Study Of The Biological Characteristics Of H9N2 Virus With The Variation Of The Glycosylation Site In HA Protein

H9N2 subtype influenza virus(IAV)was first detected in North America turkey in 1966,and then widely spread in the world,bring a huge economic losses to poultry industry.The virus had accumulated more mutations in HA gene that including the number or location changes of N-linked glycosylation sites after evolution in different kinds of animals.By analyzing and comparing the HA genes of 60 H9N2 subtype influenza virus between 1994 and 2014 year,variation of the potential glycosylation sites appeared at 313 site or 218 site in majority of H9N2 virus.In this study,we investigated the effect of changes of 313 or 218 glycosylation sites on the biological characteristics of H9N2 subtype influenza virus.(1)Rescue of the H9N2 influenza virus with increased 313 glycosylation site and the H9N2 influenza virus with deleted 218 and inceased 313 glycosylation sitesIn this study,Ck/SH/F/98(H9N2,F/98)virus were used as the bone skeleton and HA gene of F/98 as the template to construct the transcription/expression plasmid pHW204-HA/313G+ with the introduced 313 glycosylation site,and the plasmid pHW204-HA/218G'313G+ with the deleted 218 glycosylation site and introduced 313 glycosylation site by molecular cloning technique.The eight plasmid systems were respectively formed by the combination of pHW204-HA/313G+ or pHW204-HA/218G'313G+ with the remaining seven transcriptional/expression plasmids of F/98 strain pHW201-PB2,pHW202-PB1,pHW203-PA,pHW205-NP,PHW206-NA,pHW207-M,pHW208-NS,and resulted in new H9N2 virus rF/HA313G+ or rF/HA218G'/313G+,respectively,by reverse genetics technique.The migration rate of HA protein of rF/HA313G+ virus was significantly slower compared with that of the HA protein of F/98 strain,while the migration rate of HA protein of rF/HA218G-/313G+ was between that of F/98 and rF/HA313 G+ detected by Western-blot,indicating that the potential glycosylation sites at 218 or 313 site of HA protein in rF/HA313G+ or rF/HA218G-/313G+ were functional glycosylation sites.(2)Evaluation of the biological characteristics of H9N2 virus with the glycosylation site mutation of HA proteinTo study the effect of HA glycosylation site on the biological characteristics of H9N2 virus,the biological characteristics of the rescued virus rF/HA313G+ and rF/HA218G-/313G+ were identified,which include the 50%egg infectious dose(EIDso),50%egg lethal dose(ELD50),50%cell infectious dose(TCID50),pathogenicity,transmission route and antigenicity in chickens.Compared to F/98,EID50 and TCID50 of rF/HA313G+ virus increased 6.76-fold and 7.07-fold,respectively,while EID50 and TCID50 of rF/HA218G-/313G+ virus had similar titer,but decreased 5.62-fold and 6.02-fold than those of rF/HA313G+ virus,respectively.At days3 and 5 after infected with virus,the EID50 titers of rF/HA313G+ virus and lung tissue were higher 5.62-fold and 3.16-foldin trachea,and 3.16-fold and 3.16-fold in lung compared with that of F/98 strain.However,the replication titers of rF/HA218G-/313G+ virus decreased 3.16-fold and 1.78-fold in trachea and that decreased 3.16-fold and 3.16-fold in lung compared with that of rF/HA313G+,and were no any significantly different in trachea and lung compared to that-of F/98 strain.Animal experiment showed that F/98 virus,rF/HA313G+ and rF/HA218G-/313G+virus could be spread by direct and aerosol contact.The vaccine of F/98 could provide completely protection against the challenge of rF/HA313G+ and rF/HA218G-/313G+in chickens,suggesting that the changes of 313 or 218 glycosylation sites in HA of H9N2 virus did not change the antigenicity of the virus.In conclusion,the variation of the increased 313 glycosylation sites,or the increased 313 and deleted 218 glycosylation sites in HA protein could affect the infectivity and replication ability of virus in chicken,but could not induce antigenic variation of H9N2 influenza virus.