Name: production and titering of recombinant adeno-associated viral vectors
Uploaded: 2011-11-27T14:00:00
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Description: Watch this Scientific Journal Video about Production and Titering of Recombinant Adeno-associated Viral Vectors at JoVE.com

Se figur 1 för en illustration sammanfattar följande protokoll. Säkerhet OBS: Allt material som har varit i kontakt med monterat viruspartiklar måste desinficeras med Virkon-lösning eller annat lämpligt desinfektionsmedel. 1. Beredning av plasmid-DNA lager (~ 2 dagar) <ol><li> Följande plasmider krävs 1: <ul><li> pRV1 - Innehållande de AAV2 Rep och sekvenser Cap </li><li> pH21 - Innehållande de AAV1 Rep och sekvenser Cap </li><li> pFdelta6 - Adenovirus-helper plasmid </li><li> AAV plasmid som innehåller rekombinanta uttrycket kassetten flankeras av AAV2 förpackningar signaler (inverterad terminal upprepar, ITRS) </li></ul></li><li> Medan pFdelta6 bör odlas i Stbl2 behöriga celler för att förhindra delvis strykning kan pRV1 och pH21 odlas i DH5alpha behöriga celler. AAV plasmider kan odlas i Stbl2 celler om partiell strykningar förekommer i DH5alpha celler. </li><li>Plasmid DNA ska vara av hög kvalitet och fri från RNA föroreningar. Plasmider kan screenas för integritet med hjälp av följande smälter: <ul><li> pRV1 - sammandrag med XbaI att ge band på 7,5 kb och 3,8 kb </li><li> pH21 - sammandrag med EcoRI att ge band 4,5 kb, 2,8 kb och 0,2 kb </li><li> pFdelta6 - sammandrag med HindIII att ge band på 5,5 kb, 3 kb, 3 kb, 2,3 kb och 1,5 kb </li></ul></li></ol> 2. Beredning av humana embryonala njur 293 (HEK293) celler för transfektion (2 - 3 dagar) <ol><li> Tavla två 80% konfluenta 150 cm 2 flaskor med HEK293-celler i fem 15 Nunc cm i diameter vävnadskultur rätter. Celler bör vara 70 - 80% konfluenta innan transfektion (cirka 48 timmar efter plätering). Kultur celler i standarden Dulbecco ändrade Eagle medium (DMEM) med lågt glukos som innehåller 10% fetalt kalvserum och 100 U / ml penicillin / 100 mikrogram / ml streptomycin. </li><li> 3 timmar innan transfektion bort DMEM och ersätt med IscoveÄr modifierad Dulbecco på medellång (IMDM) som innehåller 5% fetalt kalvserum. </li></ol> 3. Transfektion av viral plasmider (~ 1 timme för transfektion, 3 dagar för inkubation) <ol><li> Förbered följande för ett enda parti (5 x 15 cm vävnadsodling plattor) av virus: <ul><li> 62,5 mikrogram AAV plasmid </li><li> 125 mikrogram pFdelta6 </li><li> 31,25 mikrogram pRV1 </li><li> 31,25 mikrogram pH21 </li><li> 1650 l 2,5 M CaCl 2 </li><li> 12 ml dH 2 O </li></ul></li><li> I en klass 2 vävnadsodling huva sterila filtrera transfektion blandningen i en 50 ml tub. </li><li> Även vortexa lösningen, snabbt lägga 13 ml 2 x HEPES saltlösning (pH 7,05). Sätt lock på 50 ml tub och fortsätter att skaka i 15 sekunder. Låt stå i 1 minut 45 sekunder bör en fin vit fällning form. </li><li> Tillsätt försiktigt 5 ml av transfektion lösning till varje 15 cm vävnadskultur maträtt. Snurra tallrikar att blanda och återgå till inkubatorn. </li><li> 16 timmar efter transfektion bort IMDM medellång och ersätta med DMEM. </li></ol> 4. Lysing celler och skörd av rAAVs (2 timmar) <ol><li> 72 timmar efter transfektion, ta bort materialet från plattorna cellodling och kasta. Allt avfall ska behandlas med Virkon-lösning eller annat lämpligt desinfektionsmedel. </li><li> Försiktigt tvätta cellerna i varma 1x fosfatbuffrad saltlösning (PBS, pH 7,4). </li><li> Tillsätt 25 ml varm PBS till varje tallrik och ta försiktigt bort celler med en cell skrapa. Samla suspension i 50 ml rör. </li><li> Pellets cellerna vid 800 xgi 10 minuter. </li><li> Kassera supernatanten och återsuspendera pelleten i 150 mM NaCl, 20 mM Tris pH 8,0, använda 10 ml per vävnadskultur platta. Delas i två 50 ml rör. </li><li> Bered en färsk lösning av 10% natriumdeoxikolat i dH 2 O. Tillsätt 1,25 ml av detta till varje rör i en slutlig koncentration av 0,5%. Lägg bensonas nukleas till en slutlig koncentration av 50 enheter per ml. Blanda röret ordentligt. </li><li&gt; Inkubera vid 37 ° C i 1 timme. </li><li> Ta bort cellulära skräp genom att centrifugera vid 3000 xg under 15 min. Transfer till färska 50 ml tub, se till att alla cellfragment har tagits bort för att förhindra blockering av heparin kolumner (se steg 5). I detta skede kan proverna förvaras vid -20 ° C innan du fortsätter. Vi har sparade prov i flera veckor vid -20 ° C utan en minskning av smittsamhet. </li></ol> 5. Heparin kolumnen rening av rAAVs (2 - 3 timmar) <ol><li> Setup HiTrap heparin kolumner med hjälp av en peristaltiska pumpen så att lösningarna flödet genom kolonnen med 1 ml per minut för steg från 5,2 till 5,4. Det är viktigt att se till att inga luftbubblor införs i heparin kolumnen. </li><li> Jämvikt kolonnen med 10 ml 150 mM NaCl, 20 mM Tris, pH 8,0. </li><li> Applicera 50 ml av viruset till kolonnen och låt rinna igenom. </li><li> Tvätta kolonnen med 20 ml 100 mM NaCl, 20 mM Tris, pH 8,0. </li><li> Använd en 5 ml spruta fortsätta att tvätta kolumnen wed 1 ml 200 mM NaCl, 20 mM Tris, pH 8,0, följt av 1 ml 300 mM NaCl, 20 mM Tris, pH 8,0. Kassera genomströmning. </li><li> Med 5 ml sprutor och lätt tryck eluera viruset från kolonnen genom att: <ul><li> 1,5 ml 400 mM NaCl, 20 mM Tris, pH 8,0 </li><li> 3,0 ml 450 mM NaCl, 20 mM Tris, pH 8,0 </li><li> 1,5 ml 500 mM NaCl, 20 mM Tris, pH 8,0 </li><li> Samla dessa i ett 15 ml centrifugrör. </li></ul></li></ol> 6. Koncentration och steril filtrering av rAAVs (1 timme) <ol><li> Koncentrera vektor med Amicon ultra-4 centrifugal filter enheter med en 100 tusen molekylvikt cutoff. Ladda 4 ml kolonn eluat i anrikningsverk och centrifugera vid 2000 xgi 2 minuter (vid rumstemperatur). Kasta flowthrough och ladda anrikningsverk med återstående virus lösning och upprepa centrifugering. Den koncentrerade Volymen bör vara ungefär 250 l. Om koncentrerad volym är betydligt mer än så, kasta flödet through och fortsätter att centrifugera i en minut steg tills volymen är ca 250 l. </li><li> Tillsätt 250 l PBS till virus för en slutlig volym på 500 l och ta bort från anrikningsverk. </li><li> Filtrera vektor genom ett 13 mm i diameter 0,2 ìm spruta filter. Vector bör alikvoteras och förvaras vid -80 ° C tills de behövs. </li></ol> 7. Titering av viral lager (3 dagar) Detta kräver en promotor som är aktiv i HEK293 celler köra en reporter gen eller en immunocytochemically upptäckas gen produkt. Vid produktion av en vektor som inte är kompatibel med HEK293 celler andra cellinjer eller primära cellkulturer kan användas. <ol><li> Förbered 18 poly-L-lysin belagt glas täckglas eller 18 brunnar Nunc kammare diabilder genom att seeda HEK293-celler så att de är 40 - 50% konfluenta. För titrering av Cre-beroende rAAVs använda HEK293 celler som stabilt uttrycka Cre recombinase 2. </li><li> Infektera varje brunn med seriell dilutions av AAV vektorer (Fig. 2A). Använd 3 brunnar per utspädning. Vi lägger rutinmässigt virus direkt i varje brunn. </li><li> Efter 72 timmar fixa HEK293-celler genom att lägga till en lika stor volym på 4% paraformaldehyd (PFA) till medium (vilket ger en slutlig koncentration av 2% PFA) i tio minuter i rumstemperatur. </li><li> Tvätta cell tre gånger i PBS, skölj i dH 2 O och täckglas. </li><li> Räkna antalet transduced celler från de tre brunnar som har den högsta spädningsfaktor, men ändå innehåller infekterade celler, hitta ett genomsnitt av dessa och multiplicera med spädningsfaktorn för att ge antalet infektiösa enheter per mikroliter (Fig. 2B). </li></ol> 8. Förväntade resultat HEK293 celler transduced med ett virus kodning förbättrad grönt fluorescerande protein (EGFP) visas i Figur 2B. Vi uppnår i stort sett överens titrar på ca 6 x 10 6 infektiösa partiklar per mikro liter. Vi använder rutinmässigt dessa vektorer för stereotaxic injeInsatser till vuxna gnagare hjärnan. Detta ger en kraftfull teknik för region-specifika genuttryck 2. Att kombinera denna region specificitet med celltyp selektivt genuttryck vi injicerar Cre recombinase beroende rAAVs i målregioner av Cre-transgena möss 2. Figur 2C-E visar exempel på stereotaxic injektioner av Cre-beroende rAAVs kodning EGFP i olika områden i hjärnan hos vuxna parvalbumin-Cre transgena möss 3. <img alt="Figur 1" src="/files/ftp_upload/3348/3348fig1.jpg" /> Figur 1. Schematisk illustration av protokollet för rAAV produktion. . 1) HEK293 celler pläterade och vuxit till 70 - 80% sammanflöde. 2.) AAV och hjälpare plasmider är förberedda och transfekterade i HEK293 cellerna. 3.) Medium ändras tillbaka till DMEM 16 timmar efter transfektion och HEK293-celler odlas i ytterligare 56 timmar. 4.) HEK293 celler skördas och lyseras. rAAV vektorer är separerade från cellrester av centrifugation. 5.) Viruspartiklar renas genom heparin kolumner innan koncentration och sterilisering. 6.) HEK293-celler odlas på 24 brunnar och infektera med seriella spädningar av AAV vektorer för att bestämma antalet infektiösa enheter. <img alt="Figur 2" src="/files/ftp_upload/3348/3348fig2.jpg" /> Figur 2. Titering och in vivo tillämpning av rAAV1 / 2. (A) Inställning av HEK293-celler för att mäta virus titer. HEK293 celler transduced med spädningar av rAAVs. C, oinfekterade kontroll, N, 1 l av outspädd virus lager. (B) HEK293-celler infekterade med rAAVs uttrycka EGFP. (CE) Viral EGFP uttryck är begränsad till Cre-uttryckande neuron i olika målregioner av parvalbumin-Cre transgena möss efter stereotaxic injektion av Cre-aktiverade rAAVs. Exempel på bilder visar GFP immunreaktivitet i (C) retikulära thalamus och globus pallidus, (D) dentate gyrus och (E) CA1 regionen i hippocampus. DG, dentate gyrus, RT, retikulära thalamic kärnan, GP, globus pallidus. Skala barer: B, 500 ìm, C, 100 ìm, D, 100 ìm, E 500 ìm.

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rAAV vektorer blir alltmer värdefulla för in vivo-studier på djur. Här har vi beskrivit en enkel och billig protokoll för att producera rAAVs, som kan underlätta den utbredda tillämpningen av denna nyttiga vektor genom att undvika kostsamma outsourcing av viruset produktion till företag. Det protokoll (baserat på referens 1) beskriver produktionen av chimära rAAV1 / 2 vektorer som innehåller kapsid proteiner från båda föräldrarna serotyper på lika nyckeltal 4. Rening av rAAV vektorer genom att binda till heparin kolumner bygger på uttrycket av AAV2 kapsid proteiner, men kan kombineras med serotyper andra än AAV1 beskrivs här. Dessutom har AAV6 rapporterats att binda till heparin, men med en lägre affinitet, och skulle kunna renas med heparin kolumner med hjälp av denna procedur 5,6. Noteras bör dock att andra serotyper kommer att kräva olika koncentrationer av NaCl för heparin kolumnen eluering 5,7. Förpackningar rAAV genomen har visat sig vara optimal mellan 4,1 och 4,9 kb med en kraftig minskning av förpackningar verkningsgrad på upp till 5,2 kb 8. Denna förpackning gräns kan anses vara den största nackdelen med den rAAV system, eftersom celltyp reglering av transgenens uttrycket uppnås vanligen med stora cis agerar element som inte kan rymmas inom de små AAV partiklar. För att övervinna låg titer partier, såsom sådana som härrör från att försöka paket gener som ligger nära AAV packning begränsar vi kombinerar olika virus partier koncentrerad till 250 l till ett 500 l parti, snarare än att lägga till PBS som beskrivs i steg 6,3. Pålitlig celltyp specifika transduktion kan uppnås genom att kombinera Cre-driver möss och villkorliga kassetter rAAV (se nedan) för att undvika stretching förpackningen gränsen. Att notera, medan detta protokoll försöker ge enkla att följa instruktionerna på produktion av hög titer rAAV krävs det närvaro av AAV2 kapsid protein beståndsdelarna för rening av heparin affinitetskromatografi. Serotyper än AAV2 skall renas med hjälp av alternativa förfaranden 9. En fördel med heparin kolumn rening är det tros att producera AAV vektorer som har större smittsamhet takt än som producerats med hjälp av en cesiumklorid lutning 10. Men det finns också vissa nackdelar med denna metod. Till exempel kan andra proteiner som binder heparin också vara närvarande i det renade virala lager. Medan tropism av heparin-renat vektorer kan påverkas av vektorn titer 10, vårt sätt att särskilt inrikta antingen excitatoriska 20 eller hämmande nervceller (Fig. 2) sysselsätter Cre-inducerad aktivering av AAV-medierad transgenens uttryck och är titer-oberoende. Ett alternativ till heparin kolumnen rening är att använda en iodixanol densitetsgradient, vilket ger en högre virus titer och renare förberedelser än användning aven cesiumklorid lutning 10. Denna metod kan också kombineras med heparin kolumnen rening för att producera en vektor som är större än 99% ren 11. Därför måste övervägas noga som ska vidtas av enskilda grupper om vilka virus rening metod är bäst för just deras nedströms ansökan. Det vanligaste problemet i samband med detta protokoll är låg virus-titer. Enligt vår erfarenhet här kan vanligtvis spåras till låga transfektion effektivitet, eller eluering av virus från heparin kolumner. Alla transfektion material ska i rumstemperatur innan transfektion, och testa transfections bör utföras för att hitta de optimala förhållandena i varje laboratorium. Andra metoder för transfektion, såsom lipofectamine, är mycket effektiva, men kan vara oöverkomligt dyr. Dessutom är koncentrationen och pH-värdet i heparinlösningar kolumnen eluering kritiskt för komplett eluering av virioner från heparin kolumner withoUT skador. En annan viktig punkt är att förpackningen cellerna måste följa väl till ytan på disken vävnadsodling. Om cellerna loss vid något tillfälle under förfarandet är det tillrådligt att avsluta försöket och tina en ny flaska av celler. Tid och rum styrning av transgenen uttryck hos gnagare är lätt uppnås genom korrekt anatomisk inriktning och utvecklingsstadiet av djuret. Effektiv AAV-medierad genöverföring har visats i livmodern 12,13. I den vuxna hjärnan, kan det område som är smittat med rAAV partiklar efter stereotaxic injektion justeras från mycket små utvalda regioner, till mycket större områden inte bara av förändringar i det virala titer, men också genom att ändra injektion parametrar. Dessa inkluderar volym och hastighet injektioner och införlivandet av polyol mannitol med virussuspension 14. Mannitol kan också användas för att förbättra infektion i olika vävnader efter systemisk rAAV injektion 15 &lt;/ Sup&gt;. Förpackningen kapacitet rAAV (ca 4,7 kb) är förmodligen den mest begränsande faktorn i termer av gener som kan uttryckas från dessa virala vektorer. Dock har nya studier visat att detta delvis kan övervinnas genom att dela upp större gener eller kassetter uttryck i separata rAAV vektorer och införa ett överbryggande sekvens, initiera genexpression när virioner infekterar samma cell 16. Uttryck nivåer av gener som bärs av rAAV vektorer kan också förbättras avsevärt genom att använda egen gratis rAAV vektorer 17. Stereotaxic injektion av rAAV vektorer ger en snabb, billig och kraftfull metod för att inducera genuttryck i en region-specifika sätt. I kombination med två RNA andra generationens interferens strategier 18 rAAVs kan också användas för region-specifika gener slå ner. rAAVs kan kombineras med Cre-transgena möss eller celltyp specifika projektansvariga att åstadkomma krets-och cell-typ-Specifik genuttryck, som möjliggör genetiska manipulationer med hög rumslig upplösning 2,19-21, medan montering rAAVs med t.ex. Tet Systemet lägger tidsmässiga kontroll över virus-medierad genuttryck 22,23. Dessa exempel visar den enorma combinatorial potentialen hos dessa vektorer och förutspår en snabb ökning av rAAV-baserade studier under de kommande åren.

<table border="1"><tbody><tr><td> Namn på reagens </td><td> Företag </td><td> Katalognummer </td><td> Kommentarer </td></tr><tr><td> Amicon Ultra centrifugal-filter </td><td> Millipore </td><td> UFC810024 </td><td> 100 tusen MWCO </td></tr><tr><td> Bensonas nukleas </td><td> Sigma-Aldrich </td><td> E1014 </td><td></td></tr><tr><td> Dulbeco ändrade Eagle medium (DMEM) </td><td> Invitrogen </td><td> 10567014 </td><td> Tillägg med 10% FCS och 10 enheter / ml penicillin/100 mikrogram / ml streptomycin </td></tr><tr><td> HEK293-celler </td><td> American Type Culture Collection </td><td> CRL-1573 </td><td> Använd vid passage mindre än 30 </td></tr><tr><td> HEPES saltlösning </td><td> Sigma-Aldrich </td><td> 51.558 </td><td></td></tr><tr><td> HiTrap Heparin kassett </td><td> Sigma-Aldrich </td><td> 54.836 </td><td></td></tr><tr><td> Iscove ändrade Dulbecco på medellång </td><td> Invitrogen </td><td> 12440-053 </td><td> Komplettera med 5% FCS </td></tr><tr><td> Natriumdeoxikolat </td><td> Sigma-Aldrich </td><td> D5670 </td><td> Gör ny lösning för varje parti </td></tr><tr><td> 15 cm vävnadskulturer rätter </td><td> Fisher Scientific </td><td> 157.150 </td><td></td></tr><tr><td> Stbl2 behöriga celler </td><td> Invitrogen </td><td> 10268-019 </td><td></td></tr><tr><td> Virkon-lösning </td><td> Fisher Scientific </td><td> NC9821357 </td><td> Låt desinficera natten innan kasta rinna </td></tr></tbody></table> Tabell 1. Specifika reagenser som krävs för protokollet

production and titering of recombinant adeno-associated viral vectors

Under senare år har rekombinant Adeno-associerad virala vektorer (AAV) har blivit allt värdefullt för in vivo-studier på djur, och är också för närvarande testas i kliniska prövningar. Vildtyp AAV är en icke-patogena medlem av parvoviridae familjen och natur replikering-brist. Den breda transduktion profil, lågt immunförsvar, liksom den starka och ihållande transgenen uttryck uppnås med dessa vektorer har gjort dem en populär och mångsidigt verktyg för in vitro och in vivo gen leverans. rAAVs kan enkelt och billigt som produceras i laboratorium och, baserat på deras fördelaktig säkerhetsprofil, är i allmänhet ges en låg säkerhetsklassning. Här beskriver vi en metod för produktion och titering av chimär rAAVs innehåller kapsid proteiner av både AAV1 och AAV2. Användningen av dessa sk chimär vektorer kombinerar fördelarna av båda föräldrarna serotyper som höga titrar lager (AAV1) och reningmed affinitetskromatografi (AAV2). Dessa AAV serotyper är bäst studerade av alla AAV serotyper, och individuellt har en bred smittsamhet mönster. Den chimär vektorer som beskrivs här bör ha infektiösa egenskaper AAV1 och AAV2 och därmed kan förväntas att infektera ett stort utbud av vävnader, inklusive nervceller, skelettmuskel, bukspottkörtel, njure bland annat. Den metod som beskrivs här använder heparin kolumnen rening, trodde en metod för att ge en högre virus titer och renare virus förberedelser än andra reningsmetoder, exempelvis centrifugering genom en cesiumklorid lutning. Dessutom beskriver vi hur dessa vektorer kan snabbt och enkelt titered att ge korrekt avläsning av antalet smittsamma producerade partiklar.

Watch this Scientific Journal Video about Production and Titering of Recombinant Adeno-associated Viral Vectors at JoVE.com

Production and Titering of Recombinant Adeno-associated Viral Vectors

Rekombinant Adeno-associerade virus (rAAVs) vektorer blir allt mer värdefulla för<em&gt; In vivo</em&gt; Studier på djur. Vi beskriver hur rAAVs kan framställas i laboratoriet och hur dessa vektorer kan titered att ge en korrekt avläsning av antalet smittsamma producerade partiklar.

Produktion och Titering av rekombinanta Adeno-associerad virala vektorer

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

production-and-titering-of-recombinant-adeno-associated-viral-vectors

Research

JoVE Journal

Immunology and Infection

Recombinant Retroviral Production and Infection of B Cells

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control of a heterologous expression system to facilitate the analysis of the resulting phenotype. This approach can be used to express a gene that is not normally found in the organism, to express a mutant form of a gene product, or to over-express a dominant-negative form of the gene product. It is particularly useful in the study of the hematopoetic system, where transcriptional regulation is a major control mechanism in the development and differentiation of B cells 1, reviewed in 2-4.
Mouse genetics is a powerful tool for the study of human genes and diseases. A comparative analysis of the mouse and human genome reveals conservation of synteny in over 90% of the genome 5. Also, much of the technology used in mouse models is applicable to the study of human genes, for example, gene disruptions and allelic replacement 6. However, the creation of a transgenic mouse requires a great deal of resources of both a financial and technical nature. Several projects have begun to compile libraries of knock out mouse strains (KOMP, EUCOMM, NorCOMM) or mutagenesis induced strains (RIKEN), which require large-scale efforts and collaboration 7. Therefore, it is desirable to first study the phenotype of a desired gene in a cell culture model of primary cells before progressing to a mouse model. 
Retroviral DNA integrates into the host DNA, preferably within or near transcription units or CpG islands, resulting in stable and heritable expression of the packaged gene of interest while avoiding transcriptional silencing 8 9. The genes are then transcribed under the control of a high efficiency retroviral promoter, resulting in a high efficiency of transcription and protein production. Therefore, retroviral expression can be used with cells that are difficult to transfect, provided the cells are in an active state during mitosis. Because the structural genes of the virus are contained within the packaging cell line, the expression vectors used to clone the gene of interest contain no structural genes of the virus, which both eliminates the possibility of viral revertants and increases the safety of working with viral supernatants as no infectious virions are produced 10. 
Here we present a protocol for recombinant retroviral production and subsequent infection of splenic B cells. After isolation, the cultured splenic cells are stimulated with Th derived lymphokines and anti-CD40, which induces a burst of B cell proliferation and differentiation 11. This protocol is ideal for the study of events occurring late in B cell development and differentiation, as B cells are isolated from the spleen following initial hematopoetic events but prior to antigenic stimulation to induce plasmacytic differentiation.

An efficient system of structure and function analysis of a gene in an ex vivo culture of splenic B-lymphocytes is described. This method takes advantage of recombinant retroviral production in a helper free, ecotrophic packaging cell line. Stable, heritable expression of a gene of interest within primary lymphocytes is achieved leading to generation of surface antibodies on B cells undergoing class switch recombination.

Rekombinant retroviral Produktion och infektion av B-celler

The transgenic expression of genes in eukaryotic cells is a powerful reverse genetic approach in which a gene of interest is expressed under the control ...

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.

Isolering av Fidelity Varianter av RNA-virus och karakterisering av virus mutationsfrekvensen

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 ...

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.

Beredning av viral DNA från Nucleocapsids

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

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Effektiv Rekombinant Parvovirus produktion med hjälp av adenovirus-härledda Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Engineering and Evolution of Synthetic Adeno-Associated Virus (AAV) Gene Therapy Vectors via DNA Family Shuffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique combination of beneficial properties1. These include the apathogenicity of the underlying wildtype viruses and the highly advanced methodologies for production of high-titer, high-purity and clinical-grade recombinant vectors2. A further particular advantage of the AAV system over other viruses is the availability of a wealth of naturally occurring serotypes which differ in essential properties yet can all be easily engineered as vectors using a common protocol1,2. Moreover, a number of groups including our own have recently devised strategies to use these natural viruses as templates for the creation of synthetic vectors which either combine the assets of multiple input serotypes, or which enhance the properties of a single isolate. The respective technologies to achieve these goals are either DNA family shuffling3, i.e. fragmentation of various AAV capsid genes followed by their re-assembly based on partial homologies (typically &gt;80% for most AAV serotypes), or peptide display4,5, i.e. insertion of usually seven amino acids into an exposed loop of the viral capsid where the peptide ideally mediates re-targeting to a desired cell type. For maximum success, both methods are applied in a high-throughput fashion whereby the protocols are up-scaled to yield libraries of around one million distinct capsid variants. Each clone is then comprised of a unique combination of numerous parental viruses (DNA shuffling approach) or contains a distinctive peptide within the same viral backbone (peptide display approach). The subsequent final step is iterative selection of such a library on target cells in order to enrich for individual capsids fulfilling most or ideally all requirements of the selection process. The latter preferably combines positive pressure, such as growth on a certain cell type of interest, with negative selection, for instance elimination of all capsids reacting with anti-AAV antibodies. This combination increases chances that synthetic capsids surviving the selection match the needs of the given application in a manner that would probably not have been found in any naturally occurring AAV isolate. Here, we focus on the DNA family shuffling method as the theoretically and experimentally more challenging of the two technologies. We describe and demonstrate all essential steps for the generation and selection of shuffled AAV libraries (Fig. 1), and then discuss the pitfalls and critical aspects of the protocols that one needs to be aware of in order to succeed with molecular AAV evolution.

Engineering and Evolution of Synthetic Adeno-Associated Virus AAV Gene Therapy Vectors via DNA Family Shuffling

We demonstrate the basic technique to molecularly engineer and evolve synthetic Adeno-associated viral (AAV) gene therapy vectors via DNA family shuffling. Moreover, we provide general guidelines and representative examples for selection and analysis of individual chimeric capsids with enhanced properties on target cells in culture or in mice.

Engineering och evolution av syntetisk Adeno-associerat virus (AAV) Gene Therapy Vektorer via DNA Familj Skyffling

Adeno-associated viral (AAV) vectors represent some of the most potent and promising vehicles for therapeutic human gene transfer due to a unique ...

Production and Purification of Non Replicative Canine Adenovirus Type 2 Derived Vectors

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the frequent pre-existing immunity against the classically used human adenovirus type 5, canine adenovirus type 2 (CAV2) has been proposed as an alternative vector for human gene transfer. The well-characterized biology of CAV2, together with its ease of genetic manipulation, offer major advantages, notably for gene transfer into the central nervous system, or for inducing a wide range of protective immune responses, from humoral to cellular immunity. Nowadays, CAV2 represents one of the most appealing nonhuman adenovirus for use as a vaccine vector. This protocol describes a simple method to construct, produce and titer recombinant CAV2 vectors. After cloning the expression cassette of the gene of interest into a shuttle plasmid, the recombinant genomic plasmid is obtained by homologous recombination in the E. coli BJ5183 bacterial strain. The resulting genomic plasmid is then transfected into canine kidney cells expressing the complementing CAV2-E1 genes (DK-E1). A viral amplification enables the production of a large viral stock, which is purified by ultracentrifugation through cesium chloride gradients and desalted by dialysis. The resulting viral suspension routinely has a titer of over 1010 infectious particles per ml and can be directly administrated in vivo.

These past 15 years, canine adenovirus type 2 (CAV2)-derived vectors have proven their efficiency to transduce cells in vitro&#160;and in vivo and are widely used for vaccination and gene therapy. Here, we describe a procedure to construct, produce and purify CAV2 vectors, giving rise to high-titer viral suspensions.

Adenovirus (Ad) derived vectors have been widely used for short or long-term gene transfer, both for gene therapy and vaccine applications. Because of the ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

Dissekera Innate Immun Signalering i Viral Evasion av cytokinproduktion

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

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.

Utvecklingen av lyofiliserad Loop-medie Isothermal amplifieringsreagenserna för detektion av<em&gt; Coxiella burnetii</em

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

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

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

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

Mätning influensaneuraminidas Inhibition Antikroppstitrar genom enzymkopplad lektin analys

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

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the person-to-person route or indirectly through environmental surfaces or food, contaminated fomites and inanimate surfaces are important vehicles for the spread of the virus during norovirus outbreaks.
We developed and evaluated a protocol using macrofoam swabs for the detection and typing of human noroviruses from hard surfaces. Compared with fiber-tipped swabs or antistatic wipes, macrofoam swabs allow virus recovery (range 1.2-33.6%) from toilet seat surfaces of up to 700 cm2. The protocol includes steps for the extraction of the virus from the swabs and further concentration of the viral RNA using spin columns. In total, 127 (58.5%) of 217 swab samples that had been collected from surfaces in cruise ships and long-term care facilities where norovirus gastroenteritis had been reported tested positive for GII norovirus by RT-qPCR. Of these 29 (22.8%) could be successfully genotyped. In conclusion, detection of norovirus on environmental surfaces using the protocol we developed may assist in determining the level of environmental contamination during outbreaks as well as detection of virus when clinical samples are not available; it may also facilitate monitoring of effectiveness of remediation strategies.

A macrofoam based sampling methodology was developed and evaluated for the detection and quantification of norovirus on environmental hard surfaces.

Pinnurvalsmetoden för påvisande av humana Norovirus på ytor

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...