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> Wissen von viralen Glykoprotein Spike-Struktur auf der Membran Virus ist von wesentlicher Bedeutung für das Verständnis der Virus-Replikation und die Entwicklung von Therapeutika zur Behandlung und Prävention der Infektion. Kryoelektronenmikroskopische kombiniert mit Einzelpartikellung wurde als die am häufigsten verwendete Methode, um die Strukturen der umhüllten Viruspartikel, einschließlich der Spitzen-Glykoprotein lösen entstanden. Jedoch ist dieses Verfahren beschränkt auf die symmetrische Viren ikosaedrisch. Hier durch die Anwendung der Elektronen Kryo-Tomographie und subtomogram Mittelung in Jsubtomo, haben wir ein allgemeines Protokoll zur Bestimmung Glykoprotein-Spikes auf pleomorphen umhüllte Viren, die nicht zugänglich für andere aktuelle Methoden der Strukturbiologie werden skizziert. Unsere repräsentative Ergebnisse zeigen, dass die Auflösung dieser Methode ist ausreichend, um Einblicke in die Domänenarchitektur, Oligomerisierung und höherer Ordnung Organisation des Glykoprotein-Spikes auf intakte Virionen offenbaren. jove_content &quot;&gt; Der wichtigste Schritt in diesem Protokoll baut zwei zuverlässige Ausgangsmodelle, die statistisch unabhängig voneinander. erfolgreiche Ausführung dieser Schritt setzt voraus, dass Glykoprotein Spikes sind ausreichend groß und nicht gegeneinander zu fest gepackt, so dass einzelne Spikes visuell erkannt werden, und manuell in der Schichtbilder aufgenommen und zwei unabhängige Modelle gemittelt. Wenn dies nicht möglich ist, zwei Änderungen an dem Protokoll versucht werden kann. Zuerst werden zwei unabhängige Zufalls Modelle können durch erste, die zwei Untergruppen von Zufalls subtomograms und dann konstruiert werden Mittelung der subtomograms innerhalb dieser Untergruppen 30. Zweitens, wenn eine Struktur des isolierten Spitze durch andere Mittel, durch Röntgenkristallographie abgeleitet, beispielsweise können sie als Ausgangsmodell verwendet werden. Allerdings muss darauf geachtet werden, werden im niedrigen Pass-Filter dieses Modell mit einem Low-Cut-off-Auflösung (50-70 Å), wie die beiden resultierenden Modelle in der nächsten Runde der Verfeinerung wirdstatistisch unabhängigen erst jenseits dieser Auflösung. Aufgrund dieser Einschränkung ist der erste Ansatz empfohlen. Die erzielbare Auflösung von diesem Protokoll hängt von vier Hauptfaktoren: i. Datensammlungsstrategie und die Qualität der Eingabedaten, ii. Anzahl der subtomograms, iii. Ausrichtungsgenauigkeit der subtomograms und iv. Heterogenität der Strukturen. Während die ersten und zweiten Begrenzungs weitgehend durch hoch Signal-Rausch direkten Elektronendetektoren kombiniert mit CTF korrigiert Tomographie und automatisierte Datenerfassung zu überwinden, wird die Ausrichtungsgenauigkeit weiter durch die Größe und Form der Struktur von Interesse selbst beeinflusst. Bei der Anwendung dieses Protokolls auf kleine Spitzen ohne hervorstechende Merkmale, kann es vorteilhaft sein, um Fab-Fragmente zu binden, um den Dorn, um die Ausrichtungsgenauigkeit und damit Auflösung 31 zu verbessern. Schließlich, wenn die Strukturen, die gemittelt werden, weisen mehrere Konformationen Unter tomogrUhr Klassifikationsmethoden verwendet werden, um im Durchschnitt unterschiedlichen Konformationen getrennt werden. Zu diesem Zweck integriert Jsubtomo mit dem Dynamo-Paket und bietet leistungsstarke subtomogram Klassifizierung 9. Das obige Protokoll komplementär zu Röntgenkristallographie von isolierten viralen Glycoproteine. Kristallographischen Strukturen in Teilschnittbild Schnittswerte eingesetzt, um die genaue Ausrichtung des Glykoproteins bezüglich des Virions Membran zu erhalten. Anwendung dieser Methode wird zweifellos auch Licht auf umhüllte Virus Struktur und Pathobiologie vergießen.

Elektronen Kryo-Tomographie ist ein Kryoelektronenmikroskopische Bildgebungsverfahren ermöglicht die Berechnung einer dreidimensionalen (3D) Rekonstruktion von komplexen biologischen Proben. Geeignete Proben reichen von gereinigtem makromolekularen Komplexen 1, Fäden 2, Vesikel 3 und 4 pleomorphe Membran Viren, ganze prokaryotischen Zellen 5 und auch dünne Bereiche der gesamten eukaryotischen Zellen 6. Nach der Datensammlung, einer Tilt-Serie, 3D-tomographischen Volumes oder Schichtaufnahmen kann mit mehreren etablierten Software-Pakete, einschließlich bsoft 7 und 8 IMOD berechnet werden. Zwei Aspekte inhärent auf die Untersuchung von biologischen Proben durch Elektronen Kryo-Tomographie begrenzen die biologische Interpretation der entsprechenden tomographischen Volumen. Erstens, aufgrund der begrenzten Elektronendosis, die biologischen Materialien vor der Einführung signifikanten Strahlenschäden angewendet werden kann, signal-zu-Rausch-Verhältnisse in tomographischen Daten sind typischerweise sehr gering. Zweitens, als Folge der begrenzten Stichprobenneigungsgeometrie während der Datensammlung, einige Ansichten des Objekts bleiben nicht vorhanden, was zu einer so genannten &quot;fehlt Keil&quot; Artefakt in der tomographischen Lautstärke. Allerdings können diese beiden Einschränkungen zu überwinden, wenn die tomographischen Volumen sich wiederholende identische Strukturen, wie beispielsweise makromolekularen Komplexen, die erfolgreich sein können gemittelt 9-12. Vor der Mittelung Strukturen Tomogramm Rekonstruktionen müssen Objekte von Interesse zu finden und mit der gleichen Orientierung ausgerichtet werden. Anordnen Solche Strukturen können durch Kreuzkorrelation eines Schablonenstruktur in dem tomographischen Volumens mit einem Ansatz oft als Template pass 13 genannten erzielt werden. Das in diesem Vergleichsprozeß verwendete Vorlage aus Kryoelektronenmikroskopische oder Elektronen Kryo-Tomographie in Kombination mit 3D-Rekonstruktion abgeleitet werden, oder es kann ein Dichtekarte von simulierendeneine atomare Struktur. Mehrere Rechenpakete wurden entwickelt, um diese Aufgaben zu erfüllen 11. Mittelung von Glycoprotein Spikes von Membran Viren, wie HIV-1, war ein besonders erfolgreicher Ansatz für die Untersuchung ihrer Struktur 14-16. Ein Verständnis der Struktur ist integraler für enthüllt sowohl die molekularen Grundlagen der Virus-Wirt-Interaktionen und Führungsantivirale und Impfstoff-Design-Entwicklung. Während makromolekulare Kristallographie ist die Technik der Wahl für hochauflösende (in der Regel besser als 4 Å) Strukturanalyse der einzelnen viralen Glykoproteinen und ihre Komplexe, die Röntgenkristallstrukturen von dieser Methode ergeben, sind von Proteinen aus der natürlichen Umgebung isoliert membranöse auf dem Virion . Somit wichtige Details wie die höherwertigen Architektur viraler Glykoproteine ​​im Kontext des Virions, bleiben fehlen. Auf der anderen Seite, Kryoelektronenmikroskopische und Einzelpartikelrekonstruktionder gesamte umhüllte Viren zu Virionen mit Ikosaedersymmetrie 17,18 beschränkt. Elektronen cryotomography kombiniert mit Teilvolumen Ausrichtung ist damit mehr als eine Ergänzung, die die Untersuchung von Glycoprotein Spikes von unregelmäßig geformten, pleomorphen Viren in situ entstanden. Wir haben eine Software namens Jsubtomo (www.opic.ox.ac.uk/jsubtomo) zum Nachweis, Ausrichtung und Mittelung der tomographischen Teilmengen entwickelt. Jsubtomo in der Bestimmung der Struktur einer Reihe von zellulären und viralen Strukturen 19-26 verwendet worden. Hier skizzieren wir ein ausführliches Protokoll, das die Bestimmung des viralen Oberflächenstrukturen Spike ermöglicht. Durch Korrelation Lärm zu über-Verfeinerung der gemittelten Strukturen zu umgehen, wird der &quot;Goldstandard&quot; Verfeinerung Regelung verabschiedet 10,27. Schließlich Strategien zur Visualisierung und Interpretation der typische Ergebnisse werden diskutiert.

<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

Umhüllte Viren nutzen Membranglykoproteine ​​auf ihrer Oberfläche, um den Eintritt in die Wirtszellen zu vermitteln. Dreidimensionale Strukturanalyse dieser Glykoprotein &#39;Spikes&#39; ist oft technisch anspruchsvoll, aber wichtig für das Verständnis der Pathogenese viraler und Wirkstoffdesign. Hier wird ein Protokoll für die virale Spike Strukturbestimmung durch rechnerische Mittelung der Kryo-Elektronentomographie-Daten dargestellt. Elektronen Kryo-Tomographie ist eine Technik, in der Elektronenmikroskopie verwendet, um dreidimensionale tomographische Rekonstruktionen Volumen oder Schichtaufnahmen, von pleomorphen biologischen Proben wie Viren-Membran in einer nahe der nativen, tiefgefrorenen Zustand abzuleiten. Diese Schnittbilder zeigen Strukturen von Interesse in drei Dimensionen, wenn auch in geringer Auflösung. Computational Mittelung der Teilvolumina, oder Unterschichtaufnahmen, ist notwendig, um eine höhere Auflösung Detail wiederholenden Strukturmotive, wie virale Glykoprotein Spikes erhalten. Eine detaillierte Berechnungsansatz für aligning und Mitteln Teilschnittbildern unter Verwendung des Softwarepakets Jsubtomo skizziert. Dieser Ansatz ermöglicht die Visualisierung der Struktur des viralen Glykoprotein Spikes mit einer Auflösung im Bereich von 20-40 Å und Studium der Studie höherer Ordnung Spitze-zu-Spitze-Interaktionen auf der Virion Membran. Typische Ergebnisse sind für Bunyamwera-Virus, ein umhülltes Virus aus der Familie Bunyaviridae vorgestellt. Diese Familie ist eine strukturell heterogene Gruppe von Krankheitserregern, die eine Bedrohung für die Gesundheit von Mensch und Tier.

Wir demonstrieren die Anwendung des Teillung Tomogramm Workflow oben für die Hüllglycoprotein Komplex von Bunyamwera Virus (Orthobunyavirus, Bunyaviridae) mit einem zuvor veröffentlichten Daten skizzierten 24. Datensammlung und Verfeinerungsparameter sind in Tabelle 1 aufgeführt. Ein Vertreter Tomogramm wird in 2 gezeigt. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Parameter (Einheit) </td><td> Wert </td></tr><tr><td> Datensammlung </td><td></td></tr><tr><td> Spannung (kV) </td><td> 300 </td></tr><tr><td> Kalibriert Vergrößerung (X) </td><td> 111.000 </td></tr><tr><td> Pixelgröße (Å) </td><td> 5.4 </td></tr><tr><td> Geschätzte Dosis (E - / A 2) </td><td> 100 </td></tr><tr><td> Unterfokus (um) </td><td> 4,0-4,5 </td&gt; </tr><tr><td> Erste CTF Null (A) ein </td><td> 26-30 </td></tr><tr><td> Neigebereich (°) </td><td> -60-60 </td></tr><tr><td> Tilt Probenahme (°) </td><td> 3 </td></tr><tr><td> Daten und Raffinesse </td><td></td></tr><tr><td> Schnittbilder </td><td> 11 </td></tr><tr><td> Virionen </td><td> 29 </td></tr><tr><td> Samen pro Virion </td><td> 106 </td></tr><tr><td> Gesamtzahl Samen </td><td> 3.074 </td></tr><tr><td> Spikes erkannt </td><td> 1.346 </td></tr><tr><td> Spikes nach dem Entfernen Überschneidungen </td><td> 1.401 </td></tr><tr><td> Spikes nach Kreuzkorrelationsbasierte Auswahl </td><td> 1.022 </td></tr><tr><td> Spikes in der letzten Durchschnitt ein, </td><td> 1.022 </td></tr><tr><td> Symmetrie </td><td> C3 </td></tr><tr><td> Winkel Probenahme (°) </td><td> 8 </td></tr><tr><td> AuflösungBereich in der Verfeinerung (a) </td><td> 42-334 </td></tr><tr><td> Endgültige Auflösung Schätzung (a) b </td><td> 35 </td></tr></tbody></table> Tabelle 1: Bunyamwera Datensammlung und Verfeinerung Statistik. ein CTF, Kontrastübertragungsfunktion. b Berechnet mit Fourier-Shell Korrelation zwischen zwei unabhängig raffinierten Strukturen bei einer Schwelle von 0.143. <img alt="Figur 2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> Figur 2:. Scheibe durch ein Tomogramm Bunyamwera Virionen Mehrere Spike Seitenansichten zeigt sich in der Peripherie jedes Virion sind mit Pfeilspitzen markiert. Das Schnittbild ist Low-Pass bis 60 Å gefiltert. Maßstab 100 nm auf. Zunächst wird ein erstes Modell mit 205 manuell abgeholt Spikes (raffiniertes wir <strong&gt; 3). Dreifache Symmetrie der Mitte-Spitze am meisten war klar, ohne Anwendung von Symmetrie (3B) und wurde in den folgenden Runden der Verfeinerung (3C) auferlegt. Zur automatischen Erkennung alle Spitzen auf der Virion-Oberfläche erzeugten wir 106 Samen pro Virion am Radius von 43 nm und der Abstand von 20 Grad (4A) und iterativ verfeinert ihre Positionen relativ zu der Membran (4B). <img alt="Figur 3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> Abbildung 3: Verfeinerung der ursprünglichen Template-Struktur. (A) Zylindrisch gemittelt (C100) Vorlage aus manuell definierten Positionen von Spitzen gebaut. (B), gemittelt Dichte nach fünf Runden der Verfeinerung ohne Symmetrie (C1) auferlegt zeigt eine Spitze mit drei-fachsymmetrische Funktionen. Die Auflösung des Modells ist 48 Å. (C) Durchschnittliche der Spitze wurde bei 41 A nach fünf Runden der Verfeinerung mit Dreifach-Symmetrie gelöst. <img alt="Figur 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> Abbildung 4:. Verfeinerung der Samen AB) Eine Teilmenge der Samen vor (A) und nach (B) sind auf einer Verfeinerung Virion Dichte von 2 gezeigt Seeds in (B) wurden farbcodiert auf der Grundlage der jeweiligen Quer. Korrelationskoeffizienten (blau, geringe Korrelation, rot, hohe Korrelation). Die besten korrelierende Glykoprotein Spike Patches (Top 75% nach dem Entfernen Überschneidungen; ~ 1.000 Spikes) wurden verwendet, um die endgültige Durchschnitt zu berechnen. Die durchschnittliche wurde auf 35-Å Auflösung (Abbildung 5) gelöst. Es zeigte eine trimere Struktur Dorn in der Mitte, inNeben einigen Beitrag aus sechs Nachbar Spikes. Verbund Modelle der Virionen, indem die Struktur in den bekannten Positionen berechnet wird, offenbart die Anordnung der Spikes auf der Oberfläche des Virions (6A). Gelegentlich waren vor Ort bestellt Patches von Spikes deutlich (Abbildung 6B). <img alt="Figur 5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> Abbildung 5: raffinierte Struktur eines Patches von dem Glykoprotein Spike Schicht nach Template-Matching. (AB) Zwei Karten &quot;auch&quot; und &quot;ungerade&quot;, aus zwei Hälften der Daten rekonstruiert werden angezeigt. Die beiden Karten zeigen einen bemerkenswerten Grad der Ähnlichkeit, die Überprüfung der Ihrer Gültigkeit des Ansatzes. Die Ausrichtung um die Längsachse Spitze unterscheidet, als die beiden Karten sind vollständig unabhängig voneinander rekonstruiert. (C) Durchschnitt der beiden Karten an gezeigtdie endgültige Auflösung von 35 Å. <img alt="Figur 6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> Abbildung 6: Platzierung der Spikes auf der Bunyamwera Virus. (A) Composite-Modell eines Virions. Ansicht Vektoren (Sticks) zeigen die Orientierung der Spikes. Farben zeigen die Kreuzkorrelation zwischen jedem Spike und der Template-Struktur (blau, geringe Korrelation, rot, hohe Korrelation). (B) A close-up von einer geordneten Patch von Spikes.

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.

Mittelung der Virushülle Glykoprotein-Spikes von Electron Cryotomography Rekonstruktionen mit Jsubtomo

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

12.1K Aufrufe.  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.

Serie: 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.

Mit einem Pan-Viral Microarray-Assay (Virochip) zur klinischen Proben, die für virale Erreger Bildschirm

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 ...

Forschung

Immunologie und Infektion

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.

Bestimmung von Molekülstrukturen von HIV Hüllglycoproteine ​​mit Cryo-Elektronen-Tomographie und automatisierte Sub-Tomogramm Averaging

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.

Kostengünstige Methode für Mikrobielle Source Tracking mit spezifischen menschlichen und tierischen Viren

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.

Isolierung von Fidelity Varianten von RNA-Viren und Charakterisierung von 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 ...

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.

Trennung von Einzelstrang-DNA, doppelsträngige DNA und RNA aus ökologischer Viral Gemeinschaft mit Hydroxyapatit-Chromatographie

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.

Herstellung viraler DNA aus Nukleokapside

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.

Imaging von HIV-1 Envelope-induzierte virologische Synapse und Signalisierung auf Synthetic Lipiddoppelschichten

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.

Konformationsänderungen Auswertung der HIV-1-Trimeric Hüllglycoproteine ​​mit eine Handy-basierte ELISA-Test

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

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

Isolierung der viralen Replikation Compartment angereicherte Teil nuklearen Fraktionen von Adenovirus-infizierten normalen menschlichen Zellen

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

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.

Die Entwicklung der Lyophilisierte Loop-mediated Isothermal Amplification Reagenzien zum Nachweis von<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. ...