Authors: Juno Jang SungHwan Hong Dongwon Choi KangSeuk Choi Seongman Kang IkHwan Kim
Publish Date: 2009/09/03
Volume: 85, Issue: 5, Pages: 1509-1520
Abstract
Newcastle disease virus NDV is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy The role of NDV V protein in virusproduction kinetics was investigated using DF1 cellbased production system The presence of an antiinterferon IFNalpha antibody resulted in enhanced NDV production kinetics in a dosedependent manner by blocking binding of NDVinduced IFN to its receptor To prepare DF1 cell whose cellular IFN signaling is blocked efficiently stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production while it had no effect on Japanese encephalitis virus production NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNAactivated enzymes These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strainsConstant expression of V protein during the whole NDV production period Control cells and DF1 cells expressing LaSota V or Kr102/89 V protein were seeded in sixwell plates at a density of 25 × 105 cells per well Two days later the cell monolayers were infected with the NDV LaSota at a MOI of 01 and incubated at 37°C in virusproduction medium basal MEMα containing 400 ng/ml trypsin Supernatants were harvested in 12h intervals until the cells expressing NDV V protein were almost died 48 h after infection The FLAGtagged V protein and actin were detected by using monoclonal antibodies against FLAG tag and actin respectively FLAGtagged NDV V protein was constantly expressed during the entire course of infection GIF 30 kbComparison of external proteases for NDV LaSota production a NDV production kinetics with various external proteases in the production medium DF1 cells were seeded in sixwell plates at a density of 25 × 105 cells per well Two days later the cell monolayers were infected with the NDV LaSota at a MOI of 01 and incubated at 37°C in basal MEMα containing external protease NDV production kinetics was almost the same regardless of the kind of protease when used with its optimized concentration b NDV production kinetics upon amount of allantoic fluid c NDV production kinetics upon amount of trypsin As described in the text when excessive protease is present in the production medium the protease will destroy host cell monolayers and reduce the virusproduction kinetics On the other hand insufficient protease is not able to support the rapid NDV production since it takes more time to cleave F protein of NDV newly produced from the cells GIF 63 kb
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