Effects Of Duck Plague Viruses GE Cytoplasmic Domains And Extracellular Domains On Virulence And Immune Efficacy

Duck plague is an acute infectious disease in the duck industry,and its pathogen is duck plague virus(DPV).At present,there are no reports on the effects of the intracellular and extracellular domains of the DPV glycoprotein gE(gE)on viral virulence and immune efficacy.In this study,a gE extracellular domain deletion mutant and a gE cytoplasmic domain mutant were constructed from CHv-50 in this laboratory,and the recombinant viruses were investigated,and the results are as follows:1.Construction of gE extracellular domain and gE cytoplasmic domain deletion recombinant viruses and determination its genetic stabilityBased on rescued bacterial artificial chromosome recombinant duck plague virus platform and Red homologous recombination,the DPV CHv-BAC-gE?ET and DPV CHv-BAC-gE?CT clone strains were constructed.The UL23 gene was replenished on the strains,and the gene-deletion viruses DPV CHv-gE?ET?DPV CHv-gE?CT without BAC component were constructed after rescue and plaque purification.DPV CHv-gE?ET,DPV CHv-gE?CT and parental virus CHv-50 with MOI=0.01 were inoculated into DEF cells.The results showed that gE cytoplasmic domain deleted viruses and gE extracellular domain deleted viruses showed similar proliferation patterns with the parental virus.Compared with the parental virus,the viral titers of the gE extracellular domain deleted viruses and gE cytoplasmic domain deleted viruses were obviously decreased by nearly 80 times and25 times respectively at 72 h after infection,indicating that the deletion of gE extracellular domain and gE cytoplasmicdomain weresignificantly affect theviruses proliferation on DEF cells.The DPV CHv-gE?ET?DPV CHv-gE?CT strains were serially passaged for 20 passages in DEF culture and 10 passages in ducks.We identified the genetic stability by PCR amplification analysis,observing the changes of TCID₅₀,and safety of susceptible animals.The results showed that the gene-deletion viruses were stable,no reverse mutations were found in the deleted genes,and its pathogenicity to susceptible animals did not change,which proved that the gE extracellular domain and gE cytoplasmicdomain deleted strains had good geneticstability.2.Pathogenicity assay and immuneresponseof DPVCHv-gE?ETand DPVCHv-gE?CTThe DPV CHv-gE?ET,DPV CHv-gE?CT,DPV CHv-?gE and parental strain CHv-50 were used in this study with 10⁴,10⁵,10⁶TCID₅₀by intramuscular injection challenge with 14 days old ducklings,and a control group was established at the same time.The study continued to observe for10 days.The ducklings challenged with CHv-50 at 10⁵,10⁶TCID₅₀appeared fever and death,with mortality rates of 50%and 60%respectively.The high-dose challenge group(10⁵and 10⁶TCID₅₀)of the gE cytoplasmic domain deleted strain showed 20%mortality.The gE extracellular domain deleted strain and gE deleted strain showed no clinical symptoms.Antibodies could be detected in the serum of the DPV CHv-gE?ET and DPV CHv-?gE challenge groups 10 days after inoculation.The results showed that compared with the parental strain,the virulence of gE cytoplasmic domain mutant in ducklings was decreased,while gE extracellular domain mutant showed a significant attenuation in virulence,and it could inducea significantimmuneresponse.Refer to a dose of live duck plague vaccine(10^7.7copies),14 days old ducklings were each inoculated with DPV CHv-gE?ET or DPV CHv-?gE.The neutralizing antibodies were detected as early as 7 days after immunization in the serum of ducklings,and neutralizing antibody titers increased from 7 to 28 days.Ducklings were challenged intramuscularly with 100 LD₅₀of virulent CHv at 14 days after vaccination.All ducklings in DPV CHv-gE?ET or DPV CHv-?gE immunized groups were survived,but all ducks died in the control group.It showed that DPV CHv-gE?ET and DPV CHv-?gE could effectively induce protective immune response,and it could achieve 100%protectionagainst 100 LD₅₀dose of CHv challenge.In summary,the DPV gE extracellular domain and gE cytoplasmic domain deleted strains can reduce the pathogenicity to ducklings,of which gE extracellular domain is closely related to virulence.The non-pathogenic gE extracellular domain and gE deletion viruses can stimulate ducks to produce neutralizing antibodies,which can resist thevirulent CHv attack.