Structural Gene Sequence Analysis And Serotype Identification Of Ibv Isolates In Guangxi From 2018 To 2021 And Whole Genome Sequence Analysis Of Three Representative Variants

Infectious bronchitis（IB）is an acute,highly contagious viral respiratory and genitourinary tract infectious disease of chickens caused by avian coronavirus infectious bronchitis virus（IBV）.Since the disease was reported in the United States in 1937,it has been distributed globally,causing serious economic losses to the poultry industry around the world.The genome of IBV is extremely prone to mutation.Point mutations,insertions,deletions and recombination were common in the entire genome,resulting in the complexity and variability of IBV genotypes and serotypes,which brings enormous pressure to the use and development of vaccines.Previously,our research group completed the S1 gene sequence analysis of IBV isolates in Guangxi in 2018-2019 and the serotype identification of Guangxi isolates in 2015-2017.On this basis,this study continued to analyze other structural genes and serotypes of IBV isolates in Guangxi from 2018 to 2021,and performed whole genome sequencing and analysis of three representative IBV variants in Guangxi,in order to understand the mechanism,characteristics,and laws of IBV gene and antigen variation in Guangxi in time,and continue to provide scientific basis for the effective prevention and control of the disease as well as development of the poultry industry in Guangxi.Details are as follows:1.Sequencing and analysis of structural genes of IBV isolates in Guangxi from 2018 to 2021Chicken embryo proliferation and RT-PCR techniques were used to isolate and identify IBV from suspected diseased chickens in Guangxi from 2019 to 2021.The four structural genes S1,N,M,and E of Guangxi IBV isolates from 2018 to 2021 were amplified and sequenced.Sequence similarity,phylogenetic tree,recombination,selection pressure,amino acid entropy,glycosylation sites,and S1,S2 protein cleavage sites were analyzed.The results showed that 8 strains of IBV were isolated from2019 to 2021,and the nucleotide lengths of S1,N,M and E genes of 22IBV strains from 2018 to 2021 were 1611～1638 bp,1230 bp,675～693 bp and 309～333 bp,respectively.Based on the S1 gene,the 22 IBV strains can be divided into 6 genotypes,among which the LX4-type strain accounted for the highest proportion,reaching 36.36%（8/22）,the LDT3-A type accounted for 22.73%（5/22）,and the Taiwan-type accounted for 18.18%（4/22）,CK/CH/LSC/99I-type accounted for 9.09%（2/22）,New-type 1 accounted for 9.09%（2/22）,Mass-type accounted for4.55%（1/22）.There were 3,3 and 4 genotypes based on N,M and E genes,respectively,and the dominant genotypes were LX4-type,CK/CH/LSC/99I-type,and LX4-type,respectively.The results of recombination analysis showed that 12 of the 22 IBV isolates had recombination events in the S1 or N genes.In the S1 gene recombinants,the major parent of 5 strains was the CK/CH/LSC/99I-type strain,and the minor parent was the QX-type strain.In the N gene recombinants,the major parent of 6 strains was LX4-type strain,the minor parent of 3recombinant strains was 4/91-type strain,and the minor parent of the other 3 recombinant strains was CK/CH/LSC/99I-type strain.The results of selective pressure analysis showed that the d N/d S values of the S1,N,M,and E proteins were 0.409,0.127,0.210,and 0.232,respectively,which were all less than 1,and there were 2,2,0,and 1 positive selection sites,respectively;the average value of amino acid entropy was S1>E>M>N;the S1,N,M and E proteins of 22 strains of IBV contained potential N-glycosylation sites of 11～18,1,2～3,and 2～3;the N protein of all isolates and the S1 and M proteins of a few isolates have potential O-glycosylation sites;the cleavage sites of S1 and S2 proteins were HRRRR,RRFRR,and HRRKR.2.Serotype determination of IBV isolates in Guangxi from 2018to 2021Serotype identification of 22 IBV isolates from 2018 to 2021 was performed using the tracheal organ cultures（TOC）neutralization assay.The results showed that 22 isolates could be divided into 6 serotypes,of which 8 isolates were serotype VII,6 isolates were serotype II,3 isolates were serotypes III,2 isolates were serotypes IV,2 isolates were serotypes VI,and one isolate belongs to serotype I.The present study showed that there were still multiple serotypes of IBV prevalent in Guangxi from2018 to 2021,and the dominant genotype was serotype VII,which was different from the dominant serotype II in 2015-2017,suggesting the need for continuous monitoring of serotype.3.Whole genome sequence determination and analysis of three representative IBV variants in GuangxiThree representative variants,GX-QZ181028,GX-NN200723,and GX-NN200724,were selected from 22 Guangxi IBV isolates,and their whole genome sequences were determined by high-throughput sequencing technology.The analyses of nucleotide insertion and deletion,nucleotide similarity,phylogenetic tree,recombination and RNA stem ring structure were performed.The results showed that the coincidence rate between high-throughput sequencing and next-generation sequencing was over 99%.All the three variant strains conformed to the IBV classicalgenomestructure5’UTR-Pol-1a/1ab-S-3a-3b-EM-5a-5b-N-3’UTR.Compared with vaccine strains H120,4/91,and LDT3-A,the three IBV variant strains had nucleotide insertions and deletions at 3194/3198 bp.The phylogenetic tree showed that all three variants belonged to LX4-type;the recombination analysis showed that there were 3,2,and 2,recombination events in GX-QZ181028,GX-NN200723,and GX-NN200724,respectively.The results of RNA stem ring structure analysis showed that there were two stem ring structures in the three IBV variants,among which GX-QZ181028 had the same stem ring structure as GX-NN200724,and the stem ring structure of GX-NN200723 was unique,which might have an important impact on the production of recombination events.4.Correlation between genotyping and serotyping of IBV isolates in Guangxi from 1985 to 2021Based on the results of the first and third chapters of this study and the previous results of our research group,the genotype and serotype correlation of 114 Guangxi IBV isolates from 1985 to 2021 were analyzed.At the same time,the B and T cell epitopes of S1 protein and N protein of IBV isolates were predicted,and the correlation between epitopes and serotypes was analyzed.The results showed that among 114IBV isolates in Guangxi from 1985 to 2021,the coincidence rate of S1gene-based typing and serotyping was 28.07%（32/114）,and the coincidence rate of N gene-based typing and serotyping was 16.67（19/114）,indicating that genotyping could not fully represent serotyping.Epitope prediction showed that 4 B cell epitopes(³⁴FRPPNGWHL⁴²,²⁴²GNFTDGFYPFTNSSSVNERFIVYRESSV²⁶⁹,³²⁴ESNYMYGSYHRACNFRLETINN³⁴⁵ and ³⁵⁹GPLQGGC³⁶⁵)in S1protein and 4 T cell epitopes(¹⁴¹AMRNGSLFY¹⁴⁹,¹⁶⁶CVNNFTSVY¹⁷⁴,²⁰⁷MKEFKVLAY²¹⁵ and ⁴³³ITLNTCVDY⁴⁴¹)were relatively conserved;All the N proteins of the 114 IBV isolates contained 8-10 B-cell epitopes with high similarity（86.1%-100%）.In addition,the T-cell epitope⁸⁶PVPDAWYFYY⁹⁵ at Pep86-Pep95 was also highly conserved.In summary,there were multiple genotypes and serotypes of IBV in Guangxi from 2018 to 2021,and the dominant genotype was still LX4;the dominant serotype was serotype VII,which was different from the dominant serotype II in 2015-2017.The recombination phenomenon of IBV isolates in Guangxi was very extensive,and there were multiple recombination phenomena.The major parents of the recombinant strains were LX4 dominant genotype strains;and the genotyping cannot fully represent serotyping.The present study results show that the IBV epidemic strains in Guangxi continue to mutate,and the recombination becomes more and more extensive and complex,indicating the necessity of continuous epidemiological monitoring,genetic variation analysis and development of new targeted vaccines.