الكتاب أريد أن أشكر أعضاء سابقون وحاليون في أدولفو غارسيا ساستري وبيتر باليس مختبرات لتطوير تقنيات الوراثة عكس الإنفلونزا والبلازميدات. وتمول جزئيا البحوث في مختبرات AG - S و التراث ، وهو مركز NIAID الممولة من التميز لأبحاث الأنفلونزا ومراقبة (HHSN266200700010C) ، ومنح NIAD R01AI046954 ، وU01AI070469 P01AI058113. ويمول جزئيا البحث في LM - S المختبر منح 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>

جبل سيناء كلية الطب وحقوق الملكية الفكرية في مجال فيروسات الأنفلونزا المؤتلف ، وAG - S هو المخترع في هذه الملكية الفكرية.

الانقاذ من فيروسات الانفلونزا المؤتلف من الحمض النووي البلازميد هو عملية بسيطة ومباشرة مرة واحدة يتم تنفيذ البروتوكول بشكل روتيني في المختبر ، ولكن في البداية ، يمكن للعديد من الأمور على غير ما يرام. لا بد أن يكون إعداد البلازميد جيدة لتوليد الفيروس. الصيانة السليمة...

خطوط خلية 293T (رقم الكتالوج CRL - 11268) وMDCK (رقم الكتالوج CCL - 34) تمسك خطوط الخلايا في الحاضنة 37 درجة مئوية مع نسبة 5 ٪ CO 2 في FBS 10 ٪ DMEM ، PS 1 ٪. الخلايا هي شكل متاح للثقافة الأمريكية نوع مجموعة (آي تي ​​سي سي ، 10801 جامعة بوليفارد ، ماناساس بولاية فرجينيا. 20110-2209 الولايات المتحدة). بيض الدجاج المحتوي ويمكن الحصول على المحتوي بيض الدجاج 10 يوما من عمره من مختبرات نهر تشارلز ، رسوم محددة الطيور المرضي التموين (SPAFAS) منتجات الطيور والخدمات. فرانكلين العموم ، 106 شارع 32 ، شمال فرانكلين ، CT 06254 الولايات المتحدة الأمريكية. يتم تحضين البيض عند 37 درجة مئوية السابقة وبعد العدوى الفيروسية. قبل وبعد العدوى الفيروسية ، وcandled البيض لتحديد الجدوى من الأجنة. من المهم جدا أن نبحث عن البيض الموتى قبل وبعد العدوى الفيروسية. يمكن أن تكون العدوى قبل بيضة بالرصاص رصدت بسهولة بسبب عدم وجود الأوعية الدموية ، فضلا عن غياب الحراك الجنين. عندما candled والأجنة الحية التحرك. وبعد عدوى فيروسية تكون البويضة ميتة (ربما تتعلق عدوى فيروس الأنفلونزا) رصدت بسهولة عن طريق مظهر سيئ للبويضة من وجهة نظر حجم أصغر ودموية من السوائل السقاء. يتم التخلص من المصابين ، في أكياس البيض autoclavable المزدوجة وتعقيمها وفقا للإجراءات القياسية. الدجاج خلايا الدم الحمراء (RBC) يمكن شراؤها من مزارع الدجاج RBC Truslow ، 201 شارع وادي ، Chestertown ، MD 21620. المحل في 4 درجات مئوية. لفحوصات HA ، وغسل 5 مل من RBC الدجاج مع 45 مل من 1X PBS الطرد المركزي في أنبوب 50 مل. أجهزة الطرد المركزي لمدة 5 دقائق في 1000 RPMS ، RT. تجاهل طاف بعناية واستخدام التخفيف 1:1000 من مكعبات في RBC 1X PBS (تركيز النهائي من RBC 0،5-1،0 ٪). supernatants زراعة الأنسجة وسوائل السقائي ويمكن تخزين كل من زراعة الأنسجة ، والسوائل supernatants السقائي في 4 درجات مئوية لفترة قصيرة من الزمن. بعد التأكد من فيروس الانقاذ ، يتم تخزين الفيروسات من supernatants الخلية أو السوائل في السقاء -80 درجة مئوية. البلازميدات وتعد جميع البلازميدات باستخدام طقم توصيات ماكسي البلازميد المصنعة التالية. جميع البلازميدات هي قسامة بتركيزات من 1 ميكروغرام / مل في DDH 2 O وتخزينها على -20 درجة مئوية. للتخزين على المدى القصير ، يمكن أن يكون الحفاظ على البلازميد في 4 درجات مئوية. يتم تحديد تركيز الحمض النووي البلازميد تنقيته بواسطة القياس الطيفي عند 260 نانومتر ، والتي تقدر نسبة النقاء باستخدام نانومتر 260:280. وتعتبر نسب الاستعدادات مع 1،8-2،0 نانومتر 260:280 المخصصة لأغراض الإنقاذ الفيروس. بالإضافة إلى ذلك ، ينبغي التركيز أكد البلازميد والنقاء مع agarose هلام اللوني. موضحة Ambisense pDZ البلازميدات (6) تحتوي على ثمانية جينات الأنفلونزا A/PR/8/34 الفيروسي (7) في الشكل 2. الفيروسات لا يمكن أن يؤديها في وصفها لانقاذ بروتوكول الأنفلونزا A/PR/8/34 تحت مستوى السلامة الأحيائية (BSL) 2 شروطه. ويجب تعقيم المواد الملوثة ، بما في ذلك supernatants زراعة الأنسجة والبيض المحتوي ، قبل التخلص منها. قد الانقاذ من فيروس الانفلونزا ظروف أخرى تتطلب أعلى BSL ، وبالتالي ، الشروط الخاصة / مقاييس الأمن وسوف يتعين اتباعها. زراعة الأنسجة وسائل الاعلام والحلول DMEM 10 ٪ FBS PS 1 ٪ : 445 مل Dulbecco لتعديل النسر المتوسط ​​(DMEM) ، و 50 مل من مصل بقري جنيني (FBS) ، و 5 مل من البنسلين 100X / الستبرتوميسين (PS). المحل في 4 درجات مئوية. وسيتم استخدام هذه الوسائط للحفاظ على خلايا 293T MDCK وكذلك لtransfections. DMEM BA 0.3 ٪ 1 ٪ PS : 495.7 مل من DMEM ، 4.3 مل من 35 ٪ من الأبقار الزلال (BA). المحل في 4 درجات مئوية. قبل الاستخدام ، إضافة إلى تعامل TPCK التربسين تركيز النهائي من 1 ميكروغرام / لتر. المعدية وسائل الإعلام. 10X الفوسفات مخزنة المالحة (PBS) : 80 غرام من كلوريد الصوديوم ، و 2 غرام من بوكل ، 11.5 غراما من نا 2 4 هبو 0.7 H 2 O ، 2 غرام من KH 2 PO 4. إضافة DDH 2 O يصل إلى 1 لتر. ضبط الرقم الهيدروجيني إلى 7.3. تعقيم بواسطة الأوتوكلاف. تخزين في درجة حرارة الغرفة. 1X PBS : خفف 10X مع PBS 01:10 DDH 2 O. تعقيم بواسطة الأوتوكلاف وتخزينها في درجة حرارة الغرفة.

generation of recombinant influenza virus from plasmid dna

وقد بذلت جهود من قبل عدد من المجموعات البحثية الأنفلونزا محوريا في تطوير وتحسين الأنفلونزا من الوراثة عكس الفيروس. أنشئت أصلا في عام 1999<sup&gt; 1،2</sup&gt; البلازميد المستندة إلى التقنيات الجينية العكسية لتوليد المؤتلف فيروسات أحدثت ثورة في مجال البحوث الأنفلونزا لأنه تم الرد على أسئلة محددة من قبل والمعدية وراثيا ، المؤتلف فيروسات الانفلونزا. مثل هذه الدراسات تشمل تكاثر الفيروس ، وظيفة من البروتينات الفيروسية ، ومساهمة من طفرات معينة في البروتينات الفيروسية في تكاثر الفيروس و / أو المرضية ، وأيضا ، وناقلات الفيروسية باستخدام فيروسات الأنفلونزا المؤتلف معربا عن البروتينات الخارجية<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

الانقاذ من فيروسات الانفلونزا من الحمض النووي البلازميد هو تقنية أساسية وضرورية التجريبية التي تسمح للباحثين لتوليد فيروسات الانفلونزا المؤتلف لدراسة جوانب متعددة في البيولوجيا من فيروس الانفلونزا ، وإلى أن تستخدم ناقلات المحتملة أو اللقاحات.

توليد المؤتلف فيروسات الأنفلونزا من الحمض النووي البلازميد

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

generation-of-recombinant-influenza-virus-from-plasmid-dna

Research

JoVE Journal

Immunology and Infection

30.1K Views.  University of Rochester School of Medicine and Dentistry. الانقاذ من فيروسات الانفلونزا من الحمض النووي البلازميد هو تقنية أساسية وضرورية التجريبية التي تسمح للباحثين لتوليد فيروسات الانفلونزا المؤتلف لدراسة جوانب متعددة في البيولوجيا من فيروس الانفلونزا ، وإلى أن تستخدم ناقلات المحتملة أو اللقاحات.

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.

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, ...

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.

تنقية تقارب من المجمعات الأنفلونزا فيروس بروتين نووي ريبوزي من لونين من الخلايا المصابة

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.

الانقاذ من الفيروسات المؤتلف مرض نيوكاسل من [كدنا]

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.

التعبير عن المؤتلف وظيفية راصة دموية والبروتينات النورامينيداز من الفيروسات الأنفلونزا H7N9 رواية باستخدام نظام التعبير الفيروسة العصوية

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.

نشر فيروس الانفلونزا في أجنة بيض الدجاج

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.

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 ...

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.

الجيل المؤتلف الإنسان مفتش حيدة النسيلة من مخلد خلايا التصنيف B

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.

الأنف المسحات لمكافحة الأنفلونزا A الكشف عن الفيروسات وعزل من الخنازير

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.

في المختبر التفكيك من الأنفلونزا من الفيروسات Capsids بواسطة الطرد المركزي التدرج

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.

إنقاذ وتوصيف الفيروسات المؤتلف من عالم جديد زيكا الفيروسات استنساخ العدوى

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