Substantial Attenuation of Virulence of Tembusu Virus Strain PS Is Determined by an Arginine at Residue 304 of the Envelope Protein

Abstract

The Tembusu virus (TMUV) PS strain, derived by several passages and plaque purifications in BHK-21 cells, displays remarkedly lower virulence in Pekin ducklings relative to a natural isolate of TMUV, but the potential virulence determinants and the in vivo mechanisms for substantial virulence attenuation of the passage variant remain unknown. Here, we constructed a series of chimeric and mutant viruses and assessed their virulence using a 2-day-old Pekin duckling model. We showed that residue 304 in the envelope (E) protein is the molecular determinant of TMUV virulence. Further investigations with mutant and parental viruses demonstrated that acquisition of positive charges at E protein residue 304 plays a critical role in substantial attenuation of neurovirulence and neuroinvasiveness, which is linked to enhanced binding affinity for glycosaminoglycans (GAG). In Pekin ducklings infected by subcutaneous inoculation, an Arg at residue 304 in the E protein was shown to contribute to more rapid virus clearance from the circulation, remarkedly reduced viremia, and significantly decreased viral growth in the extraneural tissues and the central neural system, relative to a Met at the corresponding residue. These findings suggest that in vivo mechanism of virulence attenuation of the TMUV passage variant closely resembles that proposed previously for GAG-binding variants of other flaviviruses. Overall, our study provides insight into the molecular basis of TMUV virulence and the in vivo consequences of acquisition of a GAG-binding determinant at residue 304 in the E protein of TMUV.IMPORTANCE TMUV related disease emerged in 2010 has a significant economic impact on the duck industry. Although the disease was originally recognized to affect adult ducks, increasing evidences have shown that TMUV also causes severe disease of young ducklings. It is, therefore, essential to investigate the pathogenesis of TMUV infection in a young duckling model. The significance of our studies is in identifying E protein residue Arg304 as the molecular determinant for TMUV virulence and in clarifying the crucial role of positive charges at E protein residue 304 in virulence attenuation of TMUV passage variant. These data will greatly enhance our understanding of the pathogenesis of TMUV infection in ducklings, and have implications for development of a safe and efficient vaccine.

The key amino acids of E protein involved in early flavivirus infection: viral entry

“…These assays demonstrated that the K291 and K295 residues are important for viral binding in human cell lines but not in insect cell lines [ 62 ]. The passage of an attenuated strain of TMUV shows that E-304 is very important to the neurovirulence and neuroinvasiveness of TMUV, and the charged condition of this amino acid plays a key role in the binding affinity between the E protein and GAGs; another study found a similar situation at the E-367 residue [ 59 , 60 ].…”

The difference in CD4+ T cell immunity between high- and low-virulence Tembusu viruses is mainly related to residues 151 and 304 in the envelope protein

“…The BHK-21 cell-derived Y and PS strains of TMUV were isolated and propagated previously, which were shown to display high- and low-virulence for 2-day-old Pekin ducklings respectively ( 11 ). Parental backbone viruses rY and rPS were rescued from the full-length cDNAs of Y and PS respectively.…”

“…To date, the role of T cells in protection from TMUV infection and the molecular basis of T cell-mediated protection against TMUV remain largely uncharacterized. Earlier works in our laboratory showed that the natural isolate Y and the plaque-purified strain PS, which exhibited distinct virulence in a 2-day-old Pekin duckling model, elicited comparable levels of neutralizing antibody ( 11 ). In this study, we describe the comparative studies on cellular immune responses to Y and PS.…”

New Insights into the Biology of the Emerging Tembusu Virus

“…Recently, the team of Yang et al worked with a variant of TMUV which developed attenuated virulence after several passages. Their work shed new light on the development of an effective vaccine against TMUV infection, determining that residue 304 of the E protein was essential for virulence and cell attachment [ 139 ]. However, despite the interest of these candidate vaccines, further investigations are needed to optimize certain aspects of vaccination such as the route of inoculation, the optimal number of inoculations to achieve the correct immunity or possible interactions with other animal vaccination programs.…”