We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Insertion of EGFP seems to result in overattenuation of the virus, while our previous experiments demonstrated that the insertion of an epitope tag into a similar position did not affect L protein function. Thus, a desirable level of attenuation could be reached by manipulating the length of the insert (in the second hinge region of the L protein), providing additional tools for optimization of controlled attenuation. This strategy for controlled attenuation may be useful for a "quick response" in vaccine development against well-known and "new" viral infections and could be combined efficiently with other strategies of vaccine development and delivery systems.Canine distemper virus (CDV) is an enveloped negativestrand RNA virus which, along with measles virus (MV) and rinderpest virus (RPV), belongs to the Morbillivirus genus, a serologically closely related genus of the Paramyxoviridae family of the order Mononegavirales. Morbilliviruses have a significant global impact on both human and animal health, and although CDV primarily infects canines, infections of other terrestrial and aquatic carnivores have been reported (13, 33). Mortality rates due to CDV infection vary among susceptible species (1) and range from 0% for domestic cats (22) to 100% for ferrets (54,56). For domestic dogs, lethality rates are approximately 50% (18), although some outbreaks in African wild dogs led to mortality rates of up to 95% (52).In natural hosts, such as ferrets or dogs, CDV infection can represent a very useful model for the study of general morbillivirus pathogenesis and immunogenesis. These are therefore generally considered to be better models than modified/ adapted MV in rodent model systems, in which systemic infection is severely restricted (27). Because ferrets are exquisitely susceptible to CDV, they also provide a useful means to assess protection after vaccination by subsequent challenge with a wild-type pathogenic virus. Ferrets are popular domestic pets, but current CDV vaccines, which were developed specifically for dogs, are often insufficiently attenuated for ferrets and can cause symptomatic and sometimes fatal infections after administration (56). This could be due to the specific way in which viruses have been attenuated for vaccine preparation. For example, for the common CDV vaccine strain Onderstepoort (CDV OP ), attenuation involved 57 passages of wild-type virus in ferrets, 208 passages in chicken embryos, 62 to 66 passages o...