Virginia-Maryland College of Veterinary Medicine, Department of Veterinary Medicine, University of Maryland, College Park, MD 20740, USA.
Poultry Diseases Department, Faculty of Veterinary Medicine, Cairo University, Giza 12211, Egypt.
J Gen Virol. 2022 Jun;103(6). doi: 10.1099/jgv.0.001761.
Newcastle disease virus (NDV) has been extensively explored as a vector for vaccine and oncolytic therapeutic development. In conventional NDV-based vectors, the transgene is arranged as a separate transcription unit in the NDV genome. Here, we expressed haemagglutinin protein (HA) of an avian influenza virus using an NDV vector design in which the transgene ORF is encoded in-frame with the ORF of an NDV gene. This arrangement does not increase the number of transcription units in the NDV genome, and imposes a selection pressure against mutations interrupting the transgene ORF. We placed the HA ORF upstream or downstream of N, M, F and HN ORFs of NDV so that both proteins are encoded in-frame and are separated by either a self-cleaving 2A peptide, furin cleavage site or both. Only constructs in which HA was placed downstream of the NDV HN were viable. These constructs expressed the transgene at a higher level compared to the vector encoding the same transgene in the same position in the NDV genome but as a separate transcription unit. Furthermore, the transgene expressed in one ORF with the NDV protein proved to be more stable over multiple passages. Thus, this design may be useful for applications where the stability of the transgene expression is highly important for a recombinant NDV vector.
新城疫病毒（NDV）已被广泛探索作为疫苗和溶瘤治疗开发的载体。在传统的基于 NDV 的载体中，转基因被排列为 NDV 基因组中的一个单独转录单元。在这里，我们使用 NDV 载体设计表达了禽流感病毒的血凝素蛋白（HA），其中转基因 ORF 与 NDV 基因的 ORF 框内编码。这种排列不会增加 NDV 基因组中转录单元的数量，并对中断转基因 ORF 的突变施加选择压力。我们将 HA ORF 置于 NDV 的 N、M、F 和 HN ORFs 的上游或下游，以便两种蛋白都以框内方式编码，并通过自我切割 2A 肽、弗林切割位点或两者分开。只有将 HA 置于 NDV HN 下游的构建体才具有活力。与在 NDV 基因组中相同位置编码相同转基因的载体相比，这些构建体以更高的水平表达了转基因，但作为单独的转录单元。此外，与在 NDV 蛋白中的同一位置编码相同转基因的载体相比，在一个 ORF 中表达的转基因在多次传代中更稳定。因此，这种设计可能对重组 NDV 载体中转基因表达的稳定性非常重要的应用非常有用。
