Development Of Detection Methods And Marked Vaccine Against H7N9 Subtype Avian Influenza Virus

H7N9 subtype avian influenza virus(AIV)is an emerging zoonotic pathogen.Since it was first reported in China in 2013,it has caused 5 waves of epidemics in humans.To date,1567 laboratory-confirmed human cases infected with avian influenza A(H7N9)virus have been reported,including 615 deaths,with mortality close to 40%.The H7N9 AIV in early stage was low pathogenic to poultry,and there were no obvious symptoms after poultry infection.At the beginning of 2017,H7N9 AIV was changed to high pathogenicity in poultry,caused many epidemics within 17 provinces across the country.In the second half of 2017,the use of the recombinant avian influenza virus(H5+H7)bivalent inactivated vaccine effectively controlled the H7N9 epidemic in poultry and significantly reduced human infections.However,H7N9 and H7N2 AIVs,which were highly pathogenic to chickens and ducks,could still be isolated in some provinces of China,which brought new challenge to the prevention and control of H7 AIVs in China.Therefore,it is imperative to develop marked vaccines that can be used to differentiate an infection from a vaccinated animal,as well as rapid diagnostic methods for the prevention and clean-up of H7 AIVs.1.Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus Nine avian influenza virus neuraminidase(NA)subtypes were discovered in poultry and wild birds.Since the reassortant occurred easily in AIVs,one hemagglutinin(HA)subtype of AIV can combine with different NA subtype.It is necessary to identify HA and NA subtypes of AIVs at same time.Here we developed a multiplex probe combination-based one-step real-time reverse transcriptase PCR(rRT-PCR)to detect nine NA subtypes of AIVs.Nine primer-probe pairs were assigned to three groups based on the different fluorescent dyes of the probes(FAM,HEX or Texas Red).The results showed that each of the primer-probe pair in the multiplex RT-PCR method had good specificity for detecting combinations of different NA subtypes without cross-reactivity.The method was highly sensitive,and the detection limit was less than 100 copies and 100 EID50 per reaction.The method was used to detect the tracheal and cloacal samples of chickens infected with H9N2 virus and the clinical samples from live poultry market.The results showed that the positive rate of the method was consistent with the results of virus isolation and gene sequencing methods,and the sensitivity was higher.In summary,the fast,specific and sensitive multiplex probe combination rRT-PCR method we established provides a new rapid detection method for NA typing.2.Development of a colloidal gold-based immunochromatographic strip for rapid detection of H7N9 influenza virusesMonoclonal antibodies were prepared using H7N9 virus(A/Chicken/Jiangsu/W 1-8/2015,W1?8)as immunogen,and antigenic epitopes were determined by antigen escape assay and HA gene sequence determination and analysis.Monoclonal antibodies with different epitopes were used to develop immunocolloidal gold test strips for H7N9 AIVs.The results showed that 13 monoclonal antibodies with hemagglutination inhibition properties were obtained,and three epitopes at positions 198,227 and 235 were identified.Immunocolloidal gold test strips were prepared for 227 and 235 monoclonal antibodies.The results showed that the test strips had good specificity,stability and sensitivity.The detection limits of swabs and tissue samples were both 2.5 logio EIDso/0.1mL.The virus detection rate of live poultry market samples was 3%(6/200),which was consistent with virus isolation results(4.5%,9/200)and HA gene PCR sequencing results(3.5%,7/200).It provides a new method for rapid detection of H7N9 AIVs on site.3.Development of inactivated marked vaccine against H7N9 subtype avian influenza virusThe specific epitope H7-12 peptide on HA2 of H7N9 AIV was successfully screened by peptide chips,and then the epitope in HA protein of H7N9 AIV was modified accordingly using A/Chicken/Huadong/JD/17(JD/17)as backbone by reverse genetics.The recombinant H7N9 AIV was named as A/Chicken/Huadong/JD-cHA/17(JD-cHA/17).The HA titer of the recombinant virus was 10 Log2 and the EID50 titer was 9.67 Log10EIDso/mL.At 3 weeks post immunization,the HI antibody titer reached 9.22±0.6,indicating that the vaccine candidate had good antigenicity and immunogenicity.The challenge results showed that the vaccine candidate had the same immune protection as the parent strain inactivated vaccine,and had 100%protection against both high pathogenicity(HP)and low pathogenicity(LP)H7N9 AIVs.Peptide chip coated with H7-12 peptide was applied successfully to detect the seroconversion at three days after infection with JD/17[Signal to noise ratio(SNR)=2.33±0.15],and the sensitivity was higher than the HI test(HI=0).When the peptide chip coated with H7-12 peptide was used to detect the JD-cHA/17 vaccination sera(HI=9.2±0.6)and JD/17 vaccination sera(HI=9.1 ±0.5),the SNR values for antibody binding to H7-12 peptide were 0.44 ±0.14 and 6.39 ± 0.13,respectively,and the difference was extremely significant,indicating that antibody against H7-12 peptide can't be detected in the inactivated marked vaccine,which had the characteristics of DIVA and broad application prospects.4.Establishment of a competitive inhibition ELISA for the differentiation of the immunized sera from marked vaccine and infected sera from wild type strainSix monoclonal antibodies were successfully prepared using the H7-specific epitope H7-12 peptide conjugated to BSA as an immunogen.The results of IFA and Western-blot showed that the 6 monoclonal antibodies only specifically recognized H7N9 AIV JD/17 with a good specificity.Monoclonal antibody 3G10 with high inhibition rate was screened successfully by competitive inhibition ELISA.To establish a competitive inhibition ELISA against H7-12 antibody,vaccination sera from JD-cHA/17 and infected sera form JD/17 were used as test sera,the H7-12 peptide was used as a coating antigen,and purified 3G10 monoclonal antibody ascites was labeled with horseradish peroxidase(HRP)as a detection antibody.The reaction conditions of the competition ELISA were optimized as following:the optimal concentration of the antigen was 50?g/mL,the optimal working concentration of the enzyme-labeled antibody was 1:200,and the optimal dilution of the serum to be tested was 1:4,the best choice for blocking was the calf serum of 8%for 90 min,and the optimal reaction time of TMB was 10 min.The inhibition rate ?20%was considered to be positive and ?16%was considered to be negative.The infected sera form JD/17 was detected as positive,while the vaccination sera from JD-cHA/17,H1,H3,H4,H5(RE-6,RE-7 and RE-8),H6,H9 and H10 subtype AIVs were all detected as negative by the ELISA method,indicating that the competitive inhibition ELISA method had good specificity and effectively can distinguish between infected serum and immunized serum.