All the experimental work described above was performed using treatments with 1000 virus particles per cell, a condition that would be unlikely to pertain in natural primary infections. To establish whether much lower virion numbers would be effective, HFFs were serum-starved for five days then mock-inoculated or inoculated with 1, 10, 100 or 1000 particles per cell of DgH HSV-1 for six hours. Infections were carried out in duplicate with mockinoculated controls that received an equivalent volume of PBS. Total RNA was purified and reverse transcribed, with the cDNA from each duplicate inoculation used in triplicate for real-time PCR.