In Vivo Characterizations Of Infectious Molecular Clone And Its Derivatives Of Donkey Leucocyte Attenuated Equine Infectious Anemia Virus

In order to investigate the molecular mechanism of the attenuation and protection of donkey leukocyte attenuated equine infectious anemia virus (DLA-EIAV), the complete genomes of DLA-EIAV and EIAV strain Liaoning (L-EIAV) proviruses were cloned and sequenced. The infectious molecular clone (pOK8266) of DLA-EIAV was construct and its derived virus was obtained (designated as vOK.8266), Based on pOK.8266, a chimeric infectious molecular clone (pOKVltr) was constructed by replaced the long terminal repeat (LTR) sequence of DLA-EIAV with the counterpart of L-EIAV. and its derived virus was generated (designated as vOKVltr).In this study, seven EIAV-negative horses were randomly divided into four groups, two horses (4# and 5#) in Group 1 were inoculated with vOKVltr, two horses (6# and 7#) in Group 2 with vOK8266. one horse in Group 3 with DLA-EIAV, and 2 horses (1# and 2#) in Group 4 were served unvaccinated control. Eight months post-inoculation, all horses were challenged with L-EIAV. Pre- and post-challenge, the body temperature of all horses was recorded. All horses showed no abnormal change pre-challenge, indicating that all viruses used were safe for horses, and the replacement of the LTR of the attenuated molecular clone by L-EIAV LTR didn't result in virulence increasing. The horses in Group 4 experienced 1 (2#)to 4 (l#)episodes of fever and died 19 days and 134 days post-challenge, respectively. The horses in Groups 1 and 3 had no abnormal changes post-challenge, indicating that the chimeric virus and vaccine virus protected the horses from challenge. In Group 2, the horse 6# showed no clinical signs, but the horse 7# experienced 7 episodes of fever and died 185 days post-challenge. Dead horses (1#, 2# and 7#) showed typical El A pathological changes at autopsy, and survived horses (4#, 5#, 6# and 8#) were euthanized 450 days post-challenge, and displayed no pathological changes.The antibodies against the EIAV structural proteins pi5, pi 1, p26, p9 and gp45 were measured by ELISA based on the recombinant proteins expressed in E.coli. The antibody levels induced by p!5, pll and p9 proteins were very low or undetectable pre-challenge, antibody against p26 was increased soon after immunization and slowly come down later, antibody to gp45 was increased slowly and persistentat high level. After challenge, the titers of antibodies against the five structural proteins in horses 1 #, 2 #and 7# increased significantly and sustained till death, antibodies to pi5, p26 and gp45 were also increased in horses 6#and 8#. Antibodies level against the five structural proteins did not show significant change in horses 4# and 5# after challenge, the lower or no remembrance response might reflect good protection. The anti-gp45 titers had no obvious difference between diseased and protected horses, so the anti-gp45 antibody seems to have no relationship with the disease progress.The EIAV provirus DNA in PBMCs and the EIAV RNA in plasma of inoculated horses wereinvestigated by real-time PCR and RT-PCR. We found that the quantity of L-EIAV provirus DNA had positive relationship with the antibodies level of anti-pi 1 and p9, and the EIAV provirus DNA content were related to antibodies against p26. The virus loads exceeded 106copies/ml plasma in diseased horses, but presented low level in protected horses, and decreased to undetectable level three months post-challenge. But the provirus DNA in PBMCs from all horses can be detected, indicating that EIAV exists as latent provirus.The variation of EIAV LTR and env may associate with the pathogenesis. By sequencing ed the provirus LTR and env gene from PBMC, we found that there are three types of LTR and env gene sequences, representing virulent type (similar to L-EIAV), attenuated type (similar to DLA-EIAV) and intermediate type (between L-EIAV and DLA-EIAV). In diseased horses the virulent type of LTR and env was dominant, but all three types could be found in protected horses. Sequence analysis demonstrated that there are two E-box motifs in LTR highly variable region of at...