Biz Virochip protokolün ilk geliştirme ve optimizasyonu için David Wang, Anatoly Urisman Amy Kistler, Kael Fischer, Patrick Tang, ve Alexander Greninger teşekkür ederim. Bu çalışma bir NIH K08 hibe ve Abbott Keşif Ödülü (CC) ve Howard Hughes Tıp Enstitüsü (JLD) tarafından desteklenmektedir.

Virochip testinin adımlar (1) klinik örneklerden nükleik asit ekstraksiyon, (2) çıkarılan RNA ters transkripsiyon ve 2. iplikçik cDNA sentezi, rastgele astarlanmalıdır cDNA (3) PCR, (3) Cy3 floresan boya dahil (4) Virochip mikroarray etiketli malzeme hibridizasyon ve (5) tarama ve analizi (Şekil 1). Aşağıda gösterilen protokol grip benzeri bir hastalığa sahip bir çocuğun bir nazal sürüntü örneği Virochip testinin kullanımını göstermek. Primer Diziler Primer bir 5&#39;-GTTTCCCAGTCACGATA-(N 9) -3 &#39; Primer B 5&#39;-GTTTCCCAGTCACGATA-3 &#39; 1. Nükleik Asit Ekstraksiyonu <ol><li> Bu örnekleri potansiyel bulaşıcı olarak klinik örneklerden nükleik asit ekstraksiyon, Biyolojik Güvenlik Seviye 2 (BSL-2) veya daha yüksek koşulları altında yapılmalıdır. </li><li> Nazal sürüntü örneği viral tutarak orta taşınır. 1.5 ml Eppendorf mikrosantrifüj tüp içine örnek kısım 200 mcL. </li><li> İsteğe Bağlı: 0.22 mikron filtre (viral parçacıkların arındırmak için) geçmesine örnek </li><li> İsteğe bağlı: Turbo DNAz kiti (Ambion) kullanarak üreticinin talimatlarına göre DNAz (ana bilgisayar genomik DNA aşağılamak ve arındırmak için korumalı, encapsidated viral nükleik asit) ile ön işlemden örnek </li><li> Zymo ZR viral RNA Ekstraksiyon Kiti veya üreticinin talimatlarına göre Qiagen Ultrasens Virus Kit kullanarak örnek ayıklayın. </li></ol> 2. Ters Transkripsiyon ve 2.-iplikçik cDNA sentezi (&quot;Yuvarlak A&quot;) <ol><li> 4 uL çıkarılan RNA 1 uL 40 mcL / pmol Primer ekleyin. Isı 65 ° C PCR (örneğin GeneAmp PCR Sistemi 9700) ve daha sonra 5 dakika boyunca oda sıcaklığında x 5 dakika soğumasını bekleyin. </li><li> 2 mcL 5X RT tampon, 1 mcL 12.5 mM dNTP, 1 mcL su, 0.5 mcL 0.1M DTT, ve 0.5 mcL SSIII RT (Invitrogen) oluşan bir ana karışımı 5 mcL ekleyin. 42 ° C&#39;de x 60 dakika. </li><li> Programlanabilir bir PCR, ısı 94 ° 10 serin C x 2 dk (iptal etmek için ters transkriptaz reaksiyonu), ° C, nabız santrifüj, ve ardından 1 mcL 5X Sequenase tampon, 3.8 mcL su oluşan 5 mcL Sequenase karışımı ekleyin, 0.15 mcL Sequenase (USB Corporation). 2. iplikçik sentez için, 10 ° C - 37 ° C 8 dk boyunca, 94, 8 dakika sonra, ısı tutun ° C x 2 dk (Sequenase inaktive etmek için) ve 10 ° C (basılı tutun) serin kadar rampa </li><li> İsteğe bağlı: Rd örnek bir cDNA -20 ° C&#39;de bu noktada saklanabilir </li></ol> 3. PCR Amplifikasyon rastgele Astarlı cDNA (&quot;Yuvarlak B&quot;) <ol><li> 5 Rd uL bir örnek, 45 mcL 5 mcL 10X PCR buffer, 1 mcL 12.5 mM dNTP, 1 mcL 100 pmol / mcL astar B, 1 mcL KlenTaq LA (Sigma), ve 37 mcL su oluşan bir ana karışımı ekleyin . </li><li> PCR protokolü aşağıdaki gibi çalıştırın: 94 ° C, 2 dakika → (25 devir 94 ° C, 30 / 50 ° C, 45 s / 72 ° C, 1 dak.) → 72 ° C, 5 dk → 10 ° C ( basılı tutun) </li><li> % 1.5 agaroz elektroforez jel üzerinde Rd B numunesi kontrol edin. A smear yaklaşık 200 - 1000 bp görüntülenmeyecektir gerekir (Şekil 2A) </li></ol> 4. Cy3 Floresan Boya Kuruluş <ol><li> İsteğe Bağlı: Cy5, floresan boya ile başka bir örnek etiketli olabilir ve aynı mikroarray için melezleşmiştir. </li><li> Aa-dNTP birleştirmek için, 5 uL 10X PCR buffer, 1 uL 12.5 mM aa-dNTP (Invitrogen), 1 uL 100 pmol / uL astar Yatak, 1 oluşan bir ana karışımı 45 uL Rd B numunesinin 5 uL 1 uL KlenTaq LA (Sigma), ve 37 uL su. PCR protokolü aşağıdaki gibi çalıştırın: 94 ° C, 2 dakika → (15 döngü 94 ° C, 30 / 50 ° C, 45 s / 72 ° C, 1 dak.) → 72 ° C, 5 dk → 10 ° C ( basılı tutun) </li><li> Rd C örnek üreticinin talimatlarına göre DNA Temiz ve Konsantratörü-5 Seti (Zymo Araştırma) ile temizleyin. 10 mcL Zehir. </li><li> 1M bikarbonat 1 uL (Sigma) ve Rd C Örnek 1 uL Cy5 (GE Healthcare) ekleyin. </li><li> Inkübe x 1 saat karanlıkta. </li><li> Üreticinin talimatlarına göre DNA Temiz ve Konsantratörü-5 Seti (Zymo Araştırma) ile Cy3 etiketli örnek temizleyin. 12 mcL Zehir. </li></ol> 5. Hibridizasyon için Virochip Mikroarray <ol><li> İsteğe bağlı: (numune normalleşmesi için), Nanodrop spektrofotometre dahil cDNA konsantrasyon ve boya miktarını kontrol 1.5 mcL </li><li> 1 mcL 25X parçalanma tampon, 5 mcL 5X engelleme tampon, 9.5 mcL Cy3-etiketli örnek (veya normale miktarı), su ve PCR şeridi tüpü 25 mcL toplam hacmi (Agilent) eklemek </li><li> 2X GEX hibridizasyon tamponu (Agilent) 25 mcL ekleyin. Kabarcıkları kısaca aşağı spin. </li><li> Conta slayt üzerine numune 40 mcL yükleyin. </li><li> Place Agilent baskılı Virochip array (Californi Üniversitesi, San Francisco / Agilent) (aşağı &quot;Agilent&quot; işaretli tarafı) ve conta slayt üst sıkıca vidayı sıkın. </li><li> 65 gecede melezleşme ° C fırında 10 devirde hız ayarı. </li><li> 37 ° C&#39;ye kadar diziler, ön ısıtma ve ön tampon 2 (Agilent) yıkamak için Tampon 1 (Agilent) conta slayt / dizi sandviç sualtı ayrı ele alalım. Tampon 1 x 1 dakika yıkayın. Önceden ısıtılmış tampon 2 x 1 dakika yıkayın. Yavaş yavaş, kabarcıkları önlemek için dikkatli olmak (kimwipe uzakta doğrudan dizi aktif yüzlerine dokunmaktan kaçınmak için dikkatli olmak ekstra nem fitil kullanın), tampon 2 slayt kaldırmak </li><li> Slayt slayt tutucunun içine yerleştirin ve dizi tarayıcı takın. </li></ol> 6. Virochip Tarama ve Analizi <ol><li> Tarama Cy3 etiketli dizi araç standart tarama protokolü (Şekil 2B) göre 5 mm veya 2 mm. </li><li> Virochip mikroarray&#39;ler görsel denetim, kümeleme analizi, ya da otomatik Virochip analiz programı tarafından analiz, 11 E-tahmin edin . Bu üç yöntemin her biri tarafından, grip virüsü burun sürüntü örneği (influenza benzeri hastalığı olan bir çocuk) (Şekil 2C) kolayca tespit </li></ol> 7. Temsilcisi Sonuçlar: Protokol doğru yapıldığında, bir karalama Rd B adım (Şekil 2A) sonra agaroz jel elektroforez görülebilir. Agilent platformu Virochip mikroarray (Şekil 2B) görsel olarak analiz etmek zor olacak, ama bir virüs varsa kolayca görsel denetim, kümeleme analizi, ve / veya E-Tahmin (Şekil 1C) tespit edilmelidir. Tayini için bir pozitif kontrol olarak hizmet; Numuneler bilinen bir virüs (MS2 bakteriyofaj örneğin tütün mozaik virüsü) nükleik asit ölçülen miktarları ile çivili olabilir. <img src="/files/ftp_upload/2536/2536fig1.jpg" alt="Şekil 1" /> Şekil 1. Virochip mikroarray işleme ve analiz şematik klinik örneklerden nükleik asit Çıkarılan rastgele amplifiye floresan boya ile işaretlenmiş ve Virochip mikroarray için melezleşmiştir. Mikroarray&#39;ler 2 mikron çözünürlükte taranır ve E-tahmin de dahil olmak üzere, çeşitli hesaplama araçları kullanılarak analiz edildi. <img src="/files/ftp_upload/2536/2536fig2.jpg" alt="Şekil 2" /> Şekil 2. Virochip tayininde Adımlar amplifikasyon sonra rastgele PCR, bir karalama 200 - 1000 bp jel elektroforezi ile görüntülendi (A) (B) Üç Virochip mikroarray&#39;ler 8 diziler / cam slayt küçük bir bölge ile gösterilir. (C) sağ alt köşesinde, içeride havaya uçurulan bir mikroarray Otomatik mikroarray viral analizi ile klinik örnek, influenza A virüsünün varlığını gösteren E-tahmin edin. Yüksek benzerlik skoru (s = 0.55), bu nedenle bu son derece önemli bir tahmin yapmak, sıfıra yakın bir p-değeri karşılık gelir.

<ol>
	<li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray-based+detection+and+genotyping+of+viral+pathogens.">Microarray-based detection and genotyping of viral pathogens.</a> Proc Natl Acad Sci U S A. 99, 15687-15692 (2002).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+a+critical+respiratory+illness+caused+by+human+metapneumovirus+by+use+of+a+pan-virus+microarray.">Diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray.</a> J Clin Microbiol. 45, 2340-2343 (2007).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarray+detection+of+human+parainfluenzavirus+4+infection+associated+with+respiratory+failure+in+an+immunocompetent+adult.">Microarray detection of human parainfluenzavirus 4 infection associated with respiratory failure in an immunocompetent adult.</a> Clin Infect Dis. 43, e71-e76 (2006).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+DNA+microarrays+for+detection+of+viruses+in+acute+respiratory+tract+infections+in+children.">Utility of DNA microarrays for detection of viruses in acute respiratory tract infections in children.</a> J Pediatr. 153, 76-83 (2008).</li><li>Kistler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pan-viral+screening+of+respiratory+tract+infections+in+adults+with+and+without+asthma+reveals+unexpected+human+coronavirus+and+human+rhinovirus+diversity.">Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity.</a> J Infect Dis. 196, 817-825 (2007).</li><li>Rota, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+a+novel+coronavirus+associated+with+severe+acute+respiratory+syndrome.">Characterization of a novel coronavirus associated with severe acute respiratory syndrome.</a> Science. 300, 1394-1399 (2003).</li><li>Wang, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+discovery+and+sequence+recovery+using+DNA+microarrays.">Viral discovery and sequence recovery using DNA microarrays.</a> PLoS Biol. 1, e2-e2 (2003).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+novel+Gammaretrovirus+in+prostate+tumors+of+patients+homozygous+for+R462Q+RNASEL+variant.">Identification of a novel Gammaretrovirus in prostate tumors of patients homozygous for R462Q RNASEL variant.</a> PLoS Pathog. 2, 25-25 (2006).</li><li>Kistler, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recovery+of+divergent+avian+bornaviruses+from+cases+of+proventricular+dilatation+disease:+identification+of+a+candidate+etiologic+agent.">Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.</a> Virol J. 5, e25-e25 (2008).</li><li>Chiu, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+cardioviruses+related+to+Theiler's+murine+encephalomyelitis+virus+in+human+infections.">Identification of cardioviruses related to Theiler's murine encephalomyelitis virus in human infections.</a> Proc Natl Acad Sci U S A. 105, 14124-14129 (2008).</li><li>Urisman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-Predict:+a+computational+strategy+for+species+identification+based+on+observed+DNA+microarray+hybridization+patterns.">E-Predict: a computational strategy for species identification based on observed DNA microarray hybridization patterns.</a> Genome Biol. 6, R78-R78 (2005).</li></ol>

Virochip protokolü burada açıklandığı gibi karmaşık ve yetenekli ve titiz bir araştırma teknisyeni gerektirir. Doğru reaktif konsantrasyonu ve PCR etiketleme koşulları ve hibridizasyon kritik öneme sahiptir. Virochip protokolü nükleik asit ekstraksiyon TRIzol (Invitrogen) gibi bir doku çıkarma yönteminin kullanımı ile hastalıklı dokuların analizi kolayca uyum sağlamak için değiştirilebilir. Problar eklendi veya istenen spektrumu ve kapsamının genişliği elde etmek için gerekli silinmiş olabilir, örneğin &quot;-the-fly&quot;, bakteri ve mantarlar gibi nonviral hedeflerin saptanması için yoklamaları dizayn edilebilir ve ekledi. Henüz geliştirme aşamasında Virochip testinin bazı uygulamalar, klinik laboratuvar, salgın araştırma, saflık taraması, ilaç ve aşılar ve yeni viral patojen keşif geniş spektrumlu bir viral teşhis içerir.

<table border="1"><tbody><tr><th> Reaktif / malzeme Adı </th><th> Reaktif / malzeme Adı </th></tr><tr><td> Primera </td><td> 5&#39;-GTTTCCCAGTCACGATA (N9) -3 &#39; </td></tr><tr><td> Primer B </td><td> 5&#39;-GTTTCCCAGTCACGATA-3 &#39; </td></tr><tr><td rowspan="5"> RT Master Mix (5 mcL) </td><td> 2 mcL 5X RT tampon </td></tr><tr><td> 1 mcL 12.5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mcL su </td></tr><tr><td> 0.5 mcL 0.1M DTT </td></tr><tr><td> 0.5 mcL SSIII RT (Invitrogen) </td></tr><tr><td rowspan="3"> Sequenase Mix (5 mcL) </td><td> 1 mcL 5X Sequenase tampon </td></tr><tr><td> 3.8 mcL su </td></tr><tr><td> 0.15 mcL Sequenase (USB Corporation&#39;ın) </td></tr><tr><td rowspan="5"> Rd B PCR Master Mix (45 mcL) </td><td> 5 mcL 10X PCR buffer </td></tr><tr><td> 1 mcL 12.5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mcL 100 mcL / B pmol astar </td></tr><tr><td> 1 mcL KlenTaq LA (Sigma) </td></tr><tr><td> 37 mcL su </td></tr><tr><td rowspan="5"> Rd C PCR Master Mix (45 mcL) </td><td> 5 uL 10X PCR buffer </td></tr><tr><td> 1 mcL 12.5 mM dNTP (Invitrogen) </td></tr><tr><td> 1 mcL 100 mcL / B pmol astar </td></tr><tr><td> 1 mcL KlenTaq LA (Sigma) </td></tr><tr><td> 37 mcL su </td></tr><tr><td rowspan="4"> Hibridizasyon tamponu (25 mcL) </td><td> 1 mcL 25X parçalanma tampon (Agilent) </td></tr><tr><td> 5 mcL 5X engelleme tamponu (Agilent) </td></tr><tr><td> Cy3-etiketli örnek (veya normale miktarı 9.5 mcL) </td></tr><tr><td> 25 mcL toplam hacim su ekleyin. </td></tr><tr><td> Agilent Virochip mikroarray </td><td> Chiu laboratuvar, UCSF edinilebilir; bekleyen lisans </td></tr></tbody></table>

using a pan-viral microarray assay (virochip) to screen clinical samples for viral pathogens

Virüslerin doğal dizisi çeşitliliği yanı sıra, geleneksel yöntemlerle algılanamaz SARS coronavirüs ve 2009 pandemik H1N1 influenza virüsü gibi roman viral patojenler, devam eden ortaya çıkması nedeniyle, pek çok bulaşıcı hastalıkların viral nedenlerin tanısı zordur. Bu zorlukları ele almak için, daha önce geliştirilmiş ve kapasitesi ile korunmuş sıra homoloji 1 dayanarak, bilinen tüm virüsleri yanı sıra roman varyantları tespit etmek için Virochip denilen bir pan-viral mikroarray platformu valide . Virochip kullanarak, tam spektrum, eşdeğer ya da geleneksel klinik test 2-5 üstün bir hassasiyet ile hastanede yatan hastalarda açıklanamayan kritik hastalık vakaları da dahil olmak üzere, solunum yolu enfeksiyonları ile ilişkili virüs tespit ettik. Virochip ayrıca, SARS coronavirüs 6,7, bir roman rinovirüs dalının 5, XMRV (prostat kanseri ile bağlantılı bir retrovirüs) 8, kuş bornavirus (papağanlar bir israf hastalığı nedeni) 9, dahil olmak üzere, yeni virüsleri tanımlamak için kullanılır olmuştur solunum ve ishal hastalık 10 ile çocuklarda ve yeni bir cardiovirus. Virochip güncel sürümü Agilent mikroarray platforma taşıdık ve ~ 36.000 probları Aralık 2009 itibariyle GenBank ~ üzerinde 1.500 virüslerden kaynaklanan oluşur. Burada örnek nükleik asit ekstraksiyon, rastgele primerler kullanılarak PCR amplifikasyonu, floresan boya dahil, ve mikroarray hibridizasyon, tarama ve analiz de dahil olmak üzere, (~ 24 saat gerçekleştirme süresi) bitirmek için baştan bir Virochip testi işleme dahil adımları ortaya koymaktadır.

Watch this Scientific Journal Video about Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens at JoVE.com

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Virochip korunmuş dizi homoloji temelinde aynı anda, bilinen tüm virüsleri yanı sıra roman virüsler tespit etmek için tasarlanmış bir pan-viral mikroarray. İşte biz, varlığı bilinen ve bilinmeyen virüslere hem de klinik örnekleri analiz etmek için bir Virochip tahlil nasıl çalıştırılacağını göstermektedir.

Viral Patojenler Klinik Örnekler Ekranına bir Pan-Viral Mikroarray Testi (Virochip)

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

using-pan-viral-microarray-assay-virochip-to-screen-clinical-samples

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

18.8K Görüntüleme.  University of California, San Francisco. Virochip korunmuş dizi homoloji temelinde aynı anda, bilinen tüm virüsleri yanı sıra roman virüsler tespit etmek için tasarlanmış bir pan-viral mikroarray. İşte biz, varlığı bilinen ve bilinmeyen virüslere hem de klinik örnekleri analiz etmek için bir Virochip tahlil nasıl çalıştırılacağını göstermektedir.

Video: Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

Microfluidic Chip Fabrication and Method to Detect Influenza

Fast and effective diagnostics play an important role in controlling infectious disease by enabling effective patient management and treatment. Here, we present an integrated microfluidic thermoplastic chip with the ability to amplify influenza A virus in patient nasopharyngeal (NP) swabs and aspirates. Upon loading the patient sample, the microfluidic device sequentially carries out on-chip cell lysis, RNA purification and concentration steps within the solid phase extraction (SPE), reverse transcription (RT) and polymerase chain reaction (PCR) in RT-PCR chambers, respectively. End-point detection is performed using an off-chip Bioanalyzer (Agilent Technologies, Santa Clara, CA). For peripherals, we used a single syringe pump to drive reagent and samples, while two thin film heaters were used as the heat sources for the RT and PCR chambers. The chip is designed to be single layer and suitable for high throughput manufacturing to reduce the fabrication time and cost. The microfluidic chip provides a platform to analyze a wide variety of virus and bacteria, limited only by changes in reagent design needed to detect new pathogens of interest.

An integrated microfluidic thermoplastic chip has been developed for use as a molecular diagnostic. The chip performs nucleic acid extraction, reverse transcriptase, and PCR. Methods for fabricating and running the chip are described.

Mikroakışkan Chip İmalat ve İnfluenza Algılama Yöntemi

Fast  and effective diagnostics play an important role in controlling infectious  disease by enabling effective patient management and treatment. Here, we ...

Biyomühendislik

Assays for the Identification of Novel Antivirals against Bluetongue Virus

To identify potential antivirals against BTV, we have developed, optimized and validated three assays presented here. The CPE-based assay was the first assay developed to evaluate whether a compound showed any antiviral efficacy and have been used to screen large compound library. Meanwhile, cytotoxicity of antivirals could also be evaluated using the CPE-based assay. The dose-response assay was designed to determine the range of efficacy for the selected antiviral, i.e. 50% inhibitory concentration (IC50) or effective concentration (EC50), as well as its range of cytotoxicity (CC50). The ToA assay was employed for the initial MoA study to determine the underlying mechanism of the novel antivirals during BTV viral lifecycle or the possible effect on host cellular machinery. These assays are vital for the evaluation of antiviral efficacy in cell culture system, and have been used for our recent researches leading to the identification of a number of novel antivirals against BTV.

Three assays, including the cytopathic effect (CPE)-based assay, dose-response assay and Time-of-Addition (ToA) assay have been developed, optimized, validated and utilized to identify novel antivirals against Bluetongue virus (BTV), as well as to determine the possible Mechanism-of-Action (MoA) for newly identified antivirals.

Bluetongue Virüs karşı Roman Antiviraller belirlenmesi için Tahliller

To identify potential antivirals against BTV, we have  developed, optimized and validated three assays presented here. The CPE-based  assay was the first ...

İmmünoloji ve Enfeksiyon

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA Virüslerinin geniş spektrumlu Kimyasal Önleyicileri için yüksek verimli tarama

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

Erken Viral Giriş Tespiti için Tahliller ve Antiviral Bileşiklerin Değerlendirilmesi

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

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 ...

High-throughput Detection of Respiratory Pathogens in Animal Specimens by Nanoscale PCR

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of combining reactions together. We designed and evaluated a panel based on this principle to rapidly identify the presence of common disease agents in dogs and horses with acute respiratory illness. This manuscript describes a nanoscale diagnostic PCR workflow for sample preparation, amplification, and analysis of target pathogen sequences, focusing on procedures that are different from microliter scale reactions. In the respiratory panel presented, 18 assays were each set up in triplicate, accommodating up to 48 samples per plate. A universal extraction and pre-amplification workflow was optimized for high-throughput sample preparation to accommodate multiple matrices and DNA and RNA based pathogens. Representative data are presented for one RNA target (influenza A matrix) and one DNA target (equine herpesvirus 1). The ability to quickly and accurately test for a comprehensive, syndrome-based group of pathogens is a valuable tool for improving efficiency and ergonomics of diagnostic testing and for acute respiratory disease diagnosis and management.

High-throughput testing of DNA and RNA based pathogens by nanoscale PCR is described using a syndromic canine and equine respiratory PCR panel.

Nano PCR ile Hayvan Örnekler Solunum Patojenler yüksek verimlilik Algılama

Nanoliter scale real-time PCR uses spatial multiplexing to allow multiple assays to be run in parallel on a single plate without the typical drawbacks of ...

Unbiased Deep Sequencing of RNA Viruses from Clinical Samples

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from clinical and biological sources. The method is unbiased and universal; it uses random primers for cDNA synthesis and requires no prior knowledge of the viral sequence content. Before library construction, selective RNase H-based digestion is used to deplete unwanted RNA &#8212; including poly(rA) carrier and ribosomal RNA &#8212; from the viral RNA sample. Selective depletion improves both the data quality and the number of unique reads in viral RNA sequencing libraries. Moreover, a transposase-based &#39;tagmentation&#39; step is used in the protocol as it reduces overall library construction time. The protocol has enabled rapid deep sequencing of over 600 Lassa and Ebola virus samples-including collections from both blood and tissue isolates-and is broadly applicable to other microbial genomics studies.

This protocol describes a rapid and broadly applicable method for unbiased RNA-sequencing of viral samples from human clinical isolates.

Klinik Örneklerden RNA Virüs tarafsız Derin Sıralama

Here we outline a next-generation RNA sequencing protocol that enables de novo assemblies and intra-host variant calls of viral genomes collected from ...

Tıp

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

Araçlar Viral Immunomodulators ve ana bilgisayar Antiviral faktörler tanımlanması için eleme olarak Abdovirüs enfeksiyonlar

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

Viral Nükleik Asitleri ve Proteinleri Görselleştirmek ve HIV, HTLV, HBV, HCV, Zika Virüsü ve İnfluenza Enfeksiyonunu İzlemek için Tek Hücreli Çoklanmış Floresan Görüntüleme

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Biyoloji

A Viral Attachment Assay for Screening of Antiviral Compounds

For the viral attachment assay, plate 1 x 104 cells per well in a 96-well plate, and incubate overnight for cell attachment. The next morning, first, chill the 96-well plate containing the cell monolayer at 4 degrees Celsius for 1 hour.
Prepare the test compounds and virus mixture on ice. For example, HCV-CHLA, HCV - 1% DMSO, or positive control HCV-heparin solutions with 102 FFU virus for each treatment well. Aspirate the medium from the cell culture wells gently. Then, immediately add 100 microliters of virus test compound mixture in triplicate wells, taking care not to raise the temperature. Incubate the plate in a refrigerator maintained at 4 degrees Celsius for three hours.
Virus particles will attach on the cell surface at 4 degrees Celsius but will not be internalized. Next, aspirate the supernatants. Gently wash the cells twice with 200 microliters of ice-cold PBS. Add 100 microliters of basal medium to each well, and then incubate at 37 degrees Celsius for 72 hours. Finally, for quantitating the released luciferase reporter from the infected cells, collect the supernatant from each sample well and perform the Gaussia luciferase assay.