Construction and In Vitro Evaluation of EIAV Full-length Infectious Clones with Site-directed Retromutations
Abstract: Based on t he infectious clone p FD328 wit hin EIAV f ull2lengt h genome , and according to the variation of structural genes during vaccine preparation , several chimeric infectious clones involved in gag and env genes , modified by overlap PCR site2 directed mutagenesis , were constructed successfully. These clones were used to t ransfect fetal donkey dermal (FDD) cell s and donkey differentiated monocyte macrop hages (DL ) , and their infectious characteristics were monitored by RT-PCR and rever set ranscriptase activity assay. The results indicated that after five generations passaged in FDD cell s t hen five generations in differentiated monocyte2macrop hages , RT activity and RT2PCR were found to be obviously positive in cell cult ure supernatant and viruses particles were al so clearly observed under electron microscope. Nevertheless , t he final viral titer s of these artificial viruses in DL culture were obviously higher than that in FDD culture. Analysis of the replicative characteristics between these chimeric viruses and t heir parental virus pL GFD328 showed that the formers slightly delayed in replication compared with the latter . All this provides a solid basis for further study of the pathogenic mechanism and immune protection against Chinese equine infectious anemia virus.