This work has been funded in part with federal funds from the Centers of Excellence for Influenza Research and Surveillance (CEIRS), National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. HHSN272201400006C.

Aşağıdaki veri toplanmasında kullanılan tüm domuz Ohio State Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Kurulu koruma altına alınmıştır (hayvan kullanım protokol numarası 2009A0134-R1). 1. Viral Transport Orta Hazırlanması ve Numune Toplama Flakon <ol><li> Saf su 900 ml beyin kalp infüzyon 37 g eklenir ve ısıtma 70 ° C, tamamen tozu çözmek için ise, bir karıştırma çubuğu ve manyetik bir karıştırıcı ile iyice karıştırın. </li><li> 15 dakika boyunca 121 ° C&#39;de suyu otoklava. Suyu kadar tezgah üstünde Serin oda sıcaklığını ulaşır. 4 ° C &#39;de bir gece buzdolabında gerekirse. </li><li> Bir manyetik karıştırma çubuğu, Penisilin G Sodyum tuzu 6,02 g, oda sıcaklığında saflaştırılmış su içinde 50 ml streptomisin sülfat, 10 g ile karıştırılarak, eritin. 100 ml toplam hacmi getirmek için ilave saf su ekleyin. Steril şişe içine 0,22 um polietersülfon zarından solüsyonu filtre. </li><li> Aseptik soğutmalı Beyin Kalp İnfüzyon Broth içine süzülmüş penisilin ve streptomisin çözüm ekleyin ve viral taşıma ortamı yapmak için iyice (karıştırma çubuğu ya da elle girdap şişesi) karıştırınız. </li><li> Aseptik viral taşıma ortamı pH testi; Gerekirse, HCI veya NaOH kullanılarak 7.4 ± 0.2 pH ayarlamak. </li><li> Zhang ve Harmon tarafından anlatıldığı gibi, RRT-PCR ile influenza A virüs matriks protein test ederek viral taşıma ortamında kalite güvence testleri gerçekleştirin. 22 </li><li> 8 ml kapasiteli steril, yüksek yoğunluklu polietilen şişeler içine viral taşıma ortamının 5 ml koyun. Alanında kullanım için şişeleri etiketleyin. </li><li> Numune toplama kadar -20 ° C&#39;de standart bir kriyo-kutularda hazır kullanım şişeleri saklayın. </li></ol> Domuz adlı Burun Mendil 2. Koleksiyonu <ol><li> Toplama örnek hemen önce şişeleri uygun sayıda çözülme; tek şişe örneklenmiş domuz başına ihtiyaç duyulacaktır. Odasında çözündüktensıcaklığı yaklaşık 30-45 dakika sürer. Çözülmüş şişeleri kullanılana kadar buz paketleri üzerinde serin tutun. </li><li> Don uygun kişisel koruyucu ekipman durumdan tarafından dikte (örn tulumlar, çizme kapakları, solunum, kulak tıkacı,) olarak. </li><li> Hayvan alanını girin. Gerekirse, küçük bir alana domuz sınırlandırmak ama dizginlemek hayır yoktur. Hayvanları dinlenme canlandırmak etmeyin. </li><li> Tek kullanımlık sınav bir çift eldiven giyin. Dokunmadan domuz, insanlar, ya da cansız nesneler tarafından eldiven kontamine etmemeye dikkat edin. </li><li> (2 ile. Büyük × 2.) 5.08 cm x 5.08 cm steril kaldırmak için bir eldivenli elinizi kullanın pamuklu gazlı bez ambalajından. Steril gazlı bez, mümkün kullanılmalıdır. Bireysel sarılı gazlı bez paket başına iki gazlı bez ile sarılmış olanlar daha kullanmak daha uygundur. </li><li> Bir elin parmak uçları örnekleme için mümkün olduğunca ped kadar açığa pamuk gazlı bez tutun. </li><li> Alarak, domuz burnu genelinde gazlı bez silinMümkünse ekstra bakım harici burun deliğine girmek için. Aynı gazlı bezle görünür burun salgıları (yaklaşık 1 ml) toplayın. </li><li> Aynı eli kullanarak, viral transport medium içeren flakon yerleştirilmesini kolaylaştırmak için kendi üzerine gazlı bez katlayın. </li><li> Diğer elinizi kullanarak, şişenin kapağını kaldırın ve şişenin içine katlanmış gazlı bez yerleştirin. Şişeyi tekrarlamak ve gazlı bezle ve viral taşıma ortamı karışmasını sağlamak için şişeyi sallayın. </li><li> Uygun bir kabın içinde eldiven ve yer çıkarın. Her domuz arasındaki eldiven değiştirin. </li><li> Şişe tanımlayıcı doğrulayın. Kayıt domuz kimlik numarası, yaş, cinsiyet, klinik bulgular ve araştırmacı tarafından gerekli görülen diğer notlar. </li><li> Toplandıktan hemen sonra numuneleri Chill. Birden donma-çözülme döngüleri önlemek için dondurmadan önce örnek hacimde yapma düşünün. En kısa sürede toplandıktan sonra mümkün olduğunca, laboratuara taşınması için kuru buz üzerinde örnekleri yerleştirin. Şişeleri de saklayın -80Test başlayana kadar ° C. </li></ol> 3. Tespit Gribi A Virüsü Nükleik Asit <ol><li> 5 dakika boyunca 37 ° C kuru boncuk banyosunda örnekleri çözülme ve daha sonra 20-30 dakika için oda sıcaklığında, bir tezgah üstünde örnekleri çözülme tamamlanır. Boncuklara birkaç flakon eş-zamanlı ilavesinin banyosu ısıya neden olur; dikkatli şişeleri ısınmasına değil gibi olunmalıdır. </li><li> RNA ekstraksiyonu için viral taşıma ortamının 100 ul çıkarın. <ol><li> Örneklerin çok sayıda RNA çıkarmak için yüksek verim (96 oyuklu plaka formatı) manyetik boncuk platformu kullanın. 31 </li></ol></li><li> Hızlı algılama influenza A virüs için gerçek zamanlı ters transkriptaz PCR deneyleri kullanın. 31 </li></ol> Grip 4. İzolasyon Burun Mendil A Virüsü <ol><li> Bölüm 3.1&#39;de tarif edildiği gibi numuneler çözülme </li><li> (1000 gentamisin sülfat ile çözülmüş örnekleri tedavi| ig / ml), amfoterisin B (22.5 ug / ml) ve kanamisin sülfat (325 ug / ml). </li><li> Madin-Darby Köpek Böbrek içine inoküle (MDCK), daha önce tarif edildiği gibi epitel hücreleri. 32 </li></ol>

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The authors have nothing to disclose.

Polyester uçlu nasal swapları ile domuz örnekleri toplanması IAV gözetim yürütülmesi yararlı olduğu kanıtlanmıştır; Ancak, nazal sürüntü prosedürünün kullanılması kısıtlama için tuzak gerekli kullanımı nedeniyle gözetim çabalarını engellemektedir. Burun mendil numune toplama sırasında insanlar ve domuzlar üzerinde stresi en aza indirmek için mevcut domuz örnekleme teknikleri bir arıtma temsil etmektedir. Yöntem için geliştirilen ve sergi domuz ayarlarında valide edilmiş olsa da ...

<html> Grip A virüsleri (IAV) evcil kuşlar, domuz ve insan da dahil olmak üzere pek çok türde, solunum hastalıklarına neden olur. Nedeniyle oluşabilir parçalı IAV genom hızlı viral evrim reassortment ve yeni IAV suşları sık sık ortaya çıkar. Domuz yaygın Kuzey Amerika domuz (H1N1, H1N2, H3N2) arasında dolaşan IAV üç ana alt tipi bulunmamaktadır. 1 birden çok ana türden iAVS yeniden yapılanması için bir karıştırma kabı olarak hizmet edebilen bir türün, ancak insan çoklu IAV tanıtımlar sahip Bu alt tipleri içinde yaygın IAV çeşitliliğe yol açtı. domuz bulaşmasını iAVS 2 Hızlı evrimi, insan, kuş ve domuz virüslerinin gen segmentleri içeren üçlü reassortant IAV 3 ABD&#39;de domuzlar arasında yaygınlaşan 1998 yılından beri belirgin olmuştur. 4 dahili gen Bu üçlü reassortant IAV gelen segmentleri şu anda domuz bulaşmasını iAVS arasında oldukça yaygın kalır. 5 &quot;&gt; Dünya, IAV tipik klinik bulgular ateş, iştahsızlık, uyuşukluk, öksürük dahil olduğu domuz solunum hastalığı önemli bir nedenidir, burun akıntısı ve kilo alımının yetersiz hapşırma, nefes darlığı. IAV nerede üreme çiftlikleri ekmek özellikle pahalıya mal olabilir nedeniyle IAV kaynaklı ateş ve zayıf doğumlu domuz yavrusu başarısızlık belgelenmiştir. 6,7 Amerika Birleşik Devletleri içinde IAV yaygın ticari domuz sürüleri ve IAV bulaşmasını domuz arasında yaygın antijenik ve genomik çeşitlilik ve sürekli evrim tespit edildiğinde, bu kontrol engel olan Virüs. 8-11 Üçlü reassortant Kuzey Amerika domuz soy ve Avrasya kuş-benzeri domuz soyundan gelen gen segmentleri içeren bir domuz-soy IAV dünya çapında bir salgın nedeniyle zaman 2009 yılında gerçekleşmiştir domuzlarda reassortment kaynaklanan bir salgın IAV soyunun ortaya çıkışı hakkında halk sağlığı kaygıları İnsanlarda. 12 pandemik virüs (A (H1N1) pdm09) beri varendemik domuz ile reassorted IAV 13,14 suşları ve bu yeni reassorted suşların bazı insanlara geri ahiren paylaşılmıştır. 15 reassorment olaylar ve pandemi potansiyeli olan yeni IAV suşlarının ortaya çıkması domuz zorunlu olarak IAV virüs dolaşımdaki aktif sürveyans yapar sıklığı, özellikle domuz-insan arayüzünde. Domuz-insan arayüzü IAV çift yönlü türler arası iletimi için önemlidir. Insan-to-domuz ticari domuz üretiminde ortaya çıkan iletim domuz popülasyonda anda mevcut IAV çeşitlilik büyük miktarda sorumludur. Tarım fuarları ABD&#39;de insanların domuz comingling için büyük ayarları ve bir varyant H3N2 IAV salgını sırasında, IAV zoonotik iletimi için siteler. 2012 yılında 15-21 bilinmektedir, vakaların% 93 katılımın bildirdi günlerde bir tarım fuarı hastalığın başlangıcında önce. 15 Genomik analizzoonotik iletim doğruladı insan izolatlar ile karşılaştırıldığında sergi domuzların viral izolatların. IAV ile enfekte 21 Sergi domuz genellikle doğrudan tanısal testler için ihtiyaç gösteren hastalık, 21-23 klinik belirtilerini göstermez. Gözle görülür hasta domuz Örnekleme yalnız başarıyla domuzlarda IAV yaygınlığını tespit olmaz ve domuz arasında çıkan IAV yeni suşları belirlemek için dayanıyordu olamaz. Aktif sürveyans IAV ortaya çıkan suşlar domuzlarda ve domuz ve halk sağlığı hem de kendi tehdidi değerlendirmek tespiti için kesinlikle gereklidir. Çoğu IAV gözetim faaliyetleri gönüllü ve bu nedenle minimal yıkıcı yöntemlere ihtiyaç vardır vardır. IAV domuz bulaştırdıkları için üç önemli ölüm öncesi numune toplama prosedürleri vardır: Burun bezlerden, sözlü sıvılar ve burun mendil. IAV listesine sentetik elyaf sokulmasını algılamak için bireysel domuz örnekleme için akım tavsiyeler tercih edilen yöntem olarak burun deliklerine bezlerden uçluburun salgıları ve epitel hücreleri toplamak için. 24,25 domuz ya elle ya da bir tuzak hayvanın büyüklüğüne bağlı olan domuz dizginlemek gerekir bu prosedürü, eğitimli personel bir ekip önlemek için deneyebilirsiniz çünkü. 26 emniyet süreci için zahmetli personel ve domuzlar için stresli. Bir yarışma hayvan eklenen stres algısı gözetim çabalarına sahipleri dirençli yapabilirsiniz Ayrıca, sergi domuz genellikle fuarda birden yarışmalarda katılıyor. 80-100 den% IAV enfekte sürüler arasında değişen IAV algılama olasılıkları ile sözlü sıvılar domuzların popülasyonlarında IAV moleküler tespiti için bezlerden burun popüler bir alternatif haline gelmiştir. 27,28 Ayrıca, ağız sıvıları daha geniş bir pencere sağlayabilir İlk enfeksiyonu takiben nazal bezlerden daha IAV algılama. Ancak, ağız sıvılardan IAV izolasyon virus izolasyonu girişimleri sadece% 50&#39;si IA ile sonuçlanan sorunlu olmuşturV kurtarma. 29 Domuzlarda IAV gözetim sırasında burun bezlerden yerine burun mendil kullanarak yukarıda açıklanan sınırlamalarının üstesinden gelir. Nazal mendiller bir yasaklama tuzak kullanımını gerektirmeyen ve prosedürün hayvan veya tanıkları zorlamadan yapılabilir. Minimal teknik eğitim gözetim örnekleri toplamak için, domuz sahipleri de dahil olmak üzere sivil veteriner profesyonelleri, izin verir burun mendil, toplamak için gereklidir. Burun silme bezleri, daha önce A virüsü 30 ve örnekleme bu invazif olmayan bir yöntem için ayrıntılı bir protokol, aşağıda tarif edilmiştir algılama ve grip izolasyonu için burun bezlerinde karşılaştırılmıştır.

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			<td height="21" style="height:21px;width:216px;">BBL Brain Heart Infusion</td>
			<td style="width:360px;">Becton, Dickinson and Company</td>
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			<td height="21" style="height:21px;width:216px;">Penicillin G Sodium Salt</td>
			<td style="width:360px;">MP Biomedicals, LLC</td>
			<td style="width:119px;">021194537</td>
			<td>1500 u/mg</td>
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			<td height="21" style="height:21px;width:216px;">Streptomycin Sulfate</td>
			<td style="width:360px;">AMRESCO LLC</td>
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nasal wipes for influenza a virus detection and isolation from swine

Influenza A virüs hızla domuz toplumlarda gelişir ve yeni suşlar ortaya çıkmaya devam ediyor, çünkü domuz influenza A virüsleri için Gözetleme insan ve hayvan sağlığına için çok önemlidir. Domuz onlara çıkması ve yeni influenza A virüsünün bakımı suşları için önemli ana yapım influenza A virüsünün farklı soy tarafından enfekte edilmesi mümkün bulunmaktadır. Böyle ticari domuz çiftlikleri, tarım fuarları ve canlı hayvan pazarlarında gibi çeşitli ortamlarda domuz Örnekleme halen IAV suşları dolaşan kapsamlı bir görünüm sağlamak için önemlidir. Bu domuzların fiziksel kısıtlama gerektirdiğinden güncel altın standart ante-mortem örnekleme tekniği (burun bezlerden yani toplama) emek yoğundur. Burun mendiller hayvanın hiçbir kısıtlama minimal ile domuz burnu genelinde kumaş parçasını ovuşturarak barındırır. Burun prosedürü gerçekleştirmek için basit ve profesyonel veteriner veya hayvan taşıma eğitimi ile personel gerektirmez silin. Wnazal bezlerden biraz daha az duyarlı hile, virüs algılama ve izolasyon oranları nazal düşük stres örnekleme yöntemleri gerektiğinde bireysel domuz örnekleme için bir alternatif mendil yapmak için yeterli. Işlem protokolü uygulanabilir bir nazal toplamak için gerekli adımlar tek bir domuz silin özetliyor.

Bu yöntemin başarılı kullanımı RRT-PCR herhangi bir çevresel enkaz numune alma sırasında aldı RNA ekstraksiyonu ve RRT-PCR sırasında iç kontrolün kullanımı ile birlikte, gösteri örnekleri PCR inhibitörleri içermiyordu, bu sonuçlar verir. Örnek aşılamadan sonra, virüs izolasyon kuyuları örnekten görülebilir çevre enkaz arındırılmış olmalıdır. PCR viral nükleik asit, ille canlı virüs tespit, çünkü PCR genellikle virüs izolasyonu daha yüksek IAV pozitif oranı getirir anlayış?...

Watch this Scientific Journal Video about Nasal Wipes for Influenza A Virus Detection and Isolation from Swine at JoVE.com

Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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

Domuz influenza A virüs Algılama ve İzolasyonu için Burun Mendil

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

nasal-wipes-for-influenza-a-virus-detection-and-isolation-from-swine

Research

JoVE Journal

Immunology and Infection

9.3K Görüntüleme.  The Ohio State University. The authors present a protocol to collect swine nasal wipes to detect and isolate influenza A viruses. 

Video: Nasal Wipes for Influenza A Virus Detection and Isolation from Swine

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.

Gribi için yüksek verimlilik Tespit Yöntemi

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

İmmünoloji ve Enfeksiyon

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.

Enfekte Hücre Kromatin gelen Gribi ribonükleoprotein Komplekslerinin Affinity Arıtma

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

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.

Embriyone Tavuk Yumurta Grip Virüsü Yayılım

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

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

Studying the interaction between proteins is key in understanding their function(s). A very powerful method that is frequently used to study interactions of proteins with other macromolecules in a complex sample is called co-immunoprecipitation. The described co-immunoprecipitation protocol allows to demonstrate and further investigate the interaction between the antiviral myxovirus resistance protein 1 (Mx1) and one of its viral targets, the influenza A virus nucleoprotein (NP). The protocol starts with transfected mammalian cells, but it is also possible to use influenza A virus infected cells as starting material. After cell lysis, the viral NP protein is pulled-down with a specific antibody and the resulting immune-complexes are precipitated with protein G beads. The successful pull-down of NP and the co-immunoprecipitation of the antiviral Mx1 protein are subsequently revealed by western blotting. A prerequisite for successful co-immunoprecipitation of Mx1 with NP is the presence of N-ethylmaleimide (NEM) in the cell lysis buffer. NEM alkylates free thiol groups. Presumably this reaction stabilizes the weak and/or transient NP&#8211;Mx1 interaction by preserving a specific conformation of Mx1, its viral target or an unknown third component. An important limitation of co-immunoprecipitation experiments is the inadvertent pull-down of contaminating proteins, caused by nonspecific binding of proteins to the protein G beads or antibodies. Therefore, it is very important to include control settings to exclude false positive results. The described co-immunoprecipitation protocol can be used to study the interaction of Mx proteins from different vertebrate species with viral proteins, any pair of proteins, or of a protein with other macromolecules. The beneficial role of NEM to stabilize weak and/or transient interactions needs to be tested for each interaction pair individually.

This co-immunoprecipitation protocol allows to study the interaction between the influenza A virus nucleoprotein and the antiviral Mx1 protein in human cells. The protocol emphasizes the importance of N-ethylmaleimide for successful co-immunoprecipitation of Mx1 and influenza A virus nucleoprotein.

İnfluenza A virüsü nükleoproteini Fare MX1 Protein Co-immunopresipitasyonu

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

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

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

Nötrofil İzolasyon ve Analiz Terapötik Ajanların için Lenfoma Hücre Duyarlılık kendi Rolü belirleme

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

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

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Influenza A Virüsü hemaglutininler aviditesi ve özgünlüğünün değerlendirilmesi için Minyatür Glycan Mikroarray Testi

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

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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.

Gradient Santrifüj tarafından İnfluenza A Virüsü Kapsidlerin Vitro Demontaj içinde

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

Rapid Molecular Detection and Differentiation of Influenza Viruses A and B

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality every year. The Influenza A and B assay was the first CLIA-waived molecular rapid flu test available. The Influenza A and B test works by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. Here, the performance of the Influenza A and B assay on frozen, archived nasopharyngeal swab (NPS) specimens stored in viral transport medium (VTM) were compared to a respiratory panel assay.
The performance of the Influenza A and B assay was evaluated by comparing the results to the respiratory panel reference method. The sensitivity for total influenza virus A was 67.5% (95% CI (CI), 56.6-78.5) and the specificity was 86.9% (CI, 71.0-100). For influenza virus B testing, the sensitivity and specificity were 90.2% (CI, 68.5-100) and 98.8% (CI, 68.5-100), respectively.
This system has the advantage of a significantly shorter test time than any other currently available molecular assay and the simple, pipette-free procedure runs on a fully integrated, closed, small-footprint system. Overall, the Influenza A and B assay evaluated in this study has the potential to serve as a point-of-care rapid influenza diagnostic test.

We describe a rapid, molecular-based Influenza A and B assay. The Influenza assay detects each target within 15 min by employing isothermal amplification with influenza-specific primers followed by target detection with molecular beacon probes. The Influenza A and B assay is user-friendly and required minimal hands-on time to perform.

Hızlı moleküler algılama ve Nezle Virüsleri A ve B farklılaşması

Influenza is a contagious respiratory illness caused by influenza viruses A and B in humans and causes a significant amount of morbidity and mortality ...

Influenza A Virus Studies in a Mouse Model of Infection

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone considering direct medical and hospitalization expenses and work absenteeism2. Animal models are crucial in Influenza A virus (IAV) studies to evaluate viral pathogenesis, host-pathogen interactions, immune responses, and the efficacy of current and/or novel vaccine approaches as well as antivirals. Mice are an advantageous small animal model because their immune system is evolutionarily similar to that found in humans, they are available from commercial vendors as genetically identical subjects, there are multiple strains that can be exploited to evaluate the genetic basis of infections, and they are relatively inexpensive and easy to manipulate. To recapitulate IAV infection in humans via the airways, mice are first anesthetized prior to intranasal inoculation with infectious IAVs under proper biosafety containment. After infection, the pathogenesis of IAVs is determined by monitoring daily the morbidity (body weight loss) and mortality (survival) rate. In addition, viral pathogenesis can also be evaluated by assessing virus replication in the upper (nasal mucosa) or lower (lungs) respiratory tract of infected mice. Humoral responses upon IAV infection can be rapidly evaluated by non-invasive bleeding and secondary antibody detection assays aimed at detecting the presence of total or neutralizing antibodies. Here, we describe the common methods used to infect mice intranasally (i.n) with IAV and evaluate pathogenesis, humoral immune responses and protection efficacy.

Influenza A viruses (IAVs) are important human respiratory pathogens. To understand the pathogenicity of IAVs and to perform preclinical testing of novel vaccine approaches, animal models mimicking human physiology are required. Here, we describe techniques to evaluate IAV pathogenesis, humoral responses and vaccine efficacy using a mouse model of infection.

Grip A virüs enfeksiyonu bir fare modeli çalışmalarda

Influenza viruses cause over 500,000 deaths worldwide1 and are associated with an annual cost of 12 - 14 billion USD in the United States alone ...

Generation of Escape Variants of Neutralizing Influenza Virus Monoclonal Antibodies

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface glycoproteins of the virus. The generation of escape variants is a powerful method in elucidating how viruses escape immune detection and in identifying critical residues required for antibody binding. Here, we describe a protocol on how to generate influenza A virus escape variants by utilizing human or murine monoclonal antibodies (mAbs) directed against the viral hemagglutinin (HA). With the use of our technique, we previously characterized critical residues required for the binding of antibodies targeting either the head or stalk of the novel avian H7N9 HA. The protocol can be easily adapted for other virus systems. Analyses of escape variants are important for modeling antigenic drift, determining single nucleotide polymorphisms (SNPs) conferring resistance and virus fitness, and in the designing of vaccines and/or therapeutics.

We describe a method by which we identify critical residues required for the binding of human or murine monoclonal antibodies that target the viral hemagglutinin of influenza A viruses. The protocol can be adapted to other virus surface glycoproteins and their corresponding neutralizing antibodies.

Grip virüsü monoklonal antikorları nötralize kaçış türevleri üretimi

Influenza viruses exhibit a remarkable ability to adapt and evade the host immune response. One way is through antigenic changes that occur on the surface ...