Molecular Basis For Different Virulence Of Tembusu Viruses

Tembusu virus(TMUV)infection is an emerging infectious disease affecting ducks,which has a significant economic impact on Chinese duck industry.So far,data regarding the molecular pathogenesis of TMUV is lacking.To shed light on the possible amino acids and nucleotides associated with pathogenicity of TMUV for ducklings,we analyzed the genetic differences among TMUV isolates.Subsequently,we identified the virulence-associated genetic loci responsible for virulence difference between different TMUV strains.In October 2014,a disease characterized by leg paralysis occurred in two flocks of egg-type ducklings aged 3 to 4 weeks.The causative agent was identified as TMUV following detailed virological investigations including PCR-based assay,virus isolation,indirect immunofluorescent assay,and animal experiments.The clinical signs observed in the field were reproduced in 2-day-old Pekin ducklings by inoculation of the third passage of duck embryo-derived isolate(GL strain)by intracerebral route.All infected ducklings died at 3-6 days post inoculation.The investigations suggested that the TMUV isolated from ducklings exhibited high pathogenicity to Pekin ducklings.To investigate the virulence difference between different TMUV isolates,laboratory infection experiments were performed by intracerebral inoculation with the PS,Y,and GL isolates,employing 2-day-old Pekin ducklings as models.Ducklings infected with GL and Y strains exhibited severe symptoms and histopathological lesions with high mortalities(70%and 80%,respectively).Ducklings infected with PS strain had no death,the slower weight gain and mild histopathological lesion were observed.These investigations demonstrated that the virulence of GL and Y strains was significantly higher than that of PS strain.Following genome sequencing and sequence comparison,3 and 4 nucleotide substitutions in noncoding regions and 16 and 19 amino acid changes in the polyprotein were detected in strains Y and GL when compared with PS strain.These findings provide strong indications that the introduction of mutations in the genome may play a critical role in determining the pathogenicity of TMUV to Pekin ducklings.To identify virulence-associated genes of TMUV,we established the reverse genetic manipulation system of TMUV PS strain.Five cDNA fragments of strain PS were amplified by RT-PCR,and cloned into plasmids.Fragmented cDNAs were amplified and assembled by fusion PCR to produce a full-length cDNA using the recombinant plasmids as templates.Subsequently,a full-length RNA was transcribed from the full-length cDNA in vitro and transfected into BHK-21 cells;infectious viral particles were rescued successfully.The growth characteristics in BHK-21 cells and virulence in mice of the rescued virus were both similar to that of the parental virus.Furthermore,the rescued virus was shown to be genetically indistinguishable from the parental virus except the genetic markers.On the basis of the rescued PS strain,we constructed four chimeric viruses,namely rPSY5'UTR,rPSY3'UTR,rPSYE,and rPSYNS1-3'UTR,which contained the genome of 5'UTR,3'UTR,E,and NS1-3'UTR of Y strain,respectively.In the animal experiments,the rPSYE caused high(70%)mortality,similar to that of Y strain,indicating that virulence of PS strain increased significantly by replacement of its E gene with that of Y strain.Therefore,the E gene may play an important role in determining the virulence difference between PS and Y strains.Using site-directed mutagenesis technology,the residue Arg at position 304 in the E protein of PS strain was mutated to Met,which is present in position 304 of the E protein of Y strain.Subsequently,the mutant virus rPSYER304M was rescued.The mortality of ducklings infected by rPSYER304M was 60%,similar to that caused by Y strain.Only 10%mortality occurred in ducklings infected with the rescued parental virus rPS.The findings suggest that residue 304 of E protein is crucial to virulence difference between PS and Y strains.