The Construction And Biological Characteristics Of A Novel Highly Pathogenic H7N9 Avian Influenza H71903 Vaccine Candidate

Avian influenza virus（AIV）belongs to Orthomyxoviridae and the genus of influenza A virus.Since 2013,the isolation of H7N9 AIV was documented as causing continuous outbreaks in various of regions of China.Until 2018,the novel H7N9 virus had caused five waves.In our laboratory,we identified a high pathogenicity H7N9 AIV named as A/Chicken/Guangdong/16876/2016（H7N9）（hereinafter referred to as 16876）in July 2016.In January 2017,two human cases infected with H7N9 virus was reported by Guangdong Provincial Center for Disease Control and Prevention,and these variants possessed an insertion of 4 amino acids,PKGR/G to PKRKRTAR/G motif,which was regarded as a marker of the characteristics of high pathogenicity（HP）in chickens.In this study,we firstly systematically identified the genomic sequence of all available HP H7N9 AIVs that have recently emerged in China.Therefore,we selected A/Chicken/Shandong/SD12/2019（H7N9）strain（hereinafter referred to as SD12）for research.The HA gene of SD12 was used as a template to site-directed mutagenesis and deleted the multi-basic amino acids PKGR/G to PKRKRTAR/G motif,which reduced the cleavage efficiency of HA protein and its virulence.Then,with the A/Duck/Guangdong/D7/2007（H5N2）eight-plasmid reverse genetic system previously constructed by our laboratory,the NA gene of SD12 and the modified HA gene were mixed with six internal genes（PB1,PB2,PA,NP,M,NS）of the D7 vaccine strain to rescue a new H7N9 subtype AIV,named H71903.The recombinant virus H71903 was inactivated to manufacture an oil emulsion vaccine.The 21-day-old SPF chickens were injected subcutaneously into the neck with H7N9 avian influenza inactivated vaccine H71903 and r GD76（the strain was 16876）,0.3 m L for each chicken.The chicken-positive serum was collected from the immunized H71903 for 21 days,and the serum crossover test was carried out together with the r GD76 chicken positive serum together with two vaccine strains and ten H7N9 subtype AIV isolated from 2016 to 2019.The antibody titer of recombinant avian influenza inactivated vaccine H71903 was higher than that of the r GD76 vaccine.The inactivated vaccine H71903 was saved into 4℃after 3 months.Then,we conducted trait test and sterility test,and the results were all compliance.Furthermore,the safety test results of the inactivated vaccine H71903 inoculated by single-dose vaccination,single-dose repeated vaccination and one-time overdose vaccination for 21 days showed that the inactivated vaccine H71903 had no obvious side effect on 21-day-old SPF chickens and10-day-old ephedra broilers,and no obvious inflammatory reaction at the injection site in different injection routes such as subcutaneous and intramuscular injection.This result proved that the vaccine had good safety for both target animals.The safety test of one-dose vaccination,single-dose repeated vaccination and one overdose vaccination of Hailan brown hens showed that the mental state of Hailan brown hens was good,and the feeding and drinking were normal.Moreover,there was no significant difference in egg rate between the test group and the control group.This result indicated that the vaccine had no effect on the laying rate of laying hens and was safety for using.After that,forty 21-day-old SPF chickens were injected subcutaneously into the neck,0.3 m L for each chicken and divided into four groups on average.At the same time,5unimmunized SPF chickens were set in each group as control.The GMT of antibody titer of immunized chicken was 7.0log2～7.5log2.Another key to remember,we have dilute the four H7subtypestrainsA/Chicken/Shandong/SD1115/2018（H7N2）,A/Chicken/Shandong/SD12/2019（H7N9）,A/Chicken/Hebei/He B1908/2019（H7N9）,A/Chicken/Liaoning/LN/2019（H7N9）into 100LD₅₀.After that,the experimental group and the control group were challenged by intranasal inoculation,0.2 m L for each chicken.The cloacal and oropharygeal swabs of all surviving chickens were collected on the 5th day after the challenge.The results of this test showed that there were no viruses isolated from the immunized chickens,while the control chickens were tested for virulence.What’s more,all of the control chickens died within 3 to 6 days.In this study,the immunized avian influenza inactivated vaccine H71903 can induce SPF chickens to produce higher antibody levels and had good safety and efficiency for ephedra broilers,SPF chickens and Hailan brown hens.Another key fact to remember,the vaccine can protect them against the H7 subtype AIV.Therefore,the recombinant virus of H71903 strain can be used as an inactivated vaccine candidate for the prevention and control of novel highly pathogenic H7N9 subtype AIV.