Urine (approximately 0.5-1 ml) was collected off plastic sheets placed underneath a colony of flying foxes (predominantly Pteropus alecto with some P. Poliocephalus in the mixed population) in Cedar Grove, South East Queensland, Australia and pooled into 2-ml tubes containing 0.5 ml of viral transport medium (SPGA: a mix of sucrose, phosphate, glutamate and albumin plus penicillin, streptomycin and fungizone). The tubes were temporarily stored on ice after collection and transported to a laboratory in Queensland, frozen at 280uC, and then shipped on dry ice to the CSIRO Australian Animal Health Laboratory (AAHL) in Geelong, Victoria for virus isolation. The samples were thawed at 4uC and centrifuged at 16,0006g for 1 min to pellet debris. Urine in the supernatant (approximately 0.5-1 ml) was diluted 1:10 in cell culture media. The diluted urine was then centrifuged at 1,2006g for 5 min and split evenly over Vero, PaKi, PaBr, PaSp and PaPl cell monolayers in 75-cm 2 tissue culture flasks. The flasks were rocked for 2 h at 37uC, 14 ml of fresh cell culture media was added and then incubated for 7 d at 37uC. The flasks were observed daily for toxicity, contamination, or viral cytopathic effect (CPE).