Rapid Titration of Measles and Other Viruses Optimization with Determination of Replication Cycle Length 英文参考文献.docVIP

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Rapid Titration of Measles and Other Viruses Optimization with Determination of Replication Cycle Length 英文参考文献.doc

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Rapid Titration of Measles and Other Viruses Optimization with Determination of Replication Cycle Length 英文参考文献

RapidTitrationofMeaslesandOtherViruses: OptimizationwithDeterminationofReplicationCycle Length BoyanGrigorov*,JessicaRabilloud,PhilipLawrence,DenisGerlier INSERM,U758,EcoleNormaleSupe′rieure deLyon,Lyon,France,Universite′ deLyon,Lyon,France Abstract Background: Measlesvirus (MV)is amemberofthe Paramyxoviridae familyandanimportant humanpathogencausing strongimmunosuppressioninaffectedindividualsandaconsiderablenumberofdeathsworldwide.Currently,measlesisa re-emergingdiseaseindevelopedcountries.MVisusuallyquantifiedininfectiousunitsasdeterminedbylimitingdilution and counting of plaque forming unit either directly (PFU method) or indirectly from random distribution in microwells (TCID50method).Bothmethodsaretime-consuming(uptoseveraldays),cumbersomeand,inthecaseofthePFUassay, possiblyoperatordependent. Methods/Findings:Arapid,optimized,accurate,andreliabletechniquefortitrationofmeaslesviruswasdevelopedbased onthedetectionofvirusinfectedcellsbyflowcytometry,singleroundofinfectionandtitercalculationaccordingtothe Poisson’slaw.Thekineticsfollowupofthenumberofinfectedcellsafterinfectionwithserialdilutionsofavirusallowed estimationofthedurationofthereplicationcycle,andconsequently,theoptimalinfectiontime.Theassaywassetupto quantifymeaslesvirus,vesicularstomatitisvirus(VSV),andhumanimmunodeficiencyvirustype1(HIV-1)usingantibody labeling of viral glycoprotein, virus encoded fluorescent reporter protein and an inducible fluorescent-reporter cell line, respectively. Conclusion:Overall,performingtheassaytakesonly24–30hoursforMVstrains,12hoursforVSV,and52hoursforHIV-1. The step-by-step procedure we have set up can be, in principle, applicable to accurately quantify any virus including lentiviralvectors,providedthatavirusencodedgeneproductcanbedetectedbyflowcytometry. Citation:GrigorovB,RabilloudJ,LawrenceP,GerlierD(2011)RapidTitrationofMeaslesandOtherViruses:OptimizationwithDeterminationofReplicationCycle Length.PLoSONE6(9):e24135.doi:10.1371/journal.pone.0024135 Editor:YanX