Comparion Of The LTR Promoter Activity Of Chinese Equine Infectious Anemia Virus And Biological Characterization Of The EIAV_FDD Attenuated Virus

The equine infectious anemia virus donkey leukocyte attenuated vaccine (EIAV_DLA) 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 EIAV_L strain. Secondly, EIAV_L strain was then passaged 100 times in donkeys resulting to gain a more virulent strain EIAV_d 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 DLA (EIAV_DLA) vaccine was developed from EIAV_D 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 EIAV_DLA in fetal donkey dermal (FDD) cells for 15 times and EIAV_FDD vaccine was proved to have more excellent effect of immune protection than that of EIAV_DLA vaccine.Long terminal repeat(LTR) presents in the two terimals of previral genome DNA. LTR contains promoter and enhancer which regulate the viral genmone transcription.In this study, the LTR of donkey-leukocyte attenuated vaccine and 1-26 passages of EIAV_FDDthat obtained from EIAV_DLV passaged in fetal donkey dermal cells was sequenced and analyzed. The LTR promoter activity of these strains was compared. EIAV exists as quasispecies, as other lentiviruses. EIAVdla and EIAV_FDD LTR had lots of types, some of them in major portion, others in small portion.The EIAV_DLA LTR had four types according to NRE sequence difference, which NRE 1 with the equal length of highly virulent EIAV LTR but point mutant was 1/3, NREⅢwith 17nt insertion and 10nt deletion was 2/3 in EIAV_DLA. NREⅡ(only 17nt insertion),NREⅣ(only 10nt deletions) were detected one clone each. In LTR enhancer region, some clones had a 16nt insertion within a binding site of macrophage-specific transcription factors PU.1. In 21 clones sequenced, transcription start site of EIAV_DLA LTR had four types of GGTC, AGAC, AAAC, GAAC, separately for 23.8%, 14.3%, 47.6%, 14.3%. The transcription start site of virulent strain was GGAC. Promoter activity of LTR with insertion in NRE was inhibited. The insertion in enhancer region increased the promoter activity slightly, not reaching significant difference. The impact of the transcription start site on the promoter activity of LTR was GGTC＞AAAC＞AGAC＞GGAC. The promoter activity of 13th and 26lh passages of EIAV_FDD LTR were higher than EIAV_DLA and EIAV_D LTRs' in equine macrophages and fetal donkey dermal cells, and the promoter activity of EIAV_D LTR was the lowest. With the viral virulence reducing in different stages during the vaccines formation, the LTR promoter activity increased gradually. It was concluded that LTR was an contribution to the cell tropism and virulence of EIAV.The sequences of LTR NRE had significant changes when EIAV_DLA was passaged in FDD cells. It had 11nt insertion (GATGACTAGCT) from 10th passage. In the 13rd passage LTR had a new mutation CCCTTATGACTAGCT, but not in the majority. It became the absolute superiority in the 23rd passage. CTCTCA was deleted in the 21st passage behind the 49nt. There were 10 A→G mutations in LTR sequences in the 23 and 26 passage, being the dominant types. As the appearance of a lot of point mutations, the nucleotides 54-68nt and 70-83nt was non-complete duplication. LThe comparison of promoter activity of LTR was indicated that the promoter activity of LTR enhanced gradually with the passages increasing in the fetal donkey dermal cells, but not in significant difference. EIAV infected cell provided the Tat, transacting the LTR promoter activity 1.5-2.7 folds. The EIAV_DLA LTR promoter activity was lower than the EIAV_FDD LTR, P＜0.01.The complete sequences of the 19th and 26th passages of EIAV_FDD were amplified by PCR in five fragments. In analyses of the complete sequences of the 19th and 26th passages of EIAV_FDD without immunologic protection, it was found that only 1 amino acid change in Gag, but the same as forgein EIAV strains 1369. Polymerase had 8 amino acid changes, 3 changes in charge. Env protein was found to several stable amino acid changes. Two N-linked glycosylation sites were destroyed. An amino acid change without charge change was in principle neutral domain of gp90. gp45 was truncated as same as the EIAV_FDD vaccine. S1 and S2 protein did not change in predicted amino acid sequences. Rev protein coded by ORF S3 changed in 3 sites, an amino acid change in basal region, and a 26aa deletion before the basal region, which needed further study of the effect of the 3 variant sites on Rev function.Three horses were inoculated with the 26th passage of EIAV_FDD. Plasma viral RNA load was very low, the virus replication ability poor, inducing week antibody reaction. LTR sequences detected in horse were not in variation with the inoculate virus.