| Equine infectious anemia virus (EIAV) is a lentivirus of the Retrovirus family and is thepathogen of equine infectious anemia (EIA). The Chinese attenuated EIAV vaccine is the onlylentiviral vaccine that was successfully applied on a large scale. EIAV’s env gene encoding thetrans-membrane protein gp45plays an important role in mediating the fusion between virus andthe target cell, the combination of the glycoprotein and viral replication and infection, and it isalso an important immunogen.The study of the mechanism of EIAV’s attenuation and immuneprotection induction based on EIAV gp45, which could provide reference information for thestudy of the immune mechanism of lentivirus and the development of other lentiviral vaccines,and this is of very important theoretical significance and application value.In this study, the gene sequences of the proviral gp45of the following strains were alignedand analyzed, and these strains include the parental virulent strain EIAVLN40of EIAV’s attenuatedvaccine strain, EIAVDLV32, EIAVDLV62, EIAVDLV92, EIAVDLV121(donkey leukocyte attenuatedvaccine) and EIAVDLV137,which were respectively the32nd,62nd,92nd,121stand137thpassages ofEIAVDV117in donkey leukocyte, and also include EIAVFDDV4, EIAVFDDV13and EIAVFDDV23,whichwere respectively the4th,13th, and23rdpassages of EIAVDLV121in fetal donkey dermal cells, aswell as EIAVFM2and EIAVFM6which were respectively the second and sixth passages ofEIAVFDDV13in peripheral blood leukocytes. The results showed that:①in the course of vitropassage of gp45of EIAVLN40mainly39nucleotide sites mutated, and16mutation sites among39mutation sites caused amino acid variation;②the average difference between the differentpassages of viruses and EIAVLN40was between2.12~2.77%;③EIAVLN40had three (50G,51Land277K) specific amino acid sites, and three variable sites (42E/K,166I/V and256L/F)which mainly occurred in the fetal donkey dermal-adapted strain (EIAVFDDV13and EIAVFDDV23),and54V (I)/T mutation site mainly occurred in the vaccine strains (EIAVDLV121and EIAVFDDV13)and its derived viruses;④783G/A mutation occurred in the nucleotide sequences of all clones offetal donkey dermal-adapted strains, and there occurred a stop codon781TGA783in gp45gene,which caused the truncation of gp45, and this mutation site existed in23/25clones of EIAVFM1indonkey peripheral blood leukocytes, and3/21clones in EIAVFM6,3/22clones in EIAVDV117, andthere did not occur this kind of mutation in the rest of the strains. To further study the variationpattern of the mutation783G/A of EIAV gp45in vivo, partial sequences of gp45of the strainsEIAVLN40, EIAVDLV121and EIAVFDDV13after infecting horses were amplified and analyzed, and theresult indicated that extremely low proportion of783G/A mutation occurred in the clones ofEIAVLN40and EIAVDLV121, and after the infection of EIAVFDDV13, with the time increasing,783G/Amutation declined gradually, even was lost completely.On the other hand, based on the analysis of crystal structure of EIAV gp45conducted by theNankai University researchers, by means of reverse genetics eight sites which affect the structuralstability of the gp45spiral hexamer were mutated respectively, and infectious clones wereconstructed to save the virus, and the replication abilities in vitro of each mutant strain andwild-type ones were compared. The results showed that the replication ability of the mutant strain T491I declined significantly and the remaining seven variable sites had no obvious effect on viralreplication. The analysis of the infection ability of virus showed that although at41℃theinfection ability of each mutant strain decreased, especially V505T and L512T, which indicatedthat there existed difference for the mutation sites’ sensitivity to temperature change, suggestingV505T and L512T mutation reduced the structural stability of gp45’s coil hexamer, while theremaining variations may increase its stability.The above results showed that: EIAV gp45had obvious characteristic of gene diversity, andgp45truncation mutation was caused by the virus’s adaptation to culture in fetal donkey dermalcells, and the mutation of V505/I in the vaccine strain may change the structure stability of gp45’sspiral hexamer and then affect the infectivity of virus. |
