Study On The Function Of Fiber1 And Fiber2 In Fowl Adenovirus Type 4 (FAdV-4) Cell Culture

Fowl Adenovirus serotype 4(FAdV-4)belongs to fowl adenovirus subgrou p I.Research on FAdV-4 can be traced back to 1971.However,because the low pathogenicity of previously discovered FAdV-4,their research progress has been slow.In 2015,the Hepatitis Hydropericardium Syndrome(HHS)caused by FAd V-4 broke out in Shandong,Henan,Zhejiang and other places,which caused a serious blow to the poultry industry in East China.This has caused China and e ven the international community to attach importance to FAdV-4.Reverse genetic modification of FAdV-4 Fiber to obtain recombinant virus.T o study the effects of two fibers on the proliferation and virulence of FAdV-4,we constructed a recombinant virus that deleted Fiber1 and named it FAV4XF1 GFP and a recombinant virus that deleted Fiber2 and named it FAV4XF2 GFP.The results show that FAV4XF1 GFP can be rescued but cannot replicate normal ly.With the help of pcDNA3-FAV4F1,FAV4XF1 GFP can replicate and proliferat e.FAV4XF2 GFP can replicate and proliferate normally,but its ability is weakene d.To study the blocking effect of Fiber Knob protein on the process of FAd V-4 infected cells.In this study,human adenovirus type 5 was used as a vector to construct eukaryotic expression vectors HAd5XE3-F1H6 and HAd5XE3-F2H6,and inoculated 293 T cells to express and purify the Knob region of Fiber1 an d Fiber2.After protein purification,the concentration was measured with BCA ki t,and then SDS-PAGE electrophoresis and Western Blot identification were perf ormed,and then a blocking experiment was performed.According to the analysis of Fiber1 protein blocking experiment results,when the protein concentration is0-1?g/mL,the amount of fluorescence is basically unchanged;At 5-10 ?g/mL,the amount of fluorescence decreased significantly,and the fluorescence ratio dec reased from about 80% to 20%.Study to the replication proliferation of FAdV-4 deleted Fiber2.The design used the parental virus FAV4 GFP as a control to determine the growth curve,v irus plaque test,and chicken embryo test of FAV4XF2 GFP.It can be seen from the growth curve that the growth cycle of FAV4XF2 GFP is the same as that of the parent virus FAV4 GFP.The virus production in the cell is 10% of the pare ntal virus FAV4 GFP.From the virus plaque test,it can be seen that the number of plaques produced by FAV4XF2 GFP and the parent virus FAV4 GFP with the same number of particles is basically the same.The plaque area was 27.1% of F AV4 GFP.The chicken embryo experiment showed that the survival rate of FAV4XF2 GFP was 40% at 14 days,and the survival rate of the parent virus FAV4 G FP was 0% at 12 days.Liver virus titers of dead chicken embryos on the same day in the FAV4 GFP group were 100 to 1000 times that of the FAV4XF2 GFP group.In summary,Fiber1 is an essential protein for replication and proliferation of FAdV-4.Fiber2 is a non-essential protein.Fiber1 is a key protein in the proces s of FAdV-4 infecting cells.