Early steps in the EIAV life cycle are inhibited by Prunella aqueous extracts. A) Time frame of extract inhibition of EIAV infection. ED cells were infected with EIAVWSU5 virus and, at times noted following infection, DMSO, Ames 27664 extract, or Ames 27748 extract was added to the cells to a final concentration of 0.2% (66 μg/mL of Ames 27664 or 62.4 μg/mL of Ames 27748). B) Time frame of extract inhibition of EIAVWSU5 that was previously bound to ED cells. Viral particles were bound to ED cells at 4°C for 1 h. Unbound virus was removed and cells were shifted to 37°C. At the times indicated, DMSO, Ames 27664 extract or Ames 27748 extract was added to the cells to a final concentration of 0.2%. The percent of infected cells was determined by dividing the number of EIAV antigen positive cells in the presence of extract compared to the number of EIAV antigen positive cells in the presence of DMSO at time zero. C) Time frame of extract inhibition of EIAVWSU5 following virion internalization. Virions were bound to ED cells at 4°C for 1 h, unbound virions were removed, fresh media was replaced, and cells were permitted to internalize at 37°C. At the time points indicated, the cells were washed with citric acid buffer to inactivate any non-internalized virions, washed and media containing DMSO, Ames 27664 extract, or Ames 27748 extract was added (0.2%). Data represent the average and standard error of three experiments performed in duplicate. Prunella extracts significantly decreased EIAV infectivity compared to DMSO control at 0, 1, 2, 3, and 4 h time points in panels A and B (p < 0.05). Differences observed at 6 and 8 h were not statistically significant.