Construction And Evaluation Of Infectious Laryngotracheitis Virus Expressing The Newcastle Disease Virus Fusion Protein

Newcastle disease(ND)is one of the highly pathogenic viral diseases of avian species,which is endemic in the whole world.The pathogen of ND is Newcastle disease virus(NDV).All NDVs belong to one serotype but can be divided into various genotypes.Nowadays,all NDV vaccines widely used belong to genotype II,while the most epidemic virulent NDVs(vNDV)in China belong to genotype ?.Despite of the prevention of clinical disease and death from genotype ? virulent NDVs,these vaccines fails to limit the replication and shedding of vNDVs.This may result in the epidemic of NDV in immunized chickens.Therefore,the development of novel vaccines against the genotype ? vNDVs is urgently needed in China.The F protein encoded by the F gene of NDV is a glycoprotein.It is a protective antigen of NDV and frequently inserted into the vectors of virally vectored vaccines.Infectious laryngotracheitis(ILT)is an upper respiratory tract disease of chickens caused by infectious laryngotracheitis virus(ILTV).The control of ILT mainly relies on strict biosafety measures and vaccination.Both commercial attenuated live vaccines and virally vectored vaccines expressing ILTV antigen are available and wildly administrated present.However the reversion of virulence of the attenuated vaccine strains and the incomplete protection by vectored vaccines have been reported frequently.In order to overcome these defects of current vaccines,great effort has been made to develop ILTV gene deletion vaccine and ILTV live vector vaccine.ILTV has a double-stranded DNA genome,which is ca.150 kbp in size.Since ILTV vaccines are suitable for mass application through aerosol or drinking water,they may also be useful vectors for expressing immunogenic proteins of other chicken pathogens.In this study,an ILTV ck/CH/LHLJ/120305(LHLJ/120305)strain was isolated.SPF chickens infected with LHLJ/120305 showed no respiratory signs,and the intratracheal pathogenicity index(ITPI)value of this strain was 0.No apparent pathological change was observed in the target tissues,namely larynx and trachea,of the infected chickens.Besides,despite of the viral replication in larynx and trachea and the shedding of virus in the oropharyngeal swabs at the early stage of infection,the viral load of LHLJ/120305 was lower than that of the virulent ILTV WG strain significantly in both tissue and swab samples.Above results suggest LHLJ/120305 as an avirulent ILTV strain.Furthermore,vaccination with LHLJ/120305 provided complete clinical protection against the challenge with the virulent ILTV WG strain with no shedding of challenged virus,which indicates this avirulent ILTV strain as a potential live vaccine strain and a vaccine vector.To test this possibility,the US9-deleted ILTV mutant was constructed through replacing the coding sequence of US9 with the expression cassette for EGFP protein via homologous recombination.The expression of EGFP protein was observed in LMH cells infected with this recombinant virus ILTV-?US9-G.Comparing with the parental LHLJ/120305 strain,this recombinant strain had similar maximum titer but delayed replication kinetics in LMH cells.To further investigate whether the effect of the introduction of exogenous genes on the pathogenicity and immunogenicity of ILTV virus,a recombinant virus expressing the F protein of genotype ? NDV(ILTV-?US9-F)was generated by replacing the EGFP gene was replaced with F gene via homologous recombination..The growth patterns of the recombinant ILTV-?US9-F and LHLJ/120305 were similar with each other.After consecutive 15 passages in LMH cells,the expression cassette of F gene was stably maintained in ILTV-?US9-F and the F protein was stably expressed as assayed by both IFA and Western blotting in ILTV-?US9-F-infected cells.Further in vivo study observed no apparent respiratory clinical signs in any chicken infected with either LHLJ/120305 or ILTV-?US9-F.Both,the ITPI values of LHLJ/120305 and ILTV-?US9-F were 0.Both LHLJ/120305and ILTV-?US9-F protected chicken from the infection of virulent ILTV WG strain completely with no clinical signs and death of infected animal throughout the whole observation period.No viral shedding in oropharyngeal swabs was detected in either vaccinated groups.In order to evaluate the efficacy of ILTV-?US9-F against vNDV,the level of antibody was detected in chickens after vaccination of ILTV-?US9-F followed by infection of homologous or heterologous vNDV.Comparing with La Sota vaccine,vaccination with 10~4 PFU ILTV-?US9-F induced significant lower levels of antibodies recognizing NDV and neutralizing antibodies against NDV.Both La Sota vaccine and ILTV-?US9-F protected chicken from the infection of a genotype IX F48E9 strain completely without the observation of either vital replication or viral shedding.Although the clinical protections against genotype ? LHLJ/01/06 were achieved by both La Sota vaccine and ILTV-?US9-F,only ILTV-?US9-F prevented the replication and shedding of ? LHLJ/01/06 effectively,suggesting that ILTV-?US9-F was more efficient in limiting the replication and shedding of genotype ? LHLJ/01/06 than La Sota.In addition,we evaluate the minimum immune dose of ILTV-?US9-F.A single vaccination with 10~3 PFU of ILTV-?US9-F provided sufficient clinical protection and a single vaccination with 10~2 PFU of ILTV-?US9-F provided 95%clinical protection.Thus the minimum immune dose of ILTV-?US9-F was determined as 10~3 PFU.The recombinant virus expressing the F protein of genotype ? NDV(ILTV-?US9-F)was generated via homologous recombination.Which provided an ideal protection against both ILTV and NDV.Our present studies suggest the ILTV-?US9-F as a bivalent vaccine candidate against both ILTV and NDV.