Paneth Iheozor-Ejiofor Rommel, Levanov Lev, Hepojoki Jussi, Strandin Tomas, Lundkvist Åke, Plyusnin Alexander, Vapalahti Olli
Department of Virology, Medicum, Helsinki, Finland.
Department of Medical Biochemistry and Microbiology, Microbiology-Immunology, Uppsala University, Sweden.
J Gen Virol. 2016 May;97(5):1052-1059. doi: 10.1099/jgv.0.000437. Epub 2016 Feb 23.
Puumala virus (PUUV) grows slowly in cell culture. To study antigenic properties of PUUV, an amenable method for their expression would be beneficial. To achieve this, a replication-defective recombinant vesicular stomatitis virus, rVSVΔG*EGFP, was rescued using BSRT7/5 and encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES)-enabled rescue plasmids. Using these particles, pseudotypes bearing PUUV Sotkamo strain glycoproteins were produced, with titres in the range 105-108, and were used in pseudotype focus reduction neutralization tests (pFRNTs) with neutralizing monoclonal antibodies and patient sera. The results were compared with those from orthodox focus reduction neutralization tests (oFRNTs) using native PUUV with the same samples and showed a strong positive correlation (rs = 0.82) between the methods. While developing the system we identified three amino acids which were mutated in the Vero E6 cell culture adapted PUUV prototype Sotkamo strain sequence, and changing these residues was critical for expression and neutralizing antibody binding of PUUV glycoproteins.
普马拉病毒（PUUV）在细胞培养中生长缓慢。为了研究PUUV的抗原特性，一种便于其表达的方法将是有益的。为实现这一目标，使用BSRT7/5和带有脑心肌炎病毒（EMCV）内部核糖体进入位点（IRES）的拯救质粒拯救了一种复制缺陷型重组水疱性口炎病毒rVSVΔG*EGFP。利用这些病毒颗粒，制备了携带PUUV索特卡莫株糖蛋白的假型病毒，滴度在105 - 108范围内，并将其用于与中和单克隆抗体和患者血清的假型焦点减少中和试验（pFRNTs）。将结果与使用相同样本的天然PUUV进行的传统焦点减少中和试验（oFRNTs）的结果进行比较，结果显示两种方法之间存在强正相关（rs = 0.82）。在开发该系统的过程中，我们鉴定出在适应Vero E6细胞培养的PUUV原型索特卡莫株序列中发生突变的三个氨基酸，改变这些残基对于PUUV糖蛋白的表达和中和抗体结合至关重要。
