Construction And Evaluation On The Immunoprotectivity Of Recombinant Duck Enteritis Virus Expressing The Major Structural Proteins Of Infectious Bronchitis Virus

Infectious bronchitis is a highly contagious viral disease of the upper respiratory and urogenital tracts in chickens that is caused by the infectious bronchitis virus(IBV). The disease was epidemic in many countries and mainly infected the respiratory and kidney. All ages of chickens could be infected and the egg quality and product droped, which caused severe economic losses.Duck enteritis virus(DEV), also was named duck plague virus(DPV), caused acute, heat and septicemic diseases in the order of Anseriformes, such as duck and goose. The disease was epidemic in many regions of countries and caused economic losses. DEV was a member of family Herpesvirus. Some genes of DEV genome were not essential for virus replication so that DEV could be used as nonreplicating vaccine vector to construct live vector vaccine carry foreign genes.In this study, the US10 gene of DEV was deleted and replaced by EGFP gene to construct recombinant virus. This recombinant virus was used as a parental virus to construct recombinant viruses that expressed the major structural proteins of IBV N, S and S1 by the method of homologous recombination. The differentiation PCR, cross PCR and sequencing was performed to determine that the IBV N, S and S1 were inserted into DEV genome properly with the replacement of US10 gene. IBV N, S and S1 were identified to be expressed in the recombinant virus by western blot and indirect immunofluorescence assay. To identify the biology property of 3 recombinant viruses, plaque shape and size was examined to compare with the parental virus. The results showed that the plaque shape and size of the three r DEVs was similar to the parental virus r DEV-EGFP and the wild virus DEV Clone-03. Compared with the wild virus DEV Clone-03, the titers of r DEV-N, r DEV-S and r DEV-S1 decreased. Multiple replication kinetics detection showed that three r DEVs was consistent with the parental virus and the wild virus. The titers of r DEVs reached the highest at 72 h post infection and decreased at 96 h post infection, indicated that the replication capacity of r DEVs was correlated with the deletion site in DEV genome but not associated with the foreign gens. The three r DEVs was passaged in CEF for 20 times, it was illustrated that the r DEVs showed genetic stability and the foreign genes were expressed stably along with the replication of r DEVs.The three r DEVs was used to immunize the one-month-old SPF chickens. Chickens were challenged on 21 days post immunization by the virulent IBV ck/CH/LDL/091022. Serum samples were collected at week 1, 2, 3, 4 and 5 to detect the antibody level of immunized chickens. At week 1 post immunization, 84% chickens showed antibody positive in r DEV-N vaccinated group, 92% sera antibody positive in r DEV-S and r DEV-S1 groups. At week 2 post vaccination, the antibody level decreased at different degree. The ratio of antibody positive in r DEV-N was 76%, 52% in r DEV-S, and 28% in r DEV-S1 group. At week 3 post vaccination, the antibodypositive ratio decreased sharply and showed 40%, 16% and 8% in r DEV-N, r DEV-S and r DEV-S1 group, respectively. Chickens showed different illness, the morbidity was 80% in the control group, 30% in r DEV-N, 20% in r DEV-S and 30% in r DEV-S1. The mortality was 40%, 30%, 10% and 30% in r DEV-N, r DEV-S and r DEV-S1 group, respectively. The oropharyngeal swabs were collected from the immunized chickens and were detected the IBV by the method of real time RT-PCR to determine the virus shedding. Virus shedding detection showed that 90% chickens shed virus in control group, 20% in r DEV-N, 30% in r DEV-S and 40% in r DEV-S1. The sera antibody of chickens post challenge was detected on days 5, 10, 15 and 20. The result of antibody level post challenge showed that the antibody titers increased at different degree in the 4 groups, and the antibody level in 3 vaccinated groups was higher than the control group, which indicated success challenge. All these results suggested that chickens vaccinated r DEVs could be protected against the virulent virus challenge, and the antibody titers increased faster than the control group, that is chickens vaccinated with r DEVs have the immunity memory.The three rDEVs conferred protections for chickens against the challenge of virulent IBV but cannot provide complete protection from the morbidity, mortality and the virus shedding after challenge with virulent IBV. The r DEV-S and r DEV-N conferred better protection than the r DEV-S1 for chickens.