Development Of Live Vaccine For Group ? Fowl Adenovirus(Type 4 And 8b)Using Heat-resistant Newcastle Disease Virus As Vector

Fowl adenovirus(FAdV)can be divided into groups of I,II and III,which can cause diseases of chickens,ducks,geese and other poultry or wild birds.Group I fowl adenovirus can be divided into 12 serotypes.Serotype 4(FAdV-4)and serotype 8b(FAdV-8b)are the main serotypes prevalent in chicken flocks in China.They cause diseases such as hydropericardium syndrome(HPS)and chicken inclusion body hepatitis(IBH),respectively,which cause serious economic losses to poultry industry.Fiber is an important structural protein of fowl adenovirus.It contains neutralizing epitopes of the virus and is also related to the infectivity of the virus.In this study,the recombinant viruses expressing FAdV-4 and FAdV-8b fiber proteins respectively were constructed using heat-resistant Newcastle disease virus(NDV)as vectors by reverse genetic technology,and the immune efficacy of the recombinant live vaccine was evaluated.1.Construction of recombinant avian adenosis virus group I(type 4 and 8b)using heat-resistant Newcastle disease virus as vectorUsing the genomic DNA of FAdV-4(GX2013 strain)and FAdV-8b(QD2016 strain)as templates,the fiber2 gene and fiber gene were amplified by PCR,and then the fiber2 gene and fiber gene were inserted into the P and M gene spacers of the whole genome plasmid rAHR09-P containing NDV/rAHR09 attenuated strain,respectively,to construct rAHR09-P-FAdV-4 fiber2 and rAHR09-P-FAdV-8b fibers.BSR-T7/5 cells were transfected into with two recombinant plasmids and plasmids(pCI-NP,pCI-P and pCI-L)expressing NDV NP,P and L genes respectively for 60 hours,and then inoculated with 9-11 day-old SPF chicken embryos.The recombinant virus was rescued by hemagglutination test.RT-PCR and sequencing showed that fiber2 gene and fiber gene were inserted into NDV/rAHR09 genome respectively.The two recombinant viruses were named rAHR09-4F2 and NDV/rAHR09-8bF.Western Blot test showed that both fiber2 gene and fiber gene inserted into rAHR09 could express,and the size of protein bands expressed by the two recombinant viruses was consistent with expectation.The results of heat tolerance test showed that rAHR09-4F2 and rAHR09-8bF still had HA activity after being treated at 56 C for 70 minutes.Both recombinant viruses and maternal viruses had good heat tolerance.EID50 test showed that the virus titers of rAHR09-4F2 and rAHR09-8bF were 10'.42 EIDso/mL and 107 EIDso/mL respectively.The two recombinant viruses had good replication ability in chicken embryos.Both recombinant viruses had good replication.The results of MDT and ICPI test showed that rAHR09-4F2 and rAHR09-8bF MDT were more than 120 hours and ICPI values were both 0.050.There was no significant difference in virulence between the two recombinant viruses and their parents,and they were still attenuated strains.2.Evaluation of immune efficacy of recombinant strainsIn order to evaluate the immune efficacy of recombinant strains rAHR09-4F2 and rAHR09-8bF,immunization challenge protection test 1 and test 2 were carried out respectively.Test I was divided into rAHR09-4F2 group,FAdV-4+FAdV-8b inactivated vaccine group and challenge control group.7-day-old SPF chickens,rAHR09-4F2 group were inoculated with rAHR09-4F2(106 EID50)by nasal eye drips and FAdV-4+FAdV-8b bivalent inactivated vaccine(0.25 mL/chicken)were subcutaneously injected in the neck within inactivated vaccine group.Serum samples were collected from chickens on days7,14,21 and 28 after immunizing.The titers of FAdV-4 and FAdV-8b neutralizing antibodies in serum were detected by serum neutralization test(SNT).Test 2 was divided into rAHR09-8bF group,FAdV-4+FAdV-8b inactivated vaccine group and challenge control group.The treatment of test 2 is consistent with test 1.In the rAHR09-4F2 and inactivated vaccine groups,the neutralizing antibody titers reached peak level 21 days after immunizing,and the titers of antibodies against FAdV-4 were 102 93 and 103.23,respectively.On the 21st day after immunizing,FAdV-4(107.5 TCIDso/mL)were challenged by intramuscular injection in each group,0.2 mL/chicken.The challenge control group died within 3 days,while the rAHR09-4F2 group died 5 chickens within 3 days.The inactivated vaccine group showed normal performance.The throat swabs and cloacal swabs of chickens in each group were collected on days 3 and 7 after challenging.The viruses that the swabs containing were detected by SYBR Green I real-time fluorescence quantitative PCR.The virues shedding of respiratory and digestive tract in rAHR09-4F2 group was significantly higher than that in inactivated vaccine group on the 3rd and 7th day after challenging(P<0.05).The level of antibody produced by rAHR09-4F2 stimulating is low,and it has 50%efficiency of death protection for FAdV-4.rAHR09-4F2 can reduce the detoxification of test chickens,but the effect is not significant.In the rAHR09-8bF group and inactivated vaccine group of test 2,neutralizing antibodies began to produce 7 days after imunizing,and reached the peak on the 21st day after immunizing.The titers of antibodies against FAdV-8b were 103.11 and 103.15,respectively.On the 21st day after immunizing,0.2 mL FAdV-8b(106.5 TCID50/mL)was injected in each chicken.After challenging,rAHR09-8bF group and inactivated vaccine group showed normal performance,while dilution occurred in the challenge control group.The viruses shedding of respiratory and digestive tract in rAHR09-8bF group and inactivated vaccine group were lower than those in control group on the 3rd and 7th day after challenging,and the difference was very significant(P<0.01).The viruses shedding of respiratory and digestive tract in rAHR09-8bF group was not significantly different from that in inactivated vaccine group(P>0.05).rAHR09-8bF can stimulate test chickens to produce higher levels of antibodies against FAdV-8b,and can significantly reduce the viruses shedding of test chickens.The protective efficiency can reach about 90%.