The West Nile virus (WNV) genome contains a single RNA-dependent RNA polymerase 
(RdRp) gene, which is responsible for replication of the viral genome and, as 
such, is an important target for antiviral therapy. Viral RdRps are known to 
lack proofreading capabilities and as a result viruses such as WNV exist as a 
mixture of viral genotypes within an infection, enabling the virus to readily 
emerge and adapt to new host environments. To test the consequences of subtle 
structural alterations remote from the RdRp active-site, the following single 
point mutations were engineered in the WNV NS5 RdRp coding region: T363N, A365N, 
and T537I; these mutations were selected in an effort to stabilize the secondary 
structural elements near the rNTP binding pocket of the RdRp. Mutant viruses 
were tested in vitro on Vero, C6/36, Culex tarsalis and DF-1 cell types and in 
vivo in one day old chickens and Culex pipiens mosquitoes. Plaque morphology was 
affected by each mutation and growth and RNA replication kinetics were altered 
as well. Our results demonstrate that subtle alteration of the RdRp protein away 
from the active site can have a significant overall biological effect on WNV 
fitness, and that this effect can be host-dependent.