Whole Genome Sequencing,Bioinformatics Analysis And Pathogenicity Study Of Four Representative Guangxi IBV Variants

Avian infectious bronchitis(IB)is an acute and highly infectious respiratory disease in chickens.The economic losses are very large in poultry industry in Guangxi.The genome of avian infectious bronchitis virus(IBV)is very easy to occur mutation.There are great differences in the genome of the virms strains prevalent in different countries or regions.In order to further elucidate the mutation mechanism and pathogenesis of IBV and effectively control the disease and promote the development of poultry industry in Guangxi,it is necessary to carry out whole-genome sequencing analysis and pathogenicity study of IBV epidemic strains in Guangxi.Therefore,complete genome sequencing analysis and pathogenicity study of four representative Guangxi IBV variants were conducted,and the obtained whole genome sequences were analyzed.SPF chickens and cherry ducks were challenged with the four IBV variants to analyze their pathogenicity,providing evidence for the prevention and control of IB in this region.Firstly,the whole genome sequencing of four IBV variants of GX-NN130048,GX-NN160421,GX-QZ170728 and GX-QZ171023 strains were performed by high-throughput sequencing technology.RNA extraction,reverse transcription,library construction,sequencing reaction and data analysis of four IBV strains were done.The results were showed as followings.(1)GX-NN130048,GX-NN160421,GX-QZ170728 and GX-QZ171023 variants were 27477bp,27751 bp,27674bp and 27663bp in length respectively;(2)The genome structures of four IBV variants were consistent with the classical structure:5'-UTR-Pol-S-3a-3b-E-M-5a-5b-N-UTR-3'.Secondly,bioinformatics analysis was carried out on the whole genome sequences of four IBV variants,including base insertion and deletion analysis of whole genome sequence,homology analysis,phylogenetic analysis,recombination analysis,S1 protein cleavage site analysis,glycosylation site analysis and RNA stem ring structure prediction.The results were showed as followings.(1)There were significant base insertion and deletion at the genome nucleotides of 2928-3428,20490-20930,21330-21550,24180-24740 and 27370-27440 in the four representative IBV variants;there were three continuous bases deletion at the genome nucleotides of 505-507 in the highly conserved N gene of GX-NN160421 strain,which was the fir-st deletion of N gene strain found in Guangxi.(2)The similarities of GX-NN130048 strain with GX-YL9,YX10 and DY07 strains were higher than 95.0%.The similarities of GX-NN 160421 strain with CKCHHB2016 and GX-NN09032 strains were higher than 96.4%.The similarities of GX-QZ170728,GX-QZ171023 and YX10 strains were 95.2%and 95.3%respectively.(3)GX-NN130048,GX-QZ170728 and GX-QZ171023 strains grouped together with YX10 and DY07 strains in the phylogenetic tree;GX-NN160421,GX-NN09032 and CK strains grouped together.The S1 genes of GX-NN130048 and GX-QZ170728 strains belonged to 4/91-type;The S1 genes of GX-NN160421 and GX-QZ171023 strains belonged to New-type and CK/CH/LSC/991-type respectively.(4)GX-NN130048 and GX-QZ170728 strains were proved to be recombinants derived from vaccine strains 4/91 and LX4 field strains.The breakpoints of recombinant fragments of GX-NN130048 strain were 1-20444,26981-27477,and those of GX-QZ170728 strain were 1-19046,26821-27674.(5)The cleavage sites of S1 protein of GX-NN130048,GX-NN160421,GX-NN160421 and GX-QZ171023 strains were RRSRR,HRRKR,RRSRR and RRFRR,respectively.The S1 genotypes of GX-NN130048 and GX-QZ170728 strains belonged to 4/91-type.(6)The N-glycosylation sites of S1 genes in GX-NN130048,GX-NN160421,GX-QZ170728 and GX-QZ171023 strains were 18,10,17 and 16,respectively,and those of N genes in the four strains were all 1.(7)Three RNA stem-ring structures were predicted using genome 5'terminal leading sequence as template,and mutation sites were found in 4/91,M41,CQ04-1,SAIBK and SC021202 strains.Two stem-ring structures were predicted using ORF1b fusion region of genome as template,mainly with mutation sites of BJ and California strains.Stem ring structure I was the main nucleotide mutation site.The present results indicated that the IBV variants in this region were mainly caused by point mutation and gene recombination.The 5'terminal leading sequence of genome and stem ring structure of ORF1b fusion region may play an important role in gene recombination,meaning that prevention and control of IB is facing more severe challenges.Thirdly,to further understand the pathogenicity of four representative IBV variants,pathogenicity tests of GX-NN130048,GX-NN160421,GX-QZ170728 and GX-QZ171023 strains in SPF chickens and Duck-Derived GX-QZ170728 strains in Cherry Valley ducks were carried out in this study.SPF chickens were randomly divided into four challenge groups and control group.Cherry Valley ducks were randomly divided into challenge group and control group.Animals of all the challenge groups at 7 days of age were challenged with 1×106 EID50 virus by eye drop and nasal drop respectively,and the control group was replaced by an equal amount of PBS.After the challenge,the clinical symptoms of each group were observed daily and the morbidity and mortality were recorded.The dead chickens were examined in time.Serum samples of 0,1,7,11,14 and 21 dpi were collected to detect IBV specific antibodies.1,3,5,7,11,14 and 21 dpi,the trachea,lungs and kidneys were collected to prepare tissue sections and observe pathological changes,and the viral loads of trachea,kidney and analswabs were detected by real-time PCR.The results were showed as followings.(1)At 24 hours after challenge,the chickens showed clinical symptom such as depression,loose feathers,weight loss,gasping,rale of trachea and increase drinking water.The tracheal mucus and hemorrhage,renal enlargement and pale and spotted kidney were found.There were no abnormal changes in control chickens,challenged and control ducks.(2)The morbidity and mortality of four IBV strains were 53.33%-100%and 2.22%-28.89%,respectively.The highest morbidity and mortality were found in GX-QZ171023 group.The mortality rate of Cherry Valley ducks challenged by the duck source GX-QZ170728 strain was 0%.(3)At 0-21 dpi,the antibody levels of chickens in the challenged group increased gradually.At 21 dpi,the antibody titer was higher than the positive value of the kit.During the whole experiment,the antibody level of chickens in the control group,ducks in the challenged and control groups remained low and unchanged.(4)Histopathological observation showed that all the four IBV strains resulted in disorder of tracheal mucosa structure,degeneration and necrosis of epithelial cells and infiltration of a large number of inflammatory cells,degeneration and necrosis of pulmonary epithelial cells,infiltration of a large number of inflammatory cells,local pulmonary congestion and atrophy of pulmonary atrophy.Renal tubular necrosis,glomerular cell necrosis and atrophy were observed in GX-QZ171023 group;there was a small amount of lymphocyte infiltration in the kidneys of GX-NN130048 group and no abnormal changes in the kidneys of the other two groups.There were no abnormal changes in pathological sections of control chickens,challenged and control ducks.(5)The results of real-time PCR showed that IBV could be detected in the trachea,kidney and anal swabs of chickens in 1-21 dpi,and the tracheal virus loads were significantly higher than the renal viral loads in GX-NN130048 and GX-NN160421 groups.The renal viral loads were significantly higher than the tracheal virus loads in GX-QZ170728 and GX-QZ171023 groups.IBV was not detected in control chickens,challenged and control ducks.The results of this study indicated that all four IBV variants were pathogenic to SPF chickens.The pathogenicity of GX-QZ171023 strain was the strongest.Duck-derived GX-QZ 170728 strain was pathogenic to SPF chickens and apathogenic to Cherry Valley ducks.The present results indicated that Guangxi IBVs were still mutating and the Guangxi IBV variants were mainly caused by point mutation and gene recombination.Four IBV variants were pathogenic to SPF chickens,among which GX-QZ171023 strain was the most pathogenic.Duck-derived GX-QZ 170728 strain was pathogenic to SPF chickens and apathogenic to Cherry Valley ducks.This study enriches the whole genome bioinformatics database of IBV and coronavirus,providing the basis for pathogenicity research and important reference for the prevention and control of IB in Guangxi and even China.