Study On Correlation Between Genotypes And Serotypes Of Avian Infectious Bronchitis Virus

Avian Infectious Bronchitis(IB), caused by Avian Infectious Bronchitis Virus(IBV) of Coronavirus, is an acute and highly contagious infectious disease which leads to significant economic loss in poultry industry. Due to discontinuity of genomic RNA replication and incomplete proofreading mechanism of RNA polymerase, genome is easy to bring about point mutation, deletion, insertion and homologous recombination among different strains. Thus, genotype, serotype, structural protein and immunogenicity of IBV are easy to change, so clinical manifestation of IBV is complex and there is partial or entirely no cross protection among distinct serotypes, hence, it is difficult to prevent IB. IBV typing is siginificant to enforce effective control on disease, make epidemiological investigation and analyse evolution of IBV. In this study, prevailed strains, vaccine strains and strains used for test were grouped by combining genotying with serotyping, then we evaluated the correlation of genotype and serotype so that the biological characteristic of IBV can be reflected more truly. This method can not only provide refernce for IBV typing, but also is helpful for IB vaccine uses and is siginificant to prevent IB.In this study, we used seventeen isolated srains, two vaccine strains(M41, W93), and one virulent strain used for challanging as subjects, and obtained S1 gene of twenty strains by RT-PCR using specific designed primers. S1 gene was inserted into vector pGEM-T Easy, then we sequenced inserted fragment after recombinant plasmid was determined as positive one by PCR. We compared nucleotide sequences and analysed homology of S1 gene between twenty strains and twenty reference strains by Clustal V method in DNAstar software, then drawn up phylogenetic tree based on S1 gene. The result showed that twenty srains, vaccine strains and reference strains were grouped into three genotypes. The first one is LX4-type, including fourteen strains isolated from chicken groups in different regions of our country(BJ-0, BJ-2, BJ-3, BJ-4, BJ-5, 333-4, 333-5, 333-6, 333-7, 333-8, 333-9, 333-10, DC07-1, DC07-2). There is a much closer genetic relationship between BJ-0, BJ-2, BJ-3, BJ-4 and BJ-5. There is also a much clsoer genetic relationship among 333-4, 333-5, 333-6, 333-7, 333-8, 333-9, 333-10 and DC07-2, while DC07-1 has a much further genetic relationship with thirteen strains mentioned above in the same genotype. The second one is Mass-type, including five strains(333-1, 333-2, 333-3, M41, W93), vaccine strains H52 and H120, and foreign isolated strains(isolated from Spain, America, Korea, and Japan et al.). 333-1 strain had a closer genetic relationship with 333-2 and 333-3, while W93 had a closer genetic relationship with IBN, and M41 owned a closer genetic relationship with Spain-98-308. The third genotype is X-type, including X strain in this study, and X strain had a closer genetic relationship with Ark99 because their nucleotides similarity is 97.8%.According to the rusult of twenty strains’ genotyping, six highly variable strains(X strain in X-type, M41, W93, 333-3 in Mass-type, DC07-1 and 333-7 in LX4-type)were selected from three genotypes in the phylogenetic tree base on S1 gene to proceed serotyping test. Purity tests were made and we determined 50% embryo infectious dose(EID50) of every strain, then we immunized three weeks old SPF chickens to prepare hyper-immune sera, and carried out cross neutralization tests. At last, we calculated relative coefficient of every IBV strain. The result indicated that six strains were pure and contained no other microbes, and their EID50 are more than 106.0EID50/0.1mL. By calculating in immunological method, relative coefficients of 7%, 14%, 8%(<25%)were gotten between Mass-type strains M41, W93, 333-3 and X strain in X-type, and relative coefficients of 6%, 13%(<25%)were gotten between LX4-type strains DC07-1、333-7 and X strain in X-type. They demonstrated that Mass-type strains M41, W93, 333-3; LX4-type strains DC07-1, 333-7 and X strain in X-type belonged to diffenrent serotypes respectively, X strain shared a low cross antigenicity with them. Relative coefficients of 97% 、 98%( >80%) were gotten between 333-3 and M41, W93 in the same genotype—Mass-type. Relative coefficients of 8%、19%(<25%)were gotten in Mass-type strain 333-3 with LX4-type strains DC07-1 and 333-7, demonstrating that 333-3、M41、W93 belonged to the same serotype, and they shared a high cross antigenicity with each other. But 333-3, together with LX4-type strains DC07-1、333-7, belonged to the different serotypes, and they shared a low cross antigenicity with each other. Mass-type strain M41 owned a relative coefficient of 35% with W93 in the same genotype, and it had relative coefficients of 7% and 21% with LX4-type strains DC07-1、333-7, indicating that M41 and W93 belonged to the same serotype, and they shared a high cross antigenicity with each other. But M41 shared a low cross antigenicity with LX4-type strains DC07-1、333-7, so they belonged to the different serotypes. W93 in the Mass-type owned relative coefficients of 13% and 21%(<25%)with LX4-type strains DC07-1、333-7, proving that they belonged to different serotypes, and they shared a low cross antigenicity with each other; DC07-1 strain in LX4-type had a relative coefficient of 65%(>25%)with 333-7, so they shared a high cross antigenicity with each other and belonged to the same serotype. From what we discussed above, X strain in X-type, M41, W93, 333-3 in Mass-type, DC07-1, 333-7 in LX4-type belonged to three serotypes respectively. M41, W93, 333-3 in Mass-type belonged to the same serotype; DC07-1, 333-7 in LX4-type belonged to the same serotype. Therefore, six strains were grouped into three serotypes by cross neutralization tests: M41, W93, 333-3 in Mass-type belonged to the same serotype, while DC07-1, 333-7 in LX4-type belonged to the same serotype, and X strain belonged to another serotype.From what we discussed above, twenty strains’ genotypes were determined and we selected six strains which had significant difference with other strains according to phylogenetic tree based on S1 gene to carry out cross neutralization tests. The result showed that there was a consistent relationship between genotypes and serotypes of IBV, and preliminarily proved that genotypes are parallel to serotypes of IBV. This result can provide reference for complicated status of IBV typing currently, and is siginificant to prevent IB.