| In 2013-2016,H7N9 subtype avian influenza virus(AIV)showed a low pathogenicity to poultry,while from late in 2016,highly pathogenic avian influenza virus(HPAIV)viruses emerged and became dominant.Compared with low pathogenic avian influenza virus(LPAIV),highly pathogenic H7N9 viruses have not only a strong pathogenicity but also a diverse genetic distance,which poses challenges to prevention of AIV.At present,eradication is an effective strategy to control HPAIV,which has been included in national medium-and long-term plan of the animal epidemic prevention formulated by the Chinese government.However,it is difficult to use serological diagnostic methods to distinguish infected from vaccinated animals(DIVA)with existing vaccines.Thus,it is urgently required to develop labeled vaccines that can be used to distinguish naturally infected animals from vaccine-immunized animals for eradication of H7N9 subtype AIV.A vaccine candidate strain A/Chicken/Huadong/JD-cHA/17(JD-cHA/17)was developed and the monoclonal antibody 3G10 was prepared to target the peptide,and the competitive inhibition ELISA method was established in our previous study.Due to the continuous variation of H7N9 subtype AIV,the immune protective effect and distinguishing ability of DIVA vaccine and its matching detection method against newly emerged variant strains are unknown.Therefore,further safety and systematic immune efficiency evaluation of this vaccine are needed.At the same time,the broad spectrum of the matching detection method was evaluated to provide an effective tool for the prevention,control and cleanup of H7N9 subtype avian influenza.1.Biological characteristics and biosafety evaluation of JD-cHA/17To evaluate the genetic reliability and safety of candidate vaccine strain JD-cHA/17,the biological characteristics of the JD-cHA/17 virus and safety of vaccine were determined in this study.The HA titer and EID50 titer of JD-cHA/17 were up to 10 log2 and 109.03/mL respectively after 5 passages in chicken embryos,which was the same as wild-type JD/17.The sequence of HA gene and marker gene in the 5th generation of JD-cHA/17 was same as that of the 1st generation of JD-cHA/17.Moreover,thermal stability assay showed that the HA titer of JD-cHA/17 remained unchanged compared with that of JD/17 after 37℃ incubation.While after 42℃ incubation,the HA titer of JD-cHA/17 decreased llog2 on the first day.After 56℃ incubation,both JD-cHA/17 and wild-type JD-HA/17 viruses lost their hemagglutination within 30 min,indicating an unstable thermostability.pH stability assays showed both JD-cHA/17 and wild-type JD-HA/17 viruses were unchanged when exposed to a pH of 5.0,7.2,and 9.0,suggesting a stable pH stability.Overdose and repeated injection of JD-cHA/17 inactivated vaccine were used to test the safety.The results showed that immunized chickens were in good appetite and spirits.The weight increase was consistent with that of the control group.The data indicate that JD-cHA/17 had a good genetic stability and high safety.2.Immune efficiency evaluation and DIVA characteristics of JD-cHA/17 vaccineIn order to clarify the immune efficacy of JD-cHA/17 inactivated vaccine,SPF chickens were vaccinated with different dose groups.The results showed that the HI titers of three different dose groups were 5.8±1.62 log2、6.5±1.05 log2,and 7.4±0.52 log2,respectively after 21 days vaccination,indicating a dose-dependent manner.All chickens survived and no obvious clinical symptoms observed when challenged with either wild-type JD/17 or another Re3 strain A/Chicken/China/Liaoning/0866/2020(LN0866).Moreover,only 10%vaccinated chickens shed viruses while 90%control chickens shed viruses when challenged with JD/17.Similarly,only 10%vaccinated chickens shed viruses while all control chickens died when challenged with LN0866,indicating the high immune efficacy of JD-cHA/17 against both the wild-type JD/17 and the epidemic strain LN0866.The competitive inhibitory ELISA was used to evaluate the DIVA properties of JD-cHA/17.The results showed that the inhibition rate of JD-cHA/17 vaccinated serum was 9.70±2.42%,which was lower than negative value.The inhibitory rates of infected serum against LN08866 and JD/17 after JD-cHA/17 vaccination were 48.08±6.82%and 33.27±7.99%,respectively,which were significantly higher than that of the negative value,indicating a successful DIVA strategy.However,the inhibitory rate against serum against LN08866 alone was 11.1±0.56%,also lower than negative value,suggesting that the matched detection method can only distinguish vaccinated serum from the JD/17 infected serum,but not LN08866 infected serum and need to be updated.3.Preparation of monoclonal antibody against HA2 specific epitope of H7N9 Re 3 like strainTo solve the previous problem of broad spectrum of competitive inhibition ELISA based on 3G10 monoclonal antibody,HA2 specific epitope of H7N9 Re3-like subtype strain LN08866 was synthesized and immunized mice by BSA coupling.The results showed that six monoclonal antibodies(4E5,6C2,6F4,6F5,6G4,and 6G6)against the specific HA2 specific epitope were successfully prepared.The titers of the six monoclonal antibodies ranged from 5.12×104 to 8.19×105.Identification of antibodies subcategory indicated that 4E5,6F4,6F5,and 6G4 were IgG1 isotypes while 6C2 and 6G6 were IgM isotypes.Indirect immunofluorescence assay(IFA)showed that 4E5,6F5,and 6G4 can react with Re1 like,Re2 like,and Re3 like H7N9 AIV while not react with JD-cHA/17 and other subtypes,including H1,H3,H4,H5,H6,H9,and H10 AIV,suggesting that the monoclonal antibodies have good specificity.Moreover,these monoclonal antibodies can distinguish JD-cHA/17 from other H7N9 viruses,which lays a foundation for establishing serum antibodies to detect broadspectrum H7N9 subtype AIV infection. |
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