| Avian Infectious Bronchitis (IB), caused by Avian Infectious Bronchitis Virus (IBV) of Coronavirus, is an acute and highly contagious infectious disease which leads to significant economic loss in poultry industry. IBV has a narrow host range, which is difficult to culture on cells but has to resort to propagate in embryos. At present, the study of IBV adaptation on cells and its biological characteristics is seldom underwent, and it is inevitable to make a deeper research on IBV especially the vaccine strains in this area. In this experiment, IBV vaccine strains were attempted to culture on CEK cell and identify the biological characteristics and the IBV Real Time Fluorogenetic Quantitative PCR (FQ-PCR) has been also established to detect the virus contents quickly during the virus passing on cells.First, Avian Infectious Bronchitis Virus (IBV) vaccine strains, H120, W93,H52,M41,were tried to culture and pass on CEK cell,CEF cell,Vero cell,Vero cell with TPCK trypsin and Vero cell with D-Glucosamine, and they had the best adaption on CEK cell.Therefore, IBV strains were tried to culture and pass on CEK cell up to 25 passages, studied its pathologic effect both in embryo and cell induced by CEK cell-adapted virus, identified the biological characters of CEK cell-adapted virus including the tests of hemagglutination,virus tittering, S1 gene sequencing and serology and compared with virus in chicken allantoid fluid correspondingly. The results indicated that the typical CPE such as shrinking circularly, assembling and the pathologic effect of embryos such as dehydration,crunch and low growth were induced by CEK cell-adapted virus,which had no hemagglutination as virus in chicken allantoid fluid after dealing with clostridium perfringens supernatant fluid. We found that the average virus titer of CEK cell-adapted viruses detected by median embryo infective dose (EID50) was a titer lower than that of allantoid fluid viruses.The virus titer is related to IBV S1 gene mutations among these four vaccine strains, and the virulence changes due to the point mutations in S1 protein.In addition, M41 shows faster cell-adaption than the other three strains which was observed the typical CPE of CEK cell in the 3rd passage. The virus titer of M41 E11P18 is 106.87EID50/0.1mL, higher than other viruses detected, and the average virus titer of cell-adapted M41 is also higher than the other three strains. M41 and W93 are less variable than H120 and H52 according to S1 gene sequencing of different passages.The ELISA S/P value induced by vaccination with cell-adapted IBV of H120 was-0.01 on the 14th day after vaccination,equal to allantoid fluid virus. It was 0.153 on the 21st day,0.282 on the 28th day, which were higher than allantoid fluid virus of 0.071,0.216. However, the S/P of H120 was 0.31,lower than 0.385 of the allantoid fluid virus on the 35th day. For CEK cell-adapted virus W93, its antibody S/P values were-0.02,0.02,0.261,on the 14th,21st,35th day after eyedrop, lower than the classic virus with S/P values of -0.001,0.039,0.37,at the same days.However, W93 and the classic virus have the same S/P on the 28th day after immunization.For CEK cell-adapted virus H52, its antibody S/P values of 0.246,0.815 and 1.044, on the 14th,21st and 35th days after eyedrop, were also lower than the classic virus with S/P values of 0.307,0.964 and 1.154 at the same days. Different to the W93 strain though, H52 had a higher S/P of 1.269 than the classic of 1.128 on the 28th day after immunization.The antibody S/P values of CEK cell-adapter M41 strain, which were 0.108,0.189, 0.544 and 0.828 on the 14th,21st,28th and 35th day after vaccination, were consistently higher than those of the classic allantoid fluid virus, which were -0.03,0.011,0.34 and 0.808,on the corresponding dates. Cell-adapted M41 shows fast cell-adaption,with higher virus titer and less variation, and produces more antibody than corresponding allantoid fluid virus, which has better cell-adaption and immunogenicity.We also adopted IBV Real Time Fluorogenetic Quantitative PCR(FQ-PCR) to test virus content after preparing standard reference with recombinant plasmid, optimizing the system and completing the standard curve. We analyzed the correlation between this new method and median embryo infective dose (EID50), and used it to monitor H120, W93,H52 and M41 virus content changes during the passages of IBV on CEK cells and to study the difference of virus contents in various culture conditions. The relevant coefficient of the new method is 0.995 for its standard curve. It is 10 times more sensitive than common PCR, with which virus nucleotide is detected successfully within a primary template of 2.916×10 copies/μL. The repeated coefficient of variation was less than 0.0805.There were no cross reactions when using the method to detect Newcastle Disease Virus, Infectious Bursal Disease Virus, Reticuloendotheliosis Virus and Avian Encephalomyelitis Virus, which demonstrates that it is sensitive, stable, accurate and special.The correlation coefficient (r) between the virus content detected by FQ-PCR and by EID50 was 0.748,which was highly significant correlation by t-test with its t value of 4.653 that was higher than t0.01 (20-2) of 3.922.A linear regression equation was derived according to the parallel relationship between the two methods.By utilizing this tool, the virus content was detected higher in the 16th-18th passages of the 1st to 25th passages and higher virus content was learned by inoculating with 0.1mL and culturing after 24-36h using M41 E11P18 with its titer of 106.87EID50/0.1mL.In summary, IBV H120, W93,H52 and M41 strains were successfully adapted and cultured on CEK cell.CEK cell-adapted viruses were attained and their biological characteristics involving hemagglutination, virulence, genetic variation and immunogenicity were identified. M41 strain has better cell-adaption and immunogenicity than the other three strains, which is helpful to further research on IBV variation in cells and cell-based vaccine development. Furthermore, IBV FQ-PCR, which was confirmed to be related closely to EID50 detecting the virus contents, was utilized effectively as a valuable method to observe the changes on virus contents during IBV passing on CEK cell and to learn and choose optimal culturing conditions.
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