Molecular Mechanism Of Difference In Host Adaptability To Waterfowl Between Japanese Encephalitis Virus Genotype ? And ?

Japanese encephalitis virus(JEV)is a member of the genus Flavivirus in the family Flaviviridae,and mainly causes damage to the central nervous system of humans and reproductive disorders in pigs.JEV is transmitted through bite by mosquitoes in nature.Waterfowls and pigs are considered the major amplifying hosts of JEV.According to the nucleotide sequence of JEV E gene or the gene encoding the complete polyprotein,JEV is phylogenetically classified into five genotypes(GI to V).GIII was the dominant genotype from the 1930 s through the end of the 20 th century throughout most countries in Asia.In the past two decades,GI has gradually replaced GIII as the dominant genotype,resulting in JEV genotype shift in most countries in Asia.However,the molecular mechanisms responsible for JEV genotype shift is still unclear.Recent studies describe that GI has an advantage of host adaptability over GIII in waterfowl hosts,causing that GI-inoculated ducklings develop higher viremia titers and display a comparatively longer viremic duration than GIII-inoculated ducklings.This difference in host adaptability to waterfowl hosts between GI and GIII has been considered to be responsible for JEV genotype shift.To explore the molecular mechanism responsible for the difference in host adaptability to waterfowl between GI and GIII,a GI/III JEV PCR-based reverse genetics system was established.The genome between GI and GIII showed approximately 88.9% homology and 12.3% difference.This difference in genomic sequences limited the choice of restriction sites,thereby resulting in a difficulty in construction of chimeric viruses of GI and GIII by use of restriction enzymes.To solve this problem,four overlapping DNA fragments covering the full length of GI-SH7 and GIII-SH15 strains were amplified using four pairs of universal primers based on the conserved sequences of JEV and cloned into the low-copy plasmid POK-12.Using the constructed low-copy plasmids containing four fragments of GI and III genomes as templates,the four fragments were amplified and spliced in order by two rounds of fusion PCR with specific universal primers to generate the full-length DNA sequences of GI and GIII containing T7 promoter,which were used to transcript in vitro the full-length RNAs.The generated RNA transcripts were transfected into BHK cells and recombinant GI(rGI)and GIII(rGIII)were rescued from the transfectants.The rescued viruses exhibited the replication characteristics and plaques morphology similar to their parental viruses on BHK cells.This GI/III JEV PCR-based reverse genetics system was used for construction of chimeric viruses and site-directed mutation viruses.The primary study suggested that the SH7 and SD12 strains of GI induced lower levels of IFN-?and IFN-? expression than the SH15 and SH19 strains of GIII in DEF cells,which was associated with the replication advantage of SH7 and SD12 strains over SH15 and SH19 strains in DEF.Similar results were also observed in duckling in vivo,in which ducklings inoculated with the GI-SH7 strain showed lower levels of IFN-? and IFN-? production in the serum as compared with ducklings inoculated with the GIII-SH15 strain.Consequently,the GI-inoculated ducklings developed higher viremia titers and displayed a comparatively longer viremic duration than the GIII-inoculated ducklings.However,this difference in IFN-I induction between GI and GIII was observed only in avian hosts,but not in othertwo host cells(ST and b End.3 cells),showing a host-specific manner.Therefore,the ability of GI and GIII JEV to induce IFN-I expression in waterfowl hosts was associated with their different host adaptability to waterfowl.To explore the molecular mechanism of difference in IFN-I induction between GI and GIII in waterfowl hosts,we compared the whole genome sequences of 50 GI and GIII strains,and eventually found a total of 53 amino acid variations between two genotypes.According to the amino acid variations,a series of chimeric viruses with exchanges of viral structural and non-structural(NS)proteins between GI and GIII were generated based on the GI/III JEV PCR-based reverse genetics system and subsequently subjected to examination of the changes in induction of IFN-I expression and replication efficiency in DEF.Among 10 viral proteins harboring the amino acid variations,NS5 was identified to be the viral determinant responsible for the differences in IFN-? and IFN-? induction and replication efficiency between GI and GIII strains.NS5 inhibited IFN-? and IFN-? production induced by poly(I:C)stimulation and harbored 11 amino acid variations distributed on three domains.To narrow the region responsible for the difference in IFN-I induction,a series of chimeric viruses with exchanges of NS5 domains were generated and the NS5-a-aRdRp region was finally identified to be responsible for the differences in IFN-? and IFN-? induction and replication efficiency in DEF.There were two amino acid variations(V372A and H386Y)present in the a-aRdRp region.The residues of V372 and H386 were highly conserved in GI strains,whereas the residues of A372 and Y386 were relatively conserved in GIII strains.Substitution of these two residues revealed that the NS5-V372 A and NS5-H386 Y variations co-contributed to the low levels of IFN-? and IFN-? induction and high levels of replication efficiency of GI strains.The NS5-V372 A and NS5-H386 Y variations were located in the nuclear localization signal(NLS)region that was involved in antagonism of IFN-I expression and the nuclear translocation of NS5.GI-NS5 containing V372 and H386 residues bounded to the nuclear transport receptor protein duKPNA4 stronger than GIII-NS5 harboring A372 and Y386 residues,thereby promoting the nuclear translocation of GI-NS5 more efficiently than GIII-NS5.In addition,the interaction between NS5 and duKPNA4 resulted in the competitive block of the interaction of du KPNA4 with the type I interferon transcription factor duIRF7,thereby inhibiting the nuclear translocation of duIRF7 and subsequent IFN-I expression.Substitution of NS5-V372 A and NS5-H386 Y residues indicated that the high ability of GI-NS5 harboring V372 and H386 residues to bind to duKPNA4 increased its ability to inhibit the nuclear translocation of du IRF7 as well as the subsequent IFN-I expression.To further explore the mechanisms responsible for the high binding ability of GI-NS5 to duKPNA4,the three-dimensional structure of NS5 was analyzed using bioinformatics software.A difference in the number of hydrogen bounds formed by NS5-372 and NS5-386 residues were observed between GI-NS5 and GIII-NS5,which may result in a difference in the NLS flexibility and subsequent nuclear translocation between GI-NS5 and GIII-NS5.To test this,a series of mutant viruses with substitution of different amino acids from same groups were generated and examined for the changes in induction of IFN-I expression.The mutant virus rGI/V372G-H386 K,rGI/V372P-H386 R,rGIII/A372G-Y386 K andrGIII/A372P-Y386 R harboring the number of hydrogen bonds same to rGI exhibited higher nuclear translocation levels of NS5 and induced lower levels of IFN-? and IFN-? expression as compared with the mutant viruses containing the number of hydrogen bonds same to rGIII.Infection of ducklings with the substitution mutant virus rGI/V372A-H386 Y and rGIII/A372V-Y386 H as well as the respective parental rGI and rGIII demonstrated that NS5-V372 A and NS5-H386 Y variations co-contributed to the differences in IFN-? and IFN-? induction and replication efficiency between the GI and GIII strains in ducklings.In summary,the GI/III JEV PCR-based reverse genetics system was established for analysis of the mechanisms of difference in host adaptability to waterfowls between GI and GIII strains.By using a series of chimeric viruses and site-directed mutation viruses,the NS5-V372 A and NS5-H386 Y variations were identified to co-contribute to the difference in adaptability to waterfowl host between GI and GIII viruses.The NS5-V372 A and NS5-H386 Y variations altered the ability of NS5 to bind to duKPNA4 and to competitively block the interaction between du KPNA4 and duIRF7,thereby affecting IFN-I expression in vitro and in vivo.Mechanically,the number of hydrogen bounds formed by NS5-V372 A and NS5-H386 Y was different between GI-NS5 and GIII-NS5,which resulted in the difference in binding ability of NS5 to duKPNA4,thereby leading to the difference in IFN-I induction and adaptability to waterfowl hosts.These results gained an insight into the mechanisms underlying the different host adaptability between GI and GIII as well as the mechanisms responsible for JEV genotype shift.