Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Switzerland.
Division of Neurological Sciences, Vetsuisse Faculty, University of Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
Virus Res. 2022 Jul 15;316:198796. doi: 10.1016/j.virusres.2022.198796. Epub 2022 May 11.
To provide insights into the biology of the attenuated canine distemper virus (CDV) Onderstepoort (OP) strain (large plaque forming variant), design next-generation multivalent vaccines, or further investigate its promising potential as an oncolytic vector, we employed contemporary modifications to establish an efficient OP-CDV-based reverse genetics platform. Successful viral rescue was obtained however only upon recovery of a completely conserved charged residue (V13E) residing at the N-terminal region of the large protein (L). Although L-V13 and L-V13E did not display drastic differences in cellular localization and physical interaction with P, efficient polymerase complex (P+ L) activity was recorded only with L-V13E. Interestingly, grafting mNeonGreen to the viral N protein via a P2A ribosomal skipping sequence (OP) and its derivative V-protein-knockout variant (OP-V) exhibited delayed replication kinetics in cultured cells. Collectively, we established an efficient OP-CDV-based reverse genetics system that enables the design of various strategies potentially contributing to veterinary medicine and research.
为了深入了解减毒犬瘟热病毒（CDV） Onderstepoort（OP）株（大斑块形成变体）的生物学特性，设计下一代多价疫苗，或进一步探索其作为溶瘤病毒载体的有前途的潜力，我们采用了当代的修饰方法来建立高效的基于 OP-CDV 的反向遗传学平台。然而，只有在恢复位于大蛋白（L）N 端区域的完全保守带电残基（V13E）时，才能成功挽救病毒。尽管 L-V13 和 L-V13E 在细胞定位和与 P 的物理相互作用方面没有明显差异，但只有 L-V13E 才能记录到有效的聚合酶复合物（P+L）活性。有趣的是，通过 P2A 核糖体跳跃序列（OP）将 mNeonGreen 嫁接至病毒 N 蛋白及其衍生的 V 蛋白敲除变体（OP-V），在培养细胞中的复制动力学延迟。总之，我们建立了一种高效的基于 OP-CDV 的反向遗传学系统，该系统可用于设计各种策略，可能有助于兽医医学和研究。
