Sequence Analysis Of S1 Gene Of IBV Isolates From Guangxi During 2018-2019 And Immunogenicity Study Of Four Representative IBV Variant Strains

Infectious bronchitis(IB)is an acute,highly contagious respiratory disease caused by infectious bronchitis virus(IBV).The IBV genome is prone to mutation,leading to the diversification of genotypes,serotypes,pathogenicity and immunogenicity.The cross-protection between different genotypes or serotypes is poor,and the failure of immunization in production often occurs,bringing great economic losses to the poultry industry.Therefore,it is of great practical significance for the prevention and control of IB to timely understand the variation of IBV epidemic strains in the region and to screen out the strains with good immunogenicity as local vaccine candidates.Previously,the molecular epidemiological investigation of IBV isolates from Guangxi by 2017and the pathogenic study of four representative IBV variants were carried out by our research group.Based on these finished studies,the continued tracking research on Guangxi IBV isolates from 2018 to 2019 and the immunogenicity analysis of the four representative IBV variants were conducted in the present study.The details are as follows.1.Sequence analysis of S1 gene of IBV isolates from Guangxi during2018-2019Chicken embryo inoculation and RT-PCR were used to isolate and identify IBVs in suspected diseased chickens from Guangxi during 2018-2019,and the S1 gene of the isolates was amplified,sequenced,and analyzed for similarity,phylogenetic tree,recombination,hypervariable region,glycation site and S protein cleavage site.The results showed that a total of 19 IBV isolates were identified,and the S1 gene ORF of the 19 IBV strains was 1611-1629 bp,encoding 537-543 amino acids.The nucleotide similarities of S1 gene among isolates were 66.2%to 99.9%,and the nucleotide similarities of S1 gene between isolates and vaccine strains 4/91,H120,QXL87,LDT3-A and LX4were 65.9%to 78.9%,65.6%to 83.2%,67.7%to 96.0%,66.2%to 99.1%and78.0%to 99.1%,respectively.Phylogenetic tree analysis showed that the 19 IBV strains were divided into 5 genotypes.LX4-type was the dominant genotype,accounting for 42.10%(8/19);LDT3-A-type accounted for 26.31%(5/19);Taiwan?-type accounted for 15.78%(3/19);Taiwan?-type accounted for10.52%(2/19);4/91-type accounted for 5.26%(1/19).Recombination analysis showed that 5 of the 19 IBV strains underwent genetic recombinaton,and all the major parents of recombinants GX-NN190716,GX-NN190923,GX-NN191006and GX-GG190815 were GX-YL161022(QX?-type)strain,the minor parent was CK/CH/LSC/99I strain.The major parent of recombinant GX-NN191213was GX-GL(Taiwan-type)strain,the minor parent was CK/CH/LSC/99I strain.The hypervariable region analysis showed that a large number of amino acids substitution,insertion and deletion occurred in all the three hypervariable regions of 19 IBV strains.Analysis of glycosylation sites showed that there were9-18 N-glycosylation sites in 19 IBV strains.There were two O-glycosylation sites in GX-YL191121,and no O-glycosylation sites in the remaining 18 strains.Analysis of S protein cleavage sites showed that 12 strains were RRFRR,6 were HRRRR,and one was HRRKR.2.Immunogenicity study of 4 representative IBV Variant strainsOn the basis of previous pathogenic study of four representative IBV variant strains GX-NN160421,GX-QZ170728,GX-QZ171023 and GX-NN130048,the immunogenicity and cross-immunoprotection tests of the 4strains were carried out.A total of 330 5-day-old yellow chickens were randomly divided into 6 groups:GX-NN160421 group,GX-QZ170728 group,GX-QZ171023 group,GX-NN130048 group,H120 group,and PBS group,respectively.All chickens were immunized for the first time at 5 days of age,and were given a second immunization at 19 days of age(2 weeks after the first immunization).And all chickens were conducted cross-challenge at 33 days of age(2 weeks after booster immunization).Partial experimental group chickens were slaughtered at 5 days after the challenge,and the observation group chickens were raised to 47 days old(14 days after the challenge).The Specific antibody and neutralizing antibody levels in serum after immunization,CD3~+,CD4~+,CD8~+T lymphocytes in peripheral blood,clinical morbidity and mortality of challenge chickens,viral loads of trachea,kidney and lung,shedding of pharyngeal and anal swabs were used as indicators to determine the cross-immunoprotection.The results of IBV specific antibody and neutralizing antibody showed that GX-NN130048 strain inactivated oil emulsion vaccine could induce a higher level of humoral immune response in chickens after immunization.The results of CD3~+,CD4~+and CD8~+T lymphocyte percentages showed that GX-NN130048 strain could induce a higher level of cellular immune response.The viral load results showed that the average viral loads of each group were the GX-NN160421 group(27)GX-QZ170728 group(27)GX-NN130048 group(27)H120 group(27)GX-QZ171023 group(27)PBS group.The statistical results of morbidity and mortality showed that GX-NN130048 strain had the best immune protection effect.The virus shedding of pharyngeal swab and anal swab were positive until 5dpi in each immunized-challenge group,and were positive until 7dpi in non-immunized-challenge group.In addition,the shedding amount of the pharyngeal swab and anal swab of the chickens in each immunized-challenge group was far lower than that of the non-immunized-challenge group.The difference between the immunized-challenge group and the non-immunized-challenge group was very significant(P<0.01).Based on all the indicators,the isolate GX-NN130048 could induce higher levels of cellular and humoral immunity in chickens,and has a good cross-immunoprotection.Therefore,it can be used as an IBV vaccine candidate strain in this region.In summary,the results of this study indicate that the genotypes of IBV in Guangxi during 2018-2019 include LX4-type,LDT3-A-type,Taiwan-type and4/91-type,among which the dominant genotype is LX4-type.The isolates GX-NN130048 has good immunogenicity and can be used as an IBV vaccine candidate in this region.