| Avian Infectious Bronchitis(AIB)is a highly contagious avian disease,caused by avian infectious bronchitis virus(IBV).IBV can cause respiratory tract,kidney and reproductive tract lesions in chickens,and cause death,leading to huge economic losses to the global poultry industry.The serotype of the virus is complex and its cross-protection ability is poor.The strategy of IBV prevention by traditional vaccine faces challenging.At present,there is no unified classification standard abroad the word,but many scholars use IBV S1 gene sequence typing method.Due to the considerable differences between tissue tropism to pathogenicity among different genotypes of IBV,therefore,further studies on pathogenesis and immunologic mechanism of IBV to control IB are significant.In this experiment,a virus was isolated from dead chickens suspected to be infected with infectious bronchitis(IB)in Shandong Province.It was identified as IBV and named SD/04/18 strain by RT-PCR identification and sequencing analysis.Phylogeny and homology analysis showed that the strain has the closest evolutionary relationship with the Chinese QX strain.The blood coagulation titer of SD/04/18 strain was measured after treatment by trypsin digestion;The clinical symptoms of 8d SPF chicken after infection by IBV SD/04/18,showed the same as natural infection.In order to systematically study IBV SD/04/18 strain,the primers were designed according to the published N gene sequence of IBV in GenBank,and the IBV Real-Time PCR detection method was established.This method can detect at least39.7 copies/ μ L viral nucleic acid.Secondly,classical respiratory IBV-M41 standard strain and isolated SD/04/18 strain were serially passaged on SPF chicken embryos,and then the blind transmission was carried out on chicken embryonic kidney primary cells(CEK)and African green monkey kidney cells(Vero),until appear obvious lesions.Simultaneously,the viral titer of each generation is quantitatively tested.The results showed that IBV-M41 strain showed obvious pathological changes in CEK primary cells and Vero cells at the 4th and 5th passage,respectively.However,SD/04/18 strain showed obvious pathological changes at the5 th and 6th passage.After continuous passage,the IBV-M41 standard strain and SD/04/18 strain which could proliferate stably was obtained.In order to investigate the pathological damage,tissue tropism,dynamic distribution,detoxification,and host antibody levels of the SD/04/18 strain on host-related organs,the following experiments were carried out.Firstly,Firstly,the infection model of IBV SD/04/18 strain was established.At 3,7,and 30 days after challenged,three chickens from each group was selected randomly to determine the pathological sections and immunohistochemical analysis of trachea,lung,kidney,spleen,thymus and bursa.Secondly,12 kinds of tissues and organs were collected at different time points after infection,and virus dynamic analysis was performed.At the same time,the virus-shedding law,statistical weight,and the serum antibody levels of infected chickens in the respiratory tract and cloaca are regularly detected.The results showed that the pathological changes in the same organ showed differences in distinct infection periods;at 30 days after challenged,the trachea,lungs,spleen,and thymus recovered well,however,the kidney and bursal lesions are still serious.Antigen aggregation was identified by immunohistochemistry in all of the above organs.During the whole experiment,the viral load in the brain and pancreas was relatively stable,and the viral load in the other organs showed a trend from more to less.Viral nucleic acid was detected in the respiratory tract and cloacal cavity on 1 and 3days after challenged,respectively.IBV antibody was detected at 5days after challenged.The serum antibody titer peaked at5 daysafter challenged;the clinical symptoms of the infected chickens were obvious,and there was significant difference in body weight between the two groups.In conclusion,through the systematic study of the isolated QX genotype IBV SD/04/18 strain,it was determined that the SD/04/18 strain belonged to renal IBV in clinical classification.This strain mainly causes kidney disease in infected chickens and it also has different degrees of damage to the host’s immune organs.The diseased chicken has a long detoxification time and a slow weight gain.The target organs of the virus carry the virus for a long time and has a high viral load.The viral load of each organ was negatively related to the level of host antibody as a whole.After the SD/04/18 strain is blindly transmitted in chicken embryos and cells,the virus could proliferate stably in vitro.Combining the above conclusions,this study will provide an ideal animal model for the study of anti-IBV drugs,and provide a theoretical basis and experimental materials for the study of relatedmechanisms of IBV. |
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