猪流行性腹泻病毒反向遗传学及其研究进展

Reverse Genetics and Its Research Progress of Porcine Epidemic Diarrhea Virus

猪流行性腹泻病毒(PEDV)是高度接触性肠道传染病猪流行性腹泻(PED)的病原,是有囊膜的单股正链RNA病毒,基因组全长约28 kb。2010年以来,PEDV G2型高致病性毒株不断发生变异,给全国乃至全球的养猪业造成巨大的经济损失。反向遗传学系统,即构建RNA病毒的全长感染性克隆。近年来,PEDV主要基于靶向RNA重组、BAC系统和体外连接3种方法来建立全长感染性cDNA克隆。文章简述了反向遗传学的原理和方法。靶向RNA重组利用冠状病毒RNA的高同源重组的特点来实现病毒的拯救;BAC系统利用pBeloBAC11载体克服PEDV基因组中含有的毒性序列所导致的cDNA在高拷贝质粒中不稳定的困难;体外连接技术主要利用PEDV基因组本身存在的限制性内切酶的酶切位点或通过改造的酶切位点在体外将病毒分片段地连接成全长的cDNA克隆。另外,文章还总结了近年来基于反向遗传学技术的PEDV相关的研究进展。PEDV反向遗传学是研究PEDV病毒基因组结构功能及设计减毒活疫苗的有效工具,利用反向遗传学技术探究S基因等毒力相关基因,探究其突变或缺失对病毒致病机制的影响,揭示PEDV毒力衰减的分子机制,有望设计出具有良好免疫原性且避免毒株返毒和重组减毒活疫苗。总之,PEDV反向遗传学是研究PEDV基因组结构及功能、病毒宿主相互作用及致病机制的一种重要方法,同时也是设计PEDV减毒活疫苗一种合理有效的途径。

Porcine epidemic diarrhea virus(PEDV)is the pathogen of porcine epidemic diarrhea(PED),a highly-contact intestinal infectious disease of pigs.PEDV is a single-stranded positive-stranded RNA virus with a cystic envelope,its genome is approximately 28 kb.Since 2010,the highly pathogenic PEDV G2 genotype strains have been continuously mutated,which has caused huge economic losses to the pig industry nationwide and even globally.Reverse genetic system is the full-length infectious clone of RNA virus construction.In recent years,PEDV is mainly based on the three methods of targeted RNA recombination,BAC system and in vitro connection to establish infectious cDNA cloning.The principle and method of reverse genetics were briefly summarized in this paper.Targeted RNA recombination utilizes the high homologous recombination of coronavirus RNA to achieve viral salvation,BAC system utilizes pBeloBAC11 vector to overcome the difficulty of cDNA instability in high-copy plasmid caused by the toxic sequence contained in PEDV genome.In vitro linkage technique mainly uses restriction endonuclease restriction sites existing in PEDV genome itself or modified restriction sites to connect virus fragments to full-length cDNA clones in vitro.In addition,this paper also summarized the recent progress in PEDV-related research based on reverse genetics technology.PEDV reverse genetics is an effective tool for studying the structure and function of PEDV virus genome and designing live attenuated vaccine,using reverse genetic technology to explore virulence related genes such as S gene,to explore the effect of mutations or deletions on the pathogenesis of the virus,to reveal the molecular mechanism of PEDV virulence attenuation,and to design live attenuated vaccine with good immunogenicity and avoid the return of virus and recombination of the virus strain.In conclusion,PEDV reverse genetics is an important method to study the genomic structure and function of PEDV,viral host interaction and pathogenic mechanism,as well as a reasonable and effective way to design a live attenuated PEDV vaccine.