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Amino Acid Substitutions in NS5 Contribute Differentially to Tembusu Virus Attenuation in Ducklings and Cell Cultures

文献类型： 外文期刊

作者： Sun, Xue 1 ; Sun, Mengxu 1 ; Zhang, Lijiao 2 ; Yu, Ziding 1 ; Li, Jinxin 1 ; Xie, Wanying 1 ; Su, Jingliang 1 ;

作者机构： 1.China Agr Univ, Minist Agr, Coll Vet Med, Key Lab Anim Epidemiol & Zoonosis, Beijing 100193, Peoples R China

2.Jiangsu Acad Agr Sci, Inst Vet Med, Nanjing 210014, Peoples R China

关键词： Tembusu virus; attenuation; RNA-dependent RNA polymerase; chimeric viruses; viral replication

期刊名称：VIRUSES-BASEL （ 影响因子：3.816； 五年影响因子：4.001 ）

ISSN：

年卷期： 2021 年 13 卷 5 期

页码：

收录情况： SCI

摘要： 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(2-4) 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(2-4), 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(RdRp)) in cells and ducklings were both markedly decreased compared with JXSP(2-4), 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(2-4) in cells, but the viral loads in JXSP-R543K-infected ducklings were significantly lower than that of JXSP(2-4) and higher than JXSP-310(RdRp). 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.

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