Evolution Of Equine Infectious Anemia Virus Part Of The Attenuated Strains In Vitro And In Vivo Analysis

The equine infectious anemia virus donkey leukocyte attenuated vaccine DLV had been used to protect against equine infectious anemia (EIA) disease for several decades in China. The vaccine had good immunogical protection and safety, providing a unique natural model system for other retroviruses including HIV. The generation of Chinese EIAV attenuated vaccine included three stages. Firstly, a carefully selected field isolate was passaged in horses for 16 generations to obtain a pathogenic strain, named EIAVL strain. Secondly, VL strain was then passaged 100 times in donkeys resulting to gain a more virulent strain EIAVD strain which yielded 100% acute EIA symptom and more than 90% mortality in experimentally infected horses in the first 50 days after inoculation; finally, the DLV vaccine was developed from EIAVD strain through 120 successive passages in vitro in primary donkey macrophages. Thereafter, by the same method, another fetal donkey dermal cells adapted vaccine was developed by passaging EIAVDLv in fetal donkey dermal FDDV cells for 15 times and FDDVvaccine was proved to have more excellent effect of immune protection than that of EIAVDLV vaccine.To discuss the FDDV genome evolution in vivo in inoculated horses, the provirus DNA of the EIAV in the FDDV vaccinated experimental horse which had been inoculated for 24 mouths were amplified by nest PCR. Based on the analysis about the obtained FDDV provirus DNA sequences, we hope to identify the FDDV genome evolution character in vivo in horses after a long time immunization, those included which regions in FDDV genome were conserved and which ones were variable. Our results showed that: Compared with the standard EIAVFDDV genome sequence come from a FDDV molecular clones FDDV3-8,the amino acids sequences predicted based on obtained DNA sequences in vivo showed a 98.8% and 96.1% homologous on average in LTR and ENV regions. We found that more variations occur in env region of the EIAVFDDV genome, and the number of N-glycoslation sites in env of the FDDV increased after a 24 months replication in vivo.To elucidate the mechanism of EIAV vaccine on the attenuated virulence and enhanced immunogenecity, the proviral genomic DNA of a DLV strain isolated at the 92th generation of attenuation was cloned by LA-PCR and analyzed. We obtained eight full genomic clones of DLV92 randomly in this experiment, and each individual gene of the genome was compared with the corresponding sequences of Chinese EIAV virulent strain LV (Genebank Acc.#: AF327877)and the vaccine strain DLV(Genebank Acc. #:AF327877. The compared results showed that the nucleotide homologous on average between DLV92 and DLV878 was 98.1%, and 97.7% homologous on average was showed between DLV92 and LV.17 N-glycoslation sites were discovered in membrane protein gp90 genes of the DLV92. This is consensus with the N-glycoslation sites in EIAV vaccine strain DLV878, but 4 less than LV877 and 2 less than DLV61. Results presented in this article provided valuable information for the intensive study of the mechanism on attenuation and inducing protective immunity of Chinese EIAV vaccine.