N9 Influenza Virus Using the Baculovirus Expression System

The baculovirus expression system is a powerful tool for expression of recombinant proteins. Here we use it to produce correctly folded and glycosylated versions of the influenza A virus surface glycoproteins - the hemagglutinin (HA) and the neuraminidase (NA). As an example, we chose the HA and NA proteins expressed by the novel H7N9 virus that recently emerged in China. However the protocol can be easily adapted for HA and NA proteins expressed by any other influenza A and B virus strains. Recombinant HA (rHA) and NA (rNA) proteins are important reagents for immunological assays such as ELISPOT and ELISA, and are also in wide use for vaccine standardization, antibody discovery, isolation and characterization. Furthermore, recombinant NA molecules can be used to screen for small molecule inhibitors and are useful for characterization of the enzymatic function of the NA, as well as its sensitivity to antivirals. Recombinant HA proteins are also being tested as experimental vaccines in animal models, and a vaccine based on recombinant HA was recently licensed by the FDA for use in humans. The method we describe here to produce these molecules is straight forward and can facilitate research in influenza laboratories, since it allows for production of large amounts of proteins fast and at a low cost. Although here we focus on influenza virus surface glycoproteins, this method can also be used to produce other viral and cellular surface proteins.

Here we describe a way to express correctly folded and functional influenza virus surface antigens derived from the novel Chinese H7N9 virus in insect cells. The technique can be adapted to express ectodomains of any viral or cellular surface proteins.

ביטוי של חלבונים רקומביננטי hemagglutinin וNeuraminidase פונקציונליים מוירוס רומן H7N9 שפעת שימוש במערכת ביטוי baculovirus

The baculovirus expression system is a powerful tool for expression of  recombinant proteins. Here we use it to produce correctly folded and  glycosylated ...

Influenza Virus Propagation in Embryonated Chicken Eggs

Influenza infection is associated with about 36,000 deaths and more than 200,000 hospitalizations every year in the United States. The continuous emergence of new influenza virus strains due to mutation and re-assortment complicates the control of the virus and necessitates the permanent development of novel drugs and vaccines. The laboratory-based study of influenza requires a reliable and cost-effective method for the propagation of the virus. Here, a comprehensive protocol is provided for influenza A virus propagation in fertile chicken eggs, which consistently yields high titer viral stocks. In brief, serum pathogen-free (SPF) fertilized chicken eggs are incubated at 37 &#176;C and 55-60% humidity for 10 &#8211; 11 days. Over this period, embryo development can be easily monitored using an egg candler. Virus inoculation is carried out by injection of virus stock into the allantoic cavity using a needle. After 2 days of incubation at 37 &#176;C, the eggs are chilled for at least 4 hr at 4 &#176;C. The eggshell above the air sac and the chorioallantoic membrane are then carefully opened, and the allantoic fluid containing the virus is harvested. The fluid is cleared from debris by centrifugation, aliquoted and transferred to -80 &#176;C for long-term storage. The large amount (5-10 ml of virus-containing fluid per egg) and high virus titer which is usually achieved with this protocol has made the usage of eggs for virus preparation our favorable method, in particular for in vitro studies which require large quantities of virus in which high dosages of the same virus stock are needed.

Fertile chicken eggs are widely used to produce large amounts of human influenza A virus as they provide a convenient and cost-effective system to prove high yields of virus.

רביה וירוס השפעת בembryonated עוף ביצים

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

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.

Intranasal המינהל של החיסונים שפעת רקומביננטי במודלי כימרי עכבר ללמוד חסינות רירית

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 ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

דור של רקומביננטי אדם IgG נוגדנים חד שבטיים מתאים הממוינים B הונצחו

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

Surveillance for influenza A viruses in swine is critical to human and animal health because influenza A virus rapidly evolves in swine populations and new strains are continually emerging. Swine are able to be infected by diverse lineages of influenza A virus making them important hosts for the emergence and maintenance of novel influenza A virus strains. Sampling pigs in diverse settings such as commercial swine farms, agricultural fairs, and live animal markets is important to provide a comprehensive view of currently circulating IAV strains. The current gold-standard ante-mortem sampling technique (i.e. collection of nasal swabs) is labor intensive because it requires physical restraint of the pigs. Nasal wipes involve rubbing a piece of fabric across the snout of the pig with minimal to no restraint of the animal. The nasal wipe procedure is simple to perform and does not require personnel with professional veterinary or animal handling training. While slightly less sensitive than nasal swabs, virus detection and isolation rates are adequate to make nasal wipes a viable alternative for sampling individual pigs when low stress sampling methods are required. The proceeding protocol outlines the steps needed to collect a viable nasal wipe from an individual pig.

The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses.

האף מגבונים לשפעת זיהוי וירוסים ובידוד מחזירים

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

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

במבחנה פירוק של וירוס שפעת capsids ידי צנטריפוגה שיפוע

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

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

הצלה אפיון של וירוס רקומביננטי מעולם חדש Zika וירוס זיהום מזוהם

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...