Construction Of A Recombinant Turkey Herpesvirus Co-Expressing The F Protein Of Genotype Ⅶ NDV And The VP2 Protein Of A Novel Variant Of IBDV

Newcastle disease(ND)is a highly contagious contact disease that causes serious economic losses to the poultry industry due to its rapid spread and high infection and mortality rates.Strategies for the control of Newcastle disease include biosecurity measures and vaccination.Vaccines are mainly divided into inactivated vaccines,attenuated vaccines and virus vector vaccines,of which herpesvirus of turkey(HVT)vector vaccines have outstanding advantages over other vaccines and can be very effective in the control of Newcastle disease.Infectious bursal disease virus(IBDV)belongs to the family of double RNA viruses and its main target organ is the bursa of the chicken.In recent years there has been an epidemic of a new variant of IBDV in China.Although the infection does not cause direct death in chickens,it can cause bursal atrophy and immunosuppression in chickens,which is a serious threat to the healthy development of the poultry industry,and the existing vaccines do not provide protection against the new variant of IBDV.In order to construct a recombinant turkey herpesvirus(rHVT)targeting both NDV and nVarIBDV,and to avoid the possible interference of vaccination with both rHVTs on animals,this study first constructed a recombinant virus rHVT-Fopt expressing only the NDV F protein using the CRISPR/Cas9-NHEJ and Cre/LoxP systems,and also characterised the in vitro biology of the recombinant virus and evaluated its immune efficacy.The results showed that the recombinant virus rHVT-Fopt foreign exogenous protein was well expressed and did not differ significantly from the in vitro replication rate of the parental strain of HVT,and that 1 day old SPF chickens immunized with this vaccine were fully protected against a virulent strain of NDV.Afterwards,using the constructed rHVT-Fopt as a backbone,the VP2 protein of nVarIBDV/SN 1905-2 Virus strains was inserted into the rHVT-Fopt recombinant virus using a similar method,and two rHVT viruses co-expressing genotype Ⅶ NDV F protein and nVarIBDV VP2 protein were successfully obtained.Both strains of rHVT have good genetic stability and have the potential to protect against both ND and IBD,and provide a basis for the subsequent construction of a multiplexed polyvalent rHVT vaccine.1.Construction of recombinant turkey herpesvirus expressing NDV F proteinTo construct the HVT-ND recombinant vaccine,a CRISPR/Cas9 gene editing plasmid was constructed,together with a wild-type and codon-optimised transfer vector plasmid for the gene type VII NDV F gene.The two plasmids were transfected separately in CEF cells and the expression levels of wild-type and codon-optimized F protein were compared by IFA and Western blot analysis.The transfer vector plasmid with high F protein expression level was selected and cotransfected with the gene editing plasmid in CEF cells.Afterwards,HVT virus was inoculated,the recombinant virus was purified by eGFP fluorescent tag screening,and PCR and IFA were performed to identify the expression of exogenous proteins to obtain a recombinant virus co-expressing eGFP and F proteins.After transfection of the pcDNA3.1Cre plasmid in CEF cells inoculated with fluorescent recombinant viruses,the non-fluorescent recombinant viruses were screened and purified,and those identified correctly by PCR and IFA were subjected to in vitro assays of genetic stability and replication kinetics.The results showed that the transfer vector plasmids of codon optimized gene Ⅶ F gene and wild type F gene were successfully constructed,which were named pT-sgA-eGFP-SV40Fopt and pT-sgA-eGFP-SV40-Fwt,respectively.The pT-sgA-eGFP-SV40-Fopt plasmid expressing a much higher level of F protein than the pT-sgA-eGFP-SV40-Fwt plasmid.Next,the pT-sgA-eGFP-SV40-Fopt plasmid and the CRISPR/Cas9 gene editing plasmid were cotransfected in CEF cells,and a recombinant virus named rHVT-eGFP-Fopt was constructed by inserting the optimized genotype VII NDV F gene expression cassette and eGFP expression cassette into the US2 region of the HVT Fc126 vaccine strain using the NHEJ repair pathway of the CRISPR/Cas9 system.The successfully edited recombinant virus was purified by pick spotting,PCR and IFA identified to the correct expression of F protein in the recombinant virus.eGFP expression cassette was knocked out by Cre/LoxP recombinase system,and PCR and IFA identification results showed that a recombinant virus named rHVT-Fopt was successfully obtained.The genetic stability and replication kinetics of this recombinant virus were measured in vitro.The virus was successfully transmitted in CEF for 15 consecutive generations with stable expression of F protein and replication rate comparable to that of wildtype HVT virus.2.Preliminary evaluation of immune efficacy of rHVT-Fopt recombinant virusNewcastle disease vaccination needs to achieve three main objectives:to prevent virus infection,to reduce morbidity and mortality,and to reduce the spread and dispersal of the virus in the flock.To investigate the protective efficacy of the rHVT-Fopt vaccine against NDV,1 day old SPF chickens were immunised with the vaccine and the serum antibody levels of the immunised flock were measured by a commercial NDV ELISA antibody kit.On the 28 day post vaccine,a virulent strain of NDV F48E8 was used for challenge.Morbidity and mortality were counted after 14 days of continuous observation,and oral and cloacal swabs were collected at specific time points to detect virus shedding.These data were combined to evaluate whether the recombinant virus could be used as a candidate vaccine against Newcastle disease in chickens.The results showed that rHVT-Fopt recombinant virus was able to produce a good humoral immune response in chickens.All vaccinated chickens showed no clinical signs and mortality during the 14-day observation period after receiving NDV F48E8 virulent virus attack,and no virus shedding was detected.rHVT-Fopt showed 100%protection against Newcastle disease virulent virus,proving that rHVT-Fopt can be used as a candidate vaccine against Newcastle disease.3.Construction of recombinant turkey herpesvirus co-expressing NDV F protein and nVarIBDV VP2 proteinIn order to develop a vaccine for the novel variant of IBDV and NDV,the present study builds on the previously constructed rHVT-Fopt and used a similar method to insert the VP2 gene expression cassette of nVarIBDV/SN1905-2 into the US2 region of rHVT-Fopt recombinant virus.A recombinant Turkey herpesvirus co-expressing genotype Ⅶ NDV F protein and nVarIBDV VP2 protein was constructed and the biological characteristics of the successful recombinant virus were identified.The results showed that two recombinant viruses co-expressing genotype Ⅶ NDV F protein and nVarIBDV VP2 protein were successfully obtained and named as rHVT-Fopt-VP2-4 and rHVT-Fopt-VP2-12.The F and VP2 proteins of both recombinant viruses can be stably expressed and both have the potential to protect against both ND and IBD.