中国株EIAV S2基因逆向突变感染性克隆的构建及体外感染性评价

Construction and in vitro Evaluation of an Infectious Clone of the Equine Infectious Anemia Virus Vaccine Strain EIAVFDDV with Four Reverse-mutated Vaccine-specific Sites in the S2 Gene

以中国株EIAV的驴强毒株EIAVDV115与疫苗株EIAVFDDV15的S2基因变化规律为依据,利用反向遗传技术对EIAV弱毒疫苗株全基因组感染性克隆pFDDV3-8的S2基因区进行逆向突变,构建了含有强毒株EIAVDV115S2基因区内4个稳定点突变的克隆质粒pFDDVS2r1-3-4-5,将该质粒转染FDD后进行体外继代盲传,经RT-PCR、逆转录酶活性、间接免疫荧光等检测后,显示经EIAV克隆质粒pFDDVS2r1-3-4-5转染的FDD盲传3代后,可在转染的FDD细胞培养物的上清液中检测到EIAV逆转录酶活性;该细胞培养物经RT-PCR和间接免疫荧光检测均呈EIAV阳性;在电镜下可观察到典型EIAV粒子。证明已成功拯救经EIAVS2基因逆向突变后的衍生病毒vpFDDVS2r1-3-4-5。比较vpFDDVS2r1-3-4-5衍生病毒与亲本克隆衍生病毒的复制动力学,表明前者的复制比后者略滞后。以上结果提示,对疫苗株S2基因的突变未明显影响EIAV的体外复制。

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 EIAVFDDV 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 vaccinespecific sites in the S2 of an EIAVFDDV infectious clone, pFDDV3-8, were reverse-mutated to the sequences of the virulent strain EIAVDV115. 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 vpFDD- VS2r1-3-4-5 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.