Construction And Evaluation Of GC Deletion Infectious Laryngotracheitis Virus Strain Expressing Newcastle Disease Virus F Protein

Newcastle disease(ND)and infectious laryngotracheitis(ILT),caused by Newcastle disease virus(NDV)and infectious laryngotracheitis virus(ILTV),respectively,are among the most important avian respiratory diseases in the poultry industry worldwide and have resulted in significant economic losses.Strict biosecurity measures and vaccination with efficacious vaccines are the principal strategies and essential tools for the control of the contagious viral diseases including ND and ILT.Though live vaccines can induce mucosal,cellular,and humoral immunity and are suitable for mass administration,the widespread maternal antibodies against NDV in chicken flocks may interfere with the development of effective immunity induced by live vaccines.In addition,the respiratory disease and egg production potentially induced by the application of live vaccines under field conditions have been reported.Inactivated vaccines can induce high level of humoral immunity but do not develop a strong mucosal,and cellular immunity and inefficiently in prevent viral shedding in NDV-infected chickens.For ILTV,attenuated chicken embryo origin(CEO)and tissue culture origin(TCO)live vaccines are effective in producing robust protection,however,these live vaccines,especially CEO vaccine,may regain virulence and become the source of clinical outbreaks.The development of novel vaccine technology is still needed for most of the pathogens,such as NDV and ILTV control.Recombinant gene-deleted live attenuated vaccines retained the protection efficacy of live vaccines with diminished possibilities of regaining virulence can be used as vectors to express other viral protective antigens and served as potential multivalent vaccine candidates.In this study,to develop an alternative potential live bivalent vaccine against ILTV and NDV,the gC gene was deleted from the genome of an avirulent ILTV strain,then a recombinant gC gene-deleted ILTV mutant expressing the F protein of the genotype ? virulent NDV was obtained.We further evaluated the growth characteristic,stability,safety,and protective efficacy of the recombinant virus against virulent ILTV and NDV challenges.To develop an alternative vaccine against Newcastle disease virus(NDV)and infectious laryngotracheitis virus(ILTV),we firstly constructed a gC-deleted ILTV mutant based on an avirulent ILTV,and then obtained a recombinant ILTV expressing the fusion protein(F)of the genotype ? NDV(designated ILTV-?gC-F)based on the gC-deleted ILTV mutant.Expression of the NDV F protein in ILTV-?gC-F-infected LMH cells was examined by indirect immunofluorescence assay(IFA)and western blotting(WB).The recombinant ILTV-?gC-F retained the growth kinetics of parental ILTV with increased replication rate.The inserted F gene was stably maintained in the genome of ILTV-?gC-F and the F protein was be stably expressed.Based on the clinical signs and viral shedding detection of experimentally infected chickens,the recombinant ILTV-?gC-F is proven to be avirulent.Vaccination of specific pathogen-free(SPF)with recombinant ILTV-?gC-F induced ILTV-specific enzyme-linked immunosorbent assay(ELISA)antibody and provided complete clinical protection against virulent ILTV although viral shedding in respiratory tract and viral replication in the target tissues were detected at the early stage of infection in only s very small number of birds.In addition,a single vaccination with ILTV-?gC-F could provide complete clinical protection against virulent genotype ? NDV challenge although the level of NDV-specific ELISA antibody were low.Notably,the numbers of birds which were positive for virus detection and the replication level of the challenged NDV in the selected target tissues were significantly reduced in ILTV-?gC-F-vaccinated chickens,comparing to those of control birds.Our results indicate that ILTV-?gC-F could be considered as a bivalent vaccine candidate against both ILTV and NDV.