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> La conoscenza della struttura glicoproteina virale punta sulla membrana virione è essenziale per comprendere la replicazione del virus e lo sviluppo di terapie per trattare e prevenire le infezioni. Electron crio-microscopia combinata con un&#39;unica media delle particelle è emerso come il metodo più utilizzato per risolvere le strutture di particelle del virus con involucro, comprese le punte glicoproteina. Tuttavia, questo metodo è limitato a icosahedrally virus simmetrici. Qui, attraverso l&#39;applicazione di elettroni tomografia crio-e subtomogram media in Jsubtomo, abbiamo delineato un protocollo generale per determinare picchi glicoproteina su pleomorfo avvolto virus che non sono suscettibili di altre correnti metodi di biologia strutturale. I nostri risultati rappresentativi dimostrano che la risoluzione di questo metodo è sufficiente a rivelare intuizioni architettura di dominio, oligomerizzazione, e l&#39;organizzazione di ordine superiore di punte glicoproteina su virioni intatti. jove_content &quot;&gt; La fase più critica all&#39;interno di questo protocollo sta costruendo due modelli di partenza affidabili che sono statisticamente indipendenti gli uni dagli altri. esecuzione di questa operazione presuppone che picchi glicoproteina sono sufficientemente grandi e non ammassata contro l&#39;altro troppo stretto, in modo che i singoli picchi può essere riconosciute visivamente e manualmente raccolte nel tomogrammi, e due modelli indipendenti media. Se ciò non è fattibile, due modifiche al protocollo possono essere tentate. Innanzitutto, due modelli casuali indipendenti possono essere costruiti in primo luogo la definizione di due sottoinsiemi casuali di subtomograms e poi media dei subtomograms all&#39;interno di questi sottoinsiemi 30. secondo luogo, se una struttura del picco isolato è stato ricavato con altri mezzi, ad esempio cristallografia a raggi X, può essere utilizzato come modello di partenza. Tuttavia, la cura deve essere presa per bassa filtro passa questo modello utilizzando un cut-off a bassa risoluzione (50-70 Å), come i due modelli risultanti nel prossimo turno di raffinatezza volontàessere statisticamente indipendenti solo oltre questa risoluzione. A causa di questo avvertimento, si raccomanda il primo approccio. La risoluzione ottenibile da questo protocollo dipende da quattro fattori principali: i. strategia di raccolta dei dati e la qualità dei dati in ingresso, ii. numero di subtomograms, iii. precisione di allineamento dei subtomograms, e iv. eterogeneità delle strutture. Mentre il primo e il secondo limite può essere ampiamente superato utilizzando rivelatori elevati segnale-rumore scalo elettroni combinati con CTF corretto tomografia e raccolta dati automatizzata, la precisione di allineamento è ulteriormente influenzata dalla dimensione e la forma della struttura di interesse stesso. Quando si applica questo protocollo sulle piccole punte prive di caratteristiche di spicco, può essere vantaggioso per legare i frammenti Fab per il picco di migliorare la precisione di allineamento e, quindi, la risoluzione 31. Infine, se le strutture siano in media presentano molteplici conformazioni, sub-TomogrMetodi di classificazione am possono essere utilizzati a media differenti conformazioni separatamente. A tal fine, Jsubtomo si integra con il pacchetto Dynamo, offrendo classificazione potente subtomogram 9. Il protocollo di cui sopra è complementare alla cristallografia a raggi X di glicoproteine ​​virali isolati. Strutture cristallografiche possono essere inseriti nelle medie sub-tomogramma per ottenere l&#39;orientamento preciso della glicoproteina rispetto alla membrana virione. L&#39;applicazione di questa metodologia sarà senza dubbio continuerà a far luce sulla struttura del virus avvolto e patobiologia.

Electron crio-tomografia è una tecnica di imaging elettronico crio-microscopia che permette il calcolo di una tridimensionale (3D) ricostruzione di campioni biologici complessi. I campioni idonei vanno da complessi macromolecolari purificate 1, 2 filamenti, vescicole rivestite 3, e virus membrana pleomorfi 4 a intere cellule procariote 5 e anche le aree sottili di intere cellule eucariotiche 6. Dopo la raccolta dei dati di un tilt-serie, volumi tomografiche 3D, o tomografie, può essere calcolato utilizzando diversi pacchetti software affermati, tra cui Bsoft 7 e IMOD 8. Due aspetti inerenti allo studio di campioni biologici dal limite elettroni tomografia crio-l&#39;interpretazione biologica dei corrispondenti volumi tomografiche. In primo luogo, a causa della dose di elettroni limitata che può essere applicato a materiali biologici prima di introdurre significativi danni da radiazioni, signaL-to-noise ratio nei dati tomografici sono in genere molto bassi. In secondo luogo, a causa della geometria del campione limitato inclinazione durante la raccolta dei dati, alcune viste dell&#39;oggetto rimangono assenti, portando ad un cosiddetto artefatto &#39;cuneo mancante&#39; nel volume tomografica. Tuttavia, entrambi questi limiti possono essere superati se il volume tomografica contiene ripetendo strutture identiche, come i complessi macromolecolari, che possono essere correttamente mediate 9-12. Prima di una media struttura di ricostruzioni tomogramma, oggetti di interesse devono essere trovate e allineati allo stesso orientamento. Individuazione tali strutture può essere raggiunto mediante cross-correlazione di una struttura modello nel volume tomografica utilizzando un approccio spesso indicato come modello di corrispondenza 13. Il modello utilizzato in questo processo di corrispondenza può essere derivata da elettroni crio-microscopia elettronica o crio-tomografia combinata con ricostruzione 3D, oppure può essere una mappa di densità simulato dauna struttura atomica. Alcuni pacchetti computazionali sono stati sviluppati per svolgere questi compiti 11. Della media dei picchi glicoproteina di membrana di virus, come l&#39;HIV-1, è stato un approccio particolarmente efficace per studiare la loro struttura 14-16. La comprensione della struttura è integrale per rivelare sia le basi molecolari delle interazioni virus-ospite e guidare lo sviluppo di design antivirali e di vaccini. Mentre la cristallografia macromolecolare è la tecnica di scelta per alta risoluzione (generalmente migliore di 4 Å) analisi strutturale delle singole glicoproteine ​​virali e loro complessi, le strutture raggi X risultanti da tale metodo di proteine ​​isolate dall&#39;ambiente membranosa naturale sul virione . Così, dettagli importanti come l&#39;architettura ordine superiore di glicoproteine ​​virali, nel contesto del virione, rimangono privi. D&#39;altra parte, elettrone crio-microscopia e singola particella ricostruzionedi intere virus con involucro è limitato a virioni con simmetria icosaedrica 17,18. Electron cryotomography combinato con l&#39;allineamento sub-volume è dunque emerso come tecnica complementare che permette lo studio dei picchi glicoproteina di forma irregolare, virus pleomorphic in situ. Abbiamo sviluppato un software chiamato Jsubtomo (www.opic.ox.ac.uk/jsubtomo) per la rilevazione, l&#39;allineamento e la media dei tomografiche sotto-volumi. Jsubtomo è stato utilizzato nella determinazione della struttura di un certo numero di strutture cellulari e virali 19-26. Qui, descriviamo un protocollo dettagliato, che permette alle strutture di picco virale di superficie determinazione. Per aggirare over-raffinamento di strutture mediati correlando rumore, il regime di perfezionamento &#39;gold standard&#39; è adottato 10,27. Infine, le strategie per la visualizzazione e l&#39;interpretazione dei risultati tipici sono discussi.

<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

Virus con involucro utilizzano glicoproteine ​​di membrana sulla superficie di mediare l&#39;ingresso nelle cellule ospiti. Tridimensionale analisi strutturale di queste glicoproteina &#39;punte&#39; è spesso tecnicamente impegnativo, ma importante per comprendere la patogenesi virale e nella progettazione di farmaci. Qui, un protocollo viene presentato per la determinazione della struttura virale picco attraverso media computazionale di elettroni dati crio-tomografia. Electron crio-tomografia è una tecnica di microscopia elettronica utilizzata per ricavare tridimensionali ricostruzioni tomografiche di volume, o tomografie, di campioni biologici pleomorfi come virus membrana in uno stato congelato-idratato quasi native. Queste tomografie rivelano strutture di interesse in tre dimensioni, anche se a bassa risoluzione. Media computazionale dei sotto-volumi, o sub-tomografie, è necessario per ottenere maggiore dettaglio la risoluzione di ripetere motivi strutturali, come ad esempio i picchi glicoproteina virale. Un approccio computazionale dettagliato per aligning e media sub-tomogrammi utilizzando il pacchetto software Jsubtomo è delineato. Questo approccio consente la visualizzazione della struttura di picchi glicoproteine ​​virali di risoluzione nell&#39;intervallo 20-40 Å e studio dello studio di ordine superiore picco-a-picco interazioni sulla membrana virione. I risultati tipici sono presentati per Bunyamwera virus, un virus con involucro della famiglia Bunyaviridae. Questa famiglia è un gruppo strutturalmente eterogeneo di agenti patogeni che costituiscono una minaccia per la salute umana e animale.

Dimostriamo l&#39;applicazione del flusso di lavoro media sub-tomogramma sopra descritta per il complesso busta glicoproteina del virus Bunyamwera (Orthobunyavirus, Bunyaviridae) utilizzando un set di dati precedentemente pubblicati 24. Parametri di raccolta dei dati e la raffinatezza sono elencate nella Tabella 1. Una tomogram rappresentativo è mostrato nella Figura 2. <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> Parametri (unità) </td><td> Valore </td></tr><tr><td> La raccolta dei dati </td><td></td></tr><tr><td> Tensione (kV) </td><td> 300 </td></tr><tr><td> Ingrandimento calibrato (X) </td><td> 111.000 </td></tr><tr><td> Dimensione del pixel (Å) </td><td> 5.4 </td></tr><tr><td> Dose stimata (e - / A 2) </td><td> 100 </td></tr><tr><td> Underfocus (micron) </td><td> 4,0-4,5 </td&gt; </tr><tr><td> Prima di zero CTF (Å) un </td><td> 26-30 </td></tr><tr><td> Intervallo inclinazione (°) </td><td> -60-60 </td></tr><tr><td> Campionamento Tilt (°) </td><td> 3 </td></tr><tr><td> Dati e raffinatezza </td><td></td></tr><tr><td> Tomogrammi </td><td> 11 </td></tr><tr><td> Virioni </td><td> 29 </td></tr><tr><td> Semi per virione </td><td> 106 </td></tr><tr><td> Numero totale di semi </td><td> 3.074 </td></tr><tr><td> Spikes rilevati </td><td> 1.346 </td></tr><tr><td> Spikes dopo la rimozione di sovrapposizioni </td><td> 1.401 </td></tr><tr><td> Spikes dopo selezione basata cross-correlazione </td><td> 1.022 </td></tr><tr><td> Spikes inclusi nella media finale </td><td> 1.022 </td></tr><tr><td> Simmetria </td><td> C3 </td></tr><tr><td> Campionamento angolare (°) </td><td> 8 </td></tr><tr><td> Risoluzioneintervallo utilizzato nella raffinatezza (Å) </td><td> 42-334 </td></tr><tr><td> Stima risoluzione finale (A) B </td><td> 35 </td></tr></tbody></table> Tabella 1: raccolta di dati e statistiche Bunyamwera raffinatezza. un CTF, funzione di trasferimento di contrasto. b Calcolato utilizzando Fourier shell correlazione tra due strutture indipendenti raffinati ad una soglia di 0.143. <img alt="Figura 2" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig2highres.jpg" width="600" /> Figura 2:. Fetta attraverso una tomografia di virioni Bunyamwera Diverse viste laterali picco evidenti nella periferia di ogni virione sono indicati con frecce. Il tomogramma è stato filtrato passa-basso a 60 Å. Barra di scala 100 nm. In primo luogo, abbiamo perfezionato un modello iniziale con 205 punte raccolte manualmente ( <strong&gt; Figura 3). Tre volte la simmetria del centro-picco più era evidente senza applicare alcuna simmetria (Figura 3B) ed è stato imposto nei successivi cicli di perfezionamento (Figura 3C). Per rilevare automaticamente tutte le punte sulle superfici virione, abbiamo generato 106 semi per ogni virione al raggio di 43 nm e la spaziatura di 20 gradi (Figura 4A) e iterativamente perfezionato le loro posizioni rispetto alla membrana (Figura 4B). <img alt="Figura 3" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig3highres.jpg" width="600" /> Figura 3: affinamento della struttura iniziale del modello. (A) cilindrica media (C100) modello costruito da posizioni definite manualmente di picchi. (B) In media densità dopo cinque turni di raffinatezza senza alcuna simmetria (C1) ha imposto mostra un picco con triplicecaratteristiche simmetriche. La risoluzione del modello è di 48 Å. (C) media del picco è stato risolto a 41 Å dopo cinque turni di raffinatezza con triplice simmetria. <img alt="Figura 4" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig4highres.jpg" width="600" /> Figura 4:. Affinamento dei semi AB) Un sottoinsieme di semi prima (A) e dopo B) affinamento (sono mostrate su una densità virione dalla figura 2 Semi in (B) sono stati codice colore in base ai rispettivi incrociata. coefficienti di correlazione (blu, bassa correlazione, rosso, alta correlazione). Le migliori patch correlando glicoproteina picco (superiore al 75% dopo la rimozione di sovrapposizioni; ~ 1.000 picchi) sono stati utilizzati per calcolare la media finale. La media è stato risolto a 35-risoluzione (Figura 5). Ha rivelato una struttura picco trimeric nel mezzo, inOltre a qualche contributo da sei picchi vicini. Modelli compositi dei virioni, calcolato ponendo la struttura nelle posizioni note, rivelato posizionamento dei picchi sulla superficie del virione (Figura 6A). Di tanto in tanto, le patch in ordine localmente di picchi erano evidenti (Figura 6B). <img alt="Figura 5" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig5highres.jpg" width="600" /> Figura 5: struttura raffinata di una patch dello strato glicoproteina picco dopo il modello corrispondente. (AB) Due mappe &#39;anche&#39; e &#39;strano&#39;, ricostruiti da due metà dei dati vengono visualizzati. Le due mappe mostrano un notevole grado di somiglianza, verificare la Validità dell&#39;approccio. L&#39;orientamento intorno all&#39;asse lungo picco è diversa in quanto le due mappe sono state ricostruite completamente indipendente l&#39;uno dall&#39;altro. (C) media delle due mappe è mostrato inla risoluzione finale di 35 Å. <img alt="Figura 6" fo:content-width="6in" src="/files/ftp_upload/51714/51714fig6highres.jpg" width="600" /> Figura 6: Posizionamento delle punte sul virione Bunyamwera. (A) modello composito di un virione. Vedi vettori (bastoni) indicano gli orientamenti delle punte. I colori indicano la cross-correlazione tra ogni picco e la struttura del modello (blu, bassa correlazione, rosso, alta correlazione). (B) A close-up di una patch ordinata di picchi.

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.

Della media di Viral Busta Glicoproteina Spikes da Electron Cryotomography Ricostruzioni con 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

12.0K Views.  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.

Utilizzando un pan-virale Assay Microarray (Virochip) allo schermo campioni clinici di patogeni virali

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 ...

Ricerca

Immunologia e infezioni

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.

Determinazione di strutture molecolari di glicoproteine ​​dell&#39;HIV Busta con Cryo-Electron Positron e automatizzato Sub-tomogramma 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.

Costo-efficace metodo per il rilevamento di origine microbica Utilizzando specifici virus umani e animali

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.

Isolamento di varianti di virus RNA Fidelity e caratterizzazione di frequenza Virus Mutation

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.

Separazione di DNA a singolo filamento, doppia elica del DNA e RNA da una Comunità Ambientale virale usando la cromatografia idrossiapatite

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.

Estrazione di DNA virale da nucleocapsidi

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 di HIV-1 Envelope indotta Synapse virologica e segnalazione sui doppi strati lipidici sintetici

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.

Valutazione conformazionale di HIV-1 trimeric busta glicoproteine ​​Utilizzando una base di Cell-ELISA Saggio

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.

Lo sviluppo del liofilizzato Loop-mediate isotermici amplificazione reagenti per la rilevazione di<em&gt; Burnetii Coxiella</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.

Misurare Influenza neuraminidasi Inibizione anticorpo I titoli da Enzyme-linked lectina Assay

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