Lack of possibility of infection or proliferation of HVJ-E in humans or experimental animals has been confirmed by means of the following three methods.
1. Assay using cultured cells
LLC-MK2 cells (monkey kidney cell line; ATCC CCL-7) were treated with appropriate amounts of HVJ-E. After incubation for two days, existence of F(fusion) envelope proteins in the cell surfaces was determined by means of immunocytochemistry using anti-F polyclonal antibody and fluorescence-labeled secondary antibody.
Live HVJ-treated groups exhibited strong fluorescence-positive features, while HVJ-E-treated groups were fluorescence-negative in this assay, suggesting that HVJ-E includes no infectious (no proliferative) live virus. This assay is performed for each production lot.
2. Assay using fertilized chicken eggs
An appropriate amount of HVJ-E is inoculated into the chorioallantoic cavity of fertilized chicken eggs. After three-day cultivation, agglutinating activity in the chorioallantoic fluid is determined using chicken erythrocytes.
Live HVJ-treated groups exhibited significant increases in hemagglutination unit, while HVJ-E-treated groups exhibited no increase in the titer, suggesting that HVJ-E includes no infectious (proliferative) live virus.
This assay is performed for each production lot.
3. Assay using mice
Mice subjected to intranasal injection of an appropriate amount of HVJ-E are bred with normal mice (in the same cages) for six weeks. Sera prepared from mice injected with HVJ-E exhibited significant levels in anti-HVJ antibody(ELISA), whereas sera isolated from co-bred normal mice exhibited no increase in the antibody level, suggesting that HVJ-E includes no infectious live virus.
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