Construction And Characteristics Of Recombinant Newcastle Disease Viruses Expressing GI And GB Genes Of Infectious Laryngotracheitis Virus

Infectious laryngotracheitis(ILT) is a highly contagious acute respiratory tract disease of chickens caused by infectious laryngotracheitis virus(ILTV) and it is one of the main infectious diseases that affect the poultry industries greatly.At present,live attenuated vaccines and recombinant viral-vectored vaccines which based on turkey herpesvirus(HVT) and fowlpox virus(FPV) are used to control ILT infection.However, live attenuated vaccines have residual virulence which can become more virulent after bird-to-bird passage and are capable of establishing life-long latent infections. As to vectored vaccines, they fail to induce completely protection although they overcome the mentioned weaknesses successfully.Therefore, a more efficacious and safer ILTV vaccine is in urgent need.The ILTV genome contains 76 Open reading frames(ORF) and at least eleven of them encode glycoproteins.The UL27 and US7 genes encode glycoprotein B(g B) and glycoprotein I(g I) respectively. g I and g E form a heterodimer that involved in cell-to-cell spread of the virus while g B is necessary for virus infectivity and functions in membrane fusion and penetration.Newcastle disease(ND),which caused by Newcastle disease virus(NDV) is a serious disease in the poulty worldwide.Its genome is a non-segmented, single strand negative-sense RNA,which containing six genes and encode eight proteins.According to its pathogenicity,NDV was classified into three groups:lentogenic,mesogenic and velogenic.At present,Lentogenic strains are wide ly used as live vaccines and they also have been evaluated as vaccine vectors for the control of animal and human diseases.ILTV LJS09 was isolated from Jiangsu Province and its complete sequence has been published in the Gen Bank(accession number: JX458822).Acorrding to the published sequence,we divided g B gene into two partially overlapped parts and designed primers to amplify them. We also designed primers to amplify full length g I gene. Three target fragments were cloned into eukaryotic expression vect or PCAGGS and we identified the correctness of recombinant plasmids by enzyme-cutting and DNA sequencing.We also identified whether three ORFs can be correctly expressed by indirect immunofluoresence(IFA)。We amplified other three target fragments and inserted them into NDV full length c DNA clone to rescue viruses.The NDV full length c DNA clone was based on La Sota and a pme I was introduced at 3 165 site,which was between P and M. Foreign genes were inserted into c DNA clones through this restrict enzyme sites. In this study,we rescued three recombinant NDVs which expressed g I and two g B fragements,designated r L-g I、r L-g BN and r L-g BC respectively.We used RT-PCR,IFA and Western Blot to confirm whether foreign genes are inserted and expressed.Our results showed that foreign genes can be expressed stably at least within 20 passages.The biological characterisitics on chicken embryos and immunity tests on SPF chickens were also studies.The titers of recombinant viruses,measured by HA and EID50,were comparable to the titers of of parent La Sota.The growth kinetics of r NDVs showed that they replicated similarly with La Sota strain and all reached the highst titers at 72 h post-inoculation. At the same time, MDT demonstrated that recombinant viruses kept low pathogenicty. The immunity tests revealed that r NDVs were effective in eliciating HI antibody and could completely protect chickens against virulent NDV strain F48E9 challenge.The mortality of immunized groups was zero while the control group was 100% and detoxification rates were also significantly lower than control group.We could not detect ILTV antibody by ELISA and all vaccinated birds showed typical clinal signs after attack by virulent ILTV strain WG.The morbidity,mortality and detoxification rates showed no significant differences with the control group.But the mortality of r L-g I immunized group was lower than control group,supposed that r L-g I have partial protection against ILTV.The reasons of poor protection of r L-g BN、r L-g BC may attributed to its inefficient envelop incorporation and low cell surface expression and the exactly mechanisms still need to be investigated.