Abstract Infectious clones of West Nile virus (WNV) have previously been generated and used to decipher the role of viral proteins in WNV virulence. The majority of molecular clones obtained to date have been derived from North American, Australian, or African isolates. Here, we describe the construction of an infectious cDNA clone of a Mediterranean WNV strain, IS-98-ST1. We characterized the biological properties of the recovered recombinant virus in cell culture and in mice. The growth kinetics of recombinant and parental WNV were similar in Vero cells. Moreover, the phenotype of recombinant and parental WNV was indistinguishable as regards viremia, viral load in the brain, and mortality in susceptible and resistant mice. Finally, the pathobiology of the infectious clone was examined in embryonated chicken eggs. The capacity of different WNV strains to replicate in embryonated chicken eggs closely paralleled their ability to replicate in mice, suggesting that inoculation of embryonated chicken eggs could provide a practical in vivo model for the study of WNV pathogenesis. In conclusion, the IS-98-ST1 infectious clone will allow assessment of the impact of selected mutations and novel genomic changes appearing in emerging European strains pathogenicity and endemic or epidemic potential. This will be invaluable in the context of an increasing number of outbreaks and enhanced severity of infections in the Mediterranean basin and Eastern Europe.