Optimization and application of titration method for varicella-zoster virus
Objective To optimize the titration method for live varicella zoster virus(VZV),and apply it for neutralizing antibody and antiviral drug detections in vitro.Methods The VZV vaccine strain Voka strain was used to infect adult retinal pigment epithelial cell line 19(ARPE-19)in a 48-well plate.The virus replication dynamics and plaque characteristics were detected after infection for 7 consecutive days.The classical plaque staining(CPS)and enzyme-linked immunospot(ELISpot)assays for live virus titration were established and optimized.The correlation between the results of both assays was analyzed.The optimized assays were both applied to the testing of anti-VZV drug(cidofovir,used in this study)inhibition effect and serum neutralizing antibody titer.Result The replication peak of VZV was reached on the sixth day after infection of ARPE-19 cells(multiplicity of infection=0.05)and formed visible plaques.For CPS,addition of 0.2 mg/ml diethylaminoethyl-dextran(DEAE-Extran)resulted in larger and clearer plaque compared to the group without adding DEAE-Extran.The result of ELISpot can be read on the third day after infection.Modification of antibody and colorimetric solution created more clear and countable plaques.Both methods were suitable for machine counting and had good correlation in testing of virus titer or in-vitro detection of anti-VZV drug efficacy and serum neutralizing antibody titers.Conclusions The optimized ELISpot assay was rapid and sensitive and can be used in place of CPS assay in testing of VZV titer and related applications,The present study provided simple and reliable experimental method for the detection of VZV and the development of vaccine and drugs.
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