番鸭小鹅瘟病毒PT株E盒基序缺失感染性克隆的构建及生物学特性分析

Construction and characterization of the E-box deleted Muscovy duck-origin goose parvovirus

为获得含有E盒基序(E-box)缺失突变体病毒全基因组的番鸭小鹅瘟病毒(MDGPV) PT株缺失病毒并分析其生物学特性,本研究以含有MDGPV PT株全长感染性DNA克隆的质粒pMDGPVPT为模板,通过重叠延伸PCR方法将5'-末端倒置重复序列(5'-ITR)第315位E-box缺失,获得感染性重组质粒pPT-Ed315-320。pPT-Ed315-320经卵黄囊接种转染9日龄番鸭胚后拯救出缺失病毒rMDGPV-Ed315-320。生物学特性试验显示,rMDGPV-Ed315-320感染番鸭胚后能够引起其特征性病变,与其亲本病毒株PT相比,rMDGPV-Ed315-320的毒价及其在番鸭胚中增殖能力均有所下降,但在番鸭胚成纤维细胞中的复制能力增强。本研究为进一步了解5'-ITR中E-box的缺失与MDGPV致病力之间的关系奠定了基础。

In order to generate E-box motif（315 CACGTG320） deleted Muscovy duck-origin goose parvovirus（MDGPV） and biologically characterize the recombinant virus, the E-box motif at nucleotide position 315 on 5＇-inverted terminal repeats（5＇-ITR）in recombinant plasmid pMDGPVPT containing full-length infectious DNA clone of MDGPV PT strain was deleted using the overlap extension PCR to create the recombinant plasmid pPT-Ed315-320. Subsequently, the E-box motif deleted MDGPV of rMDGPV-Ed315-320 was rescued by transfecting pPT-Ed315-320 into the 9-day-old Muscovy duck embryos via yolk sac inoculation. The biological characteristics indicated that rMDGPV-Ed315-320 was still able to cause Muscovy duck embryo dwarf.Compared with its parental virus strain PT, the virulence of rMDGPV-Ed315-320 and its proliferation ability in Muscovy duck embryos were attenuated, however the replication ability of rMDGPV-Ed351-320 in MDEF cells was enhanced. This study has laid the foundation for the further studies on the relationship between the E-box deletion in 5＇-ITR and pathogenicity in MDGPV.