| Fowl adenovirus 4(FAdV-4)is a double-stranded DNA non capsular virus,which mianly causes chicken pericardial effusion hepatitis hydropericardium syndrome(HHS).The outbreak of FAdV-4 in China in 2015 caused huge economic losses to the domestic poultry industry,and it is widely distributed in China.In 2014,Duck adenovirus 3(DAdV-3)was isolated from dead Muscovy ducks in China.It is a linear segmentless double stranded DNA virus with strong pathogenicity.It can mainly cause pericardial effusion,liver fibrosis and liver hemorrhage to different degress.In recent years,DAdV-3 has been detected in many farms in our country,which has caused huge economic losses to our waterfowl industry.Therefore,there is an urgent need to analyze the pathogenic mechanisms of both viruses and develop a dual vaccine that can protect against both FADV-4 and DADV-3.In the experiment,FAdV-4 and DAdV-3 were isolated and identified from the disease materials collected from the fram,and the circular plasmids of FAdV-4 and DAdV-3 were constructed by homologous recombination.The FAdV-4 and DAdV-3 circular plasmids were constructed by Red/ET homologous recombination to replace Hexon and Fiber-2,respectively.The recombinant plasmids D3-F4/Hexon,D3F4/Fiber2,F4-D3/Hexon and F4-D3/Fiber2 were constructed.Several circular plasmids of DAdV-3,FAdV-4,D3-F4/Hexon,D3-F4/Fiber2,F4D3/Hexon,F4-D3/Fiber2 were subjected to enzymatic tangent modification by using PacI enzyme,and then were electrotransferred into LMH cells to rescue the virus.LMH cells were infected with MOI=0.001,and cell supernatants wedrawre collected every 12h until 120h to draw virus growth curves.The six rescued viruses were tested for pathogenicity in SPF chickens and Cherry Valley ducks,respectively,to analyze the effects of Hexon and Fiber2.Construct inactivated vaccines of several viruses and conduct immune challenge tests on SPF chickens and Cherry Valley ducks.The results show:(1)The virus isolated from the infected material were identified as FAdV-4 and DAdV-3 by PCR and sequencing.(2)The plasmid DAdV-3,FAdV-4,D3-F4/Hexon,D3-F4/Fiber2,F4-D3/Hexon and F4-D3/Fiber2 were successfully constructed by PCR,sequencing and enzyme digestion.(3)The highest titer of DAdV-3 strain was significantly lower than that of FAdV-4 and the other four recombinant viruses.(4)After inoculating SPF chicks,the virus titer in heart,liver,spleen,lung and kidney of D3-F4/Hexon virus group was significantly higher than that of DAdV-3 virus group(P<0.05);The virus titers in heart,liver,spleen,lung,kidney and brain of the D3-F4/Fiber2 virus group were not significantly different from those of the DAdV-3 virus group(P>0.05);The virus titers in heart,liver,spleen,lung,kidney and brain of F4-D3/Hexon and F4-D3/Fiber2 virus groups were significantly lower than those of FAdV-4 virus group(P<0.05).(5)The pathogenicity test of avian adenovirus showed that the virus titer in heart,liver,spleen,lung and brain of D3-F4/Hexon virus group was significantly lower than that of DAdV-3 virus group after inoculation of Cherry Valley Duck(P<0.05);The virus titers in heart,liver,spleen,lung and brain of the D3-F4/Fiber2 virus group were not significantly different from those of the DAdV-3 virus group(P>0.05);The virus titers in kidney of D3-F4/Hexon virus group and D3-F4/Fiber2 virus group were significantly lower than those of DAdV-3 virus group(P<0.05);The virus titers in heart,liver and spleen of F4-D3/Hexon virus group were significantiy higher than those of FAdV-4 virus group(P<0.05);The virus titers in heart,liver and spleen of F4-D3/Fiber2 virus group were not significantly different from those of FAdV-4 virus group(P>0.05);The virus titers of F4-D3/Hexon virus group and F4D3/Fiber2 virus group were significantly higher than those of FAdV-4 virus group in lung and brain,and the difference was significant(P<0.05),the virus titers in kidney of F4-D3/Hexon virus group and F4-D3/Fiber2 virus group were not significantly different from those of the FAdV-3 virus group(P>0.05);(6)After immunizing SPF chicken with FAdV-4,F4-D3/Fiber2,F4-D3/Hexon,D3-F4/Fiber2 and D3F4/Hexon inactivated vaccines and PBS group,none of them died after intramuscular injection of FAdV-4 0.2mL 104TCID50,and the survival rate was 100%;After challenge with DAdV-3 inactivated vaccine,the survival rate was 60%,and that of PBS group was 50%;The protective rate of F4-D3/Fiber2,D3-F4/Hexon and FAdV-4 inactivated vaccines against FAdV-4 was 100%;The protection rate of F4-D3/Hexon inactivated vaccine against FAdV-4 was 28.6%;The protection rate of D3-F4/Fiber2 inactivated vaccine against FAdV-4 was 14.3%;The protection rate of DAdV-3 inactivated vaccine and PBS group against FAdV-4 was 0%.(7)The results of avian adenovirus cross-protection test showed that Cherry Valley ducks were immunized with FAdV-4,F4D3/Fiber2,F4-D3/Hexon,D3-F4/Fiber2,D3-F4/Hexon and DAdV-3 inactivated vaccines and PBS.Intramuscular injection of DAdV-3 0.2mL 104TCID50 animal did not result in death,and the survival rate was 100%;The protection rates of F4-D3/Hexon,F4-D3/Fiber2,D3-F4/Fiber2 and DAdV-3 inactivated vaccines against DAdV-3 were 100%;The protection rate of D3-F4/Hexon inactivated vaccine against DAdV-3 was 85.7%;The protective rateof FAdV-4 inactivated vaccine against DAdV-3 was 71.4%;The protection rate of PBS group against DAdV-3 was 0%.Conclusion:Two fowl adenovirus strains were isolated and purified from the infected material in this study,which were identified as FAdV4 and DAdV-3,respectively;It is proved that Hexon is the main antigenic protein of fowl adenoviruses,and Fiber2 also has partial antigenic effect.The inactivated vaccine obtained by inactivating the recombinant virus F4-D3/Fiber2 has good immune effect on FAdV-4 and DAdV-3;Fiber2 protein can affects the growth of adenovirus on different cells,which is an important species recognition protein. |
PDF Full Text Request