马传染性贫血病毒囊膜基因gp 90 V4区糖基化回复突变感染性克隆的构建

The Chinese equine infectious anemia virus (EIAV) donkey-leukocyte attenuated vaccine (DLV) provides a unique model system for study of the attenuation mechanism and the immunological control of lentivirus replication. In this study, we compared gp90 sequences of the env gene from both virulent and attenuated EIAV strains and found that all attenuated strains lost the potential N-linked glycosylation sites in the V4 region. To determine the action of this mutation, we constructed the infectious clone pLGFDg9 to restore the potential N-linked glycosylation site. This clone was transfected into fetal donkey dermal (FDD) cells and virus replication was monitored by RT-PCR, indirect immune fluorescence and reverse transcriptase activity assay. The results showed that, after 3 passages in FDD cells, virus replication in the cell cultures supernatant was detected by all three methods and viral particles were clearly observed by electron microscopy. The N-glycosylation reverse-mutation infectious clone provides a solid base for further study of the mechanism of attenuation and increased immune protection of EIAV attenuated vaccines.