The Molecular Basis On The Pathogenicity Of Tembusu Viruses In Ducks And Mice

Since 2010,Novel duck Tembusu virus?TMUV?disease has spread widely in China,causing huge economic losses in the duck breeding industry.DTMUV,like other zoonotic mosquito-borne flaviviruses such as Japanese encephalitis virus?JEV?and West Nile virus?WNV?,can be transmitted vector-independently.To date,why these flaviviruses can be transmitted without mosquito vectors remains poorly understood.The recent duck-origin TMUV FX2010 replicated systemically and was transmitted efficiently in ducks while the early mosquito-origin TMUV MM1775 displayed limited replication and could not be transmitted directly among birds.To explore the key molecular basis of flavivirus transmissibility,two chimeric viruses,MM/FXE containing the FX2010 E protein in the MM1775 background and FX/MME containing the MM1775 E protein in the FX2010 background,were rescued by mutually repaced the envelope?E?protein of FX2010 and MM1775 by using TMUV reverse genetic technology and DF-1 cells were infected with chimeric or parental viruses,respectively.Viral titers of infected-cell supernatants were determined every 12 hours post infection.We found that the E protein-exchanged chimeric viruses replicated similarly with their parental viruses on DF-1 cells but quite different in ducks.Compared to parental FX2010,FX/MME was detected in spleens,kidneys and ovaries,but not in the lungs of ducks inoculated intramuscularly?i.m.?at 3 days post inoculation?d.p.i?.On the contrary,the MM/FXE with FX2010 E protein obtained the ability to replicate in the lungs and kidneys of i.m.inoculated ducks in contrast to its parental MM1775.These data indicated E protein influence TMUV replication in ducks.To further confirm transmissibility of chimeric viruses,sero-conversion of contact ducks was determined by an enzyme linked immunosorbent assay?ELISA?.The sera of contact ducks in either FX2010 or MM/FXE groups were positive to TMUV at tested dates,while the sera of contact ducks in either MM1775 or FX/MME groups were negative to TMUV even at 14 days post contact?d.p.c.?,which suggested E protein plays a key role in TMUV transmissibility.Furthermore,E protein was divided into two parts:domain I and II?DI&II?and domain III and transmembrane helix?DIII&Th?,and corresponding chimeric viruses were rescued.Duck experments showed that DI&II was crucial for the replication and transmissibility in ducks.DI of the E protein was proved to be responsible for efficient transmissibility of tissue tropisms of TMUVs in the following studies.There are 9 different amino acids in DI of E protein between FX2010 and MM1775,nine mutant viruses with single amino acid change in the background of FX2010 were rescued and further characterized in ducks.E_S156P156P mutation restricted TMUV replication and plays a critical role in virus tropism in ducks.By western blotting assay,it was proved that E_S156P mutation demolished the N-linked glycosylation at position 154.Analyses of available E protein sequences of different flaviviruses show that most of viruses contain N154?or N153?glycosylation sites,suggesting that this glycosylation site might be important for flaviviruses in transmission in natural bird hosts.Our previous work found that MM1775 and FX2010 show completely different pathogenicity in mice and in ducks.MM1775 was lethal to mice inoculated intranasally?i.n.?,in contrast,FX2010 was non-pathogenic to i.n.inoculated mice.In order to study the molecular basis of different pathogenicity in mice,E gene-exchanged chimeric virus MM/FXE and FX/MME were rescued and studied in mice.MM/FXE,lost the lethality to i.n.inoculated mice,whereas FX/MME obtained the lethality to i.n.inoculated mice,which indicated that E protein is critical to the pathogenicity of TMUVs in mice.To map which part of TMUV E protein is critical for pathogenicity difference,the E protein was divided into four parts:domain I?DI?,domain II?DII?,domain III?DIII?and Transmembrane helix?Th?,and 8chimeric viruses were rescued,in the background of either MM1775 or FX2010,respectively,and characterized in mice.Compared to MM1775,MM/FXE-DI and MM/FXE-DII was not detected in mice at either 4 or 6 d.p.i.and mice in these two groups survived.MM/FXE-DIII and MM/FXE-Th,as similar as their parental virus MM1775,could be detected in brains of i.n.inoculated mice at both 4d.p.i and 6 d.p.i,and kept the lethality to mice.Among four chimeric viruses on the backbone of FX2010,only FX/MME-DI could replicate in brain of mice and showed lethality to i.n.inoculated mice.FX/MME-DII,FX/MME-DIII and FX/MME-Th,were similar to thier parental virus FX2010,caused no disease symptoms in i.n.inoculated mice.The data indicated DI of E protein is the critical domain for TMUV virulence in mice.To determine which amino acids contribute to TMUV virulence in mice.Nine single amino acid muatant viruses on the backbone of MM1775,were rescued and characterized in mice.Mutation of valine to alanine at position 149?V149A?,mutation of proline to serine at position 156?P156S?,and mutation of glycine to glutamic acid at position 181?G181E?could decrease the virulence in i.n.inoculated mice,respectively.Low titers of MM-E_V149A and MM-E_P156S156S could be detected in one of i.n.inoculated mouse at 4 and 6 d.p.i.,respectively.The surviving rate of mice inoculated with MM-E_V149A and MM-E_P156S156S was 80%and 100%,respectively.MM-E_G181E was not detected in the brain if i.n.inoculated mice and all mice inoculated with MM-E_G181E survived and did not show any clinical symptoms.Further studies showed that V149A and G181E mutations influence the virus replication in the brain of the mice inoculated intracerebrally?i.c.?.Compared to the parental virus MM1775,the median lethal dose(MLD₅₀)of both V149A and G181E mutant viruses increased more than 30-fold in5-week-old mice inoculated i.c..However,the replication of P156S mutant virus was only restricted in brain during early period post i.c.inoculation,the MLD₅₀ of MM-E_P156S and MM1775 were equal.We found the molecular basis of Tembusu virus in tissue tropism and transmissibility in ducks and provided a virus model for the studies on vector-independent transmission of flaviviruses among animals.In addition,the molecular basis on TMUV pathogenicity in mice established a foundation for further study of pathogenesis mechanism of TMUV in mammals.