| Avian infectious bronchitis(IB),caused by avian infectious bronchitis virus(IBV),is an acute globally distributed domestic avian disease of major economic importance throughout the world,which has also endangered the health of breeding poultry industry in China.At present,several commercial vaccines are used for immunoprophylaxis of IB.However,due to the high variability of IBV virus and the lack of cross protection between different serotypes,IB has not been effectively controlled.There are still a lot of problems in the comprehensive prevention and control of IBV.In view of this,IBV positive samples were collected for virus isolation,gene sequencing and genetic evolution analysis.Based on the isolates,two rapid detection methods of IBV were developed,including a real-time fluorescence quantitative RT-PCR method and an indirect ELISA for antibody detection.Using those detection methods,an epidemiological investigation throughout Shandong province was carried out.Several isolated virus strains were used for cell culture and optimizing the cultivation methodology.With the cell cultured virus,inactivated IBV vaccine was developed and tested for immune protect effect evaluation.thus established a basic research platform for IBV early diagnosis,epidemiological investigation,pathogenic mechanism study,genetic variation,vaccine preparation and quality inspection for comprehensive prevention and control measures to IBV in Shandong area.For the isolation and identification of clinical cases of suspected IBV infection pathogens in Shandong,and the study of biological and evolutional characteristics of isolated strains,virus isolation,hemagglutination test,chick embryo pathogenicity test,and animal regression test were performed in this study,which showed a total of 48 isolated strains with different EID50 values from 10-5.0/0.1 m L to 10-5.83/0.1 m L.All the 48 isolates could cause classical "dwarf embryo" symptoms of SPF embryo,with lethality rates ranged from 30% ~ 60%.Phylogenetic analysis of S1 sequences showed that 46 of the 48 isolates belonged to QX lineage,while the other two strains belonged to Mass lineage.It is indicated that the QX genotype has become the dominant lineage of IBV in Shandong.For the early diagnosis and epidemiological investigation of IBV infection,organ tropic evaluation,and virus shedding detection,an IBV real-time RT-PCR test method and its kit were established according to the information of QX-type isolates.The results showed a good standard curve(correlation coefficient R2 is 0.9988)of the IBV SYBR Green I Real-time RT-PCR method,and a detection limit of at least 7.5 x 103copies/ ?L viral nucleic acid,which was 100 times higher than the sensitivity of conventional RT-PCR.The specificity detection of MDV,NDV,IBDV,ILTV and other avian virus samples with this method showed negative results.The coefficient of variation values of inter-batch and intra-batch difference were less than 3%,192 clinical samples were detected using this kit,with a positive rate of 25%(48/192),which was significantly higher than that of common PCR detection method.These results showed that the IBV real-time RT-PCR detection kit was a highly sensitive,specific and reproducible method,which could be used for the quantitative detection,early diagnosis and epidemiological investigation of IBV infection.To provide a new serological diagnostic technique for IBV immunization antibody monitoring,rapid diagnosis of clinical wildtype virus infection and epidemiological investigation,a 867 basepair fragement target the main antigen domain of isolated IBV S1 gene was amplified by RT-PCR,cloned into the p ET30 prokaryotic expression vector,and transformed Rossetta expression bacteria.After IPTG induction,IBV recombinant S1 protein expressed in inclusion body was obtained.On this basis,the recombinant S1 protein was purified and used as coating antigen to establish an indirect ELISA method for detecting IBV antibody.Compared with commercial kit from IDEXX,the coincidence rates were 94.55%,95.42% and 92.96%,respectively.The ELISA antibody detection kit provides a new serological diagnostic technique for supporting the development of IBV vaccine.It was also helpful for IBV epidemiological investigation and clinical disease diagnosis.In order to determine the best cell line for IBV cultivation,four IBV strains including GQ/2015 strain,WF/2015 strain,M41 strain and M491 strain were inoculated to Vero,Vero-E6,BHK-21,Macr-145 and DF-1 cells,and blind passaged to the fifth generation.The best virus and cell line were selected according to the EID50 values.Basing on the results,the culture conditions such as cell denesity,growth curve of cell line,virus proliferation accelerant and the application of small bioreactor were investigated.The results showed that only Vero cells form the 5 cell lines could be used for the proliferation of those 4 IBV strains,in which GQ/2015 strain and M491 strain showed best proliferation rate,with titers of 10-4.83/0.1 m L and 10-4.5/0.1 m L,respectively;Marc-145,BHK-21 and DF-1 were only suitable for one IBV strain proliferation with significantly lower titers.On the other hand,the optimization of culture conditions for GQ/2015 strain and M491 strain showed that the optimal inoculation volume of GQ/2015 and M491 was 1% volume of cell culture medium,the best serum concentration was 2%,the best inoculation way was step by step seed,and the best time to harvest the virus was 60 h to 72 h.finally,GQ/2015 and M491 strains were cultivated in bioreactor with immoblized paper carrier,indicating that the EID50 value of GQ/2015 strain and M491 strain could be significantly improved to 10-6.375/0.1 m L and 10-5.83/0.1 m L,respectively.Therefore,these data could be used as reference for the application of large-scale production cell line for IBV proliferation.For the development of a novel vaccine against epidemic IBV strains,IBV GQ/2015 strain was used as vaccine strain to prepare inactivated IBV vaccine using cell culture method and white oil adjuvant.The inactivated vaccine showed good safety and immune effeciency.Animals inoculated with 0.1m L and 0.3 m L IBV inactivated vaccine showed highest ELISA antibody level(S/P value)21 d after vaccination.The cross immunization test showed a 100% protection rate as well.The results showed that the IBV GQ/2015 strain oil emulsion inactivated vaccine had good safety and immunogenicity,which laid the foundation for the development of IBV vaccine against epidemic strains with independent intellectual property rights. |
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