[Show abstract][Hide abstract] ABSTRACT: The requirement to replicate in both vertebrate and invertebrate hosts is thought to limit the introduction of genetic changes into the genome of arboviruses. Serial passage under laboratory conditions will overcome this limitation allowing for genetic changes to be introduced and affecting the virulence of the virus for animals. In the studies detailed here, the consequence of removing the restriction of alternate replication was demonstrated to be different depending on the virus. Passing Venezuelan equine encephalitis virus in tissue culture cells, eggs or mice resulted in up to 11 nucleotide or amino acid changes but no significant change in the virulence of the virus for mice. Passing Japanese encephalitis virus (JEV) under the identical conditions resulted in as many as 22 nucleotide or amino acid changes that often resulted in improved survival probabilities. For JEV, most genetic changes along with the attenuated phenotype were selected within 5 passes.