State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, No. 2 Yuanmingyuan West Road, Beijing, 100193, China.
School of Medicine, The Chinese University of Hong Kong, Shenzhen (CUHK-Shenzhen), Shenzhen, 518172, Guangdong, People's Republic of China.
Virus Genes. 2023 Aug;59(4):572-581. doi: 10.1007/s11262-023-01999-9. Epub 2023 Apr 27.
Newcastle disease (ND) is the most important infectious disease in poultry, which is caused by avian orthoavulavirus type 1 (AOAV-1), previously known as Newcastle disease virus (NDV). In this study, an NDV strain SD19 (GenBank accession number OP797800) was isolated, and phylogenetic analysis suggested the virus belongs to the class II genotype VII. After generating wild-type rescued SD19 (rSD19), the attenuating strain (raSD19) was generated by mutating the F protein cleavage site. To explore the potential role of the transmembrane protease, serine S1 member 2 (TMPRSS2), the TMPRSS2 gene was inserted into the region between the P and M genes of raSD19 to generate raSD19-TMPRSS2. Besides, the coding sequence of the enhanced green fluorescent protein (EGFP) gene was inserted in the same region as a control (rSD19-EGFP and raSD19-EGFP). The Western blot, indirect immunofluorescence assay (IFA), and real-time quantitative PCR were employed to determine the replication activity of these constructs. The results reveal that all the rescued viruses can replicate in chicken embryo fibroblast (DF-1) cells; however, the proliferation of raSD19 and raSD19-EGFP needs additional trypsin. We next evaluated the virulence of these constructs, and our results reveal that the SD19, rSD19, and rSD19-EGFP are velogenic; the raSD19 and raSD19-EGFP are lentogenic; and the raSD19-TMPRSS2 are mesogenic. Moreover, due to the enzymatic hydrolysis of serine protease, the raSD19-TMPRSS2 can support itself to proliferate in the DF-1 cells without adding exogenous trypsin. These results may provide a new method for the NDV cell culture and contribute to ND's vaccine development.
新城疫（ND）是家禽中最重要的传染病，由禽正黏病毒 1 型（AOAV-1）引起，以前称为新城疫病毒（NDV）。本研究分离出一株 NDV 株 SD19（GenBank 登录号 OP797800），系统进化分析表明该病毒属于 II 类基因型 VII。生成野生型拯救株 SD19（rSD19）后，通过突变 F 蛋白裂解位点生成减毒株（raSD19）。为了探索跨膜蛋白酶丝氨酸 S1 成员 2（TMPRSS2）的潜在作用，将 TMPRSS2 基因插入 raSD19 的 P 和 M 基因之间的区域，生成 raSD19-TMPRSS2。此外，将增强型绿色荧光蛋白（EGFP）基因的编码序列插入同一区域作为对照（rSD19-EGFP 和 raSD19-EGFP）。Western blot、间接免疫荧光法（IFA）和实时定量 PCR 用于确定这些构建体的复制活性。结果表明，所有拯救的病毒都可以在鸡胚成纤维细胞（DF-1）中复制；然而，raSD19 和 raSD19-EGFP 的增殖需要额外的胰酶。我们接下来评估了这些构建体的毒力，结果表明 SD19、rSD19 和 rSD19-EGFP 是强毒力的；raSD19 和 raSD19-EGFP 是弱毒力的；raSD19-TMPRSS2 是中毒力的。此外，由于丝氨酸蛋白酶的酶解作用，raSD19-TMPRSS2 可以在没有添加外源性胰酶的情况下自行在 DF-1 细胞中增殖。这些结果可能为 NDV 细胞培养提供新方法，并有助于 ND 疫苗的开发。
