Molecular Epidemiology And Pathogenicity Of IBV In Eastern China During 2001?2016,and The Innate Immune Responses In Primary Chicken Kidney Cell Induced By Viral S1 Protein

Infectious bronchitis(IB)is an acute and highly contagious disease caused by infectious bronchitis virus(IBV).IBV causes respiratory and nephritic diseases in chicken is one of the economically important viral pathogens to poultry industry worldwide.Although the IBV vaccine plays a vital role in controlling IB,it is still an epidemic across the world.IBV variants have been continuously emerging,and result in huge economic loss in the affected flocks.Serotypic and genotypic diversity is a characteristic of IBV and little or no cross-protection occurs between different serotypes of IBV,and IBV undergo a high frequency of mutation and homologous RNA recombination result in IBV new genotypes and serotypes continuously emerged.All these make it difficult to prevent and control IB.It indicates that dynamic surveillance of IBV and the detailed epidemiological information of new genotype or viraint strains of IBV are very important for better development of effective anti-IBV strategies in IB controlling.The clinical types of IBV contain respiratory pathogenic type,nephropathogenic type,reproductive tract pathogenic type and glandular stomach pathogenic type and so on,and nep hrop at ho ge nic IBV is the predominate clinical type in recent years in china.The nephropathogenic IBV mainly caused the enlargement of the chicken kidney and the deposition of urate in the kidney,which leads to an increase in the mortality in infected chicken.Viral S proteins played a determine role in IBV serotyping,genotyping,viral tissue tropism and pathogenicity,whereas the S protein neutralizing site and receptor binding region were located on the S1 subunit.Thus,studying the interaction between IBV S1 protein and host can provide a better understanding for the mechanism of viral tissue tropism and pathogenicity.The main contents of this study include the following three parts.1.Molecular epidemiology of IBV in eastern China during 2001?2016A total of 36 IBV field strains were isolated from chicken flocks suspected of infection with IB in eastern China during 2010?2016.Of the 36 strains collected,35 were isolated from broiler flocks and 1 was isolated from layer flock.We found one case was caused by co-infection with IBV and H9 subtype avian influenza viruses(AIV)in broiler chicken flock.The S1 genes of 36 isolates were cloned and sequenced,revealing 4 different S1 genes,including 1620/540(28 isolates),1617/539(4 isolates),1611/537(3 isolates)and 1638/546(1 isolate),and 5 cleavage recognition sites of S protein,including HRRRR(25 isolates),RRFRR(6 isolates),RRSRR(3 isolates),RRLRR(1 isolate)and HRRKR(1 isolate).Sequence comparison showed similarities in nucleotide sequences among the 36 isolates of the S1 gene were 65.2%?99.9%and 65.0%?99.9%among 36 isolates and reference strains.Phylogenetic analysis of S1 gene showed 36 isolates during 2010?2016 were grouped into seven genotypes,including QX(26 isolates),Mass(3 isolates),4/91(3 isolates),tl/CH/LDT3/03(1 isolate),TW-I(1 isolate),TC07-2(1 isolate)and a newly emerging genotype New-I(1 isolate).To better understand the distribution of the different genotypes in eastern China.S1 gene sequences of 396 isolates between 2001 and 2016 collected from GenBank in eastern China(?)ere combined for further phylogenetic analysis.Genotyping results revealed 432 isolates could be clustered into thirteen genotypes,including QX(321 strains),tl/CH/LDT3/03(25 strains),TW-I(20 strains),4/91(18 strains),Mass(18 strains),CK/CH/LSC/99I(7 strains),TC07-2(4 strains),TW-II(2 strains),Ck/CH/LDL/97I(1 strain),Arkansas(1 strain),Vis S(1 strain),newly emerging genotype New-I(8 strains)and New-II(3 strains),and 2 varints.It was notable that ck/CH/LSD/110712 belonging USA branch was found in China in this study.Recombination analysis revealed the S1 gene of New-I genotype IBVs was originated from recombination between 4/91-and tl/CH/LDT3/03-type virus,while New-II genotype was originated from recombination between tl/CH/LDT3/03-and QX-type IBV.Our data demonstrated that multiple genotypes of IBV were co-circulated in eastern China during 2001?2016,and novel IBV genotypic clusters and variants could be emerged through recombination.The detailed epidemiological information in this work is of significance for better development of vaccine and anti-IBV strategies in China.2.Recombination analysis of genomic sequences of IBV from 2001-2016 in ChinaTo investigate the characteristics of recombination in genomes of IBV,especially the frequency of recombination between vaccine and field IBVs in China,whole-genome sequences of three IBVs,CK/CH/2010/JT-1,CK/CH/2014/FJ14 and CK/CH/2014/QL1403,were sequenced and the genomic sequences of 55 strains isolated during 2001?2016 in China were collected from GenBank in this study.The genomic sequences of these strains were analyzed with reference strains by bio informatics.Recombination analysis revealed that three recombination events could be found in the genome of CK/CH/2010/JT-1 at positions 24709-365,17160-19811 and 21136-21770,while one recombination event could be identified in the genome of CK/CH/2014/QL1403 at position 20395-24840.Further whole-genome sequence analysis showed that CK/CH/2010/JT-1 originated from multiple template switches among QX-,CK/CH/LSC/99I-,tl/CH/LDT3/03-and 4/91-type IBVs,CK/CH/2014/QL1403 originated from recombination between the QX-like and TC07-2-type IBVs,while CK/CH/2014/FJ14 was a typical QX-type strain.Here,with analysis of whole genomes of 55 IBV isolates,52 recombinants were identified through RDP4.The partial sequence of vaccine strains were found in 25/52 IBV isolates.The recombination events were further confirmed by SimPlot bio informatics analysis.Bioinformatics analysis results demonstrated that numbers of recombination events and novel IBV genotypic clusters or variants could be emerged through recombination which frequently occurring between field and vaccine strains.It indicates the adverse effects of IBV vaccine on controlling of IB in China should be paid more attention.3.Pathogenicity and cross-protection studies of IBV isolates in ChinaTo determine the pathogenic characteristics of isolates CK/CH/2010/JT-1,CK/CH/2014/FJ14 and CK/CH/2014/QL1403,and the antigenic relationship between different strains,pathogenicity,cross-neutralization and cross-protection studies were performed in this study.The resluts revealed CK/CH/2010/JT-1 and CK/CH/2014/FJ14 could cause 43.75%and 50%mortality with respiratory and severe renal lesions in inoculated SPF chicken,while CK/CH/2014/QL1403 strain is non-virulent with no obvious clinical sign,pathological change and death in infected chicken.The cross-neutralization test results showed that sera against Mass-and 4/91-type IBV could not completely neutralize isolates CK/CH/2010/JT-1 and CK/CH/2014/FJ14,while serum against QX-type IBV CK/CH/2014/FJ14 could neutralize isolate CK/CH/2014/QL1403 effectively.Cross-protection studies suggested chicken vaccinated with strain CK/CH/2014/QL1403 could confer protection against challenge of QX-type strain CK/CH/2014/FJ14.All these data demonstrated that CK/CH/2010/JT-1 and CK/CH/2014/FJ14 are highly virulent,and H120 and 4/91 vaccine strains could not provide effective protection against this two isolates,while CK/CH/2014/QL1403 is non-virulent which can be used as vaccine candidate to prevent QX type IBV.4.The innate immune response induced by IBV S1 protein in chicken embryo kidney cellTo better understand the host innate immune response induced by IBV S1 protein in chicken embryo kidney cell(CEKC),especially the differences of innate immune response induced by S1 proteins of nephropathic and standard strains in CEKC.We construct the eukaryotic expression vectors of S1 gene of nephropathic IBV CK/CH/2010/JT1 and CK/CH/2014/FJ14,and non-nephropathic strain M41,and then transfected into CEKC.The results showed that IBV S1 proteins were successfully expressed in CEKC and distributed throughout the whole cell.At 12 h posttransfection,the S1 proteins of nephropathic strains CK/CH/2010/JT1 and CK/CH/2014/FJ14 induced the expression of TLR3 and MDA5 genes were significantly higher than non-nephropathic strain M41 at mRNA level;at 24 h posttransfection,compared to M41,the S1 proteins of nephropathic strains CK/CH/2010/JT1 and CK/CH/2014/FJ14 caused greater induction of TLR3,IFN-?,MDA5 and IL-6.The differences of induction of TLR3,IFN-? and IL-6 at 24 h posttransfection were further comfirmed at protein level.At 24 h posttransfection,the differences of innate immune response induced by S1 protein of different nephropathic strains deacribed above were not expanded by stimulating with ligand poly(I:C)and LPS.These results shows that the S1 proteins of nephropathic strains CK/CH/2010/JT1 and CK/CH/2014/FJ14 caused greater induction of TLR3,IFN-?,MDA5 and IL-6 than M41,suggesting greater levels of TLR3,IFN-?,MDA5 and IL-6 expression are associated with increased pathogenicity of IBV strains in renal tissue.