| From 2013 to 2016,the H7N9 subtype avian influenza virus(AIV)showed a low pathogenicity to poultry,but after the end of 2016,it was gradually replaced by the H7N9 subtype highly pathogenic avian influenza virus(HPAIV).Compared with the H7N9 subtype low pathogenic avian influenza virus(LPAIV),The H7N9 subtype HPAIV not only has strong pathogenicity,but also has an increasing genetic distance.Although the current H7 inactivated vaccine has been updated from Rel to Re3,it is still difficult to achieve the coverage for epidemic strains,therefore it is necessary to develop a highly efficacy and broad-spectrum H7N9 subtype AIV vaccine.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 naturally infected animals from vaccine-immunized animals with existing vaccines.Therefore,there is an urgent need to develop labeled vaccines that can be used to distinguish naturally infected animals from vaccine-immunized animals(DIVA)for the eradication of H7N9 subtype AIV.1.Screening of HPAIV HA and NA gene donor virus of H7N9 subtypeThree H7N9 subtype HPAIVs,including A/Chicken/Guangdong/GD4/2017(GD4),A/Chicken/Guangdong/HZLH2/2017(HZLH2),and A/Chicken/Hebei/XT-3/2017(XT-3)were used for plaque purification and genetic evolution analysis.The results showed that the HA genes of the three virus all belonged to the branch of the Yangtze River Delta line A/Duck/Zhejiang/12/2011(H7N3),and the genetic distance was close to the HA gene donor virus A/Chicken/Guangxi/SD098,which is as currentH7-Re2 vaccine strain.The HA protein cleavage sites(CS)of the three strains all have the insertion of continuous basic amino acids,including two highly pathogenic virus CS patterns,PEIPKGKRTAR ↓ GLF and PEIPKRKRTAR ↓ GLF,among them,GD4 and HZLH2 belong to the first CS mode,and XT-3 belongs to the second.186V and 226Q were found in the receptor binding sites of the three strains of HA protein.The assay of goose red blood cell agglutination proved that these viruses all have α2,3 and α2,6 dual receptor preference.The results of the thermal stabilityassay showed that the HA titer of GD4 and HZLH2 remain unchanged at 37℃ and 42℃,but the HA titer(log2)of XT-3 decreased from 9 to 8;At 56℃ for 90 min,the HA titer(log2)of GD4,HZLH2,and XT-3 were reduced from the original 10,10,and 9 to 7,8,and 2,respectively.It was determined that GD4,HZLH2,and XT-3 belong to moderate heat stable,heat stable,and heat unstable virus,respectively.In the assay of pH stability,the HA titer of the three virus did not decrease significantly in the environment of pH 3.0,5.0,and 9.0,showing good pH stability.The results of biological characterizations showed that GD4 had higher HA titer and EID50 titer,reaching 10 loge and 8.17 log10/0.1 mL,respectively.Intravenous vaccination pathogenicity index(IVPI)assay showed that the pathogenicity index of GD4 was 2.55,which was in line with HPAIV characteristics.SPF chickens were immunized with oil emulsion inactivated vaccine based on GD4,Antiserum was prepared for cross-hemagglutination inhibition assay.The results showed that the H7-Re2 serum had insufficient cross-reactivity against HZLH2 and XT-3 HPAIVs.By contrast,GD4 immune serum has better cross-reactivity against H7-Re2 standard antigen,GD4 strain,LPAIV JD/17,and HPAIV HZLH2and XT-3.Therefore,GD4 strain was selected as the donor strain to provide HA and NA genes for the subsequent construction of vaccine candidate.2.Construction and biological characteristics analysis of H7N9 subtype recombinant virus rGD4HALo-mH3-NA-TXIn this study,the H7N9 subtype HPAIV A/Chicken/Guangdong/GD4/2017(GD4)was selected as the HA and NA gene donor virus,and the consecutive basic amino acids at the CS of the HA gene were deleted for attenuating.According to the previ ous screening results for the H7 subtype specific epitope,the 12 peptides(HA2-12)(463ADSEMDKLYERVKRQLRENA482)in the HA2 region were replaced by the corres ponding peptides of the H3 subtype(463ADSEMNKLFEKTKKQLRENA482)as the ma rker of the DIVA vaccine.Combining with the internal skeleton(PB2,PB1,PA,NP,M,and NS)of a highly chicken embryo adapted H9N2 subtype AIV A/Chicken/Taixi ng/10/2010(TX),a H7N9 subtype recombinant strain was rescued by reverse genetic technology and named rGD4HALo-mH3-NA-TX.The results of indirect immunofluorescen ce assay showed that the strain had a negative response to monoclonal antibody 3G10,which is against the H7 subtype HA2-12 peptide,whereas the wild-type strain GD 4 had a positive response,indicating that the 12 peptide was successfully replaced an d can be used as a DIVA marker;After five consecutive passaged,the HA titer of r GD4HALo-mH3-NA-TX was 10 log2,and the EID50 value was 8.50 log10/0.1 mL,which was equivalent to the parent virus rGD4.In the assay of chicken embryo infection,GD4 can casue death of chicken embryos about 36 h after inoculation,while rGD4H ALo-mH3-NA-TX was not lethal for chicken embryos at different dilutions.The results of the IVPIassay showed that all chickens inoculated with rGD4HALo-mH3-NA-TX survived without obvious clinical symptoms,and the pathogenicity index was 0.01,indicating that the recombinant virus was attenuated successfully.3.Evaluation of immune efficacy of H7N9 subtype recombinant virus rGD4HALo-m H3-NA-TX and identification of DIVA characteristicsThe cross-HI assay showed that the immune serum from the immunization group of H7N9 subtype recombinant virus rGD4HALo-mH3-NA-TX targeted H7-Re2 standard antigen,GD4 strain,LPAIV JD/17,and HPAIV HZLH2and XT-3,which had a better cross-reactivity compared with H7-Re2 serum;an oil emulsion inactivated vaccine was prepared based on the H7N9 subtype recombinant virus strain rGD4HALo-mH3-NA-TX for and immunized with 3-week-old SPF chickens,and the weight change,HI antibody level and its growth and decline were evaluated.The results showed that the weight change of rGD4HALo-mH3-NA-TX immunization within 20 days was equivalent to that of the PBS immunization group,indicating that the inactivated vaccine had higher safety.2 weeks after first immunization,the HI titer(log2)of the rGD4HALo-mH3-NA-TX immunization group was 7.15± 1.63,which was higher than that of the GD4(6.10± 1.57)and JD-cHA/17(5.40± 1.64)immunization groups.3 weeks after first immunization,the HI titer(log2)of the rGD4HALo-mH3-NA-TX immunization group(8.60±1.60)was similar with the GD4 immunization group(8.70± 1.22),and higher than the JD-cHA/17 immunization group(8.00±1.72).From the analysis of antibody duration,it was found that the HI level of rGD4HALo-mH3-NA-TX immunized group reached a peak value(9.80±0.45 loge)in the fifth week,and then slowly decreased,but it still reached 7.40±0.55 log2 at 11 weeks after first immunization.The results of the cross-challenge protection assay showed that when JD/17 and GD4 were used to challenge with 106EID50 via ophthalmic and nasal drops,the weight gain of the rGD4HALo-mH3-NA-TX immunization group was greater than that of the GD4,JD-cHA/17,and PBS immunization groups;The rGD4HALo-mH3-NA-TX immune group can provide stronger cross-protection levels against HPAIV(GD4)and LPAIV(JD/17)with 90%and 80%,respectively,while JD-cHA/17 was with 60%and 80%,GD4 immunization group was with 80%and 70%,respectively.The established competitive inhibition ELISA method was used to evaluate the DIVA characteristics of rGD4HALo-mH3-NA-TX.The results showed that the inhibition rate of GD4(56.79±3.76%),JD/17(55.33±3.06%),and H7-Re2(48.56±3.98%)serum was significantly higher than the negative value(16.14%).However,the inhibition rate of the immune serum against rGD4HALo-m3-NA-TX was 11.71±0.37%,which was lower than that of the negative control,suggesting that the immune serum of the recombinant strain has obvious DIVA characteristics that can be distinguished from the infection serum of the wild strain.The above studies demonstrated that the H7N9 subtype recombinant virus rGD4HALo-mH3-NA-TX had a strong reproductive performance,high safety,and DIVA characteristics,which can provide an efficient cross-immune protection against H7N9 subtypes LPAIV and HPAIV,and can be used as a ideal candidate strain of H7N9 subtype AIV DIVA inactivated vaccine. |
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