Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China; Institute of Preventive Veterinary Medicine, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Wenjiang District, Chengdu City, Sichuan Province, 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Wenjiang District, Chengdu City, Sichuan Province, 611130, China.
Vet Microbiol. 2021 Oct;261:109215. doi: 10.1016/j.vetmic.2021.109215. Epub 2021 Aug 25.
Duck Tembusu virus (DTMUV) is an emerging mosquito-borne flavivirus that has caused acute egg-drop syndrome in egg-laying ducks. DTMUV nonstructural protein 1 (NS1) contains three potential predicted N-linked glycosylation sites at residues 130, 175 and 207. In this study, we found that mutations at these sites affect the molecular weight of recombinant NS1, as assessed by western blot assays; however, the mutations do not affect their subcellular localization in the cytoplasm, as assessed by colocalization assays. Four recombinant viruses substituting the asparagine (N) residues at N130, N175, N207 or N130/N175/N207 of NS1 with alanine (A) residues were generated using rDTMUV-i, an infectious cDNA clone of the DTMUV CQW1 strain. Deglycosylation assays of the mutant virus NS1 were performed using endoglycosidases Endo H or PNGase F treatment in both mammalian and avian cells. The NS1-WT, NS1-N130A, NS1-N175A and NS1-N207A showed a shift in migration to 37 kDa after digestion with both endoglycosidases, which further confirmed that N130, N175 and N207 were the glycosylation sites of DTMUV NS1. Compared to the parental rDTMUV, the single mutants impaired viral multiplication in vitro, while the nonglycosylated virus rDTMUV-NS1-N130A/N175A/N207A showed a 5-fold to 178-fold decrease in viral titers and smaller plaque sizes. Notably, all mutant viruses were still highly virulent to duck embryos, but the embryos inoculated with rDTMUV-NS1-N130A/N175A/N207A started to die on the fourth day, which exhibited a prolonged time to death compared to that of rDTMUV. Moreover, rDTMUV-NS1-N130A/N175A/N207A was attenuated in vivo, showing no mortality and producing significantly lower viral titers in heart, spleen, kidney, brain and thymus as well as 2-fold to 3-fold lower viremia at 3 and 5 days post infection. Overall, our results indicated that N130, N175 and N207 are N-linked glycosylation sites of DTMUV NS1, which play crucial roles in viral multiplication, viremia and virulence in vitro and in vivo.
鸭坦布苏病毒（DTMUV）是一种新兴的蚊媒黄病毒，可导致产蛋鸭产蛋急性下降综合征。DTMUV 非结构蛋白 1（NS1）在残基 130、175 和 207 处含有三个潜在的预测 N 连接糖基化位点。在这项研究中，我们发现这些位点的突变会影响 NS1 的相对分子质量，这可以通过 Western blot 分析来评估；然而，通过共定位分析，这些突变不影响它们在细胞质中的亚细胞定位。使用 rDTMUV-i（鸭坦布苏病毒 CQW1 株的传染性 cDNA 克隆）生成了四个替代 NS1 中 N130、N175、N207 或 N130/N175/N207 处天冬酰胺（N）残基的丙氨酸（A）残基的重组病毒。使用哺乳动物和禽类细胞中的内切糖苷酶 Endo H 或 PNGase F 处理对突变病毒 NS1 进行了去糖基化分析。NS1-WT、NS1-N130A、NS1-N175A 和 NS1-N207A 在两种内切糖苷酶消化后迁移到 37 kDa 处发生变化，这进一步证实了 N130、N175 和 N207 是 DTMUV NS1 的糖基化位点。与亲本 rDTMUV 相比，单突变体在体外病毒繁殖中受到损害，而非糖基化病毒 rDTMUV-NS1-N130A/N175A/N207A 的病毒滴度降低了 5 到 178 倍，噬菌斑尺寸变小。值得注意的是，所有突变病毒对鸭胚仍具有高度致病性，但接种 rDTMUV-NS1-N130A/N175A/N207A 的胚胎在第四天开始死亡，与 rDTMUV 相比，死亡时间延长。此外，rDTMUV-NS1-N130A/N175A/N207A 在体内减毒，无死亡率，心脏、脾脏、肾脏、大脑和胸腺中的病毒滴度显著降低，感染后 3 天和 5 天的病毒血症低 2 到 3 倍。总体而言，我们的结果表明，N130、N175 和 N207 是 DTMUV NS1 的 N 连接糖基化位点，在病毒繁殖、病毒血症和体内外毒力中发挥着关键作用。
