Development And Characterization Of A Pathogenic Molecular Clone Of Chinese Equine Infectious Anemia Virus Donkey-adapted Strain

Equine infectious anemia virus (EIAV), a macrophage-tropic lentivirus, causes persistent infections of horses characterized by rapid development of acute disease and following a long chronic and persistent phase. Chinese equine infectious anemia virus donkey-leukocyte attenuated strain (EIAV-DLA) is the unique lentivirus vaccine which has been used in large range for more then 20 years in China mainland. The donkey-adapted strain of EIAV (DV116), parental strain of EIAV-DLA, is a highly virulent isolate which was developed by sequential passaging a virulent EIAV strain in donkeys in 1970s. The donkeys inoculated with fatal doses of EIAV D strain can always be killed within in the first acute disease period.In this study, we constructed a full-length provirus DNA clone of DV116 by PCR. Sequence analysis shows that genes of DV, as well as other Chinese EIAV strains used in development of vaccine, have low degree of homology with Wyoming-like strains reported previously. The divergences of gag and env gene are 22.3%-23.1% and 29.6%-31.6% with corresponding fragments. These findings indicate the Chinese EIAV strains, according to the phylogenetic tree analysis, would represent a new subtype distinct from other known strains.An infectious EIAV clone was recovered by transfecting fatal donkey dermal (FDD) cell cultures and donkey leukocyte (DL) culture in vitro with the full-length gene clone of DV. The virus (designed pD70344V) derived from the third passage in DL culture was observed by electron microscope and the reverse transcriptase (RT) activity was determined. Two donkeys were injected intravenously with the pD70344V stock and developed acute diseases at 9 day and 12 day after injection, respectively. One animal (donkey 03008) was euthanized due to the severity of the clinical symptoms on day 17 after injection and the other donkey (03009) died on day 18 after injection. We observed high fever, weigh lost, anorexia, depression, thrombocytopenia and high plasma viremia levels during the courses. This result indicated that the virus pD70344V derived from the infectious molecular clone is a highly virulent strain which had inherited main characters from its parental virus DV.