West Nile virus is classified under the family Flaviviridae and is clustered with the Japanese Encephalitis serogroup. The first epidemic of West Nile virus in the Western hemisphere occurred in New York in 1999 (WNVNY99), since then it has spread across the entire United States and into neighbouring countries and have become endemic in the region. Infection with WNVNY99 usually causes a febrile illness. In severe cases, a neuroinvasive disease called West Nile encephalitis develops and may lead to death or a recovery marked by long-term neurological sequalae. In Australia, a closely related strain of WNV called Kunjin virus (WNVKUN), is endemic in the Northern parts of the country. Compared to WNVNY99, WNVKUN is mildly virulent and have never been associated with any human outbreaks; although in recent years, equine outbreaks of WNVKUN have been reported. WNVNY99 remains a potential threat to Australia given the suitability of Australian corvids and mosquito vectors for supporting WNV replication and spread, and the development of specific serological assays that can accurately distinguish infections with WNVNY99 and WNVKUN are vital to maintain avid surveillance. It has been proposed that the cross-reactive E antigen used in current serological assays could be replaced by the less cross-reactive E domain III antigen, or with NS1 or NS5 to allow more specific diagnosis. The prM protein has also been identified as a suitable antigen to accurately distinguish infections with JEV and WNV; and preliminary data from our lab have demonstrated that prM-specific antibodies were only detected in sera from WNVNY99-infected horses, but not in WNVKUN-infected horses, making prM a potential antigen for specific serodiagnosis of WNVKUN and WNVNY99. The first aim of the research project was to develop a suitable strategy for the expression of an antigenically authentic prM antigen with an affinity tag for purification purposes. During protein expression, we encountered complications with the antigenic presentation of recombinant WNVKUN prM. Further investigations identified specific residues in WNV prM that was vital for correct presentation of a prM epitope. This discovery expanded the scope of the project to investigate the residues in WNV prM (by comparing the prM residues of WNVNY99 with WNVKUN) that was associated with the virulence of WNVNY99. Our investigations revealed that the Val22 and Ser72 residues in the ectodomain of prM, when substituted into WNVKUN prM, resulted in a mutant virus that yielded higher viral titers and enhanced the secretion of sub-viral particles. Correspondingly, we also demonstrated that the WNVKUN prM mutant virus displayed increased virulence in mice. In summary, our investigations identified a suitable strategy for the large-scale expression of recombinant WNV prM antigen with an affinity tag for purification purposes. At the same time, we identified residues in WNV prM that have significant influence in particle secretion and virulence.