Construction And Immune Efficacy Evaluation Of Recombinant Duck Plague Virus Expressing The E Protein Of Duck Tambusu Virus

Duck plague caused by Duck Plague virus（DPV）and Duck Tambusu disease caused by Duck Tambusu virus（DTMUV）both seriously endanger the livestock and poultry industry.This article uses the chicken embryo weakened strain of duck distemper virus to express the truncated envelope E protein of duck Tambusu virus,and evaluates the genetic stability and immune efficacy of the recombinant strain.The results are as follows.1.Construction and Genetic Stability of Recombinant Duck Plague Virus Expressing the E Protein of Duck Tambusu VirusUsing the DPV bacterial artificial chromosome platform and Red homologous recombination technology established in our laboratory,the DPV CHa strain was used as a vector to connect the DTMUV truncated E protein to the C-terminus of the DPV high expression protein p UL49 with the duck hepatitis A virus 2A1 protein.An infectious clone expressing the DTMUV truncated E protein was successfully constructed.The recombinant virus strain[r DPV-E（UL49）]was obtained by transfection into duck embryo fibroblast（DEF）cells.The multi-step growth curve measurement results showed that r DPV-E（UL49）,like its parent virus DPV CHa,gradually increased in titer from24 to 72 hours of infection with DEF,reaching a peak of about 10^6.5TCID₅₀/0.1 m L at 72 hours of infection,and then decreased.After 96 hours of infection,it decreased to about 10^5.6TCID₅₀/0.1 m L,and there was no significant difference between the two（P>0.05）,indicating that inserting the E gene did not affect the proliferation of DPV CHa in host cells.RDPV-E（UL49）was subcultured 25 times in DEF and 5 times in ducks,respectively.The insertion and expression of the E gene were confirmed to be stable through virus titer measurement,PCR identification and sequence analysis,Western blot（WB）,and indirect fluorescence antibody test（IFA）,indicating that r DPV-E（UL49）has good genetic stability in both in and out of ducklings.2.Evaluation of the immune efficacy of recombinant duck plague virus expressing the E protein of duck Tambusu virus（1）Antibody levelsa.Neutralizing antibodiesDTMUV and DPV antibodies can be detected at 1 week,with antibody titers reaching 2^3.63and2^3.61at 2 weeks,and peak values reaching 2^7.19and 2^7.11at 6 weeks,respectively;Afterwards,the antibody titers(2^4.01and 2^3.99,respectively)were still higher at 18 weeks than at 2 weeks,and the antibody levels of the recombinant virus at different time points were not significantly different from those of the vaccine（P>0.05）.b.ELISAantibodies:DTMUV(OD₄₅₀>0.2)and DPV（OD450>0.289）antibodies were detected positive at 1 week.At 2 weeks,the OD₄₅₀values were 0.336 and 0.573,and at 8 weeks,the OD₄₅₀peak values were 1.07 and 1.017,followed by a decrease.At 18 weeks,the OD₄₅₀values were 0.633 and 0.496,still higher than at 2 weeks.The antibody levels at each time point were not significantly different from the vaccine（P>0.05）.（2）Attack Poison Protectiona.After DTMUV strong toxicity attack,the non immunized control group ducks experienced mental depression,elevated body temperature,claudication,and death,with a mortality rate of 30%.However,r DPV-E（UL49）,like ducks immunized with duck plague vaccine,did not show any symptoms of Tambusu’s disease,had normal body temperature,and had a 100%protection rate.This indicates that r DPV-E（UL49）immunization can protect ducks against 10⁸TCID₅₀DTMUV virulence attacks.b.After being attacked by strong DPV toxin,non immunized control ducks showed an increase in body temperature and died,all of whom died after 6 days;RDPV-E（UL49）,like ducks immunized with duck plague vaccine,has a normal body temperature and all survive,with a protection rate of 100%.This indicates that r DPV-E（UL49）immunization can protect ducks against DPV virulence attacks of 100 LD₅₀.（3）Immunized duck strong toxin clearance effecta.After DTMUV strong toxicity attack,the brain,spleen,and liver load of non immunized ducks were 10^5.5,10^7.2,and 10^8.2copies/mg respectively on day 1,10^8-10.5copies/mg on day 3 to 7,and decreased to 10^4-7copies/mg on day 9;The copy numbers in various tissues of ducks immunized with r DPV-E（UL49）and duck tambusine vaccine within 9 days were 10^2-4copies/mg,which showed significant differences（P<0.05）or extremely significant（P<0.01）compared to the non immunized control ducks.This indicates that the recombinant virus can inhibit the proliferation of DTMUV in ducks and clear some of the DTMUV virulence in ducks.b.After DPV strong virus attack,non immunized control ducks could detect a strong virus load of 10^8-9copies/mg in various tissues within 1 day,and the virus load in each tissue reached a maximum of about 10^10-13copies/mg after 5 days,until death;However,r DPV-E（UL49）,like commercially available duck plague vaccine immunized ducks,had a copy number of 10^8-9copies/mg in each tissue after 1 day.All tissues and organs except for the liver were significantly（P<0.05）or extremely significant（P<0.01）lower than the non immunized blank control ducks,with a decrease after 3 days and a decrease to 10^3-5copies/mg after 9 days.This indicates that r DPV-E（UL49）,like commercially available duck plague vaccine immunized ducks,can effectively inhibit the proliferation of DPV virulence in the body and clear some DPV virulence in the body.（4）Observation and Microscopic Changes of Tissues after Immunized Ducks Infected with Strong Poisona.After DTMUV strong toxicity attack,non immune control ducks showed obvious visual lesions（liver bleeding,splenomegaly,etc.）and tissue microscopic lesions（liver cell degeneration and necrosis,spleen cell necrosis and fragmentation,brain neuron cell degeneration and necrosis,etc.）;However,r DPV-E（UL49）,like ducks immunized with Tambusu disease vaccine,showed normal liver and spleen structures and no significant cell necrosis.This indicates that recombinant virus immunization of ducks can resist the damage of DTMUV strong toxicity to tissues and organs.b.After DPV strong toxicity attack,non immune control ducks showed liver bleeding,splenomegaly,intestinal mucosal necrosis and detachment,intestinal wall thinning,atrophy and bleeding of the bursa and thymus,and all died within 6 days;However,r DPV-E（UL49）and duck plague vaccine group ducks showed no significant pathological changes.This indicates that recombinant virus immunization of ducks can resist the damage of DPV strong toxicity to tissues and organs.（5）Antibody levels after immunization with DTMUV in ducksa.Neutralizing antibodies:r DPV-E（UL49）,like ducks immunized with Tambous disease vaccine,detected antibodies at 1 day(2^4.22and 2^4.24),followed by an increase in antibody levels,reaching 2^5.38and 2^5.42at 9 days,respectively.There was no significant difference in antibody levels between the two at different times（P>0.05）;Unimmunized blank control ducks,like ducks immunized with duck plague vaccine,can detect low levels of antibodies within 1 day.The difference in DTMUV antibody titer between r DPV-E（UL49）and ducks immunized with Tambusu vaccine at different times was extremely significant（P<0.0001）,compared to non immunized blank control ducks and ducks immunized with duck plague vaccine.b.ELISA antibody:r DPV-E（UL49）was detected in ducks immunized with duck Tambous disease vaccine,with antibodies detected at 1day(OD₄₅₀mean 0.533 and 0.55)and 1.01 and 0.99 at 9 days.There was no significant difference between the two at different time points（P>0.05）;Unimmunized blank control ducks,like ducks vaccinated with duck plague vaccine,can detect low levels of antibodies within 1 day.r DPV-E（UL49）is similar to ducks vaccinated with Tambusu vaccine,and there is a significant（P<0.05）or extremely significant（P<0.01）difference in antibody titer at different time points compared to non immunized blank control ducks and ducks vaccinated with duck plague vaccine.It indicates that after the attack of DTMUV,non immunized blank control ducks and duck plague vaccine immunized ducks also produced DTMUV neutralizing antibodies and ELISA antibodies,while recombinant virus immunized ducks and duck Tambous disease vaccine immunized ducks had higher antibody levels after the attack compared to the non immunized blank group and duck plague vaccine immunized ducks.In summary,the r DPV-E（UL49）constructed in this study is stably inherited in both DEF cells and ducks;Immunizing ducks with 10⁶TCID₅₀r DPV-E（UL49）can induce the production of neutralizing and ELISA antibodies that are equivalent to those of commercially available duck plague vaccines and duck Tambous disease vaccines.It can resist the strong toxicity of 10⁸TCID₅₀DTMUV and 100 LD₅₀DPV,and has good immune protection effects.It has the potential to develop a live vaccine against duck Tambous disease.