Construction Of Bacterial Artificial Chromosome Of Chick Embryo Attenuated Duck Plague Virus CHa Strain And Preliminary Evaluation Of Its Vaccine Potential

Duck plaque virus（DPV）,also known as Duck enteritis virus（DEV）,mainly causes infectious and fatal diseases of geese,such as ducks,geese and swans.DPV genome contains a large number of replication unnecessary regions,which can accommodate the simultaneous insertion of multiple exogenous genes,and its host spectrum is narrow,so it has the potential to develop into vector vaccine.Based on this,this study took the attenuated duck plague virus strain CHa,which has been continuously passed on chicken embryos in our laboratory,as the object,constructed the recombinant duck plague virus with bacterial artificial chromosome（BAC）and evaluated its potential as a vaccine vector.The main research contents and results of this paper are as follows:1.Study on construction of infectious clone of DPV CHa strain and stability testFirstly,a transfer vector containing homologous arms on both sides of the insertion site,BAC vector functional sequences mini-F and EGFP expression cassette was constructed,which was inserted into the UL55 gene of DPV CHa strain through intracellular homologous recombination,and purified by spot-picking and limited dilution to obtain recombinant virus CHa-Ins5-BAC.Then the cyclized recombinant viral DNA was extracted and transferred to E.coli GS1783.Through PCR identification and RFLP analysis,infectious clone pDPV-CHa-BAC containing the whole genome of DPV CHa strain was obtained.The genetic stability of pDPV-CHa-BAC was tested after 20 passages on LB/Cm plate and every 5passages.The results showed that pDPV-CHa-BAC had stable genetic characteristics and could be used as a platform forDPV gene manipulation.2.Full sequence determination and analysis of infectious clone of DPV CHa strainThe infectious clone pDPV-CHa-BAC plasmid was sequenced for the next generation and sequence spliced to obtain the whole genome sequence of pDPV-CHa-BAC.Sequence comparison analysis of BLAST and the attenuated vaccine strains DPV C-KCE and Vac on NCBI found that the sequence similarity of pDPV-CHa-BAC compared with C-KCE and Vac was 99.98%and 99.96%,respectively,and the GC content was 45%;the ORF of the three strains is 77,and the whole genome of the virus is highly homologous.It shows that the insertion of BAC will not cause large genetic mutations in the DPV CHa genome,and has the potential to be a vector vaccine platform.3.Infectious clone rescue of DPV CHa strain and study on biological characteristics of the virusThe infectious clone pDPV-CHa-BAC DNA was transfected with DEF to obtain the rescued recombinant virus rDPV-CHa-BAC.The recombinant virus was successfully constructed by PCR and IFA.The growth characteristics of the parent strain DPV CHa and the rescue recombinant virus rDPV-CHa-BAC were studied through a one-step growth curve.It was found that the virus amount gradually increased with time,and the growth curve of the recombinant virus and the parent strain was similar to the one-step growth curve,without significance difference.In the multi-step growth curve,the titer of the recombinant virus rDPV-CHa-BAC was significantly lower than that of the parent strain DPV CHa at 24～96 h.4.Analysis of infection characteristics of recombinant virus rDPV-CHa-BAC on duck embryos and ducks.Duck embryos were infected with rDPV-CHa-BAC,CHa and CHv at doses of 10⁴,10⁵and 10⁶ TCID₅₀.Except for rDPV-CHa-BAC infected with 10⁴ TCID₅₀,all the other groups died in different degrees,and the mortality rate of CHv and CHa infected groups reached100%.In contrast,the death rate of duck embryo infected by virulent strain CHv is faster.10^8.63 copies/0.2 m L recombinant virus rDPV-CHa-BAC and CHa infected duck embryos were tested for virus proliferation,safety and innate immunity.The results showed that virus replication could be detected in liver,brain and allantoic fluid of duck embryos infected by rDPV-CHa-BAC,which indicated that the replication of recombinant virus rDPV-CHa-BAC in duck embryos was not affected by inserting BAC vector sequence into DPV UL55 gene.After inoculation for 1 d,3 d and 5 d,all groups could cause significant pathological changes in liver and brain,and the degree of pathological changes increased with the extension of exposure time.Transcription level of some natural immune-related genes in brain and liver tissues of duck embryos increased with time,which indicated that replication of virus genome could induce natural immune antiviral response in embryos.