A virus stock of Langat virus strain TP21 (LGTV) (kindly provided by Dr. Alexander Pletnev, NIH/NIAID) was prepared by infection of Vero cell cultures at a multiplicity of infection (MOI) of 0.005 [38, 39] . For virus titration by immunofocus assay, 1610 5 Vero cells were seeded in complete DMEM into wells of twelve-well plates. Ten-fold dilutions of LGTV were adsorbed for 60 minutes at 37uC, 5% CO 2 with constant rocking. Infected cells were then washed twice with Dulbecco's phosphate buffered saline (DPBS, Invitrogen) and fresh complete DMEM added. After three days incubation, cells were washed twice with DPBS and fixed with 100% methanol for 30 minutes. Cells were rinsed with DPBS, blocked with OptiMEM supplemented with 2% FCS for 10 minutes, and incubated with Russian Spring-Summer Encephalitis immune ascites fluid (ATCC) diluted 1:1000 in OptiMEM/2% FCS. Following two DPBS washes, cells were incubated with 1:1000 dilution of anti-mouse horseradish peroxidase antibody (Dako) in OptiMEM/2% FCS. Foci were visualized using peroxidase substrate containing 0.4 mg/ml 3, 39 diaminobenzidine (Sigma Aldrich) and 0.0135% H 2 O 2 (JT Baker) in PBS. For all experiments, Vero and ISE6 cells were infected at an MOI of 10.