The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique natural model system to study the attenuation mechanism and immunological control of lentivirus replication. Critical consensus mutations were identified between virulent Chinese EIAV strains and vaccine strains. Based on a full-length infectious clone of EIAV vaccine strain pLGFD3, two molecular clones, mFD5-4-7 and mFD7-2-11, were successfully constructed, in which 4 and 6 critical consensus mutations in the env gene of the vaccine strain were point-mutated to the wild-type sequence, respectively by an overlap PCR mutagenesis strategy. The infectivity, virulence, and pathogenesis of the constructed clones were investigated in vitro using a reverse transcriptase assay, an indirect immunofluorescence assay, observation of cytopathogenic effect, and virion observation as well as in vivo by inoculation of animals with the resulting infectious clones. The pathogenic symptoms in horses inoculated with mFD7-2-11 were more severe than those inoculated with mFD5-4-7, whereas no pathogenic symptoms were detected in animals inoculated with their parental clone pLGFD3 strain. The results indicate that the consensus mutation residues of the env region involved in this study play significant roles in the virulence and pathogenicity of EIAV. This will contribute to the elucidation of the attenuating and protective mechanisms of the Chinese EIAV vaccine.