Basic Amino Acid Substitution at Residue 367 of the Envelope Protein of Tembusu Virus Plays a Critical Role in Pathogenesis

Abstract

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 in vitro 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 in vitro and in vivo infection of TMUV. IMPORTANCE 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.

Genome Analysis of a Novel Tembusu Virus in Taiwan

“…Except for TMUV-MM1775, all the other TMUVs, including TMUV-TP1906, contain 156-Ser which would not disrupt 154-Asn glycosylation and thus may not affect the glycosylation-associated infectivity of TMUVs in avian species. A recent study showed that the Thr to Lys mutation at residue 367 of the E protein plays a predominant role in viral cell-adaptation and virulence attenuation in ducks [25]. TMUV-TP1906 contains 367-Thr of the E protein and thus may retain the virulence in the host.…”

Sexual Transmission of Arboviruses: A Systematic Review

“…Naïve female mice became infected with TBEV after mating with infected males, with viral RNA detected in embryonal tissues of two of 11 litters [134]. Tembusu virus, a mosquito-transmitted pathogen of poultry, replicates in the ovaries and testes of infected birds [135][136][137][138].…”

Innate immune responses to duck Tembusu virus infection

“…Yan et al reported that a single mutation at amino acid residue 156 (S-P) reduced the ability of viral replication and transmission in ducks, and further analysis confirmed that the potential mechanism was composed by the disruption of N-linked glycosylation at position 154 and changes in the conformation of the “150 loop” of the E protein [ 51 ]. Recently, it has been found that the threonine-to-lysine mutation of residue 367 in E protein can attenuate DTMUV [ 52 ]. As research continues, the effects of other proteins on viral replication will be discovered.…”