Defective interfering viral particles are readily produced in cell culture after a high multiplicity of infection with many animal RNA viruses. Due to defects that they carry in their genomes, their life cycle needs to be complemented by the helper functions provided by a parental virus which makes them both dependent on and competitive with the parental virus. In many instances, this may cause the abrogation of a lytic cycle of the parental virus, leading to a persistent infection. In this paper, we describe for the first time the presence of truncated or defective interfering viral RNAs produced in Vero cells persistently infected with the flavivirus Murray Valley encephalitis virus. While these RNAs have not been detected in acutely infected Vero cells, their appearance coincided with the establishment of persistent infection. We also show for the first time that the defective viral RNAs replicate well in both cell culture and cell-free virus replication systems, indicating that they may interfere with the replication of parental virus at the level of viral RNA synthesis. Significantly, structural analyses of these RNA species including nucleotide sequencing have revealed that they carry similar nucleotide deletions encompassing the genes coding for the prM and E proteins and various gene segments coding for the N terminus of the NS1 protein. These deletions are in frame, allowing the synthesis of truncated NS1 proteins to occur in persistently infected cells. This may have further implications for the interference with the parental virus at the level of viral RNA synthesis in addition to a major one at the level of virion assembly and release.