| Avian Infectious bronchitis(IB)is an acute,highly contagious avian disease caused by infectious bronchitis virus(IBV),which has severely affects the development of the world poultry industry.The genome of IBV has a very high mutation rate and recombination rate and is more prone to mutation when host selection pressure and vaccine immunological pressure existing,which leads to numerous serotypes and genotypes.The poor cross-protection between different serotypes and genotypes of IBV makes IB one of the most difficult diseases to control in poultry production.In recent years,TW IBV has become the prevalent genotype in China.In order to solve the above problems,the following experiments were carried out in this study:133 IBV strains were isolated from broiler farms in 13 provinces,autonomous region and municipality during 2016~2018.Phylogenetic analysis the S1 gene of IBV isolates showed that 122 isolates were clustered with reference strains of 8 genotypes.QX type was the predominant genotype,which accounted for 62.4%.TW type accounted for 9.8% of the isolates.The number of the 4/91 type isolates accounted for 9.8%.The similiarity of four 4/91 type isolates and 4/91 vaccine strain was more than 99.7%.Whether the strains were 4/91 vaccine strains need further verification.4/91 type were widely involved in recombination.11 mutant strains accounting for 9% formed separate evolutionary branches.Recombinant analysis showed all mutants were recombinant ones and the main recombinant strains were produced by 4/91,QX and TW type strains trans-recombinanting.The 4/91 type strains which involed in recombination accounted for 63.6% of all the mutant strains.Different IBV attenuated vaccines were selected for evaluation of the immune protection towards to QX type IBV and TW type IBV respectively.The results showed that two vaccinations of the same vaccine provided a lower protection than combined vaccine immunization.Such as CAB or AMI two vaccinations provided 86.6% and 73.3% protection rate for QX-type IBV respectively,but it boosted to 93.3% when combining immunization.However,all type vaccination could not provide effective protection for TW-type IBV.The protection rate was less than 50%.TW-type IBV had become the second dominate genotype in China,it was necessary to carry out TW-type IBV vaccine research.A TW type attenuated strain GX15F105 was obtained by continuous passaging.Pathogenicity studies of GX15 strain in different generations revealed that GX15F105 had no pathogenicity to 10 day-old chickens after F80.Histopathology study showd GX15F95 had a slight pathogenicity in bronchitis,lung and kidney,but GX15F105 had the same microstructure with the control group.IBV challage assays showd GX15F105 could provide 80% protection for GX15F5.Tracheal cilium stasis experiment indicated that F105(103.5EID50),F105(104.5EID50)and F95(103.5EID50)group had a perfect cilium activity as control group.Virus load assay showed copy numbers of field strain in different tissues of F95 and F105 groups were lower dramaticly than control-F5.The whole genome sequencing analysis of different generations of GX15 strain showed that F50,F80,F95,F105 and F120 strains producing many nucleotide mutations,inserts and deficiencies at different positions steadily,which caused amino acid substitution at the corresponding positions.These amino acid substitutioning might be responsible for the reduced pathogenicity of GXF105.In this study,molecular epidemiology of IBV were performed in south China and the most optimal vaccine combination were screening out towards QX genotype IBV,and a TW type attenuated strain GX15F105 was obtained by continuous passaging,evaluation experiments indicatied GX15F105 which had good safety and immunogenicity could be considered as a potential vaccine candidate strain. |
