Evaluation Of The Protection Conferred By Attenuated Heterologous Isolates Against The CK/CH/LDL/97I Strain Of Infectious Bronchitis Coronavirus

Avian infectious bronchitis attenuated vaccines play an important role in the control of IBV all over the world. Many commercial attenuated vaccines derived from local IBV field isolates have been used to control IBV in China besides Massachusetts serotype vaccines H120 and H52 that were imported. The molecular backgrounds of most of the domestic vaccines in China have not been clarified yet. It has been known for many years that IBV exists as dozens of serotypes. Furthermore, little or no cross protection occurs between different serotypes of IBV. So the IBV strains used for developing vaccines should be best from the epidemic serotype. The entire S1 protein gene of five infectious bronchitis (IB) commercial vaccine strains (JAAS, IBN, Jilin, J9,H120) used in China and three attenuated homologous and heterologous strains (CK/CH/LHLJ/04â…¤F110,tl/CH/LDT3/03 F120 and CK/CH/LDL/97â… F115)were compared with that of the IB field isolate CK/CH/LDL/97â… present in China. The sequence identity between the genes of vaccine strains and corresponding genes of IBV reference strains was analyzed by multiple alignment. The phylogenetic trees were also constructed based on the nucleotide sequences of the structural protein genes and those of the nonstructural protein genes to determine IBV genotypes exist in China, and the phylogenetic relationships between Chinese isolates and reference strains.Due to the important role of S1 protein in inducing virus neutralizing antibody, tissue tropism and immunity, most of the recent studys on pathogenicity of IBV focused on the S1 subunit. Phylogenetic analysis and the comparison of S1 showed that CK/CH/LDL/97I-type virus might be a new serotype compared to vaccine strains and other types of IBV isolates in China. Protection efficacy was evaluated by serum antibody detection, morbidity, mortality, clinical symptom, pathological lesion and virus re-isolation from the kidney and trachea of the challenged chicks. The nucleotide and deduced amino acid similarities between the five IB vaccine strains and the field strain, CK/CH/LDL/97â… , were not more than 76.4% and 78.7%,respectively. Phylogenetic analysis based on the amino acid sequence of S1 protein gene indicated that they belonged to the same cluster. Taken together, these results suggested that CK/CH/LDL/97â… and the three isolates, Q1, J2, T3, might be a new genotype of IBV in recent years in China.According to phylogenetic relationships of S1 gene sequences derived from IBV vaccine strains and reference strains, the most representative five vaccine strains (JAAS, IBN, Jilin, H120, J9) and attenuated homologous and heterologous strains were selected to evaluate the protective efficacy against field IBV strain. Nine groups of 20 White Leghorn SPF chickens were housed in isolators under negative pressure. At 15 day of age, chickens of groups 1 to 8 were inoculated intranasally with eight vaccines, respectively. The chickens of group 9 served as negative control. The antibodies in serum specimens were detected by indirect ELISA 20 days post inoculation (PI).The result indicated that commercial vaccine and attenuated strain virus was capable of stimulating systemic immunity in chicks. At 35 days of age, all chickens were challenged with the heterologous virulent strain CK/CH/LDL/97â… . At 3 days PI, clinical signs were observed in some vaccinated chickens. In total, the morbidity ranged from 0 to 100% and no mortality, whereas the unvaccinated chickens showed 100% morbidity and 10% mortality. The nephritis was observed in both the vaccinated and non-vaccinated dead chickens and was characterized by enlarged, pale and marbled kidneys, frequently with urate deposits in the ureters and cloaca. The organ samples at 5 days post challenge showed that the detection rates were 50%~90% and 10%~30% for trachea and kidney, respectively. However, the results shown that complete protection was provided by the homologous strain CK/CH/LDL/97â… F115. The protective efficacy of vaccines showed that the live attenuated vaccines only provided partial protection to the vaccinated chickens against heterologous IBV infection.