Construction Of Recombinant ILTV Expressing Spike Protein Of IBV And Its Protective Efficacy Evaluation

Infectious bronchitis(IB)is a highly contagious viral disease of the chicken caused by infectious bronchitis virus(IBV).It is possibly the most economically important viral respiratory disease of chickens in regions everywhere that commercial chickens are kept.It is necessary to develop a new vaccine based on the results of epidemiological studies for circulating strains in China.The spike protein is an essential glycoprotein on the surface of the IBV virion,which playing an important role in virus invasion and receptor binding.While S1 subunit of S protein contains the primary neutralizing epitopes,thereby making it a major target of immune therapy and vaccine design for IBV.Infectious laryngotracheitis(ILT)is another severe contagious viral disease threating poultry production,infectious laryngotracheitis virus(ILTV)is the etiologic agent of ILT.The disease used to be regional endemic,currently,the continuous outbreaks of ILT are observed in China and causes large economic losses.As an effective means to prevent and control the disease,attenuated vaccine was the main prevention and control method.Infectious laryngotracheitis virus and infectious bronchitis virus are both important avian respiratory disease viruses.It is necessary to develop a combined vaccine to prevent and control the two diseases simultaneously due to the similarity of the infection site and immune mechanism.ILTV has the potential to serve as vector for the expression of protective antigens of other pathogens.To sum up,it is the best choice for the prevention and control of IB and ILT to construct a recombinant avirulent ILTV expressing the major immunogens of IBV.In this study,avirulent recombinant ILTV named ILTV-?US9-G,which was a US9-deleted and insert EGFP ILTV mutant isolate was used as parent virus.Two novel recombinant viruses expressing the S protein(ILTV-?US9-S)and S1 protein(ILTV-?US9-S1)of the GI-19(QX)genotype virulent IBV CK/CH/LDL/091022 were constructed based on the ILTV-?US9-G.The inserted S gene and S1 gene did not significantly affect the two recombinant ILTV replication in vitro.And the ILTV-?US9-S1 showed an increased virus replication ability.The two recombine viruses were passaged 15 generations(P1?P15)in LMH cells,and the monoclonal antibody against IBV S1 protein prepared in this study was used for IFA detection,the results showed that S protein and S1 protein was stably expressed in ILTV-?US9-S and ILTV-?US9-S1,but showing the low protein expression level.SPF chickens were used to evaluate efficacy and safety of the two recombinant viruses as vaccine for chickens.Firstly,SPF chickens did not show clinical symptoms after high-dose inoculation,which indicated that the two recombinant viruses are safe to chickens.Secondly,to evaluate the immune protection effect of two recombinant viruses against virulent ILTV,the serum antibodies of immunized chickens were detected by indirect ELISA.The two recombinant viruses induce a high level of serum antibody at 7 dpv and the antibody level of ILTV-?US9-S1 was higher by contrast.From the clinical performance after challenging with virulent ILTV WG strain,the chickens immunized with the two recombinant viruses shows no respiratory symptoms,viral load of larynx and trachea of the immuned chickens were much lower than that of control chickens,and viral shedding in the oropharyngeal swabs showed that no virus was detected in the ILTV-?US9-S immuned chickens at 8 dpc while the ILTV-?US9-S immuned chickens at 12 dpc.The above results indicated both recombinant viruses provided complete protection against virulent ILTV.Thirdly,to evaluate the protective efficacy of the two recombinant viruses against a virulent IBV CK/CH/LDL/091022,s Ig A ELISA and IFN-? ELISpot testes show that both recombinant viruses induce limited specific mucosal and cellular immune responses in partial chickens.To assess the level of antibodies against IBV in serum samples of chickens,the ELISA test result showed that antibody was detected in few serum samples of chickens at14 d after immunization.There was no significant difference observed in viral load of the trachea and kidneys between the immunized groups and the control group at 5 dpc with virulent IBV strain.Compared with the control group,which showed 80% virus secretion rates of swab at 8 dpc and 20% at12 dpc.The ILTV-?US9-S and ILTV-?US9-S1 immuned chickens showed 50% and 60% virus secretion rates of swab at 8 dpv,10% and 0 at 12 dpv.These results indicated the two recombinant viruses can accelerate the clearance of the virus in the respiratory tract of immunized chickens to a certain extent after being immunized,but the immune protection stimulated is weak.In summary,two recombinant ILTV expressing S protein or S1 protein of GI-19(QX)genotype IBV,named ILTV-?US9-S and ILTV-?US9-S1,were constructed.The two recombinant viruses provide adequate protection against virulent ILTV challenge.Also stimulated immune response against the S1 protein of GI-19(QX)genotype IBV.However,the recombinant viruses cannot provide complete immune protection against virulent IBV.It is necessary to optimize the expression of exogenous immunogen genes so that improving the potential value of ILTV as a recombinant gene engineering vector.