| It has been known for many years that IBV exists as dozens of serotypes. Furthermore, little orno cross protection occurs between different serotypes of IBV. So the IBV strains used fordeveloping vaccines should be best from the epidemic serotype. Now there are at least three IBVserotypes circulating in China, including Mass serotype, LX4 serotype and t1/CH/LDT3/03 serotype.Moreover, IB vaccines used in China induced a poor protection against the t1/CH/LDT3/03 strain,indicating the necessity of developing vaccines from local IBV strains of this serotype in China.The t1/CH/LDT3/03 strain for attenuation was passaged by inoculating three 9 to 11-day-oldembryonating SPF chicken eggs by chorioallantoic sac route. Inoculated eggs were incubated for 72h at 37℃, and the chorioallantoic fluid was harvested for subsequent passage. The surviving onewith typical pathological changes would be best for the subsequent passaging. On the base of deathtime and pathological changes of chicken embryos, 10-1 to 10-4 dilution was made beforeinoculation.The attenuation of t1/CH/LDT3/03 was examined in 15-day-old SPF leghorn chicks by clinicalresponse and mortality. The chicks inoculated with t1/CH/LDT3/03 P3 strain showed morbidity andmortality as high as100% and 80%, respectively; while no clinical response was observed when thet1/CH/LDT3/03 P120 were inoculated. So the t1/CH/LDT3/03 P120 strain was attenuated andapathogenic to chickens.The efficacy of t1/CH/LDT3/03 P120 strain was tested by serum antibody detection,vaccination-challenge test and organ protection in this study. All the birds showed positive serumantibody response at day 20 postinoculation in t1/CH/LDT3/03 strain inoculated groups, whereas,the birds in control group showed negative serum antibody response. The serum antibodyproduction detected by ELISA in this study clearly indicated that attenuated t1/CH/LDT3/03 P120strain virus was capable of stimulating systemic immunity in chicks. The vaccination-challenge testshowed that the attenuated t1/CH/LDT3/03 P120 strain could afford protection against challengewith the homologous virus, pathogenic t1/CH/LDT3/03 P3 strain, by mortality and clinical signs. Allthe chickens in control group showed respiratory signs and there are two and four chickens died ondays 4 and day 5 postchallenge, respectively. Then, all the chicks were sacrificed at days 5postchallenge Tracheals and kidneys were collected for virus recovery. In our study, 5 tracheals and5 kidneys were positive for virus recovery by RT-PCR detection, while all the tracheals and kidneys from chickens in control group were positive, showing that t1/CH/LDT3/03 P120 strain could wellprotect against the respiratory tract and renal damage. This might be associated withsystemic immunity and mucosal immune induced by t1/CH/LDT3/03 P120 strain. In all, theattenuated t1/CH/LDT3/03 P120 strain with good immunogenicity could provide a satisfactoryimmune protection and would be developed as a attenuated vaccine.Due to the important role of S1 protein in inducing virus neutralizing antibody, tissue tropismand immunity, most of the recent studys on pathogenicity of IBV focused on the S1 subunit. Inorder to find the correlation between S1 gene and IBV pathogenicity, t1/CH/LDT3/03 P3 strain andP120 strain S1 gene were sequenced and then compared. Two nucleotide substitutions (position 361and 656) were found, leading to the Gly substituted by Trp at residue 121, which was in areported hypervariable region, and Gin substituted by Arg at residue 219 of S1 subunit. Itmight need a further investigation to confirm whether the two amino acid residues werecorrelated with IBV pathogenicity.
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