Development Of H9N2 Subtype Live Attenuated Vaccine By Exchanging The Packaging Signals Between HA Gene And NS1 Gene

H9N2 subtype avian influenza virus(AIV)is a low-pathogenic virus,and wide distributed in our country.It can infect both poultry and wild bird,resulting in huge economic losses to the poultry industry.H9N2 subtype AIV can directly infect human or donate partial or even whole internal genes to generate novel reassortment viruses,posing a potential threat to public health.Currently,only inactivated vaccines are licensed for using to control H9N2 subtype avian influenza in China.However,inactivated vaccines poccess high costs,inconvenience,single-induced immune responses,and adverse effect to epidemiological monitors.Therefore,it is necessary to develop the live attenuated vaccines with a better immune response.However,there is indeed concern about the risks associated with reassortment events that may occur between vaccine strains and circulating wild-type viruses.In this study,we exchanged the packaging signals between HA gene and partial deletion NS 1 gene(retaining 1-128 amino acids)and evaluated the reassortment probability and immune efficacy of vaccine strain.Our study laid a foundation for the development of new avian influenza vaccines.1 Rescue of packaging signal-replaced virus and evaluation of the vital biological characteristicsTo reduce the recombination chance between the live attenuated vaccine strains and wild strains.The packaging sequences of H9N2 rTX HA and NS1-128 ORF were performed synonymous mutations,and then the mutated ATGs and splice site were exchanged for constructing two recombinant plasmids.The rTX-NA1-128(mut)virus was successfully rescued by transfecting above two plasmids and six TX segments.The HA titer of rTX-NS1-128(mut)is 6 log2.The recombinant virus replicated well in SPF chicken embryos with a titer of 7.83±0.35 Log10EID50/0.1 mL,while the replication ability in MDCK cells was lower than that of wild type virus.The recombinant virus was passaged for 20 generations in SPF chicken embryos,and the genetic stability was verified by sequencing.The results showed that the recombinant virus had a good genetic stability.The MDCK cells were co-infected with rTX-NS1-128(mut)and NS1 gene-deleted virus rTX-NS1-128/wild-type TX strain at MOI=10,the cell supernatant was plaque purified,and then the recombination probability was determined.The results showed that the HA and NS fragments of rTX-NS1-128(mut)did not independently reassort with their parent strain or wild-type strain.Therefore,H9N2 rTX-NS1-128(mut)with exchanged the packaging signals between the HA gene and partly deleted NS1 gene was successfully rescued,which laid a foundation for a further research.2 Immune efficacy of the packaging signal-replaced virus(rTX-NS1-128(mut))To evaluate whether rTX-NS 1-128(mut)could be as a potential candidate vaccine strain,the pathogenicity,transmissibility,and host immune responses were evaluated.SPF chickens(4-weeks old)were inoculated intranasally with 106 EID50 of recombinant virus,the oropharyngeal and cloacal swabs was collected and inoculated into embryonated SPF eggs for virus titration,the tracheas and lungs were collected and titrated for viral infectivity by EID50 assay.The results showed that the shedding rates of rTX-NS1-128(mut)group were similar to that of NS 1 gene-deleted viruses.HI titer reached a peak 3 weeks after vaccination,and the average value was 7.89±0.87 log2,and then reached 3.85±0.25 log2 after 11 weeks of immunization.The results of mucosal and cellular immunity assay showed that the recombinant virus rTX-NS1-128(mut)induced higher levels of mucosal and cellular immunity than inactivated virus.Three weeks after inoculation,vaccinated chickens were challenged intranasally with 106 EID50 of H9 subtype virus strain(TX or F98)to determine protective efficacy.The results showed that the recombinant virus rTX-NS1-128(mut)lost the contact-transmissibility among chickens and was only able to replicate in trachea at low levels.Furthermore,rTX-NS1-128(mut)provided a 100%protection against homologous virus strain(TX)and 80%protection against heterologous virus strain(F98)challenge,in spite of one-dose vaccination.Collectedly,the results were consistent with that of the NS1 gene deletion virus,indicating that the exchange of the packaging signals did not affect viral pathogenicity,transmissibility,and host immune responses.3 Establishment of a quantative real-time PCR for differentiation between the H9N2 virus with partly deleted NS1 and wildtype strainIn order to differentiate vaccine strain from wildtype strain,a nucleic acid detection method was established to identify NS1 gene.The specific qPCR primers for AIV M gene and deletion NS1 gene were designed,respectively.Standard quality plasmids were successfully constructed,and the standard curves were generated.The results showed that this method can identify the M and the deletion NS1 genes between the wild strains and the vaccine candidate rTX-NS1-128(mut).The results of sensitivity assay showed that the detection limit of this method for H5,H7,and H9 subtype AIV is 1000?100 EID50/?L;The results of the swab samples showed that this method was similar with virus isolation.These data indicated that the detection method can distinguish the H9N2 virus with deletion NS1 gene and wild type virus with a high sensitivity and specificity.