Gao Xu, Jiang Cheng-Gang, Han Xiu-E, Zhao Li-Ping, Shen Rong-Xian, Xiang Wen-Hua, Zhou Jian-Hua
State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150001, China.
Bing Du Xue Bao. 2009 Jul;25(4):309-15.
To elucidate the function of the S2 gene in equine infectious anemia virus (EIAV) and its role in the attenuation of the Chinese attenuated EIAV vaccine strains, the S2 in the EIAV vaccine strain EIAV (FDDV) was reverse-mutated and the in vitro replication character of the resultant virus was evaluated. Based on the sequence variation of the S2 gene between the EIAV virulent strains and vaccine strains, all the four vaccine-specific sites in the S2 of an EIAV(FDDV) infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAV(DV115). The reverse-mutated molecular clone pFDDVS2r1-3-4-5 was used to transfect fetal donkey dermal (FDD) cells for rescuing the derived virus vpFDDVS2r1-3-4-5. The production and replication of vpFDDVS2r1-3-4-5 in FDD cells were proved by RT-PCR, immune fluorescence assay and reverse transcriptase activity assay. Typical virons of EIAV were clearly observed under the electron microscopy. The parallel analysis of the dynamic replication of the reverse-mutated viral clone vpFDDVS2r1-3-4-5 and its parental virus vpFDDV3-8 showed that the virus with four reverse mutations in the S2 replicated only slightly slower than its parental vaccine strain in FDD cells. This result implicates that the mutations in the S2 of the EIAV vaccine strains did not significantly alter the viral replication in vitro. Further studies on the in vivo replication of the reverse-mutated viral clone are required for understanding the relationship between the S2 and the attenuated pathogenesis of EIAV attenuated vaccines.
为阐明马传染性贫血病毒（EIAV）中S2基因的功能及其在中国EIAV减毒疫苗株致弱过程中的作用，对EIAV疫苗株EIAV（FDDV）中的S2进行了反向突变，并评估了所得病毒的体外复制特性。基于EIAV强毒株和疫苗株之间S2基因的序列变异，将EIAV（FDDV）感染性克隆pFDDV3-8的S2中所有四个疫苗特异性位点反向突变为强毒株EIAV（DV115）的序列。用反向突变的分子克隆pFDDVS2r1-3-4-5转染驴胎儿皮肤（FDD）细胞，以拯救衍生病毒vpFDDVS2r1-3-4-5。通过RT-PCR、免疫荧光分析和逆转录酶活性分析证实了vpFDDVS2r1-3-4-5在FDD细胞中的产生和复制。在电子显微镜下清晰观察到典型的EIAV病毒粒子。对反向突变病毒克隆vpFDDVS2r1-3-4-5及其亲本病毒vpFDDV3-8的动态复制进行平行分析表明，S2中有四个反向突变的病毒在FDD细胞中的复制速度仅比其亲本疫苗株略慢。这一结果表明，EIAV疫苗株S2中的突变并未显著改变病毒的体外复制。为了解S2与EIAV减毒疫苗致弱发病机制之间的关系，需要对反向突变病毒克隆的体内复制进行进一步研究。
