Attenuation Of An Infectious Bronchitis Virus CK/CH/LHLJ/04V Strain And The Immune Protection Against Homologous Pathogenic Virus

Avian infectious bronchitis virus (IBV) attenuated vaccine plays an important role in avianinfectious bronchitis (IB) control in China. it is well known that little or no cross protection occursbetween different serotypes of IBV, and emergence of new variant strains occurs frequently, whichmaybe one of the reasons leading to outbreaks of IB occur frequently in vaccinated flocks. Therefore, todevelop new attenuated vaccines from local strains based on molecular epidemiology study is a must forIB control.In this study, a field strain of IBV CK/CH/LHLJ/04V was isolated from kidney of the affectedyoung broiler flocks in Helongjiang province, China. Characterization of the virus, includingpathological role to chicken embryo, hemagglutination activity, morphological characterization andvirus pathogenicity to specific-pathogen-free (SPF) chicken, were studied. Sequence analysis of the S Iprotein gene demonstrated that the virus was a representative of one of the major type of IBV in recentyears in China. The strain was passaged 110 times (designed as P₁₁₀) through SPF embryonated chickeneggs, to develop a potential attenuated vaccine.During the passage of CK/CH/LHLJ/04V strain, the virus titer rose gradually, causing more severepathological changes to SPF embryonated chicken eggs. On the other hand, the pathogenicity of thevirus to SPF chicken was weaken. When CK/CH/LHLJ/04V P70 was inoculated, no death was found,but complete protection against homologous virus P₅ strain was observed.After serial passage for 110 times, the attenuation of CK/CH/LHLJ/04V strain was complete.When the P₁₁₀ strain was given to 15-day-old SPF chicks, no clinical signs were observed. The efficacyof CK/CH/LHLJ/04V P₁₁₀ strain was tested by serum antibody detection, vaccination-challenge test andorgan protection in this study. Only 60%～70% of the birds showed positive serum antibody response at20 days post-inoculation. The chicks inoculated with CK/CH/LHLJ/04V P₁₁₀ strain showed morbidityand mortality as high as 20%～40% and 20%～30% respectively after challenging with virulent P₅ strain.The organ samples at 5 days post-challenge were detected and the results showed that the protection ratewas only 40%～50% for kidney and trachea. Obviously, after serial passages in SPF embryonatedchicken eggs, the CK/CH/LHLJ/04V P₁₁₀ strain has been attenuated, but the immunity was also weaken.While the attenuation of CK/CH/LHLJ/04V P₇₀ with good immunity was not complete. So it isnecessary to select an attenuated passage virus with good immunity from P₇₀ to P₁₁₀ as a potentialattenuated vaccine.Most studies on the pathogenicity of IBV focused on the S1 subunit. In order to find the correlationbetween S1 gene and IBV pathogenicity, S1 gene of CK/CH/LHLJ/04V P₅ strain and P₁₁₀ strain weresequenced and compared. Three nucleotide mutations were found, leading to three amino acid residuesubstitutions. The variation of the virulence of IBV possibly was influenced by many factors and waslonely or collectively caused by several genes. So it might need a further study to confirm whether the three amino acid residue substitutions were correlated with IBV pathogenicity and immunity.