A novel toolbox for the in vitro assay of hepatitis D virus infection.

Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of hepatitis B virus (HBV) for its life cycle. The in vitro HDV infection system is widely used as a surrogate model to study cellular infection with both viruses owing to its practical feasibility. However, previous methods for running this system were less efficient for high-throughput screening and large-scale studies. Here, we developed a novel method for the production of infectious HDV by adenoviral vector (AdV)-mediated transduction. We demonstrated that the AdV-based method yields 10-fold higher viral titers than the transient-transfection approach. The HDV-containing supernatant derived from AdV-infected Huh7 cells can be used as the inoculum in infectivity assays without requiring further concentration prior to use. Furthermore, we devloped a chemiluminescent immunoassay (HDV-CLEIA) to quantitatively determine intracellular HDAg with a dynamic range of 5–11,000 pg/mL. HDV-CLEIA can be used as an alternative approach to assess HDV infection. The advantages of our updated methodology were demonstrated through in vitro HDV infection of HepaRG cells and by evaluating the neutralization activity using antibodies that target various regions of the HBV/HDV envelope proteins. Together, the methods presented here comprise a novel toolbox of in vitro assays for studying HDV infection.

丁型肝炎病毒（HDV）是一种缺陷型RNA病毒，其生命周期需要乙型肝炎病毒（HBV）的存在。由于其实用可行性，体外HDV感染系统被广泛用作研究这两种病毒细胞感染的替代模型。然而，以往运行该系统的方法在高通量筛选和大规模研究方面效率较低。在此，我们开发了一种通过腺病毒载体（AdV）介导的转导来产生感染性HDV的新方法。我们证明，基于AdV的方法所产生的病毒滴度比瞬时转染方法高10倍。来自AdV感染的Huh7细胞的含HDV上清液可在感染性测定中用作接种物，使用前无需进一步浓缩。此外，我们开发了一种化学发光免疫分析（HDV - CLEIA），用于定量测定细胞内的丁型肝炎病毒抗原（HDAg），其动态范围为5 - 11000 pg/mL。HDV - CLEIA可作为评估HDV感染的一种替代方法。我们更新的方法的优势通过HepaRG细胞的体外HDV感染以及使用针对HBV/HDV包膜蛋白不同区域的抗体评估中和活性得以证明。总之，这里介绍的方法构成了一个用于研究HDV感染的新型体外检测工具包。