Isolation Of Proventriculus Pathogenic IBV And Comparison Of Tissue Tropism On Three Different Pathogenic IBV Strains

Infectious bronchitis virus (IBV) is universally acknowleged as one of representative virus of coronavirus,being probably endemic in all regions with intensive poultry production. The continuous emergence of new IBV serotypes has complicated the design of appropriate control programs due to the antigenic variation and the low degree of cross protection observed among IBV serotypes. Moreover, IBV has varied in pathogenicity and tissue tropism. After IBV was first recognized as causing upper-respiratory tract disease,it has been reported nephrosis,enterion and proventriculus pathogenic strains were isolated and identified sequentially. So the traditional vaccines are difficult to control and eliminate IBV efficiently, which caused great economic losses to the poultry industry worldwide. Especially the proventriculus pathogenic IBV became new a hot spot of research.in recent years. Meanwhile many researchers focused on the mechanism of various tissue tropism of IBV, and hope to provide referenced date for other Coronavirus.This study starts with tissue tropism of IBV, taking proventriculus pathogenic IBV as main target,and strive to explore the pathogenesis mechanism of IB through three aspects research:the relationship of S1 gene and various pathogenicity of IBV, the binding capacity on different pathogenic IBV and chicken aminopeptidase N (chAPN), and the dynamic distribution of virus and chAPN. The methods and results are as follows:1. Isolation and identification of a strain of proventriculus pathogenic IBVA strain of virus with coronavirus characteristics which was isolated from dead chickens in a farm of Heilongjiang Province caused proventiculus disease characterized by swelling. After passage through the chicken embryo, the virus was detected by hemagglutination assay, electron microscopy, RT-PCR and regression experiment. The result demonstrated that the isolated virus is a strain of proventriculus pathogenic IBV.2. Sequence analysis of S1 gene of IBV A2-2 isolatesStudy the tissue tropism of IBV from its S1 gene. The result of sequence analysis of S1 gene of IBV A2-2 isolates shows proventriculus pathogenic IBV strains are divided into 3 groups. The first group includes A2-2, QXIBV,D971 and GE. Although A2-2 has high mortality with classic proventriculus pathogenic strains, so does with other pathogenic strains, like LX4 and CK-CH-LSD-07V, which were isolated typically from China in recent years. This shows the strains in group 1 are more related to time and regional epidemicity. The second group includes ZJ971, 1-98 and 3-97,the three proventriculus pathogenic strains has high mortality with vaccine strains such as H120,H52 and W93. That suggests vaccine may be a important source of proventriculus pathogenic IBV. Q1 and J2 belong to the third group. They have low mortality with many IBV strains. All above proventriculus pathogenic IBV may has different sources, and IBV strans with high mortality in S1 gene can differ in pathogenicity.3. Comparison of the binding capacity on three different pathogenic IBV with the chAPN in VitroThe fusion protein His-chAPN was expressed, purified and identified. The binding capacity on different pathogenic IBV strains and the possible cellular receptor chicken Aminopeptidase N (chAPN) in vitro was compared.by ELISA method,in order to investigate multi-tissue tropism mechanism of IBV through a new aspect. ELISA data demonstrated the binding capacity on different pathogenic IBV strains and chAPN in vitro was different to a certain extant, A2-2>S-03>M41. Because the three IBV strains were different pathogenic,this result suggested the binding capacity is related to tissue mechanism of IBV.4. Dynamic distribution of three different pathogenic IBV and chAPN in infected SPF chickensMonitoring of dynamic distribution of M41(respiratory pathogenicity), S-03 (nephrosis pathogenicity) and A2-2 (proventriculus pathogenicity) in SPF chickens was performed by real time PCR and IFA respectively. The result shows all three IBV strains positioned on trachea first, and M41 has high dependence with trachea. The strength of M41 expresses more apparently in lung. S-03 shows high capacity of propagation in kidney, and A2-2 can propagate well in kidney, which coincide with clinical observation. A2-2 is the dominant strain adapting to proventriculus, S-03 and M41 are hardly detected in proventriculus. In addition,three IBV strains display low difference in liver, ileum and bursa of Fabricius,with a low level in virus propagation. The clinical characterize of chicken infected by different pathogenic IBV shows time relativity with the change of virus titers, through the analysis on the propagation situation of three strains and the result of animal regression test.In this study,monitoring of dynamic distribution of chAPN is performed with two methods used on IBV. The result shows chAPN has wide distribution with the different quantity in different tissue of chicken. The distribution characteristic is consistent with the various tissue tropism and their target organs display a high and stable protein expression. It shows relatively similar changes of the chAPN quantity in chickens infected by three different pathogenic IBV: chAPN expresses quite highly in trachea,highly in proventriculus,lower in kidney,and the expression level declines with the increase of age in trachea and proventriculus,but is stable in kidney.,which suggest chAPN is related to tissue tropism of IBV. Meanwhile,some other organs contain chAPN relatively stable,like ileum,even more than kidney,where IBV propagate not well. That indicate distribution of chAPN has wider range than tissue tropism of IBV. And there was no remarkable difference among the distribution of chAPN in chickens infected by different pathogenic IBV.