| In recent years, as the animal-derived biologicals increased, human are facing agrowing risk of animal-derived viral infection. Iatrogenic infection issues becomeincreasingly urgent as well, however, the quality control of animal-derived biologicalmaterials were paid little attention by related divisions. Verification of procedure foranimal-derived virus removal/inactivation in biologics is an important measure toensure product safety.Choosing the eligible indicator virus, establishing a stable virusinfectious titer method for determination and acquiring high titer indicator virus areprerequisites of the technical evaluation.This study intends to establish the method for determination of vesicularstomatitis virus (VSV) infectious titer and prepare high infectious titer of VSV toprovide evidence for virus deactivation/removal in biologicals. Given to thesensitivity of African green monkey kidney cell (Vero) to VSV, a stable medianinfective dose of cell culture（TCID50）method for determination of VSV titer wasdeveloped by optimizing the concentration of Vero cells for inoculum, inoculumconcentration of VSV, virus adsorption time and cell infection time Then, high titerVSV was prepared with Vero cells, and the effects of multiplicity of infection (MOI),time for virus harvest and form of harvested virus on VSV titer as well as stability ofvirus were analyzed. Finally, the method for determination of VSV infectious titer andthe prepared high titer VSV were used in verification of procedure for virus removal/inactivation in biologicals.The optimal Vero cell concentration for inoculation were5×103cellsï¼well,virus culture time were48h and virus adsorption time were2h respectively. Thedeveloped TCID50method showed good repeatability. The VSV titer in precipitate ofVero cells was significantly higher than that in supernatant, which reached a maximumvalue48h after infection with a MOI of10. The prepared VSV was at a mean titer of 10-6.88ï¼0.1ml, and showed good stability after storage at4and25℃for7d or3cycles of freeze-thawing. Besides, in the verification of procedure for virusremoval/inactivation in biologicals, the cells grow normally in the control groups.Obvious cytopathy was observed in the deactivated VSV group, and viral infectionwas lost in injection and original fluid. The reduced titer is over4Log, which showedeffective in the procedure.In this study, a method for determination of infectious titer of VSV wasdeveloped, and high titer VSV was prepared, which applicated well in verification ofprocedure for virus removal/inactivation in biologics and provide evidence for virusdeactivation and removal using VSV as an indicator. |
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