Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing, China.
J Virol. 2020 Mar 31;94(8). doi: 10.1128/JVI.02011-19.
Tembusu virus (TMUV) is a flavivirus responsible for panzootic outbreaks of severe egg-drop and fatal encephalitis of domestic waterfowl in China. Although TMUV can be attenuated by passaging, experimental evidence supporting the role of specific genetic changes in virulence attenuation is currently lacking. Here, we performed site-directed mutagenesis on five envelope (E) protein amino acid residues in accordance with the attenuated TMUV generated in our recent study. Our results showed that the Thr-to-Lys mutation of residue 367 in E protein (E367) plays a predominant role in viral cell adaptation and virulence attenuation in ducks compared with mutations in other residues. We further demonstrated that the positively charged basic amino acid substitution at E367 enhanced the viral binding affinity for glycosaminoglycans (GAGs) and reduced viremia levels and the efficiency of replication in major target organs in subcutaneously inoculated ducks. Interestingly, the T367K mutation increased viral neutralization sensitivity to the early immune sera. Together, our findings provide the first evidence that a basic amino acid substitution at E367 strongly impacts the and infection of TMUV. Outbreaks of Tembusu virus (TMUV) infection have caused huge economic losses in the production of domestic waterfowl since the virus was first recognized in China in 2010. To control TMUV infection, a live-attenuated vaccine candidate of TMUV was developed in our previous study, but the mechanisms of virulence attenuation are not fully understood. Here, we found that the Thr-to-Lys substitution at E367 is a crucial determinant of TMUV virulence attenuation in ducks. We demonstrated that the T367K mutation attenuates TMUV through reducing viral replication in the blood, brain, heart (ducklings), and ovaries. These data provide new insights into understanding the pathogenesis of TMUV and the rational development of novel TMUV vaccines.
坦布苏病毒（TMUV）是一种黄病毒，可引起中国家禽严重产蛋下降和致命脑炎的大流行暴发。尽管 TMUV 可以通过传代而减毒，但目前缺乏支持特定遗传变化在毒力减毒中作用的实验证据。在这里，我们根据我们最近的研究中生成的减毒 TMUV，对 5 个包膜（E）蛋白氨基酸残基进行了定点突变。我们的结果表明，E 蛋白残基 367 位的苏氨酸到赖氨酸突变（E367）在鸭中比其他残基的突变更能促进病毒细胞适应和毒力衰减。我们进一步证明，E367 处的正电荷碱性氨基酸取代增强了病毒对糖胺聚糖（GAGs）的结合亲和力，并降低了皮下接种鸭中的病毒血症水平和主要靶器官中的复制效率。有趣的是，T367K 突变增加了病毒对早期免疫血清的中和敏感性。总之，我们的研究结果首次证明，E367 处的碱性氨基酸取代强烈影响 TMUV 的 和 感染。自 2010 年在中国首次发现坦布苏病毒（TMUV）感染以来，该病毒的暴发已给家禽生产造成了巨大的经济损失。为了控制 TMUV 感染，我们在之前的研究中开发了 TMUV 的活疫苗候选株，但病毒毒力衰减的机制尚不完全清楚。在这里，我们发现 E367 处的苏氨酸到赖氨酸取代是 TMUV 在鸭中致弱的关键决定因素。我们证明，T367K 突变通过减少血液、大脑、心脏（雏鸭）和卵巢中的病毒复制来减弱 TMUV。这些数据为深入了解 TMUV 的发病机制和合理开发新型 TMUV 疫苗提供了新的见解。
