人类丁型肝炎病毒(HDV)是一种卫星RNA病毒,它依赖于乙型肝炎病毒(HBV)表面蛋白(HBsAg)组装成针对HBV相同器官(肝脏)的感染性病毒粒子。直到最近,HDV的进化起源在很大程度上仍然未知。生物信息学在整个序列数据库中的应用导致了HDV样因子(DLA)的发现,并揭示了HDV的进化,扩大我们对HDV生物学的理解。在脊椎动物和无脊椎动物的异质群体中鉴定出DLA,强调HDV的演变,由八种不同的基因型代表,比以前预见到的更广泛、更复杂。在这项研究中,我们专注于在土拨鼠(Marmotamonax)中发现的三种哺乳动物DLA的表征,白尾鹿(Odocoileusvirginianus),和较小的狗一样的蝙蝠(peropteryxmacrotis)在复制方面,细胞类型的宽容,和传播途径。我们产生了表达每种DLA的1.1倍超长反基因组RNA的可复制构建体。通过将cDNA转染到人体内来启动复制(HuH7,HeLa,HEK293T,A549)和非人(VeroE6,CHO,PaKi,LMH)细胞系。转染和复制建立后,没有一个DLA表达大的δ抗原。细胞分裂介导的病毒扩增试验证明了非人DLA在肝和非肝组织中复制和繁殖的能力。不需要来自辅助病毒的包膜蛋白。值得注意的是,来自HDV的L-HDAg而不是S-HDAg可以通过HBsAg人工介导WoDV和DeDV核糖核蛋白(RNP)的包膜形成感染性颗粒,如通过将HuH7细胞与相应的DLA表达构建体和编码HBV包膜蛋白的质粒共转染所证明的。这些嵌合病毒对HDV进入抑制剂敏感,并且允许同步感染用于比较复制研究。我们的结果提供了对分子生物学的更详细的理解,进化,以及与HDV有关的这一独特的动物类病毒样物质组的病毒-宿主相互作用。
The human hepatitis delta virus (HDV) is a satellite RNA virus that depends on hepatitis B virus (HBV) surface proteins (HBsAg) to assemble into infectious virions targeting the same organ (liver) as HBV. Until recently, the evolutionary origin of HDV remained largely unknown. The application of bioinformatics on whole sequence databases lead to discoveries of HDV-like agents (DLA) and shed light on HDV\'s evolution, expanding our understanding of HDV biology. DLA were identified in heterogeneous groups of vertebrates and invertebrates, highlighting that the evolution of HDV, represented by eight distinct genotypes, is broader and more complex than previously foreseen. In this study, we focused on the characterization of three mammalian DLA discovered in woodchuck (Marmota monax), white-tailed deer (Odocoileus virginianus), and lesser dog-like bat (Peropteryx macrotis) in terms of replication, cell-type permissiveness, and spreading pathways. We generated replication-competent constructs expressing 1.1-fold over-length antigenomic RNA of each DLA. Replication was initiated by transfecting the cDNAs into human (HuH7, HeLa, HEK293T, A549) and non-human (Vero E6, CHO, PaKi, LMH) cell lines. Upon transfection and replication establishment, none of the DLA expressed a large delta antigen. A cell division-mediated viral amplification assay demonstrated the capability of non-human DLA to replicate and propagate in hepatic and non-hepatic tissues, without the requirement of envelope proteins from a helper virus. Remarkably L-HDAg but not S-HDAg from HDV can artificially mediate envelopment of WoDV and DeDV ribonucleoproteins (RNPs) by HBsAg to form infectious particles, as demonstrated by co-transfection of HuH7 cells with the respective DLA expression constructs and a plasmid encoding HBV envelope proteins. These chimeric viruses are sensitive to HDV entry inhibitors and allow synchronized infections for comparative replication studies. Our results provide a more detailed understanding of the molecular biology, evolution, and virus-host interaction of this unique group of animal viroid-like agents in relation to HDV.