| An attenuated equine infectious anemia virus (EIAV) vaccine was developed by scientists led by Rongxian Shen of Harbin Veterinary Research Institute in the 1970s. The pandemic of equine infectious anemia (EIA) in China, which caused the death of millions horses, was successfully controlled by the country-wide application of this vaccine from 1980-1990. A First-Class National Invention Prize was awarded for the development of this novel lentiviral vaccine in 1982. This vaccine is the first lentiviral vaccine, which elicits broad and effective immunity against the infection of pathogenic strains. The efficacy of this EIAV vaccine implicates a hidden mechanism that covers the attenuation of virulence and induction of protective immunity of lentiviruses. The development of vaccines for AIDS is still a global challenge. Due to the similarity between EIAV and HIV, decoding the mechanism of EIAV vaccine on inducing protective immunity will provide insight to the development of other lentiviral vaccines, such as HIV vaccine.The attenuated EIAV vaccine was developed through successive passages of a wild-type virulent strain firstly in horses and donkeys to enhance the virulence and immunogenicity. A consistently passaging of this virus in donkey peripheral blood mononuclear cells (PBMC) and in fetal donkey dermal (FDD) cell derived fibroblasts in vitro was performed thereafter to attenuate the virulence of the virus. To better understand the changes in characterizations of this vaccine during the above developing process, genomic evolution of the vaccine strain was examined in this study.Proviral genomes were prepared from the parental virulent strain (EIAVLN40), the fetal donkey dermal cell-adapted vaccine strain (EIAVFDDV13) and strains of representative intermediate attenuating steps, and were amplified by PCR. In addition, S2 and gp90 gene fragments were amplified from EIAVLN40 and EIAVDLV121 isolated from horses infected up to five months by RT-PCR. All these amplified fragments were sequenced and compared for sequence alterations.Results of sequence analysis demonstrated that:(1) Mutations generated during attenuation were mostly identified in LTR, env and S2. The genetic distances of these cell-adapted EIAV strains to the parental virulent strain were gradually enlarged with the increase of passages. There were multiple stable mutations in multiple genes during EIAV attenuation process in vitro, including substitutions of 100A/T,103T/S and 484D/N in Gag, 16K/E,598K/R and 619N/D in Pol,46A/E,98G/R,103H/Y,189K/E,190E/K 193S/N, 236D/-,237N/K,247E/K and 321K/E in gp90,7R/H in Tat,41T/I,51T/I and 55Q/K in S2, 74V/1 in Rev and change of transcription-factor AP-1 binding motif in the negative regulating element (NRE), transcription factor binding motif E-box within the enhancer, the transcription start position and the first residue of trans-activation responsive (TAR) element of LTR. (2) Phylogenetic analysis showed that the sequences of viruses isolated from EIAVLN40-infected horses were obviously split into two major branches. Viruses isolated in the early stage and in the asymptomatic stage of infection in all EIAVLN4o-infected horses clustered in to a branche different from EIAVLN40. Viruses isolated after disease onset remained in the same branch as EIAVLN40.The variations of gp90 were mostly concentrated in eight highly variable regions V1-V8. Changes in the glycosylation sites within V3, V4 and V5 regions were usually associated with the disease status. Specifically, glycosylation sites 191NSSN194.237NNTW240, and 280NDTS283 within these three regions were present in EIAVLN40 and most of the quasispecies isolated after, but not before. disease onset. During the evolution of EIAVLN40, over 12% substitutions of amino acid residues presented in S2. which associated with the disease status. (3) Phylogenetic analysis showed that the gp90 sequences of viruses isolated from EIAVDLV121-infected horses were obviously split into three major branches. The divergences of gp90 sequences isolated from EIAVDLV121-inoculated horses were 3.88%-6.73% compared with EIAVLN40, and were 0.91%-6.49% when compared with EIAVDLV121. The S2 sequences of viruses isolated from EIAVDLV121-infected horses were obviously split into two branches, the average differences between the S2 sequences from EIAVDLV121-inoculated horses and EIAVLN40 were from0.79%-8.83%, and were 3.08%-8.14% when compared with EIAVDLV121.Conclusion:A series of consensus substitutions in multiple genes of the attenuated EIAV vaccine strain were generated during attenuation process. The mutations in gp90 and S2 were associated with the disease status. The vaccine strain replicates in a relatively low level in inoculated horses, which enabled to the vaccine quasispecies to evolve continuous and to increase diversity of gp90. |
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