Gudima Severin, He Yiping, Meier Anja, Chang Jinhong, Chen Rongji, Jarnik Michal, Nicolas Emmanuelle, Bruss Volker, Taylor John
Fox Chase Cancer Center, 333 Cottman Avenue, Philadelphia, PA 19111-2497, USA.
J Virol. 2007 Apr;81(7):3608-17. doi: 10.1128/JVI.02277-06. Epub 2007 Jan 17.
Efficient assembly of hepatitis delta virus (HDV) was achieved by cotransfection of Huh7 cells with two plasmids: one to provide expression of the large, middle, and small envelope proteins of hepatitis B virus (HBV), the natural helper of HDV, and another to initiate replication of the HDV RNA genome. HDV released into the media was assayed for HDV RNA and HBV envelope proteins and characterized by rate-zonal sedimentation, immunoaffinity purification, electron microscopy, and the ability to infect primary human hepatocytes. Among the novel findings were that (i) immunostaining for delta antigen 6 days after infection with 300 genome equivalents (GE) per cell showed only 1% of cells as infected, but this was increased to 16% when 5% polyethylene glycol was present during infection; (ii) uninfected cells did not differ from infected cells in terms of albumin accumulation or the presence of E-cadherin at cell junctions; and (iii) sensitive quantitative real-time PCR assays detected HDV replication even when the multiplicity of infection was 0.2 GE/cell. In the future, this HDV assembly and infection system can be further developed to better understand the mechanisms shared by HBV and HDV for attachment and entry into host cells.
通过将两种质粒共转染Huh7细胞实现了丁型肝炎病毒（HDV）的高效组装：一种质粒用于表达HDV的天然辅助病毒乙型肝炎病毒（HBV）的大、中、小包膜蛋白，另一种质粒用于启动HDV RNA基因组的复制。对释放到培养基中的HDV进行HDV RNA和HBV包膜蛋白检测，并通过速率区带沉降、免疫亲和纯化、电子显微镜以及感染原代人肝细胞的能力进行表征。其中的新发现包括：（i）每细胞感染300个基因组当量（GE）后6天对δ抗原进行免疫染色，结果显示只有1%的细胞被感染，但在感染期间存在5%聚乙二醇时，这一比例增加到了16%；（ii）未感染细胞在白蛋白积累或细胞连接处E-钙黏蛋白的存在方面与感染细胞没有差异；（iii）即使感染复数为0.2 GE/细胞，灵敏的定量实时PCR检测也能检测到HDV复制。未来，这种HDV组装和感染系统可进一步开发，以更好地理解HBV和HDV在附着和进入宿主细胞方面共有的机制。
