These studies were funded by US Public Health Services grants P01AI057005, R01AI047734, R01AI111806 and the Functional Profiling and Computational Biology Core of the University of Washington&#8217;s Center for AIDS Research (P30 AI027757).

<ol>
	<li>Domingo, E., Holland, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+virus+mutations+and+fitness+for+survival.">RNA virus mutations and fitness for survival.</a> Annu Rev Microbiol. 51, 151-178 (1997).</li><li>Domingo, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+viral+emergence.">Mechanisms of viral emergence.</a> Veterinary research. 41 (6), 38 (2010).</li><li>Martinez-Picado, J., Martinez, M. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Variable+fitness+impact+of+HIV-1+escape+mutations+to+cytotoxic+T+lymphocyte+(CTL)+response.">Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response.</a> PLoS Pathog. 5 (4), e1000365 (2009).</li><li>Rolland, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-acid+co-variation+in+HIV-1+Gag+subtype+C:+HLA-mediated+selection+pressure+and+compensatory+dynamics.">Amino-acid co-variation in HIV-1 Gag subtype C: HLA-mediated selection pressure and compensatory dynamics.</a> PLoS One. 5 (9), e12463 (2010).</li><li>Abraha, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CCR5-+and+CXCR4-tropic+subtype+C+human+immunodeficiency+virus+type+1+isolates+have+a+lower+level+of+pathogenic+fitness+than+other+dominant+group+M+subtypes:+implications+for+the+epidemic.">CCR5- and CXCR4-tropic subtype C human immunodeficiency virus type 1 isolates have a lower level of pathogenic fitness than other dominant group M subtypes: implications for the epidemic.</a> J Virol. 83 (11), 5592-5605 (2009).</li><li>Quinones-Mateu, M. 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Yazarlar hiçbir rakip mali çıkarlarını olduğunu beyan ederim. Burada ifade edilen görüşler yazarlarına aittir ve resmi ya da ABD Savunma Bakanlığı ya da Ordu Bölümü görüşlerini temsil olarak ele alınmamalıdır.

rekombinant HIV-1 molekül klonlar ve büyüme rekabet deneyleri yapımı: sunulan protokoller iki ana bölümden oluşuyordu. Bir ikili olarak enfekte edilmiş hücre kültürü içinde iki virüs ayırt etmek amacıyla, rekabet eden moleküler klonlar, bir RT-qPCR primeri-prob deneyi ile veya Sanger dizileme ile tespit edilebilir dizi etiketleri, ihtiva etmesi önemlidir. Bu protokol, HIV-1 NL4-3 Vif aynı bölgesini işgal eder ve aynı amino asit dizisini kodlayan, ancak altı ayn mutasyonla Vifa ve VifB etiketler, kullanır. Bu mutasyonlar etkilemediği gösterilmiştir edildi Viral replikasyon spor 20. Bu protokolde kullanılan PCR bazlı klonlama yöntemi kısıtlama sitesi tabanlı klonlama ile karşılaştırıldığında, klonlama siteleri seçiminde daha fazla esneklik sağlar. Bununla birlikte, PCR klonlama etkinliği uç boyutu arttıkça azalmaktadır. insert büyüklüğü akım limiti ~ 5 kb 34 olduğunu. PCR bazlı klonlama ve bölgeye yönelik mutagenesi içins, yüksek sadakat DNA polimeraz kullanılması ekstra mutasyonların olasılığını azaltmak için çok önemlidir. Ürünlerin yeterli miktarda elde etmek üzere gerekli olan PCR döngüsü az sayıda kullanılması da tavsiye edilir. Deneyimlerimize göre, burada tarif edilen PCR bazlı klonlama ve site-directed mutagenesis adımlardan sonra, herhangi bir ilave mutasyonlar tespit etmedi. Yine PCR ürünleri istenmeyen mutasyonlar kontrol etmek için sıralı olmalıdır. İdeal olarak, tüm HIV kodlama bölgesi yeniden dizilmesi gerekir. En az üç numune alma zaman noktaları üstel büyüme fazında 9 içinde muayene edilmelidir. Viral büyüme kinetiği ilk büyüme yarışma için uygun kültür dönemi ve örnekleme zamanı noktalarını belirlemek için günlük örnekleme yöntemiyle kurulmalıdır. Viral büyüme kinetiği etkileyen bir faktör enfeksiyonu (İçişleri Bakanlığı) çokluğu olduğunu. Daha ro elde etmek üzere gösterildiği gibi bu protokol, monoinfection ve büyüme rekabet her ikisi için 0.005 bir toplam MOI kullanırAlt Mois 9 daha büstü sonuçlar. Bununla birlikte, alt Mois gerekirse daha uzun bir üstel büyüme fazı elde etmek için kullanılan, ancak sonuç kıvamında pahasına yapılabilir. Bu protokol virüslerinin spor farkları önceden genellikle bilinmemektedir olduğunu varsayarak, 50:50 başlangıç ​​enfeksiyon oranını göstermektedir. Viral replikasyon kinetiği önemli farklılıklar gösteren ilk veriler olduğunda Ancak, eşitsiz girdi oranlarının kullanılması uygundur. Bu gibi durumlarda, 70:30 bir enfeksiyon oranı daha az uygun bir virüs fazlalığı 9 yerleştirilir büyük bir spor farkın tespiti için ayrılması tavsiye edilir. viral büyüme kinetikleri, ve HIV-1 alt tip B kullanılarak rekabet tahlilleri optimize edilmiştir büyüme gerçekleştirmek için TCID50 belirlenmesi için protokol, PBMC, tek bir donörden NL4-3 COTB-p24 ve PBMC&#39;ler, HIV tutarlı duyarlılık göstermiştir in vitro olarak 1 enfeksiyonu. Kültür dönemive bu protokolde yer alan numune zaman noktaları, insan PBMC içinde, HIV-1 M grubu virüsleri incelemek için uygun olması muhtemeldir. Tek bir kaynaktan elde edilmiş PBMC&#39;ler kullanılarak yüksek viral replikasyon farklı bir donörden hücreleri 35 değişebilir tutarlı sonuçlar elde etmek için önerilmektedir. Bir ikame, aynı havuzu bütün deneylerde boyunca kullanıldığı sürece daha az tercih edilmesine rağmen, birden fazla vericilerden alınma toplanmış PBMC kullanılabilir. Diğer bir alternatif hücre çizgilerinin kullanımı. Burada sunulan protokol T-hücresi hattı CEMx174 23,36 ile başarılı bir şekilde kullanılmıştır. Bununla birlikte, tohumlanmıştır hücre sayısı yeniden optimize uyumlu hücre büyümesini başarmak için olması önemlidir. Büyüme rekabet adımlarla uygun örnekleme zamanı noktalarını belirlemek için, farklı hücre hatları değişir muhtemelen viral büyüme kinetikleri de, yeniden kurulmalıdır. Uygunluğu hesaplamak için viral oranını belirlemek için iki farklı yöntem protoc dahildirol. ilk önce, her bir numune toplama noktasında viral cDNA kopyası sayısı ölçmek için RT-qPCR kullanır. Viral replikasyon spor sonra cDNA kopyası sayısına bağlı viral oranı hesaplanmıştır. Alternatif olarak, virüs oranı VifAB etiketi bölgelerinde kromatogram tepe yüksekliğine oranı esas alınarak tespit edilebilir. iki yöntem benzer sonuçlar (Şekil 4) elde edildi. kromatogram doruk yüksekliği yöntemi tasarlanmış sekans etiketi olmadan diğer HIV-1 suşlarının uygulanabilir. RT-qPCR viral varyantları ayırt etmek primerler veya probların kullanımı ilk dikkatle değerlendirilmesi gerekmektedir İçin (20 bakınız). Bununla birlikte, RT-qPCR gibi üstel büyüme fazı içinde birinci zaman noktasında elde edilenler gibi viral RNA&#39;nın, az bir miktarda olan örnekler için daha iyi bir hassasiyet sağlar. Viral cDNA&#39;nın doğrudan ölçümü RNA ekstraksiyonu ve cDNA sentezi ile ilgili ortaya çıkabilecek teknik sorunlar saptanmasını sağlar. Dizi etiketleri ile moleküler klonlar kullanma maliyetli bir çözüm sunar tRT-qPCR yöntemleri o gibi primerlerin ve probların iki çifti birden fazla virüs incelemek için ihtiyaç vardır. Bu strateji aynı zamanda sıralama çalışmada tüm virüsler arasında aynı sitede yapıldığı gibi sırayla tepe yüksekliği varyasyon problemi, komşu üsleri 37 nedeniyle kromatogra- önler. RT-qPCR Sanger sekanslanması için üretilen PCR ürünleri aynı zamanda başka yöntemler ile kullanımı, tek tek klonlann 12,13 toplu dizilemesi STD 4,7,17,19 veya oligonükleotid ligasyon tahlili (OLA) 38 gibi viral oranını belirlemek için olabilir .

Viral replikasyon spor belirli bir ortamda 1 enfeksiyon döl üretmek için bir virüs kapasitesi olarak tanımlanır ve zaman üzerinde 2 nüfus düzeyinde bir virüs çeşidi yaygınlığını belirlemede önemli bir faktör olduğunu. In vivo spor çalışmaları, HIV-1 olarak patojenik insan virüsler, çeşitli in vitro ve birlikte mümkün değildir ex vivo çoğaltma spor deneyleri ilaç direnci ve bağışıklık kaçış mutasyonları, epistazi ve evrim kaynaklanan fitness etkilerini incelemek için geliştirilmiştir viral popülasyonları 3-6. Farklı spor deneyleri arasında, büyüme rekabet tahlilleri in vivo 1,7,8 oluşur olarak iki veya daha fazla viral varyantları, tam olarak aynı çevre koşullarında aynı hücre popülasyonu için rekabet olarak, spor farklılıkları daha duyarlı ve geçerli önlemleri elde etmek için kabul edilmektedir. Büyüme rekabet deneyleri başlayarak birkaç variab önceles enfeksiyonunun farklı çokluklar (İçişleri Bakanlığı), viral giriş oranı, ve analiz için numune alma zamanlaması kullanımı dahil, tespit edilmesi gerekmektedir. Biz viral büyüme kinetiği üzerine ve rekabet deneyleri sonucu bu parametrelerin etkilerini inceledik ve hücre kültürü 9 HIV-1 fitness sağlam ölçümleri için gerekli anahtar faktörleri tespit ettik. Deney değişkenleri ek olarak, büyüme rekabet deneyleri viral varyantları ölçülmesi için çeşitli yöntemler vardır. 10,11 Toplu veya klonal sıralama 12,13 faiz sitesinde (ler) nükleotid frekansları dayalı rekabet virüslerin oranını belirlemek için kullanılır olmuştur. Bağıl spor zaman bu oran değişiklik elde edilir. DNA dizileme hizmetleri yaygın olarak bulunmaktadır Bu yöntem uygundur. Paralel alel-spesifik dizilim (PASS) yöntemi birden fazla sitelerde sıralama ve rekombinantların 14 saptanmasına olanak </sup&gt;, Ama aynı zamanda özel olarak geliştirilmiş reaktifler ve saptama sistemlerini gerektirmektedir. Esas olarak, bu yöntemler, söz konusu bir bölge nükleotid farklılıklar az sayıda virüs suşu için geliştirilmiştir. Diğer yöntemler, küçük bir raportör gen sıralama ile yarışan virüsleri, heterodubleks izleme tahlilleri (HTA) 4,7,17,19 ayırt veya transkripsiyon kantitatif gerçek zamanlı polimeraz zincir reaksiyonu ters etiketleri gibi 15 veya eşanlamlı mutasyonları 4,16-18 kullanmak bağımsız olarak sekans benzerliği suşları rekabet çalışma için geçerli yapılabilir her biri (RT-qPCR) 15,16,18,20. Ek bir adım viral genomları içine etiketleri tanıtmak için gerekli ve RT-qPCR tahlil ayrıca belirli maddeler ve enstrümantasyon gerektirir. Biz bulduk toplu Sanger dizileme verimleri karşılaştırılabilir sonuçlar 9. Büyüme yarışma sonrasında, viral replikasyon spor göreli uygunluk veya t arasında fitness oranı olarak sunulmuşturvira varyantlar WO. bir virüs göreli spor aşı maddesi, ilk oran ya da iki rakip virüsler arasında net büyüme oranı farkı olarak normalleştirilmiş bir viral varyantın nihai oran olarak tanımlanabilmektedir. Biz ikinci yöntemi, sadece üstel büyüme fazında içinde uzunlamasına veri noktalarını kullanarak, en sağlam sonuçlar 9,20 ürettiğini gördük. Vitro fitness deneyleri, biyolojik klonlar 6-8 ve HIV-1 bulaşıcı moleküler klonlar incelemek için öncelikle kullanılır. İkincisi, genetik manipülasyon için uygun olan, genellikle belirli bir mutasyon veya ilgi duyulan 3-5,21,22 spesifik dizilerinden uygunluk üzerindeki etkisini incelemek için kullanılmıştır. Aşağıdaki protokoller viral büyüme kinetikleri, bir dizi etiketi içeren HIV-1 vektörleri kullanarak yeni tam uzunlukta bulaşıcı HIV-1 molekül klonlar inşa ilgi mutasyonlar tanıtarak, viral stokları yapma ve kurma açısından bir iş akışını açıklarGöreli uygunluğu büyüme rekabet tahlili yapılması ve hesaplanması için (Şekil 1). Optimize prosedürler kullanılarak, üç rekombinant HIV-1 mutantlar yarattı ve çoğaltma zindeliği belirledi. yeniden birleştirici molekül klonun ilk sentetik COTB (Merkez-of ile pNL4-3, HİV-1 laboratuar sarmalı NL4-3 tam uzunlukta enfeksiyon genomunu ihtiva eden bir plazmid HIV-1 Gag-p24 gen bölgesine değiştirilmesiyle yapılmıştır Ağaç, alt tip B) gag-p24 dizisi 23 prototip gerginlik yaratır. Tek amino değişiklikleri (T186M, T242N ve I256V) daha sonra üç mutant klonlar oluşturmak tanıtıldı. Her mutant verilen genetik arka planda her mutasyonun fitness etkisini gözlemlemek için prototip virüsüne karşı yarıştı edildi. Üç mutantlar prototip virüsü daha hafif anlamlı derecede düşük çoğaltma fsitness çeşitli düzeylerde göstermişlerdir. T242N mutasyonu, daha önce bir orta fitne olduğu bildirilmiştirss Bu çalışmada gösterilen sonuca benzer 24-26, maliyet. Diğer iki mutasyonun spor maliyeti, daha önce bildirilmemiştir.

<table border="0" cellpadding="0" cellspacing="0" width="1172">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Name of Material/ Equipment</td>
			<td style="width:484px;">Company</td>
			<td style="width:137px;">Catalog Number</td>
			<td style="width:196px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Construction of recombinant clones</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Chimeric primer/Mutagenic/Sequencing primers</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom DNA oligos</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">pNL4-3VifA and pNL4-3VifB plasmid</td>
			<td style="width:484px;">N/A</td>
			<td style="width:137px;">N/A</td>
			<td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-Fidelity DNA polymerase</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">High-fidelity buffer</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>F-549S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Thermal cycler</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems&#174; GeneAmp&#174; PCR System 9700</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA loading dye</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td style="width:137px;">R0631</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">1kb Plus DNA ladder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">10787-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAquick PCR Purification Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAquick gel extraction kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">28704</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">DpnI enzyme</td>
			<td style="width:484px;">New England Biolabs</td>
			<td>R0176S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">TOP10 chemically competent Escherichia coli</td>
			<td style="width:484px;">Invitrogen</td>
			<td>C4040-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Luria Broth base powder</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">12795-084</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Carbenicillin</td>
			<td style="width:484px;">Research Products International</td>
			<td style="width:137px;">C46000-25.0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAprep Spin Miniprep kit&#160;</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">27104</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">EndoFree Plasmid Maxi Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">12362</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Transfection</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">X-tremeGENE 9 DNA transfection reagent</td>
			<td style="width:484px;">Roche</td>
			<td>6365787001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HEK 293T-17</td>
			<td style="width:484px;">ATCC</td>
			<td style="width:137px;">CRL-11268</td>
			<td>http://www.atcc.org/</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">0.22um filter top tube</td>
			<td style="width:484px;">VWR International</td>
			<td>89220-716&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>10566016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Iscove&#8242;s Modified Dulbecco&#8242;s Medium (IMDM)</td>
			<td style="width:484px;">Life Technologies</td>
			<td>31980-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RPMI-1640 media</td>
			<td style="width:484px;">Life Technologies</td>
			<td>61870-036</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phytohemagglutinin (PHA)</td>
			<td style="width:484px;">Thermo Scientific</td>
			<td>R30852801</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Human Interleukin-2</td>
			<td style="width:484px;">Roche</td>
			<td>11147528001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum</td>
			<td style="width:484px;">JR Scientific</td>
			<td style="width:137px;">43640</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Penicillin and Streptomycin</td>
			<td style="width:484px;">Corning Cellgro</td>
			<td>30-001-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">73520-906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">48 well plate</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">62407-338</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well flat-bottomed plate</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td style="width:137px;">T-3015-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">96 well round-bottomed plate</td>
			<td style="width:484px;">BD Falcon</td>
			<td style="width:137px;">353077</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 ml Microcentrifuge Tube</td>
			<td style="width:484px;">Mt. Baker Bio</td>
			<td style="width:137px;">MBD-1500</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">1.5 mL tubes w/ O-ring</td>
			<td style="width:484px;">VWR Scientific</td>
			<td style="width:137px;">89004-290</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:355px;">50 mL conical tube</td>
			<td style="width:484px;">ISC Bioexpress</td>
			<td>C-3317-6</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ELISA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Triton-X 100</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Phosphate buffer saline (PBS)</td>
			<td style="width:484px;">Invitrogen</td>
			<td style="width:137px;">14190-250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Fetal bovine serum (FBS)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>F0392</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">glycerol</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td style="width:137px;">G5516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Tween 20</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:484px;">Sigma-Aldrich</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Rabbit anti-HIV-1 SF2 p24 antiserum</td>
			<td style="width:484px;">NIH AIDS Reagent Program</td>
			<td style="width:137px;">4250</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Goat anti-rabbit HRP</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">474-1516</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TMB Microwell Peroxidase Substrate</td>
			<td style="width:484px;">KPL</td>
			<td style="width:137px;">52-00-01</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">H2SO4</td>
			<td>Fisher Scientific</td>
			<td>A300-500</td>
			<td>Causes severe burns by all exposure routes. Use personal protective equipment. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 minutes.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">HIV p24 standard</td>
			<td style="width:484px;">AIDS and Cancer Virus program (NCI-Frederick)</td>
			<td style="width:137px;">N/A</td>
			<td>For order details please contact Julian Bess, Jr. (bessjw@mail.nih.gov); http://ncifrederick.cancer.gov/Programs/Science/Acvp/bio/Bess.aspx</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Microplate reader</td>
			<td style="width:484px;">Molecular Devices</td>
			<td style="width:137px;">N/A</td>
			<td>VMax Kinetic ELISA Microplate Reader</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNA isolation cDNA synthesis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxtractor</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">N/A</td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">QIAamp Viral RNA Mini Kit</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;">52906</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">dNTP</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-39026</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">SuperscriptIII</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">First-strand buffer&#160;</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dithiothreitol (DTT)</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18080-085</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Rnase inhibitor</td>
			<td style="width:484px;">Roche</td>
			<td>3335402001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">RnaseH</td>
			<td style="width:484px;">Invitrogen</td>
			<td>18021-071</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Real-time qPCR machine</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Applied Biosystems 7300 Real-Time PCR&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan&#174; Gene Expression Master Mix</td>
			<td style="width:484px;">Life Technologies</td>
			<td>4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Forward, reverse primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Probe</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">optical 96 well reaction plate</td>
			<td style="width:484px;">Life Technologies</td>
			<td style="width:137px;">I19N3Q216</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PCR+sequencing</td>
			<td style="width:484px;"></td>
			<td style="width:137px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Taq DNA polymerase (Biolase)</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NH4 buffer&#160;</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">MgCl2</td>
			<td style="width:484px;">Bioline</td>
			<td style="width:137px;">Bio-21043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">Sequencing primer</td>
			<td style="width:484px;">IDT</td>
			<td style="width:137px;">N/A</td>
			<td>Custom order</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:355px;">QIAxcel</td>
			<td style="width:484px;">Qiagen</td>
			<td style="width:137px;"></td>
			<td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DNA sequencing service provider</td>
			<td>http://www.htseq.org/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">GRC web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/grc/</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ChromatQuan web tool</td>
			<td>http://indra.mullins.microbiol.washington.edu/cgi-bin/chromatquant.cgi</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

HIV-1 Gag-p24 olarak tek bir amino asit değişikliklerinin spor etkisini incelemek için, HIV-1 COTB-p24 geni 23,32,33 ihtiva eden bir pNL4-3 plazmide mutasyonların katılması için oligonükleotid yönlendirilmiş mutagenez kullanılmıştır. Viral stoklar 293T hücrelerinin transfeksiyonu ile üretilir ve 48 saat sonra hasat edilmiştir. Biz Reed-Muench yöntemi 31, her bir virüs stokunun% 50 doku kültürü enfeksiyon dozu (TCID50) tahmin edilmiştir. Prototip TCID50 ve mutant virüsler, 10 4 10 5 IU / ml (Şekil 3A) arasında değişmektedir. Rekombinant virüslerin büyüme kinetikleri 0.005 MOl&#39;de tek bir donörden alınan PBMC içinde kurulmuştur. RT-qPCR altı gün boyunca günlük olarak, viral cDNA kopyası sayısını ölçmek için kullanılmıştır. Bütün mutant ve prototip virüsleri &lt;Viral RNA kopya sayısı artış azalan bir eğimi (belirtildiği gibi viral büyüme yavaşladı 2. ve 4. günde, aşağıdakiler arasında katlanarak büyüyenstrong&gt; Şekil 3B). cDNA azalma (inokulum karşılık gelir) gün 0 ve 2 arasında sayı hücreleri virüs emilmesi ve gün 1 yıkama (aşama 5.1.6) bağlanmamış virionlann çıkarılması nedeniyle kopyalama. Üslü büyüme fazında, üç mutant prototip virüsü (Şekil 3C), daha yavaş bir büyüme oranını (g) vardı. Her üç mutantlar 0.005 toplam İçişleri Bakanlığı büyüme rekabet deneylerinde prototip virüsüne karşı yarıştı bulundu. Ikili olarak enfekte kültürlerden viral büyüme kinetikleri monoinfection benzer olduğu; Viral üstel büyüme fazı gün 2 ve 4 arasında, ve virüs büyümesi günde 5 (Şekil 4A) etrafında bir düzlüğe ulaşmıştır. Viral büyüme oranı farklılıkları, zaman içinde, viral oranı değişikliği elde edilmiştir. Viral oran cDNA kopyası başvuru sayısı ve RT-qPCR kullanarak mutant virüslere ve karşılaştırarak zirve yükseklikleri ile hesaplanmıştırsırayla iki virüs (Şekil 4B) ayırt edici nükleotit siteleri kromatogra-. doruk yüksekliğini ve viral RNA kopya sayısı yöntemleri kullanılarak saptanmıştır büyüme oranı farklılıkları, benzer sonuçlar (Şekil 4C) vermiştir. Her üç mutantlar düşük uygunluğu (Şekil 4C) sahip mutant I256V ile prototip virüs daha düşük çoğaltma uygunluk vardı. <img alt="Şekil 1" src="/files/ftp_upload/52610/52610fig1.jpg" /> Şekil 1: Bu yazıda sunulan protokollerin akış diyagramı, viral stokların Virüs hazırlanması protokolleri endişe HIV-1 rekombinant klonların yapımı ve üretimi.. Fitness Tahliller protokoller viral büyüme kinetiği kurulması ve nispi viral uygunluğunu belirlemek için vardır. Kesik çizgiler protokoller için alternatif akımlarını gösterir. Örneğin, bir mutasyonun HIV-1 NL4-3 doğrudan sokulabilirbir rekombinant klonu oluşturmadan molekül klonu. <img alt="Şekil 2," src="/files/ftp_upload/52610/52610fig2.jpg" /> Şekil 2:. Örtüşme uzatma PCR (A) kullanılarak HIV-1 NL4-3 COTB Gag-p24 yeniden birleştirici molekül klonlarının kurulması kimerik primerler dizayn. 5 &#39;primer yarıları NL4-3 vektör dizisi içeren ve 3&#39; yarım uç dizisinin uçlarını içerir. (B), kimerik primerler uç fragmanını COTB Gag-p24 amplifiye edilmesi için kullanılır. (C) uç parçaları yeni bir rekombinant plazmidi üretmek için ikinci bir PCR için primerler olarak kullanılmıştır. <img alt="Şekil 3," src="/files/ftp_upload/52610/52610fig3.jpg" /> Şekil 3:. PBMC&#39;lerde viral büyüme özellikleri (A) Log 10 TCID 50 </sub&gt; viral stokların. (B) MOl&#39;de = 0.005 başlamış monoinfections viral büyüme kinetiği. Panel (B) elde edilen üslü büyüme fazında (gün 2-4) de dahil olmak üzere, altı gün boyunca (° C) Viral büyüme oranları. Gösterilen değerler, bir deneyden üç kopyanın ortalamasını temsil eder. Hata çubukları,% 95&#39;lik güven aralıklarını gösterir. <img alt="Şekil 4," src="/files/ftp_upload/52610/52610fig4.jpg" /> Şekil 4: Viral büyüme ve bağıl spor tayinleri (A) dually enfekte PBMC kültürlerinde viral büyüme.. T242N ve prototip virüs (B) Oranlar RT-qPCR veya sıralama tepe yüksekliği metodu kullanılarak belirlenmiştir. (A) &#39;da gösterildiği gibi, (C) mutant ve ikili olarak enfekte kültürlere prototip virüsler arasında net, viral büyüme oranı farklılıkları (d),. d değerleri f hesaplandı(B) &#39;de gösterildiği viral oranı verilerini rom. Gösterilen değerler, bir deneyden üç kopyanın ortalamasını temsil eder. Hata çubukları,% 95&#39;lik güven aralıklarını gösterir.

Watch this Scientific Journal Video about Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses at JoVE.com

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

İnsan İmmün Yetmezlik Virüs çoğaltma Fitness belirlenmesi için İkili Büyüme Rekabet Analizi

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

pairwise-growth-competition-assay-for-determining-replication-fitness

Research

JoVE Journal

Immunology and Infection

11.1K Görüntüleme.  University of Washington. Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

Video: Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Determining the Reactivity and Titre of Serum using a Haemagglutination Assay

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood cells are particularly useful targets as they are readily available and agglutination is observable using the naked eye. This technique is commonly used to determine the titre of an antibody (Ab), for blood grouping and viral quantification. In this video, the steps involved in preparing and performing a haemagglutination assay is demonstrated using antibodies specific to blood group A-antigens added to red blood cells (Revercells). The antiserum is serially diluted in a 96 well U-bottom microtitre tray, to which is added a suspension of Revercells. The samples are mixed and then incubated at 37&deg;C for 60 minutes. After this time, the samples can then be easily scored for  ve, +ve and intermediate (-/+) haemagglutination reactions. This approach allows for the reactivity and titre of a serum sample to be assessed using a rapid and simple technique. The video will cover the theory behind the assay, how the results are read and interpreted, how the titre is determined, how the assay can be modified and any issues associated with the use of this technique.

Haemagglutination is a form of agglutination where antibodies bind to red blood cells. Red blood cells are both readily available and the results are readily observable using the naked eye. This video demonstrates the steps involved in a haemagglutination assay, interpreting the results and determining the titre.

Hemaglütinasyon Testi kullanarak Reaktivite ve Serum Titre Belirlenmesi

Haemagglutination is a specific form of agglutination and is used when antibodies bind to red blood cells, which act as a particulate antigen. Red blood ...

İmmünoloji ve Enfeksiyon

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.
Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.
In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.
The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.

The study describes a cost-effective method for the identification of the source of fecal/urine contamination or contamination by nitrates in water using qPCR for the specific quantification of human/porcine/bovine DNA viruses, adenoviruses and polyomaviruses, proposed as MST tools.

Özgül İnsan ve Hayvan Virüsler Mikrobiyal Kaynak Takip için maliyet-etkin Yöntemi

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant ...

Determining the Phagocytic Activity of Clinical Antibody Samples

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as pathology of disease. While the properties of the variable domains of antibodies have long been considered critical to in vivo function, the ability of antibodies to recruit innate immune cells via their Fc domains has become increasingly appreciated as a major factor in their efficacy, both in the setting of recombinant monoclonal antibody therapy, as well as in the course of natural infection or vaccination1-3.
Importantly, despite its nomenclature as a constant domain, the antibody Fc domain does not have constant function, and is strongly modulated by IgG subclass (IgG1-4) and glycosylation at Asparagine 2974-6. Thus, this method to study functional differences of antigen-specific antibodies in clinical samples will facilitate correlation of the phagocytic potential of antibodies to disease state, susceptibility to infection, progression, or clinical outcome.
Furthermore, this effector function is particularly important in light of the documented ability of antibodies to enhance infection by providing pathogens access into host cells via Fc receptor-driven phagocytosis7. Additionally, there is some evidence that phagocytic uptake of immune complexes can impact the Th1/Th2 polarization of the immune response8.
Here, we describe an assay designed to detect differences in antibody-induced phagocytosis, which may be caused by differential IgG subclass, glycan structure at Asn297, as well as the ability to form immune complexes of antigen-specific antibodies in a high-throughput fashion. To this end, 1 &mu;m fluorescent beads are coated with antigen, then incubated with clinical antibody samples, generating fluorescent antigen specific immune complexes. These antibody-opsonized beads are then incubated with a monocytic cell line expressing multiple Fc&gamma;Rs, including both inhibitory and activating. Assay output can include phagocytic activity, cytokine secretion, and patterns of Fc&gamma;Rs usage, and are determined in a standardized manner, making this a highly useful system for parsing differences in this antibody-dependent effector function in both infection and vaccine-mediated protection9.

We present a high-throughput flow cytometric assay to determine the phagocytic activity of antigen-specific antibodies from clinical samples, utilizing fluorescent antigen-coated beads and a monocytic cell line expressing multiple Fc receptors&#x2014;providing receptor usage and phagocytic activity determinations in a standardized and reproducible fashion for any antigen of interest.

Klinik Antikor Örnekleri fagositik Aktivitesi Belirlenmesi

Antibody-driven phagocytosis is induced via the engagement of Fc receptors on professional phagocytes, and can contribute to both clearance as well as ...

Determining Optimal Cytotoxic Activity of Human Her2neu Specific CD8 T cells by Comparing the Cr51 Release Assay to the xCELLigence System

Cytotoxic CD8 T cells constitute a subgroup of T cells that are capable of inducing the death of infected or malignant host cells1. These cells express a specialized receptor, called the T cell receptor (TCR), which can recognize a specific antigenic peptide bound to HLA class I molecules2. Engagement of infected cells or tumor cells through their HLA class I molecule results in production of lytic molecules such as granzymes and perforin resulting in target cell death. While it is useful to determine frequencies of antigen-specific CD8 T cells using assays such as the ELIspot or flow cytometry, it is also helpful to ascertain the strength of CD8 T cell responses using cytotoxicity assays3. The most recognizable assay for assessing cytotoxic function is the Chromium Release Assay (CRA), which is considered a standard assay 4. The CRA has several limitations, including exposure of cells to gamma radiation, lack of reproducibility, and a requirement for large numbers of cells. Over the past decade, there has been interest in adopting new strategies to overcome these limitations. Newer approaches include those that measure caspase 
release 4, BLT esterase activity 5 and surface expression of CD107 6. The impedance-based assay, using the Roche xCelligence system, was examined in the present paper for its potential as an alternative to the CRA. Impedance or opposition to an electric current occurs when adherent tumor cells bind to electrode plates. Tumor cells detach following killing and electrical impedance is reduced which can be measured by the xCelligence system. The ability to adapt the impedance-based approach to assess cell-mediated killing rests on the observation that T cells do not adhere tightly to most surfaces and do not appear to have much impact on impedance thus diminishing any concern of direct interference of the T cells with the measurement. Results show that the impedance-based assay can detect changes in the levels of antigen-specific cytotoxic CD8 T cells with increased sensitivity relative to the standard CRA. Based on these results, impedance-based approaches may be good alternatives to CRAs or other approaches that aim to measure cytotoxic CD8 T cell functionality.

The chromium release assay, a common assay for detecting cytotoxic T cell activity, has several limitations. Using antigen-specific CD8 T cells and the human breast cancer tumor line, SKBR3, in the present article, an impedance-based approach was examined for the capability of detecting cell killing.

XCELLigence Sistemi Cr51 Yayın Assay Karşılaştırılması tarafından İnsan Her2neu Özgül CD8 T hücrelerinin Optimal Sitotoksik Aktivite Belirlenmesi

Cytotoxic CD8 T cells constitute a subgroup of T  cells that are capable of inducing the death of infected or malignant host  cells1. These cells express ...

Determining Soil-transmitted Helminth Infection Status and Physical Fitness of School-aged Children

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty prevails and hygiene behavior, water supply, and sanitation are often deficient1,2. Ascaris lumbricoides, Trichuris trichiura, and the two hookworm species, Ancylostoma duodenale and Necator americanus, are the most prevalent STHs3. The estimated global burden due to hookworm disease, ascariasis, and trichuriasis is 22.1, 10.5, and 6.4 million disability-adjusted life years (DALYs), respectively4. Furthermore, an estimated 30-100 million people are infected with Strongyloides stercoralis, the most neglected STH species of global significance which arguably also causes a considerable public health impact5,6. Multiple-species infections (i.e., different STHs harbored in a single individual) are common, and infections have been linked to lowered productivity and thus economic outlook of developing countries1,3.
For the diagnosis of common STHs, the World Health Organization (WHO) recommends the Kato-Katz technique7,8, which is a relatively straightforward method for determining the prevalence and intensity of such infections. It facilitates the detection of parasite eggs that infected subjects pass in their feces.
With regard to the diagnosis of S.stercoralis, there is currently no simple and accurate tool available. The Baermann technique is the most widely employed method for its diagnosis. The principle behind the Baermann technique is that active S.stercoralis larvae migrate out of an illuminated fresh fecal sample as the larvae are phototactic9. It requires less sophisticated laboratory materials and is less time consuming than culture and immunological methods5.
Morbidities associated with STH infections range from acute but common symptoms, such as abdominal pain, diarrhea, and pruritus, to chronic symptoms, such as anemia, under- and malnutrition, and cognitive impairment10. Since the symptoms are generally unspecific and subtle, they often go unnoticed, are considered a normal condition by affected individuals, or are treated as symptoms of other diseases that might be more common in a given setting. Hence, it is conceivable that the true burden of STH infections is underestimated by assessment tools relying on self-declared signs and symptoms as is usually the case in population-based surveys.
In the late 1980s and early 1990s, Stephenson and colleagues highlighted the possibility of STH infections lowering the physical fitness of boys aged 6-12 years11,12. This line of scientific inquiry gained new momentum recently13,14,15. The 20-meter (m) shuttle run test was developed and validated by L&eacute;ger et al.16 and is used worldwide to measure the aerobic fitness of children17. The test is easy to standardize and can be performed wherever a 20-m long and flat running course and an audio source are available, making its use attractive in resource-constrained settings13. To facilitate and standardize attempts at assessing whether STH infections have an effect on the physical fitness of school-aged children, we present methodologies that diagnose STH infections or measure physical fitness that are simple to execute and yet, provide accurate and reproducible outcomes. This will help to generate new evidence regarding the health impact of STH infections.

Chronic infection with soil-transmitted helminths (STHs) causes malabsorption, stunting, and wasting in the growing child. Hence, it is plausible that these infections also reduce the physical fitness of children. Here, we visualize two techniques for the diagnosis of STHs and the 20-meter shuttle run test for assessing children's physical fitness.

Belirlenmesi Toprak bulaşan Helmint Enfeksiyon Durum ve Okul Çağındaki Çocuklarda Fiziksel Uygunluk

Soil-transmitted helminth (STH) infections are common. Indeed, more than 1 billion people are affected, mainly in the developing world where poverty ...

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

T6SS aracılı Bakteriyel Rekabet Monitör A Görsel Assay

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Polarize İnsan Havayolu bir sıvı kaplı Kültürünün Oluşturulması Solunum Patojenler için Host Hücre Yanıtı Okumak Epitel Calu-3 Hücreler<em&gt; In vitro</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

Bir Kısıtlama Enzim Temelli Klonlama Yöntemi değerlendirin<em&gt; In vitro</em&gt; HIV-1 Tipi C Gag-MJ4 Kimerik Virüslerin Çoğaltma Kapasitesi

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

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.

Kimerik fare modellerinde Rekombinant Grip aşıların burun içinde yoluyla mukozal bağışıklık Okumak

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 ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

Bir Hepatit B Virüsü Muhabir Sisteminin Geliştirilmesi Çoğaltma Döngüsü Erken Aşamaları Monitör

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...