Key Laboratory of Animal Epidemiology and Zoonosis, The Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China.
Viruses. 2021 May 16;13(5):921. doi: 10.3390/v13050921.
Tembusu virus (TMUV), a highly infectious pathogenic flavivirus, causes severe egg-drop and encephalitis in domestic waterfowl, while the determinants responsible for viral pathogenicity are largely unknown. In our previous studies, virulent strain JXSP had been completely attenuated by successive passages in BHK-21 cells and the avirulent strain was designated as JXSP-310. Based on the backbone of JXSP, a series of chimeric viruses were generated according to the amino acid substitutions in NS5 and their infectivities were also analyzed in cell cultures and ducklings. The results showed that the viral titers of RNA-dependent RNA polymerase (RdRp) domain-swapped cheimeric mutant (JXSP-310) in cells and ducklings were both markedly decreased compared with JXSP, indicating that mutations in the RdRp domain affected viral replication. There are R543K and V711A two amino acid substitutions in the RdRp domain. Further site-directed mutagenesis showed that single-point R543K mutant (JXSP-R543K) exhibited similar replication efficacy compared with JXSP in cells, but the viral loads in JXSP-R543K-infected ducklings were significantly lower than that of JXSP and higher than JXSP-310. Surprisingly, the single-point V711A mutation we introduced rapidly reverted. In addition, qRT-PCR and Western blot confirmed that the mutations in the RdRp domain significantly affected the replication of the virus. Taken together, these results show that R543K substitution in the RdRp domain impairs the in vivo growth of TMUV, but sustaining its attenuated infectivity requires the concurrent presence of the V711A mutation.
腾布苏病毒（TMUV）是一种高度传染性的致病性黄病毒，可导致家禽产蛋量严重下降和脑炎，而导致病毒致病性的决定因素在很大程度上尚不清楚。在我们之前的研究中，通过在 BHK-21 细胞中连续传代，强毒株 JXSP 已完全减毒，而无毒株则被命名为 JXSP-310。基于 JXSP 的骨架，根据 NS5 中的氨基酸替换生成了一系列嵌合病毒，并在细胞培养物和雏鸭中分析了它们的感染性。结果表明，与 JXSP 相比，RNA 依赖性 RNA 聚合酶（RdRp）结构域交换嵌合突变体（JXSP-310）在细胞和雏鸭中的病毒滴度均明显降低，表明 RdRp 结构域中的突变影响了病毒复制。RdRp 结构域中有两个氨基酸替换 R543K 和 V711A。进一步的定点诱变表明，单点 R543K 突变体（JXSP-R543K）在细胞中的复制效率与 JXSP 相似，但 JXSP-R543K 感染的雏鸭中的病毒载量明显低于 JXSP，高于 JXSP-310。令人惊讶的是，我们引入的单点 V711A 突变迅速回复。此外，qRT-PCR 和 Western blot 证实 RdRp 结构域中的突变显著影响了病毒的复制。总之，这些结果表明 RdRp 结构域中的 R543K 替换削弱了 TMUV 的体内生长，但维持其减毒感染性需要同时存在 V711A 突变。
