Sequence Analysis Of The N Gene Of 793/B And Establishment And Application For Detection Of The IBV By Digoxigenin-Labeled Probe And Multiplex RT-PCR

Infectious Bronchitis(IB) is a severe acute respiratory disease of poultry, caused by Infectious bronchitis virus (IBV)。Since broke out in USA in the 1930s, IB have been an important disease of poultry around the world. IBV is a sRNA virus , the gene of the virus always mutated owing to it's point-mutation and gene recombination, so the virus have many serotypes. The known serotypes can be classified into respiratory symptomatic serotypes such as Conn, Iowa97, JMK, Florida, Arkansas99 and nephritic symptomatic serotypes such as M41, Holte, Gray, Australia'T'.Gough reported a new serotype of IBV named 793/B (also known as 4/91 and CR88) in UK in 1992,793/B isolates have a little serological relation with other serotypes of IBV, the whole S1 gene of 793/B isolates differed by 21% to 25% from those of other 17 European isolates. Youxiang Diao, Jiehua Yang et.al.(2003) isolated an IBV 793/B strain named IBV TA03 from layer flock in a chicken house of Shandong province. In recent years, the 793/B serotype of IBV, harmful to the healthy development of poultry breeding, prevailed and spread in Spain, Germany, Holland,China ,Italy and Thailand, too.According to the genomic sequences of N gene of infectious bronchitis virus (IBV) published in Genbank,one pairs of primers were designed for amplifying the 582 bp fragment in RT-PCR experiments. The PCR product was labeled with digoxigenin as DNA probe for detection of IBV. The hybridization assay result of specificity showed all RNA of IBV strains were positive, but other nucleotide extracted from NDV, GPV were negative. The sensitivity result showed that as few as 10 pg RNA amount of IBV could be detected by DIG-labeled probe. So the DIG-labeled probe could be used to detect the IBV.A Multiplex RT-PCR was optimized to simultaneously detect IBV and 793/B,for saving time of detection. Two sets of primers were designed according to the sequences of 793/B and other IBV at the GenBank. The product of 582bp and 891bp were generated only with RNA from 793/B, where as 582bp product only with RNA from other IBV. However, the RT-PCR failed to detect NDV, ILTV and GPV. The results showed that the established Multiplex RT-PCR technique provided a more sensitive method for diagnosis and epizootic study of the IBV.According to the genomic sequences of Nucleocapsid gene of IBV published in Genbank , a pair of primers were designed. The nucleocapsid protein(N) gene was amplified by RT-PCR. The results of sequencing showed that the complete genome of nucleocapsid of IBV isolate (793/B) consisted of 1229 nucleotides. Sequence analysis showed that 88 point mutations were found in N gene, and one nucleotides was deletion in 991 position. The homology of nucleotide sequence were 86.9—91.4%, and the homology of deduced aminoacids were 75.8 -77.5% compared with the 11 strains published in GenBank. The results showed that these were major variance in the N gene of 793/B.