Construction Of Duck Plague Virus ICP27 Deletion Strain And Evaluation Of Its Immune Effect

Duck plague is one of the important infectious diseases that seriously endanger the duck industry in our country.Most of the existing commercialized duck plague vaccines are inactivated vaccines and attenuated vaccines,which cannot distinguish natural infection from vaccine-immunized animals,which prevents the eradication of duck plague.Gene deletion vaccines which attenuate virulence are ideal candidates for the development of duck plague marker vaccines.In this paper,targeting the DPV ICP27 gene,a recombinant virus with ICP27 gene deletion was constructed and its immune effect as a marker vaccine candidate strain was evaluated.The results are as follows:1.Construction and rescue of DPV CHv-ΔICP27 recombinant virusUsing RED/ET and intracellular homologous recombination technology,based on the GS1783-p BAC-CHv strain constructed earlier in the laboratory,CHv-ΔICP27 and CHv-ΔICP27-without any foreign fragments were successfully constructed and rescued.R recombinant virus.ICP27 deletion strains can be quickly distinguished from parental strains by conventional molecular biology methods such as PCR,IFA,RFLP,and Western blot.2.In vitro biological characteristics analysis of DPV CHv-ΔICP27 recombinant virusThe effect of ICP27 deletion on the growth characteristics of DPV in vitro was studied by comparing the growth curve,virus copy number,viral gene m RNA expression level and promoter activity of wild strain CHv and ICP27 deletion strain.One-step and multi-step growth curve results showed that deletion of ICP27 significantly reduced the production of DPV progeny virions.Dual-luciferase reporter experiments found that ICP27 can activate the promoter activity of other viral genes,but its transcriptional regulation of viral genes is inhibited in the early stage of DPV infection,and promoted in the late stage.CHv-ΔICP27 can be stably inherited for at least 12 generations in vitro,the integrity and expression level of its adjacent genes will not be affected,and its titer remains stable within the tested generations,indicating that the ICP27 deletion strain has good genetic stability in vitro.3.Safety assessment of DPV CHv-ΔICP27 recombinant virusInfect 14-day-old ducklings with CHv-ΔICP27,CHv and CHv-ΔICP27-R with 10⁴,10⁵ and 10⁶TCID₅₀,respectively,and compare the clinical symptoms,organ indexes and mortality of ducklings inoculated with different infection titers of the three strains,detoxification,and tissue loading.The results showed that different doses of CHv-ΔICP27 infection did not cause morbidity and death in ducklings,but the body temperature of vaccinated ducks rose to 43°C 2-5 days after infection;while the ducklings infected with CHv and CHv-ΔICP27-R The morbidity and mortality were positively correlated with the inoculation dose,and the body temperature remained high at 43°C during the peak of the onset.The necropsy results found that the clinical lesions of ducklings infected with CHv-ΔICP27 recombinant virus were milder than those of CHv,with only slight enlargement and white pinpoint necrosis in the spleen tissue;in contrast,CHv and CHv-ΔICP27-R infection The ducklings in the group had splenomegaly with mottled lesions,severe atrophy and hemorrhage of the thymus,thinning of the intestinal wall of the duodenum,and some lesions such as hemorrhage of the mesentery.The test results of tissue load and shedding showed that the CHv-ΔICP27 recombinant virus was highly attenuated in ducks.4.Immunogenicity evaluation of DPV CHv-ΔICP27 recombinant virusIn order to screen out safe and effective immunization doses,30 14-day-old ducklings were divided into three groups,immunized with 10⁴,10⁵ and 10⁶ TCID₅₀ of CHv-ΔICP27 respectively,and jugular vein blood was collected on the 7th,14th and 21st days after immunization Detection of neutralizing antibody levels.The results showed that there was no significant difference in the level of neutralizing antibodies produced by immunization with 10⁴,10⁵ and 10⁶ TCID₅₀ CHv-ΔICP27.The lowest dose(10⁴ TCID₅₀)of CHv-ΔICP27 was selected to vaccinate ducklings,and the neutralizing antibody levels after vaccinating animals with commercial attenuated vaccine VAC were compared.The levels of neutralizing antibodies in serum at 4,5,and 6 weeks were comparable to VAC,indicating that 10⁴ TCID₅₀ is a relatively safe and effective immune dose of CHv-ΔICP27.5.Evaluation of the protective effect of DPV-CHv-ΔICP27 recombinant virus challengeThe 14-day-old ducklings were immunized with 10⁴ TCID₅₀ CHv-ΔICP27,2 commercial vaccines and 1ml PBS respectively.On the 14th day after immunization,100LD₅₀ of duck plague was used to attack,and the incidence,death,disease,and viral load of ducklings in each group were observed.The results showed that ducklings in the vaccine and CHv-ΔICP27 immunized groups did not suffer from morbidity and death after the virulent challenge,and their body temperature and body weight had the same trend,and there were no obvious gross and microscopic pathological changes in organs.However,the body temperature of the PBS blank control group showed high fever 3-6 days after the challenge,and the body weight dropped significantly,and all died;the ducklings in the blank group that died after the challenge were found to have a large area of hemorrhage and congestion in the liver;the spleen was swollen and the color changed.Black;thymus atrophy and hemorrhage;duodenum has obvious bleeding ring lesions,etc.Viral load detection found that the content of DPV in the tissues,organs and cloaca of ducks immunized with CHv-ΔICP27 and vaccine group was significantly lower than that of the blank control group,indicating that a single immunization with 10⁴TCID₅₀ CHv-ΔICP27 recombinant virus can effectively protect ducklings from virulence attack.In summary,the deletion marker of DPV CHv-ΔICP27 constructed in this study can be stably inherited in vitro,and can be quickly distinguished from wild strains by RFLP,PCR,WB and IFA.The deletion of ICP27 gene can significantly reduce the pathogenicity of DPV in vitro and in vivo.After a single immunization of ducklings with 10⁴TCID₅₀ CHv-ΔICP27,the level of neutralizing antibody and protection comparable to commercial vaccines can be rapidly produced.It has the potential to be further developed into a gene deletion marker vaccine.