Construction Of Recombinant Duck Enteritis Virus Expressing GB Gene Of Avian Infectious Laryngotracheitis Virus And Determination Of Its Immune Efficacy

Infectious laryngotracheitis is a highly contagious upper respiratory infectious disease caused by infectious laryngotracheitis virus.The virus belongs to ?-herpesvirus family.The infected chickens mainly show conjunctivitis,hemoptysis,dyspnea and other symptoms.It is one of the important diseases in poultry breeding industry.At present,the prevention and treatment against the disease is mainly through the inoculation of attenuated vaccines,which can produce good immune protection.However,when used in clinical practice,it is found that the attenuated vaccine had a strong virulence,resulting in periodic infection of chickens and causing heavy economic losses.With the development of biotechnology,recombinant virus vaccine has gradually become a research hotspot.At present,there are two different recombinant virus vaccines have been applied in farms in the United States.They are chickenpox virus vaccine expressing ILTV gB/UL32 double gene and HVT recombinant vaccine expressing ILTV g I/g D respectively,both of which can produce specific protection after immunization.In China,Tong et al.developed a recombinant chickenpox virus expressing ILTV gB gene,which could resist the attack of ILTV after immunization.Nevertheless,chickenpox virus was susceptible to maternal antibodies in application,resulting in immune failure and unable to resist virus infection.Therefore,it is urgent to develop a safe and reliable vaccine.Duck enteritis virus is also a member of the ?-herpesvirus family.Its genome is large and contains multiple replication non-essential genes and gene spacers,which can express multiple exogenous genes.At the same time,the virus can be cultured on chicken fibroblast cells and released from cells.Therefore,the duck enteritis virus is considered as a good viral vector which is easy to be prepared and stored.At present,researchers have constructed several recombinant duck enteritis viruses expressing different foreign genes,which can produce specific protection against relative pathogens after immunization.In this study,based on the technologies of CRISPR/Cas9 genome editing and homologous recombination,a recombinant duck enteritis virus expressing gB protein of infectious laryngotracheal virus was constructed.Western blot showed that the gB protein was expressed correctly.The gB gene was still detectable on the 20 th generation after continuous passage.The growth capability of the recombinant virus was the same as that of the maternal virus.To test the immune potency of the recombinant virus against infectious laryngotracheitis,SPF chickens were innoculated with the recombinant virus.On the14 th days post innoculation,the chickens were challenged by innoculation of the virulent virus intratracheally.The results showed that the morbidity of the chickens innoculated with recombinant virus was 30%,and the mortality was 20%.Seventy percentof chickens innoculated with recombinant virus showed mild symptoms after challenge with a clinical index score of 0.8 and a protection index of 77.8%.However in the control group,the morbidity was 100%,the clinical index score was 3.7,and the mortality was 90%.The results showed that the recombinant virus had the potential to be used as a vaccine candidate strain.In this study,recombinant duck enteritis virus expressing gB protein of infectious laryngotracheitis virus was constructed successfully and showed a potential usage in vaccine development.The study provided novel approach and methodology in the development of new vaccines against infectious laryngotracheitis.