Changes Of Humoral Immunity Of EIAV FDDV Inoculated Horses And Establishment Of A 293-Cell Line Containing Luciferase Reporter For EIAV Receptor And LTR Functions

In order to investigate the humoral immune mechanism of the attenuation and protection of donkey embryo-skin cells attenuated equine infectious anemia virus (FDDV-EIAV), In this study, five EIAV-negative horses were randomly divided into two groups, four horses (6#, 7#, 8# and 9#) in Group 1 were inoculated with 1 ml of FDDV-EIAV, one horses (5#) in Group 2 were served unvaccinated control. Within 210 days post-inoculation, seclected horse's serum and then dected p26 and env-specific antibody, avidity of antibody and env-specific antibody epitope conformation dependence. Found that p26 and env-specific antibody emergerenced after 3 weeks post-inoculation, the titer of p26- and env-specific antibody changed with different horses and the titers were lower. The avidity of p26 and env-specific antibody was unstable, but standed in a high stable. The conformation index of env-specific antibody changede around 2.0, suggested that env-specific antibody was predominantly conformational epitope specificity. After 100 days post-inoculation, the titer of env-specific antibody was still stable, the avidity of p26- specific antibody was high though the titer was low. Seven months post-inoculation, all horses were challenged with EIAV L-21, all EIAV FDDV11 inoculated horses showed no abnormal change post-challengee and control horse experienced episodes of fever 10 days and died 16 days post-challenge, indicating that EIAV FDDV11 can protect horses from attacting by EIAV L-21. The reasech suggest that the humoral immune response to FDDV-EIAV is distinguished to the response to virienct EIAV and should to further reasech the effect of the humoral immune response to EIAV. At same time, the reaseth can serve as the design vaccines to other lentivirus.To accurately and conveniently detect neutralizing antibodies and receptor binding affinities of different EIAV strains, the cDNA of EIAV receptor, ELR1, was cloned and inserted in an eukaryotic expression vector pcDNA3.1(+). This recombinant plasmid was designated as pELR1. And then, the transcription regulatory region, long terminal repeat (LTR), of an EIAV vaccine strain and a reporter of firefly luciferase gene were tandemly cloned into pELR1. The resultant expression vector, which was designated as pELR1-LTR-Luc, was used to transfect 293 cells. A transfected cell line, ELR1-LTR-Luc (293E), which consistently expressed ELR1 and expressed luciferase under the regulation of LTR was isolated and further characterized. EIAV was able to enter ELR1-LTR-Luc (293E) cells by binding to ELR1 on the cell membrane and to replicate in the cells. The viral trans-activates transcription (Tat) thus trans activated LTR and sharply enhanced the expression of firefly luciferase gene. The luciferase activity in the cell line treated with 1000 TCID50 for 24h was 2.15 folds higher than the activity in untreated cells. The replication of viruses in the ELR1-LTR-Luc (293E) cells by inoculated with EIAV vaccine strain FDDV was verified by IFA. The tranfected genes in ELR1-LTR-Luc (293E) were consistently expressed during continuously passing for 35 generations. This cell line can be used to set up valuable systems for further study of interaction between EIAV strains and the receptor, as well as to evaluate neutralizing antibodies raised by EIAV strains.