Molecular Epidemiological Investigation Of Infectious Bronchitis Virus In Some Areas Of China During 2018 To 2021,and Establishment And Application Of ELISA Method For Detecting Antibodies

Infectious bronchitis virus（IBV）has a wide range of tissue tropism and a high variability.Infectious bronchitis（IB）caused by IBV can not only cause respiratory symptoms,but also cause interstitial nephritis and reproductive system dysfunction.China is a large country of poultry breeding and consumption.In recent years,the prevalence of IB in China is diverse and complex,and the situation of prevention and control is still severe.This research collected clinical samples of chickens suspected to be infected with IBV in farms in some provinces of China from 2018 to 2021,and virus isolation,identification and analysis were performed to provide epidemiological data support for the prevention and control of IB in our country.In addition,the ELISA method to detect IBV antibody end-point titer was established,and IBV N protein as the detection antigen was expressed by prokaryotic system.This method can effectively reduce the cost of monitoring chicken antibodies in the farm.1.Molecular epidemiological investigation of IBV in some areas of China during 2018 to 2021.From 2018 to 2021,tissue and swab samples from chicken flocks suffering from IB in Jiangsu,Anhui,Hebei,Shandong,Jilin,Liaoning,Zhejiang,Henan,Guangxi and Ningxia etc.were collected.IBV surveillance and molecular epidemiology research based on S1 gene were conducted in some areas of China in recent 4 years through RTqPCR,virus isolation,RT-PCR,S1 gene cloning,sequencing and S1 gene sequence analysis.102 strains of IBV were isolated and identified from clinical samples.Phylogenetic analysis based on S1 gene showed that 69 isolates belonged to GI-19（QX）genotype,accounting for 68.27%of the total strains,which was still the dominant genotype.9 isolates belonged to GVI-1（TC07-2）genotype,accounting for 8.65%.3 strains belonged to GI-13（7/93B or 4/91）genotype,accounting for 2.94%.4 strains belonged to LDT3-A type,accounting for 3.92%.A total of 5 strains belonged to GI-1（Mass）genotype,accounting for 4.90%.21 vaccine strains were isolated,accounting for 20.59%of the total isolates,included 11 QX vaccine strains,5 Mass vaccine strains,3 LDT3-A vaccine strains and 2 4/91 vaccine strains.The remaining 12 isolates accounted for 11.76%,which were not classified as known genotypes for time being.The nucleotide and amino acid homology of all isolates were 64%～99.9%and 57.9%～100%,respectively.The length of S1 sequences ranged from 1611～1638bp（537～546aa）,of which 1620bp was the most common length,accounting for 70.87%.Seven cleavage site motifs were identified:HRRRR（65,62.5%）,RR（S/F/L）RR（17,16.50%）,HRRKR（11,10.68%）,HRHRR（10,9.71%）and HRFRR（1,0.98%）.Nglycosylation sites were predicted in all strains and had obvious type specificity.Only two strains had 1 O-glycosylation sites respectively.2.Establishment and application of ELISA method for detecting IBV antibodies.Using the N protein prepared by prokaryotic expression as the detection antigen,the negative and positive serum were prepared,the test conditions were continuously optimized,and the ELISA operation process was finally determined.Using the PNT（positive-negative threshold）baseline,the linear regression equation between the corrected absorbance（S/P）at 1:500 dilution and the end-point titer of IBV antibodies was established:log（ET）=1.222×log（S/P）+3.286（P<0.0001）,and the correlation coefficient（R2）reached 0.9593,This can well predict the end-point titer of serum samples.This method has good repeatability,and the intra and inter-batches repeatition coefficient of variation were less than 10%.Its specificity was good and there was no cross reaction with common pathogenic serum.Through the optimization and selection of the storage conditions of this kit,the developed rIBVN-ELISA kit can be stored at 4℃ and-20℃ for half a year.To verify and evaluate the test effect of the rIBVN-ELISA method,461 serum samples collected from two chicken flocks were detected simultaneously by the rIBVN-ELISA method established in this subject and commercial kits.Compared with the commercial kits,coincidence rate of negative and positive of this method was 93.06%,the rise and fall tendency of antibodies titer was highly consistent,the coefficient of variation of serum detection results at different ages were close,and the distribution of antibodies titer was relatively consistent.ROC curve analysis showed that the sensitivity and specificity of the rIBVN-ELISA were 99.75%and 100%,respectively（P<0.0001）.It was verified that the rIBVN-ELISA method has good detection effect and can be applied to evaluate vaccine immunization effect and field virus monitoring in chicken farm.