非洲猪瘟病毒E184L基因缺失毒株的构建及评价

Generation and evaluation of E184L gene-deleted African swine fever virus

为阐明更多非洲猪瘟病毒(African swine fever virus,ASFV)未知基因的功能,深入探究ASFV的致病机制,为研制针对ASFV流行毒株的疫苗提供候选毒株。采用同源重组的方法构建E184L基因缺失的非洲猪瘟单基因缺失毒株,在猪肺泡巨噬细胞(porcine alveolar macrophages,PAMs)中评价其复制特性,并在猪体内比较ASFVΔE184L与亲本毒株ASFV CN/GS/2018对猪的致病力以及在动物体内诱导免疫应答的差异。成功获得了E184L单基因缺失突变毒株ASFVΔE184L,经PCR鉴定、荧光观察及测序鉴定,证实该毒株E184L基因被成功缺失。复制特性研究发现,E184L基因的缺失削弱了ASFV在PAMs中的复制能力。动物体内试验显示,ASFVΔE184L接种猪在观察期内死亡1头,其余全部存活,而ASFV WT接种猪全部死亡,说明E184L的缺失导致ASFV的毒力显著下降。此外,与亲本毒株相比,ASFVΔE184L可诱导机体ASFV特异性抗体的产生及抗病毒因子干扰素β(interferon-β,IFN-β)的分泌。E184L为ASFV毒力相关基因及免疫抑制相关基因,其基因缺失毒株在猪体内的致病力显著减弱并能诱导机体内更强的免疫应答。以上结果为ASFV未知基因功能及其致病机制的阐明提供了新的理论依据,更重要的是,为在候选疫苗基因组中进一步缺失E184L基因片段构建安全有效的双基因或多基因缺失候选疫苗株奠定了重要基础。

To elucidate the function of more unknown African swine fever virus(ASFV)genes,further explore intensively the pathogenic mechanism of ASFV,and obtain a candidate vaccine strain against ASFV.E184L-deficient recombinant ASFV(ASFV ΔE184L)was constructed by homologous recombination,and the replication capacity was evaluated in porcine alveolar macrophages(PAMs).The virulence and induced immune response of ASFVΔE184L also were compared with the parent strain ASFV CN/GS/2018 in vivo.ASFV ΔE184L was obtained through homologous recombination,and the deletion of E184L gene was confirmed by PCR,fluorescence observation and sequencing.The deletion of E184L gene weakened the replication ability of ASFV in PAMs.All but one died inoculated with ASFVΔE184L survived on 20 days post-inoculation,while all the pigs inoculated with ASFV WT died.The results demonstrated that the deletion of E184L attenuated the virulence of ASFV.In addition,compared with ASFV WT,ASFV ΔE184L successfully induced the production of ASFV-specific antibodies and the secretion of IFN-β in pigs.ASFV E184L was a virulence and immunosuppression related gene.The virulence of E184L-deleted ASFV in pigs was significant attenuated and the stronger immune response was induced by E184L-deleted ASFV in pigs.Our findings provide a new theoretical basis for elucidating the function of unknown ASFV gene and strong support for constructing safe and effective double or multi-gene deletion candidate vaccine strains by further deletion of E184L gene fragment in the candidate vaccine genome.