rapid titration of measles and other viruses optimization with determination of replication cycle length快速滴定的麻疹和其他病毒复制周期长度的优化与决心.pdfVIP

Rapid Titration of Measles and Other Viruses: Optimization with Determination of Replication Cycle Length Boyan Grigorov*, Jessica Rabilloud, Philip Lawrence, Denis Gerlier ´ ´ INSERM, U758, Ecole Normale Superieure de Lyon, Lyon, France, Universite de Lyon, Lyon, France Abstract Background: Measles virus (MV) is a member of the Paramyxoviridae family and an important human pathogen causing strong immunosuppression in affected individuals and a considerable number of deaths worldwide. Currently, measles is a re-emerging disease in developed countries. MV is usually quantified in infectious units as determined by limiting dilution and counting of plaque forming unit either directly (PFU method) or indirectly from random distribution in microwells (TCID50 method). Both methods are time-consuming (up to several days), cumbersome and, in the case of the PFU assay, possibly operator dependent. Methods/Findings: A rapid, optimized, accurate, and reliable technique for titration of measles virus was developed based on the detection of virus infected cells by flow cytometry, single round of infection and titer calculation according to the Poisson’s law. The kinetics follow up of the number of infected cells after infection with serial dilutions of a virus allowed estimation of the duration of the replication cycle, and consequently, the optimal infection time. The assay was set up to quantify measles virus, vesicular stomatitis virus (VSV), and human immunodeficiency virus type 1 (HIV-1) using antibody labeling of viral glycoprotein, virus encoded fluorescent reporter protein and an inducible fluorescent-reporter cell line, respectively. Conclusion: Overall, performing the assay takes only 24–30 hours for MV strains, 12 hours for VSV, and 52 hours for HIV-1. The st