West Nile virus (WNV), a mosquito-borne virus, is the causative agent of an outbreak in New York City in 1999 (WNVNY99) responsible for the most extensive spread of arboviral encephalitis in the Americas. In Australia, an endemic and mildly virulent strain of WNV, Kunjin virus (WNVKUN), circulates in the northern parts of the country. WNVKUN infection is usually benign and asymptomatic, but in 2011, a newly emerged virulent strain of WNVKUN appeared in southeastern Australia (WNVNSW2011) and resulted in more than a thousand equine cases with a 10-15% case mortality. An unusual aspect of the WNVNSW2011 outbreak was the lack of human cases, given its high prevalence and lethality in horses. Our research focuses on the hypothesis that further accumulations of genetic mutations could render WNVNSW2011 human-virulent. To test the hypothesis, we first generated chimeras between WNVNSW2011 and WNVNY99 and compared the growth and virulence of these chimeras in mouse and human cells, and in mice. We found that the chimera encoding all non-structural (NS) genes (NS1-NS5) of WNVNY99 on WNVNSW2011 background produced enlarged viral plaques, similar to WNVNY99, and had improved growth kinetics compared to the WNVNSW2011 parental strain. It was also observed that the NS3 chimera (NS3 of WNVNY99 on WNVNSW2011 background) exhibited enhanced replication. Both the NS and NS3 chimera showed increased virulence in mice. The data indicate that further mutations in the NS region of WNVNSW2011 have the potential to increase the virulence of WNVNSW2011 significantly.