We thank F. Chisari for providing Huh-7.5.1 cell line. We would like to thank Justine Ho for editing the manuscript. This work was supported by Cedars-Sinai Medical Center Institutional Programmatic Research Award and National Center for Advancing Translational Sciences, Grant UL1TR000124 to V.A.

Ein allgemeiner Überblick über das Protokoll ist in Fig. 1 dargestellt. 1. Cells <ol><li> Vorbereitung vollständige Wachstumsmedium, das 10-15% fötalem Rinderserum (FBS), 10 mM nicht-essentiellen Aminosäuren, 10 mM Hepes, Penicillin (100 Einheiten / ml), Streptomycin (100 mg / ml) und 2 mM L-Glutamin enthält. </li><li> Pflegen Huh-7.5.1-Zellen in 13 komplette Wachstumsmedium, das die oben genannten Ergänzungen für in-vitro-Analyse von Hepatitis-C-Virusreplikationszyklus. </li><li> Kultur Virusstämme in Huh-7.5.1-Zellen mit den angegebenen Wachstumsmedium ergänzt bei 37 ° C mit 5% CO 2. </li></ol> 2. Viren und Plasmid-Konstrukte <ol><li> Erzeugen, die komplementäre DNA (cDNA)-Form von HCV-RNA und klonen in einen Plasmidvektor für einfache genetische Manipulation. Hinweis: Die Entdeckung der japanischen fulminante Hepatitis 1, JFH-1 (HCV Genotyp 2a), Isolat HCV kritisch für Forschung gewesen undin erster Linie verwendet, um die HCV-Replikationszyklus 11-13 studieren. Inter-und intragenotypic chimären Viren basierend auf JFH-1 HCV sind häufig in Forschungs 14-18 verwendet. Konstruktion von synthetischen intragenotype 2a chimären Virus, FNX-HCV und einem monocistronischen chimären Reporterstamm wurde zuvor 19 und konstruktive Details sind nicht Gegenstand dieses Protokolls beschrieben. </li><li> Verwenden Sie die pFNX-HCV intragenotype 2a Virus als es enthält eine 5&#39;-NTR-, Struktur-Regionen und P7, und ein Teil der NS2 nicht-strukturellen Regionen (Nukleotide 1-2878) des J6CF Elternstamm (NCBI Zugangsnummer. AF177036), als sowie die nicht-strukturellen Komponenten des JFH-1-Virusstamm (NCBI Zugangsnummer. AB047639). Hinweis: FNX-HCV wurde basierend auf der Sequenz von Jc1 intragenotype 2a chimären HCV synthetisiert zuvor berichtet 15. </li><li> Generieren Sie eine monocistronischen chimären Reporter virales Konstrukt, das pFNX-Rluc, auf der Grundlage der pFNX-HCV-Stamm (oben in Schritt 2.2), indem Sie einen Renilla Luciferase-Gen zwischen der 5 &#39;NTR und Core-Gen (nt zwischen 388 und 389). Anschließend verbinden Sie das Luciferase-Gen-Gen-und Kern dieses Konstrukts mit einem Maul-und Klauenseuche-Virus 2A (F2A) Peptidsequenz, die als Spaltsignal funktionieren würde. </li><li> Ingenieur die RNA-Polymerase-Null (Pol-) virales Konstrukt entweder mit dem pFNX-HCV oder pFNX-Rluc viralen Hintergrund. Ersetzen die katalytischen Reste NS5B Polymerase GDD (aa 2759-2761; nt 8615-8623), der eine dieser viralen Rückgrat mit AAG Aminosäureresten. </li></ol> 3. In-vitro-HCV-RNA-Transkription <ol><li> Verwenden Sie ein intragenotype 2a chimären Virus FNX-HCV und HCV-FNX Pol null (Pol-) HCV für die Bewertung der viralen Replikation (Abbildung 2A). </li><li> Linearisieren die viralen Plasmide mit XbaI Restriktionsenzym und dann mit Mungobohnennuclease um glatte Enden zu erzeugen behandeln. Reinigung der verdauten Plasmide durch Anionenaustauschchromatographie. Überprüfen Sie die Integrität of Das linearisierte Plasmid indem DNA-Gelelektrophorese (Fig. 2B) Agarose. </li><li> Übertragen Sie die linearisierte Plasmide mit Hilfe der viralen T7-RNA-Polymerase. </li><li> Reinige den neu synthetisierten DNase behandelte RNA unter Verwendung eines RNA-Reinigungs-Kits. </li><li> Überprüfen Sie, ob die RNA-Produktion durch Agarose-Gelelektrophorese (Abbildung 2C). </li><li> Quantifizierung der RNA durch Spektrophotometrie. </li><li> Lagern Sie den erzeugten RNA in -80 ° C in 10 ug Arbeits Aliquots. Hinweis: Um die Veränderung der RNA-Qualität innerhalb der einzelnen Versuchsplanung zu minimieren durch Transkription aller RNA für jede Virusprobe und Kontrolle zugleich. </li></ol> 4. HCV-RNA-Transfektion und Probenentnahme <ol><li> Ernten Sie die Huh-7.5.1-Zellen mit Trypsin. </li><li> Um Zellen, Zentrifuge spülen und resuspendieren Suspension zweimal mit kaltem niedrigen Serum-Medien. Resuspendieren der Zellen in Medien mit niedrigem Serumgehalt 1 x 10 7 Zellen pro ml beträgt. </li><li> Mischen Sie insgesamt 10 &amp; #956; g transkribierte Virus-RNA mit 400 ul der resuspendierten Zellen (4 × 10 6 Zellen) in einer 0,4-cm-Elektroporationsküvette transferiert. Transfektion von Zellen mittels Elektroporation bei 270 V, 100 Ω und 950 uF. </li><li> Resuspendieren der Elektroporation Zellen in 10 ml Vollwachstumsmedium mit 15% FBS. Hinweis: Erhöhte Überlebensfähigkeit von Zellen elektroporiert wird gesehen, wenn die Huh-7.5.1-Zellen wurden in 15% fötalem Rinderserum kultiviert. </li><li> Platte, die Zellen in beiden T-25-Kolben (~ 1,2 × 10 6 Zellen pro Kolben) und 48-Well-Platten (1 × 10 4 Zellen pro Vertiefung) für jede der folgenden Zeitpunkten: 4, 48 und 96 Stunden. </li><li> Ersetzen Sie die Medien bei 4-8 Stunden nach der Transfektion mit frischem ergänzten Wachstumsmedium mit 10% FBS, um abgestorbene Zellreste aus den kultivierten Flaschen und Platten entfernen. </li><li> Erntezellkulturüberständen an den 48, 96 h Zeitpunkte und Entfernen von Zelltrümmern von gesammelten Proben durch Zentrifugation der Zellen bei 1500 UpM für 10 min bei 4 º C. </li><li> Bewahren Sie die zellfreien Überstände bei -80 º C. Lyse der Zellen, die für Protein-und RNA-Analyse durch Western-Blot und Reverse Transkription-PCR quantitativ zu den angegebenen Zeitpunkten. </li></ol> 5. Reverse Transkription-quantitative PCR (RT-qPCR) für die Beurteilung der HCV-Genom-Kopien <ol><li> Führen eine Zwei-Schritt-RT-qPCR die genomische HCV-RNA Kopienzahl zu bestimmen. </li><li> Revers transkribieren 1 ug zelluläre Gesamt-RNA unter Verwendung von reverser Transkriptase und eines Primers spezifisch für HCV-sense-Strang, der 5 &#39;NTR (JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3&#39;) bindet, oder ein Primer, die spezifisch für Haushaltsgens Peptidylprolyl Isomerase G (R ppig : 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3 &#39;). Auch Reverse Transkription FNX-HCV-RNA (in Schritt 3 erzeugte) bekannter Genomkopien (10. Januar-10. September Standard) mit HCV-Sense-Strang Primer. </li><li> Führen Sie qPCR unter Verwendung von 50 ng der resultierenden cDNA transkribiert mit spezifischen HCV-Primer (JFH RTQF: 5&#39;CTGGGTCCTTTCTTGGATAA-3; JFH RTQ R: 5&#39;CCTATCAGGCAGTACCACA-3 &#39;) und DNA-Bindung grüne Farbstoff, der qPCR Super-Mix. Führen qPCR für Housekeeping-Gen sowie ppig (ppig Primer F: 5&#39;-GAAGAGTGCGATCAAGAACCCATGAC-3 &#39;; ppig R: 5&#39;-GTCTCTCCTCCTTCTCCTCCTATCTTT-3&#39;) Anmerkung: Für die Berechnung der genauen intrazellulären HCV-RNA-Ebene über die Stichproben verwenden zellulären Housekeeping-Gen, ppig , Expressionsniveau (Ct-Zyklus) für die Normalisierung. Verwenden Sie die Kopienzahl des FNX-HCV-Genom als Standard für die Bestimmung der Kopienzahl. </li><li> Verwenden Sie die folgenden Bedingungen bei der Ausführung von qPCR auf HCV-RNA-Kopienzahl zu bestimmen: 95 ° C für 15 Sekunden und 60 &#160; º C für 30 Sekunden (40 Zyklen) mit Echtzeit-PCR-System. </li><li> Siehe Fig. 3A und 3B für die Genomreplikation Ergebnisse. </li></ol> 6. Western Blot-Analyse zum Nachweis von HCV-Protein-Expression (Fig. 3C) <ol><li> Verwenden Zelllysate from Virus-RNA bei 96 Stunden nach der Transfektion für Protein-Western-Blot-Analyse transfiziert. </li><li> Lösen das Zelllysat unter Verwendung von SDS-PAGE und Transfer auf Polyvinylidendifluorid (PVDF)-Membran. </li><li> Blockieren der Membran mit einer Blockierungslösung, enthaltend 5% Magermilch, 0,2% Tween 20 in phosphatgepufferter Salzlösung (PBS). </li><li> Inkubation der Membran mit primären monoklonalen Maus-Antikörper NS3 (1 von 1.000 Verdünnung) und beta-Aktin (1 in 5000 Verdünnung). </li><li> Hinzufügen Ziege-anti-Maus-IgG, das mit Meerrettich-Peroxidase (1 in 5.000 verdünnt) konjugiert und durch Chemolumineszenz nachzuweisen. </li></ol> 7. Immunfluoreszenz-Assay (IFA) <ol><li> Beheben die HCV-infizierten und transfizierten Zellen unter Verwendung von Methanol für 30 min bei -20 º C für Immunfluoreszenz-Assay. </li><li> Waschen Sie die Zellen dreimal mit PBS und Block mit IFA Blockierungspuffer. </li><li> Verwenden polyklonalen Kaninchen anti-NS5A in erster Linie Antikörper oder monoklonalen Maus-Anti-DSR (freundlicherweise von Dr. Dasgupta, UCLA Verfügung gestellt)NA J2 Antikörper bei einer Verdünnung von 1:200 (1 ug / ml) und Inkubation für 5 h bis über Nacht bei 4 º C. </li><li> Dreimal nach dem primären Antikörper Waschen der Zellen mit PBS. </li><li> Hinzufügen Ziegen-Anti-Kaninchen-IgG-Sekundär 488 polyklonalen Antikörper oder Ziege-anti-Maus-IgG-Sekundär 594 polyklonalen Antikörper in einer 1:1000 Verdünnung (1 ug / ml) und Inkubation für 1 h bei Raumtemperatur. </li><li> Waschen der Zellen mit PBS dreimal und Fleckenkerne mit Hoechst-Farbstoff und Ansicht mit Fluoreszenzmikroskop (Fig. 3D). </li></ol> 8. Mess Virus Titer <ol><li> Platte naiv Huh-7.5.1-Zellen bei etwa 3 x 10 3 Zellen / Well unter Verwendung eines 96-Well-Platte. Am nächsten Tag, führen das 10-fache serielle Verdünnungen der zellfreien Kulturüberstand von HCV-RNA-transfizierten Zellen mit Wachstumsmedium geerntet und in dreifacher Ausfertigung auf impfen Huh-7.5.1-Zellen. </li><li> Fix-Zellen bei 72 Stunden nach der Infektion mit Methanol (30 min bei -20 </sup&gt; º C) und immunostain für HCV NS5A-Protein, wie in Abschnitt 7 angegeben. </li><li> Verwenden Sie die höchste Verdünnung, die für die NS5A positive Foci zählen und berechnen die durchschnittliche Anzahl der Schwerpunkt Bildungseinheit (FFU) pro Milliliter. Siehe Abbildung 4 für die HCV-Titer. </li></ol> 9. Renilla Luciferase Reporter Assay für virale Genom-Replikation und Infektiosität <ol><li> Für die Beurteilung viralen Genomreplikation, Platte HCV-RNA-Elektroporation Huh-7.5.1-Zellen in dreifacher Ausfertigung in 48-Well-Platten (1 x 10 4 Zellen / well). </li><li> Lyse die Zellen mit 75 ul der passiven Lyse-Puffer an der 6 h, 48 h und 96 h nach der Transfektion. </li><li> Schaukeln Sie die Platten vorsichtig für 15 Minuten bei Raumtemperatur und bei -80 º C. </li><li> Zur Messung der Renilla-Luciferase-Enzymaktivität, die Protein-Lysat auftauen und Renilla-Luciferase-Assay-Reagenzien auf Raumtemperatur. </li><li> In 20 ul Proteinlysat pro Vertiefung einer 96-wel<html> l weiße Lumineszenz Platte und legen Sie die Platte in einem Luminometer. </li><li> Pipettieren von 100 ul Renilla Luciferase-Assay-Reagenz (Coelenterazin Substrat + Puffer) pro Vertiefung und nach einer 2 Sek. vor, lesen Verzögerung; integrieren, das emittierte Licht für 10 Sekunden. </li><li> Mittelwert und Standardabweichung für jede Probe aus den Luciferase-Werte der biologischen Triplikaten und unterziehen die Daten einer statistischen Analyse (Abbildung 5). </li><li> Zur Beurteilung Infektiosität inokulieren 500 ul zellfreier Überstand aus HCV-RNA-transfizierten Zellen für die angegebenen Zeitpunkten (48 h und 96 h) in dreifacher Ausfertigung in 48-Well-Platten auf naiven Huh-7.5.1-Zellen erhalten. </li><li> Ersetzen Sie die viralen Inokulum mit 500 ml frisches Medium pro Well nach 6 Stunden nach der Infektion. </li><li> Lyse der Zellen bei 48 Stunden nach der Infektion und nach Renilla-Luciferase-Assay, wie in Schritten von 8,2 bis 8,7 (Fig. 5) beschrieben. </li></ol>itel &quot;&gt; 10. Statistische Analyse <ol><li> Die Fehlerbalken in den Diagrammen zeigen die Standardabweichungen (SD). Die P-Werte wurden mit dem ungepaarten t-Test berechnet. </li></ol>

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The authors have nothing to disclose.

Diese Darstellung beschreibt ein Verfahren zum Analysieren des Hepatitis-C-Virus-Replikationszyklus. HCV ist ein humanes Pathogen und die vorgeschriebene Biosafety-Protokoll müssen strikt eingehalten werden. Infektiöse HCV-Zellkultursysteme wurden zuvor 11-13,16,17 beschrieben. Es gibt nur wenige wichtige Punkte zu implementieren, wenn wir nach dem Protokoll dargestellt. Erstens, von hoher Bedeutung ist es, gute Qualität von intakten voller Länge viralen genomischen RNA für nachgeschaltete Studien. Die E...

Hepatitis-C-Virus (HCV) verursacht Leberzirrhose und Leberkrebs. Es betrifft 170 Millionen Menschen weltweit mit 350.000 Menschen sterben jährlich 1-3. HCV ist ein positiver Strang-RNA-Virus mit einer Genomgröße von 9,6 kb. Das HCV-Genom als ein einzelnes Polyprotein von ca. 3000 Aminosäuren, das proteolytisch durch verschiedene zelluläre und virale Proteasen gespalten, in 10-Polypeptide translatiert wird. HCV-Virus ist der Prototyp der Gattung Hepacivirus und gehört zur Familie der Flaviviridae 4. Bei der Belichtung stellt chronischer HCV-Infektion in 80% der Individuen. Die Infektion ist meist asymptomatisch und rechtzeitige Diagnose kann die therapeutische Intervention zu Leber Verschlechterung verhindern können. Die derzeitige Behandlung nicht optimal sein und kein Impfstoff verfügbar ist 5,6. Die Ätiologie der Hepatitis C wurde erstmals 1989 7 beschrieben. Studieren HCV-Replikation ist wichtig für die Hepatitis-C-Impfstoff-und Behandlungsforschung, aber es warlang durch das Fehlen eines effizienten Viruskultursystem behindert. Ein molekularer Klon des HCV Infektions wurde gezeigt, bei Schimpansen auf intra-Impfung 8 sein. Anschließend wurden HCV subgenomische Replikon beschrieben, die das virale Genom-Replikation Stufe in einem Zellkultursystem erlaubt 9,10 sezieren. Entdeckung einer Genotyp HCV 2a isolieren JFH-1 (Japanese Fulminante Hepatitis-1), infizieren Zellkultur eröffnet neue Wege für die HCV-Replikation Forschungs 11-13. Genotyp 2a Stamm JFH-1-Basis inter-und intra-und genotypische chimären Viren vom Genotyp 1 HCV Infektions basierend Kultursysteme sind auch 14 bis 18 erhältlich. Wir haben erfolgreich JFH-1-Stamm-und HCV-intragenotype 2a chimären Virus verwendet, um die hochauflösenden funktionellen Profilierung Karte von Proteindomänen und cis-aktiven RNA-Elemente 19,20 zu erhalten. Demnach beschreiben wir eine effektive Kultursystem routinemäßig verwendet, die ermöglichtStudium der verschiedenen Stadien des HCV-Replikationszyklus und Wirt-Pathogen-Interaktion. Wir präsentieren virologische Untersuchungen auf virale Genom-Replikation und de novo Infektiosität von HCV intragenotype 2a und einem Renilla-Luciferase Reporter basierend HCV beurteilen.

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			<td>The cell line was kindly provided by Dr. Francis Chisari to Dr. Arumugaswami under executed MTA between The Scripps Research Institute and Cedars-Sinai Medical Center</td>
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			<td style="width:267px;">Cedars-Sinai Medical Center</td>
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			<td>The HCV plasmids were synthesized by Dr. Arumugaswami using overlapping oligo-nucleotides.&#160;</td>
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			<td>English &#38; Scientific Consulting Kft.</td>
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			<td height="21" style="height:21px;width:569px;">GloMax-Multi Detection System (Luminometer)</td>
			<td style="width:267px;">Promega</td>
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a protocol for analyzing hepatitis c virus replication

Hepatitis-C-Virus (HCV) betrifft 3% der Weltbevölkerung und verursacht schwere Lebererkrankungen wie chronische Hepatitis, Leberzirrhose und Leberzellkarzinom. HCV ist ein umhülltes RNA-Virus aus der Familie Flaviviridae. Strombehandlung nicht wirksam ist und bewirkt, dass nachteilige Nebenwirkungen. Es gibt keine HCV-Impfstoff zur Verfügung. So wird fortgesetzt Aufwand für die Entwicklung eines Impfstoffes und eine bessere Therapie erforderlich. Einem HCV-Zellkultursystems ist kritisch für das Studium verschiedener Stadien der HCV Wachstum einschließlich Viruseintritt, Genom-Replikation, Verpackung und Austritt. In der vorgestellten aktuellen Prozedur verwendeten wir eine Wildtyp-intragenotype 2a chimären Virus, FNX-HCV, und ein rekombinantes FNX-Rluc Virus, eine Renilla-Luciferase-Reportergens um die Virusreplikation zu studieren. Eine menschliche Hepatom-Zelllinie (Huh-7-Basis) wurde für die Transfektion von in vitro transkribierten genomischen HCV-RNAs. Zellfreie Kulturüberstände, Protein-Lysate und total RNA wurden zu verschiedenen Zeitpunkten nach der Ernte Transfektion HCV Wachstum zu beurteilen. HCV-Genom-Replikation Status wurde durch quantitative RT-PCR und Visualisierung der Gegenwart von HCV-RNA-Doppelstrang ausgewertet. Die HCV-Proteinexpression wurde durch Western-Blot und Immunfluoreszenz unter Verwendung von für HCV-NS3-und NS5A-Proteine ​​Antikörper verifiziert. HCV-RNA-transfizierten Zellen freigesetzt infektiöse Partikel in Kulturüberstand und der Virustiter wurde bestimmt. Luciferase-Assays wurden verwendet, um die Replikationsebene und Infektiosität von HCV-Reporter beurteilen. Zusammenfassend stellen wir verschiedene virologische Tests zur Charakterisierung von verschiedenen Stufen des HCV-Replikationszyklus.

Hepatitis C-Virus ist ein RNA-Virus ist. Somit zur genetischen Manipulation Zweck hat die genomische HCV-cDNA in einen bakteriellen Plasmid-Vektor kloniert. Ein T7-RNA-Polymerase-Promotorsequenz wurde unmittelbar vor dem 5&#39;-Ende des HCV-Genoms eingeführt. Eine allgemeine Übersicht der HCV-Analyse-Workflow ist in Abbildung 1 dargestellt. Um genomische HCV-RNA mit präziser 3-Ende erzeugen, wird das HCV-Genom-Plasmid, das mit XbaI Restriktionsenzym und die erzeugte Einzelstrangüberhang wur...

Watch this Scientific Journal Video about A Protocol for Analyzing Hepatitis C Virus Replication at JoVE.com

A Protocol for Analyzing Hepatitis C Virus Replication

Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle.

Ein Protokoll für die Analyse von Hepatitis-C-Virus-Replikation

Hepatitis C Virus (HCV) affects 3% of the world&#8217;s population and causes serious liver ailments including&#160;chronic hepatitis, cirrhosis, and ...

a-protocol-for-analyzing-hepatitis-c-virus-replication

Research

JoVE Journal

Immunology and Infection

23.9K Aufrufe.  Cedars-Sinai Medical Center. Hepatitis C Virus (HCV) is a major human pathogen that causes liver disorders, including cirrhosis and cancer. An HCV infectious cell culture system is essential for understanding the molecular mechanism of HCV replication and developing new therapeutic approaches. Here we describe a protocol to investigate various stages of the HCV replication cycle. 

Serie: A Protocol for Analyzing Hepatitis C Virus Replication

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of infection with influenza virus alone. Instead, most individuals are thought to have succumbed to a secondary bacterial infection, predominately caused by the bacterium Streptococcus pneumoniae (the pneumococcus). The synergistic relationship between infections caused by influenza virus and the pneumococcus has subsequently been observed during the 1957 Asian influenza virus pandemic, as well as during seasonal outbreaks of the virus (reviewed in 1, 2). Here, we describe a protocol used to investigate the mechanism(s) that may be involved in increased morbidity as a result of concurrent influenza A virus and S. pneumoniae infection. We have developed an infant murine model to reliably and reproducibly demonstrate the effects of influenza virus infection of mice colonised with S. pneumoniae. Using this protocol, we have provided the first insight into the kinetics of pneumococcal transmission between co-housed, neonatal mice using in vivo imaging 3.

Using Bioluminescent Imaging to Investigate Synergism Between Streptococcus pneumoniae and Influenza A Virus in Infant Mice

A concurrent infection with influenza A virus is one of the factors implicated in the induction of invasive pneumococcal disease during asymptomatic Streptococcus pneumoniae carriage. Here we describe a mixed infection method using infant mice to investigate the synergism between these two respiratory pathogens.

Mit Bioluminescent Imaging, um Synergie von Untersuchen Streptococcus pneumoniae Und Influenza A Virus in Infant Mäuse

During the 1918 influenza virus pandemic, which killed approximately 50 million people worldwide, the majority of fatalities were not the result of ...

Forschung

Immunologie und Infektion

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

Sezieren Host-Virus-Interaktion in Lytic Replikation von einem Modell Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Eine primäre Neuron Culture System zur Erforschung des Herpes Simplex Virus Latenz und Reaktivierung

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying Salmonella-host interactions are&#160;important to elucidate the molecular pathogenesis of Salmonella infection. The Gentamicin protection assay to phenotype Salmonella association, invasion and replication in phagocytic cells was adapted to allow high-throughput screening to define the roles of deletion mutants of Salmonella enterica serotype Typhimurium in host interactions using RAW 264.7 murine macrophages. Under this protocol, the variance in measurements is significantly reduced compared to the standard protocol, because wild-type and multiple mutant strains can be tested in the same culture dish and at the same time. The use of multichannel pipettes increases the throughput and enhances precision. Furthermore, concerns related to using less host cells per well in 96-well culture dish were addressed. Here, the protocol of the modified in vitro Salmonella invasion assay using phagocytic cells was successfully employed to phenotype 38 individual Salmonella deletion mutants for association, invasion and intracellular replication. The in vitro phenotypes are presented, some of which were subsequently confirmed to have in vivo phenotypes in an animal model. Thus, the modified, standardized assay to phenotype Salmonella association, invasion and replication in macrophages with high-throughput capacity could be utilized more broadly to study bacterial-host interactions.

High-throughput Assay to Phenotype Salmonella enterica Typhimurium Association, Invasion, and Replication in Macrophages

A high-throughput assay to in vitro phenotype Salmonella or other bacterial association, invasion, and replication in phagocytic cells with high-throughput capacity was developed. The method was employed to evaluate Salmonella gene knockout mutant strains for their involvements in host-pathogen interactions.

Hochdurchsatz-Assay zum Phänotyp<em&gt; Salmonella enterica</em&gt; Typhimurium Association, Invasion und Replikation in Makrophagen

Salmonella species are zoonotic pathogens and leading causes of food borne illnesses in humans and livestock1. Understanding the mechanisms underlying ...

Immunofluorescence to Monitor the Cellular Uptake of Human Lactoferrin and its Associated Antiviral Activity Against the Hepatitis C Virus

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or localization of a target molecule in cells and tissues. Immunofluorescence relies on the specificity of fluorescent-labelled antibodies against their corresponding antigens within a cell. Both direct and indirect immunofluorescence approaches can be used which rely on the use of antibodies linked with a fluorochrome. Direct immunofluorescence is less frequently used because it provides lower signal, involves higher cost and less flexibility. In contrast, indirect immunofluorescence is more commonly used because of its high sensitivity and provides an amplified signal since more than one secondary antibody can attach to each primary antibody. In this manuscript, both epifluorescence microscopy and confocal microscopy were used to monitor the internalization of human lactoferrin, an important component of the immune system, into hepatic cells. Moreover, we monitored the inhibitory potential of hLF on the intracellular replication of the Hepatitis C virus using immunofluorescence. Both the advantages and disadvantages associated with these approaches are discussed.

The human lactoferrin (hLF) is a component of the immune system. In this study, immunofluorescence assays are used to demonstrate both the hepatocellular uptake of hLF and a qualitative reduction in the hepatitis C virus replication upon treatment with hLF.

Immunfluoreszenz, um die zelluläre Aufnahme von menschlichem Lactoferrin und die damit verbundenen antivirale Aktivität gegen das Hepatitis C Virus-Monitor

Immunofluorescence is a laboratory technique commonly used to study many aspects of biology. It is typically used to visualize the distribution and/or ...

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

Ein miniaturisiertes Glycan Microarray-Test zur Beurteilung der Avidität und Spezifität von Influenza-A-Virus Hämagglutininarten

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

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

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

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

Entwicklung eines Systems Hepatitis-B-Virus Reporter den frühen Stadien des Replikationszyklus zu überwachen

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

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

Rettung und Charakterisierung des rekombinanten Virus aus einer neuen Welt Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

"Liver-on-a-Chip" Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally infected chimpanzees, most in vitro models require several hundreds to thousands of viral genomes per cell in order to initiate a transient infection. Additionally, static 2D cultures of primary human hepatocytes (PHH) allow only short-term studies due to their rapid dedifferentiation. Here, we describe 3D liver-on-a-chip cultures of PHH, either in monocultures or in cocultures with other nonparenchymal liver-resident cells. These offer a significant improvement to studying long-term HBV infections with physiological host cell responses. In addition to facilitating drug efficacy studies, toxicological analysis, and investigations into pathogenesis, these microfluidic culture systems enable the evaluation of curative therapies for HBV infection aimed at eliminating covalently closed, circular (ccc)DNA. This presented method describes the set-up of PHH monocultures and PHH/Kupffer cell co-cultures, their infection with purified HBV, and the analysis of host responses. This method is particularly applicable to the evaluation of long-term effects of HBV infection, treatment combinations, and pathogenesis.

&quot;Liver-on-a-Chip&quot; Cultures of Primary Hepatocytes and Kupffer Cells for Hepatitis B Virus Infection

The goal of this protocol is to provide a step-by-step guide to perform 3-D &#34;liver-on-a-chip&#34; infection experiments with the hepatitis B virus.

"Leber-on-a-Chip" Kulturen der primäre Hepatozyten und Kupffer Zellen für Hepatitis B-Virus-Infektion

Despite the exceptional infectivity of the hepatitis B virus (HBV) in vivo, where only three viral genomes can result in a chronicity of experimentally ...

Identification of Coding and Non-coding RNA Classes Expressed in Swine Whole Blood

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying gene expression related to biological processes of interest. These innovations not only allow researchers to observe expression from the mRNA sequences that code for genes that effect cellular function, but also the non-coding RNA (ncRNA) molecules that remain untranslated, but still have regulatory functions. Although researchers have the ability to observe both mRNA and ncRNA expression, it has been customary for a study to focus on one or the other. However, when studies are interested in both mRNA and ncRNA expression, many times they use separate samples to examine either coding or non-coding RNAs due to the difference in library preparations. This can lead to the need for more samples which can increase time, consumables, and animal stress. Additionally, it may cause researchers to decide to prepare samples for only one analysis, usually the mRNA, limiting the number of biological questions that can be investigated. However, ncRNAs span multiple classes with regulatory roles that effect mRNA expression. Because ncRNA are important to fundamental biologic processes and disorder of these processes in during infection, they may, therefore, make attractive targets for therapeutics. This manuscript demonstrates a modified protocol for the generation mRNA and non-coding RNA expression libraries, including viral RNA, from a single sample of whole blood. Optimization of this protocol, improved RNA purity, increased ligation for recovery of methylated RNAs, and omitted&#160;size selection, to allow capture of more RNA species.

Here, we present a protocol optimized for the processing of coding (mRNA) and non-coding&#160;(ncRNA) globin reduced RNA-seq libraries from a single whole blood sample.

Identifizierung der Codierung und nicht-kodierende RNA-Klassen ausgedrückt bei Schweinen Vollblut

The advent of innovative and increasingly powerful next generation sequencing techniques has opened new avenues into the ability to examine the underlying ...