West Nile virus (WNV) is a mosquito transmitted flavivirus which is the leading cause of arboviral neuroinvasive disease. The positive sense RNA virus genome of WNV encodes a single long polyprotein which is cleaved into three structural (C, prM and E) and seven nonstructural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. Our previous studies identified the role of WNV NS2A in immunomodulation, viral RNA replication and packaging. However, further analysis of NS2A functions has been hindered by the lack of highly specific anti-NS2A antibodies. To facilitate specific detection of NS2A and its interacting partners in the context of virus infection, we have engineered a WNV encoding NS2A protein fused to a Human influenza hemagglutinin (HA) epitope tag. Inserted HA-tag was stably maintained throughout multiple virus passages in cells and the plaque morphology of HA-tagged virus was similar to that of the wild-type WNV. Using this molecular tool, we showed by western blot and protein localization studies that the inserted HA-tag did not affect processing at NS1-NS2A junction and localisation of NS2A during infection. Furthermore, we showed co-localisation of tagged-NS2A with the viral envelope protein and double-stranded RNA, thus confirming the role of NS2A in viral RNA replication and packaging. Pull-down of HA-tagged NS2A in infected cells using agarose beads coupled with anti-HA antibodies pulled-down multimeric isoforms of tagged-NS2A, suggesting possible roles of these isoforms in virus infection. Current studies are focused on employing HA-tagged NS2A virus to identify interacting partners of NS2A in infected mammalian and insect cells.