Construction of an infectious clone of equine infectious anemia virus by N-glycosylation reverse-mutations]

To elucidate the role of N-glycosylation in fetal donkey dermal cell (FDD)-attenuated equine infectious anemia virus (EIAV), we constructed an N-glycosylation reverse-mutation molecular clone, pLGN191N236N246. This viral molecular clone was derived from the infectious clone pLGFD3-8 by site-directed mutagenesis. This clone was used to transfect fetal donkey dermal (FDD) cells. Infectious characteristics of transfectants were monitored by RT-PCR, indirect immune fluorescence and reverse transcriptase activity assay. After three passages in FDD cells, viral replications in the supernatant of cell cultures were detected by all the above three methods and viral particles were clearly observed by electron microscopy. The construction of the N-glycosylation reverse-mutation infectious clone provides a solid basis for further study of the mechanism of attenuated pathogenesis and increased immune protection of EIAV attenuated vaccines.