Mechanisms of anti-hantaviral activities of bovine lactoferrin (LF) and ribavirin (Rbv) were investigated. Hantavirus focus formation at 48 hr was 15% of the control in cells treated with 400 microg/ml LF for 1 hr at 37 degrees C prior to viral infection. Post infection treatment with 100 microg/ml Rbv also inhibited the focus formation to 2.5% of the control. Combined LF pre- and Rbv post-infection treatment completely inhibited focus formation. Viral glycoprotein (G2) and nucleocapsid protein (NP) syntheses were delayed in LF pretreated cells up to 24 hr post infection (hpi) but became comparable to the control by 48 hpi. Further, LF inhibited viral shedding at 24 hpi but did not inhibit shedding after 48 hpi. However, Rbv was able to inhibit synthesis of viral proteins, (+) and (-) strand RNAs also inhibited viral shedding after 24 hr. These results suggest that LF inhibits viral adsorption to cells, while Rbv inhibits viral RNA synthesis. For in vivo trials of LF and Rbv, LF pre- and Rbv post-treatment were evaluated in suckling mice infected with hantavirus, of which 7% survived. LF concentrations of 40 and 160 mg/kg administered prior to viral challenge improved survival rates to 15% and 70%, respectively for single administration and 85% and 94%, respectively, for double administration. Rbv concentrations of 25 and 50 mg/kg gave survival rates of 68% and 81%, respectively. This suggests that both LF and Rbv are efficacious in hantavirus infection in vivo.