Pooled organ tissue from each bat was used for RNA/DNA extraction (PureLink TM Viral RNA/DNA Mini Kit, Invitrogen, Germany) and further cDNA synthesis according to the manufacturer's instructions (TaqManH Reverse Transcription Reagents, Applied Biosystems, Germany). Broadly reactive paramyxovirusspecific RT-PCR assays were applied [21] , yielding amplicons of 538 base pairs (PAR primers) and 486 base pairs (RES-MOR-HEN primers) located across domains I and II of the RNA polymerase (L)-coding sequence, a region of the genome suitable for phylogenetic analyses [22] . Since cDNA was readily available, PCR conditions were modified using the optimization method by Taguchi [23] . For this, based on the use of orthogonal arrays representing individual reactions with components at different concentration levels, a minimal number of experiments is allowed. To increase PCR sensitivity, the product yield for each reaction is used to calculate the optimal concentration of each reaction component. By using this method, a novel PCR reaction mixture was determined and henceforth used. For first-round PCR in the seminested assay, PCR mixtures contained 3 pmol each of forward and reverse primers, 16PlatinumH Taq buffer (Invitrogen), 250 nmol MgCl 2 (Invitrogen), 2.5 pmol desoxynucleoside triphosphates (Invitrogen), 2 ml of cDNA, and 1.25 U of PlatinumH Taq polymerase (Invitrogen). Water was then added to a final volume of 25 ml. The PCR mixture was sequentially incubated at 94uC for 2 min for denaturation, and then 40 cycles at 94uC for 15 s, 50uC for 30 s, 72uC for 30 s, and a final extension at 72uC for 7 min. For the second amplification in the seminested PCR assay, 16PlatinumH Taq buffer, 25 nmol MgCl 2 , 2.5 pmol desoxynucleoside triphosphates, 3 pmol each of forward and reverse primers, 1.25 U PlatinumH Taq, 1 ml PCR product from the first reaction, adding water to a final volume of 25 ml. The cycling conditions were identical to the ones of the first round.