Abstract
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 JXSP2-4 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 JXSP2-4, 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-310RdRp) in cells and ducklings were both markedly decreased compared with JXSP2-4, 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 JXSP2-4 in cells, but the viral loads in JXSP-R543K-infected ducklings were significantly lower than that of JXSP2-4 and higher than JXSP-310RdRp. 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.
Highlights
We found that R543K/V711A substitutions in the viral RNA-dependent RNA polymerase (RdRp) domain were crucially important for viral infectivity in ducklings
The data from our previous study suggested that amino acid substitutions in NS5 contributed to virulence attenuation of Tembusu virus (TMUV) JXSP2-4 [13]
The recombinant viruses were detected by immunofluorescence staining with a monoclonal antibody against the TMUV E protein (Figure 1B) and verified by genomic sequencing, resulting in JXSP-310MTase, JXSP-310Linker and JXSP-310RdRp chimeric viruses for the MTase, Linker and RdRp domains, respectively
Summary
Tembusu virus (TMUV) was first isolated from Culex tritaeniorhynchus mosquitoes in Malaysia in 1955 [1]. The large, single precursor polyprotein encoded by the viral genome is processed into three structural proteins: the capsid (C), the premembrane/membrane protein (prM), the envelope (E) and seven nonstructural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 [1,2,3]. TMUV was associated with the explosive epizootics that occurred in commercial duck flocks in China in 2010 and in Southeast Asian countries later on [4,5,6]. The virus is transmitted among susceptible duck flocks by multiple routes (e.g., mosquito bites, infectious aerosol inhalation and eating contaminated feed) and results in severe egg drop and fatal encephalitis [7,8,9]
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