In the United States, West Nile virus (WNV) infects approximately 2500 people per year, of which 100-200 cases are fatal. No antiviral drug or vaccine is currently available for WNV. In this study, we designed gamma-modified peptide nucleic acid (γPNA) oligomers to target a newly identified guanine-rich gene sequence in the WNV genome. The target is found in the NS5 protein-coding region and was previously predicted to fold into a G-quadruplex (GQ) structure. Biophysical techniques such as UV melting analysis, circular dichroism spectroscopy, and fluorescence spectroscopy demonstrated that the target RNA indeed folds into a moderately stable GQ structure at physiological temperature and potassium concentration. Successful invasion of the GQ by three complementary γPNAs was also characterized by the above-mentioned biophysical techniques. The γPNAs showed very strong binding to the target with low femtomolar affinity at physiological temperature. Targeting this potential guanine quadruplex forming sequence (PQS) and other related sequences with γPNA may represent a new approach for inhibiting both WNV replication and transcription, thereby representing a generally useful antiviral strategy.