State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Life Science &School of Public Health, Xiamen University, Xiamen 361102, PR China.
National Institute of Diagnostics and Vaccine Development in Infectious Diseases, School of Life Science &School of Public Health, Xiamen University, Xiamen 361102, PR China.
Sci Rep. 2017 Jan 12;7:40199. doi: 10.1038/srep40199.
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。由于其实际可行性，体外 HDV 感染系统被广泛用作研究两种病毒的细胞感染的替代模型。然而，以前运行该系统的方法对于高通量筛选和大规模研究效率较低。在这里，我们开发了一种通过腺病毒载体（AdV）介导转导生产感染性 HDV 的新方法。我们证明，基于 AdV 的方法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 的上清液可作为感染性测定中的接种物使用，而无需在使用前进一步浓缩。此外，我们开发了一种化学发光免疫测定法（HDV-CLEIA），以定量测定 5-11,000pg/mL 动态范围内的细胞内 HDAg。HDV-CLEIA 可作为评估 HDV 感染的替代方法。通过 HepaRG 细胞的体外 HDV 感染以及使用针对 HBV/HDV 包膜蛋白各个区域的抗体评估中和活性，证明了我们更新的方法的优势。总之，这里提出的方法构成了研究 HDV 感染的新型体外检测工具箱。
