This work was supported by the Academy of Finland (130750 and 218080 to J.T.H.), the Wellcome Trust (090532/Z/09/Z; 089026/Z/09/Z to T.A.B.), and by the MRC (MR/J007897/1 to J.T.H and T.A.B; MR/L009528/1 to T.A.B.).

<ol>
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The authors have nothing to disclose.

<html> Viryon membran viral glikoprotein başak yapısının ortaya çıkarılmasıyla, virüs kopyalanmasını anlamak ve tedavi etmek ve enfeksiyonu önlemek için terapötik maddelerin geliştirilmesi için esastır. Tek bir parçacık ortalama ile bir araya Elektron mikroskobu kriyo-glikoprotein dahil olmak üzere sivri zarflı virüs partiküllerinin, yapılarını çözmek için en çok kullanılan yöntem olarak ortaya çıkmıştır. Bununla birlikte, bu yöntem, simetrik virüsleri icosahedrally sınırlıdır. Burada, Jsubtomo elektron cryo-tomografi ve subtomogram ortalamanın uygulaması ile, biz pleomorfik diğer mevcut yapısal biyoloji yöntemleri uygun değildir virüsleri glikoprotein sivri zarflı belirlenmesi için genel bir protokol ana hatlarıyla. Bizim için temsili sonuçlar, bu yöntemin çözünürlüğü etki mimari, oligomerizasyonu ve sağlam viryonları üzerindeki glikoprotein sivri yüksek dereceli kuruluşa bakış açısı ortaya çıkarmak için yeterli olduğunu göstermektedir. jove_content &quot;&gt; Bu protokol çerçevesinde en kritik adım, bu aşamada başka. Başarılı yürütme glikoprotein kramponları, böylece yeterince büyük ve çok sıkı birbirlerine karşı değil dolu olduğunu varsayar birinden istatistiksel olarak bağımsız iki güvenilir bir başlangıç ​​modelleri oluşturmak bireysel sivri yapabilirsiniz görsel tanınan ve el tomogramları toplanmış ve iki bağımsız modelleri ortalaması alınmalıdır., bu mümkün değilse, protokol iki değişiklik teşebbüs edilebilir. Birincisi, iki bağımsız random modeller sonra ilk subtomograms rastgele iki alt kümelerini tanımlama ve inşa edilebilir Bu alt-gruplar içinde 30 subtomograms ortalama. İkinci olarak, izole edilmiş bir sivri yapı, X-ışını kristalografisi ile, örneğin başka yollarla elde edilmiş olsa bile, bu başlangıç ​​modeli olarak kullanılabilir. Ancak, bakım, düşük alınmalıdır arıtmanın bir sonraki turda olacak iki sonuçlanan modelleri gibi, düşük çözünürlüklü cut-off (50-70 Å) kullanarak filtreyi Bu modeli geçmekSadece bu çözünürlükte ötesinde istatistiksel bağımsız olması. Bu ihtar nedeniyle, eski yaklaşım tavsiye edilir. Bu protokol elde çözünürlük, dört ana faktöre bağlıdır: i. veri toplama stratejisi ve girdi verilerinin kalitesi, ii. subtomograms sayısı, III. hizalama subtomograms doğruluğu ve iv. yapıların heterojen. Birinci ve ikinci sınırlama, büyük ölçüde yüksek sinyal-gürültü doğrudan elektron CTF ile kombine dedektörleri tomografi giderilmiştir ve otomatik veri toplanmasını kullanılarak aşılabilir birlikte, hizalama hassasiyeti daha da ilgi kendisinin yapısının boyutu ve şekli etkilenir. Belirgin özellikleri göstermeyen küçük sivri bu protokolü uygulanır, bu hizalama doğruluk ve böylece çözünürlüğü arttırmak için 31 sivri Fab fragmanlarını bağlamak için avantajlı olabilir. Son olarak, bu yapılar için ortalama halinde sergi çoklu biçimlerde, alt-tomam sınıflandırma yöntemi ayrıca farklı konformasyonlara ortalama için kullanılabilir. Bu amaçla, Jsubtomo güçlü subtomogram sınıflandırma 9 sunan, Dinamo paketi ile bütünleştirir. Yukarıdaki protokol izole edilmiş viral glikoproteinler de, X-ışını kristalografisi tamamlayıcıdır. Kristalografik yapılar viryon zanna göre glikoproteinin Tam bir yönlendirme elde etmek için alt-tomogram ortalamalar içine monte edilebilir. Bu metodolojinin uygulama şüphesiz zarflı virüs yapısında ve patobiyolojisi üzerine ışık tutmaya devam edecektir.

Elektron cryo-tomografi karmaşık biyolojik örneklerin bir üç boyutlu (3D) yeniden hesaplanmasını sağlayan bir elektron cryo-mikroskopi görüntüleme tekniğidir. Müsait örnekler bütün prokaryotik hücreler 5 ve bütün ökaryotik hücreler 6 bile ince alanlara saflaştırılmış makro-moleküler kompleksler 1, filaman 2, kaplamalı veziküller 3 ve pleomorfik membran virüs 4 arasında değişir. Bir tilt-dizi, 3D tomografik hacimleri, ya tomogramları veri toplama ardından, Bsoft 7 ve imod 8 olmak üzere birkaç kurulan yazılım paketleri kullanılarak hesaplanabilir. Elektron cryo-tomografi sınırı gelen tomografik hacimlerinin biyolojik yorumlanması biyolojik örneklerin çalışmaya doğasında iki yönü. Bağlı olarak büyük radyasyon hasarı verilmesinden önce, biyolojik malzemelerden uygulanabilir sınırlı elektron doz signa, İlktomografi veri L-gürültü oranları tipik olarak çok düşüktür. İkinci olarak, veri toplama sırasında kısıtlı örnek eğim geometrisinin bir sonucu olarak, nesnenin bazı incelemeler tomografik hacimde bir sözde &#39;eksik kama&#39; artefakt yol mevcut kalır. Tomografik birim halinde başarılı olarak 9-12 ortalama böyle makromoleküler kompleksler olarak özdeş yapılar, tekrar içerir, ancak, bu dezavantajların her ikisi de aşılabilir. Tomogram rekonstrüksiyonlarda gelen yapıları ortalama önce, ilgi nesneler bulundu ve aynı yönde hizalanmış olmalıdır. Bu tür yapıları yerlerinin çoğu zaman, şablon olarak 13 eşleşen anılan bir yaklaşım kullanarak tomografik hacminde bir şablon yapının çapraz korelasyon elde edilebilir. Bu eşleştirme işleminde kullanılan şablon elektron cryo-mikroskobu veya 3D rekonstrüksiyon ile birlikte elektron cryo-tomografi elde edilebilir, ya da gelen simüle bir yoğunluk haritası olabilirBir atom yapısı. Birkaç bilgisayar paketler 11, bu görevleri yerine getirmek üzere geliştirilmiştir. HIV-1 gibi membran virüsler, glikoprotein sivri ortalama, onların yapısını 14-16 çalışmak için özellikle başarılı bir yaklaşım olmuştur. Yapının, virüs konakçı etkileşimleri hem de moleküler temeli ortaya çıkartılması ve anti-viral ve aşı tasarımının gelişimi yönlendirmek için ayrılmaz bir parçasıdır. Makromoleküler glikoproteinler kristalografi ayrı ayrı virütik ve komplekslerin yapı analizine (4, bir zaman daha iyi) yüksek çözünürlüklü için tercih edilen bir tekniktir, ancak bu yöntemle elde edilen X-ışını yapısı, viryonun üzerindeki doğal ortamdan izole membran proteinleri vardır . Bu durumda, bu virion bağlamında viral glikoproteinlerin daha yüksek dereceli mimari, önemli bilgileri, eksik kalır. Öte yandan, elektron mikroskobu ve kriyo-tek parçacık yenidenbütün kılıflı virüsler ikosahedral simetri 17,18 ile virionlann sınırlandırılmıştır. Alt ses uyumu ile birlikte elektron cryotomography böylece yerinde düzensiz şekilli, pleomorfik virüslerin glikoprotein sivri çalışma izin tamamlayıcı bir teknik olarak ortaya çıkmıştır. Biz Jsubtomo tomografik alt birimlerin tespiti, hizalama ve ortalama için (www.opic.ox.ac.uk/jsubtomo) adında bir yazılım geliştirdik. Jsubtomo hücresel ve viral yapının 19-26 bir dizi yapı belirlemelerinde kullanılmıştır. Burada, biz belirlenmesi viral yüzey başak yapıları sağlayan ayrıntılı bir protokol, anahat. Gürültü ilişkilendirerek aşırı arıtma ortalama yapıların aşmak için, &#39;altın standart&#39; arıtma şeması 10,27 benimsenmiştir. Son olarak, görselleştirme ve tipik sonuçların yorumlanması için stratejiler tartışılmıştır.

<table border="0" cellpadding="0" cellspacing="0" width="686">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:184px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Jsubtomo (ver 1.3.1)</td>
			<td style="width:184px;">University of Oxford</td>
			<td style="width:119px;">n/a</td>
			<td>www.opic.ac.uk/jsubtomo</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bsoft (ver 1.8.7)</td>
			<td style="width:184px;">NIAMS, NIH</td>
			<td style="width:119px;">n/a</td>
			<td>bsoft.ws</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UCSF Chimera</td>
			<td style="width:184px;">UCSF</td>
			<td style="width:119px;">n/a</td>
			<td>www.cgl.ucsf.edu/chimera</td>
		</tr>
	</tbody>
</table>

averaging of viral envelope glycoprotein spikes from electron cryotomography reconstructions using jsubtomo

Zarflı virüsler konakçı hücrelere giriş aracılık etmek ile ilgili yüzeyi üzerindeki zar glikoproteinleri kullanmaktadır. Bu glikoprotein &#39;sivri&#39; Üç-boyutlu yapısal analizler çoğunlukla viral patogenezi anlamak için ve ilaç tasarımında teknik açıdan zor ama önemlidir. Burada, bir protokol elektron cryo-tomografi verileri hesaplama ortalamaya yoluyla viral başak yapı tayini için sunulmuştur. Elektron cryo tomografi bir doğala yakın, dondurulmuş sulu halde membran virüsler gibi biyolojik örneklerin pleomorfik, üç boyutlu hacimli tomografik rekonstrüksiyon veya tomografi, elde etmek için kullanılan elektron mikroskobu bir tekniktir. Bu tomografi düşük çözünürlükte olsa, üç boyutta ilgi yapılar ortaya çıkarmak. Alt birimlerine veya alt tomografi Hesaplamalı ortalama, viral glikoprotein sivri gibi yapısal motiflerini, tekrar eden yüksek çözünürlüklü detay elde etmek için gereklidir. Ali için ayrıntılı bir hesaplama yaklaşımıgning ve Jsubtomo yazılım paketi kullanarak, alt-tomogram ortalama özetlenmiştir. Bu yaklaşım, virion zar üzerinde yüksek seviyeli başak sivri uç etkileşimlerin bir çalışma 20-40 Â çözünürlük aralığında ve çalışma viral glikoprotein sivri yapısının görselleştirme sağlar. Tipik sonuçlar Bunyamwera virüsü, Bunyaviridae ailesinden zarflı virüs için sunulmuştur. Bu aile, insan ve hayvan sağlığı için bir tehdit patojenlerin yapısal olarak farklı bir gruptur.

Biz 24 set daha önce yayınlanmış verileri kullanılarak Bunyamwera virüsü (Orthobunyavirus, Bunyaviridae) zarf glikoprotein kompleksi için yukarıda belirtilen alt tomogram ortalama iş akışı uygulama göstermektedir. Veri toplama ve arıtım parametreleri izleyen Tablo 1 &#39;de listelenmiştir. Bir temsilci tomogram, Şekil 2&#39;de gösterilmiştir. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Parametre (birim) </td><td> Değer </td></tr><tr><td> Veri toplama </td><td></td></tr><tr><td> Gerilimi (kV) </td><td> 300 </td></tr><tr><td> Kalibre büyütme (X) </td><td> 111.000 </td></tr><tr><td> Piksel boyutu (Å) </td><td> 5.4 </td></tr><tr><td> Tahmini doz (e - / Å 2) </td><td> 100 </td></tr><tr><td> Underfocus (um) </td><td> 4.0-4.5 </tD&gt; </tr><tr><td> İlk CTF sıfır (Å) bir </td><td> 26-30 </td></tr><tr><td> Eğim aralığı (°) </td><td> -60-60 </td></tr><tr><td> Tilt örnekleme (°) </td><td> 3 </td></tr><tr><td> Veri ve arıtma </td><td></td></tr><tr><td> Tomografi </td><td> 11 </td></tr><tr><td> Virionları </td><td> 29 </td></tr><tr><td> Viryonunun başına Tohumlar </td><td> 106 </td></tr><tr><td> Tohum sayısı </td><td> 3.074 </td></tr><tr><td> Spike tespit </td><td> 1.346 </td></tr><tr><td> Örtüşmeler çıkarıldıktan sonra ani </td><td> 1.401 </td></tr><tr><td> Çapraz-korelasyon sivri tabanlı değil seçimi </td><td> 1.022 </td></tr><tr><td> Spike son ortalamaya dahil </td><td> 1.022 </td></tr><tr><td> Simetri </td><td> C3 </td></tr><tr><td> Açısal örnekleme (°) </td><td> 8 </td></tr><tr><td> Çözünürlükarıtmada kullanılan aralığı (Å) </td><td> 42-334 </td></tr><tr><td> Nihai çözünürlük tahmini (a) b </td><td> 35 </td></tr></tbody></table> Tablo 1: Bunyamwera veri toplama ve arıtma istatistikleri. Bir CTF, kontrast transfer fonksiyonu. b 0.143 bir eşik iki bağımsız rafine yapılar arasındaki Fourier kabuk korelasyon kullanılarak hesaplanmıştır. <img alt="Şekil 2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> Şekil 2:. Bunyamwera virionlann bir tomografisinde yoluyla Dilim her viryonunun çevresi belirgin Çeşitli başak yan görüntüleridir ok uçları ile gösterilir. Tomogram 60 Â Filtresinde düşük geçiş olmuştur. Ölçek çubuğu 100 nm. Öncelikle, biz 205 elle aldı sivri kullanarak bir başlangıç ​​modeli (rafine <strong&gt; Şekil 3). Merkez-en başak üç kat simetri herhangi bir simetri (Şekil 3B) uygulamadan açıktı ve arıtma sonraki turlarda (Şekil 3C) empoze edildi. Viryon yüzeyler üzerinde otomatik olarak sivri tespit için, 43 nm ile 20 ° (Şekil 4A) arasında aralığın yarıçapta her bir virion 106 tohumları üretilir ve yinelemeli membran (Şekil 4B) göre konumlarını rafine. <img alt="Şekil 3," fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> Şekil 3: ilk şablon yapısı iyileştirildi. (A) Silindir şeklinde sivri elle tanımlanmış pozisyonlarda inşa (C100) şablonu ortalama. (B) Ortalama yoğunluğu arıtma beş tur sonra herhangi bir simetri olmadan (C1), üç kat ile görüntüler bir başak empozesimetrik özellikleri. Modelin çözünürlüğü 48 Å. Başak (C) Ortalama üç kat simetri ile arıtma beş turdan sonra 41 Å de çözüldü. <img alt="Şekil 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> Şekil 4:.. Tohumların Arıtma ab) A, (daha önce tohumların bir alt) ve sonra (B) &#39;arıtma Şekil 2&#39; den bir viryon yoğunluğuna (B) &#39;de gösterilmektedir tohumlar olmuştur renk kodlu ilgili Çapraz göre korelasyon katsayıları (mavi, düşük korelasyon; kırmızı, yüksek korelasyon). (Örtüşmelerin çıkardıktan sonra üst% 75; ~ 1,000 kramponları) iyi correlating glikoprotein başak yamalar son ortalamasını hesaplamak için kullanıldı. Ortalama 35-Å çözünürlük (Şekil 5) karar verildi. Bu, ortada bir trimetik başak yapı ortaya çıkardıaltı komşu sivri bazı katkı ilave. Bilinen pozisyonlarda yapısını yerleştirerek hesaplanan virionlar, kompozit modelleri, viryon yüzeyi (Şekil 6) üzerine sivri yerleştirilmesini saptandı. Bazen, sivri yerel emretti yamalar (Şekil 6B) belirgindir. <img alt="Şekil 5," fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> Şekil 5: şablon eşleştirme sonra glikoprotein başak tabakasının bir yama Rafine yapısı. (AB) &#39;bile&#39; ve &#39;garip&#39;, verilerin iki yarısı yeniden inşa İki haritalar gösterilmektedir. Iki harita yaklaşımın validness doğrulayan bir benzerlik dikkat çekici derecesini göstermektedir. Iki harita birbirinden bağımsız olarak tamamen yeniden inşa edilmiş olarak başak uzun ekseni etrafında yönelim farklı. Iki harita (C) ortalama ile gösterilir35 Å nihai kararı. <img alt="Şekil 6," fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> Şekil 6: Bunyamwera virion üzerindeki sivri Yerleştirme. (A), viryonun Bileşik modeli. Görünüm vektörleri (sopa) sivri yönelimleri göstermektedir. Renkler her başak ve şablon yapısı (mavi, düşük korelasyon; kırmızı, yüksek korelasyon) arasındaki çapraz korelasyon göstermektedir. Sivri bir sipariş yama (B) A close-up.

Watch this Scientific Journal Video about Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo at JoVE.com

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Jsubtomo kullanılarak Elektron Cryotomography Rekonstrüksiyonunda ikinci viral zarf glikoproteini Spikes ortalamasını

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

averaging-viral-envelope-glycoprotein-spikes-from-electron

Research

JoVE Journal

Immunology and Infection

12.0K Görüntüleme.  University of Oxford. An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

Video: Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

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

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 of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

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

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

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 ...

İmmünoloji ve Enfeksiyon

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

Cryo-Elektron Tomografi ve Otomatik Sub-tomogram Ortalaması kullanarak HIV Zarf Glikoproteinler Moleküler Yapılarının Belirlenmesi

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

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 ...

Isolation of Fidelity Variants of RNA Viruses and Characterization of Virus Mutation Frequency

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the generation of extreme population diversity that facilitates virus adaptation and evolution. Increasing evidence shows that the intrinsic error rates, and the resulting mutation frequencies, of RNA viruses can be modulated by subtle amino acid changes to the viral polymerase. Although biochemical assays exist for some viral RNA polymerases that permit quantitative measure of incorporation fidelity, here we describe a simple method of measuring mutation frequencies of RNA viruses that has proven to be as accurate as biochemical approaches in identifying fidelity altering mutations. The approach uses conventional virological and sequencing techniques that can be performed in most biology laboratories. Based on our experience with a number of different viruses, we have identified the key steps that must be optimized to increase the likelihood of isolating fidelity variants and generating data of statistical significance. The isolation and characterization of fidelity altering mutations can provide new insights into polymerase structure and function1-3. Furthermore, these fidelity variants can be useful tools in characterizing mechanisms of virus adaptation and evolution4-7.

The present article describes the steps required to isolate and characterize RNA polymerase fidelity variants of RNA viruses and how to use mutation frequency data to confirm fidelity changes in tissue culture.

Virüs mutasyon Frekans RNA Virüsler ve Karakterizasyonu Fidelity Varyantlar izolasyonu

RNA viruses use RNA dependent RNA polymerases to replicate their genomes. The intrinsically high error rate of these enzymes is a large contributor to the ...

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

Tek iplikli DNA, çift iplikli DNA ve RNA Hidroksiapatit Kromatografisi kullanarak bir Çevre Viral Topluluk ayrılması

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

Nucleocapsids gelen Viral DNA hazırlanması

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Görüntülemede HIV-1 Zarf bağlı Virolojik Synapse ve Sentetik Lipid Bilayers üzerine Sinyal

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

Bir Hücre temelli bir ELISA testi kullanarak, HIV-1 zarf trimerik Glikoproteinler konformasyonel Değerlendirilmesi

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

tespiti için Liyofilize Döngü aracılı İzotermal Amplifikasyon Reaktifler Gelişimi<em&gt; Coxiella burnetii</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

Enzim-linked Lektin Testi tarafından Grip Nöraminidaz İnhibisyon antikor titreleri Ölçme

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...