Abstracts This study aims to determine the importance of conserved GDN motif in domain III and GXGXG motif in domain VI in Nipah virus (NiV) L protein. Four mutated L genes produced in an earlier study were inserted individually into plasmid pCITE. Optimised transfection protocol was successful in transfecting these plasmids, two helper plasmids (coding for N and P protein), NiV minigenome containing chloramphenicol acetyltransferase (CAT) reporter gene and T7 promoter. Successful in vitro transcription/translation in the NiV minireplicon system was monitored by CAT expression. In conclusion, GXGXG motif was important in the NiV minireplicon system but change of GDN motif does not affect L protein.