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Hepatitis A virus (HAV) is less well-characterized than other picornaviruses due to its slow and inefficient replication. In order to gain a greater understanding of HAV-receptor interactions we have used the recovery of cell-bound, infectious virus particles to measure the effects of temperature, pH and divalent cations on the binding of HAV to susceptible cells. Viral attachment to cultured cells proceeded at similar rates between 4 degrees C and 37 degrees C, with a slight increase in the total amount of virus attached at 4 degrees C. In contrast, both acidic pH and the presence of calcium ions independently caused greater than 20-fold increases in the cell attachment of infectious HAV diluted in buffered sodium chloride solutions, to a level approaching that of binding in culture medium, whereas magnesium led to a slight enhancement and zinc had no effect. The increased levels of binding observed with low temperature, low pH and the presence of calcium coincided with reduced rates of virus elution under similar conditions, suggesting that these conditions lead to a strengthening of the virus-receptor binding. The addition of calcium to highly purified HAV in buffered sodium chloride reduced the stability of virus during protracted incubation at 37 degrees C, as measured by immunoblotting of capsid proteins. The results suggest that the major effect of calcium in promoting HAV-receptor interactions is through a direct effect on the conformation of the viral capsid.