Study On The Pathway Of Fowl Adenovirus Serotype 4 To Enter Into LMH Cells

Hydropericardium-hepatitis syndrome?HHS?is an acute highly pathogenic infectious disease caused by Fowl adenovirus serotype 4?FAdV-4?.Since the outbreak of our country in 2015,it has spread rapidly to the whole country.At present,the research on FAdV-4mainly focuses on viral genome analysis and pathogen detection.It is still unclear about the proliferation characteristics and invasion pathway of FAdV-4 on host cells.In this study,Leghorn male hepatocellular cells?LMH cells?were selected as the research object,and the proliferation and invasion pathway of FAdV-4 on LMH cells were studied,which provided a theoretical study on the mechanism of FAdV-4 invasion of host cells.The results are as follows:?1?The proliferation of FAdV-4 with different infection numbers on LMH is quite different.After infection of LMH cells with FAdV-4,FAdV-4 virus copy number and Hexon protein expression were detected by Real-time PCR and Western blot.The study found that FAdV-4 virus copy number and Hexon protein expression reached the highest after infection at 72 after infection of LMH cells with MOI=1.After LMH cells were infected with MOI=10,the expression of Hexon protein increased continuously within 12 h after infection,and reached the highest at 12 h,and decreased at 24 h.Through intrusion kinetics experiments and laser confocal microscopy,it was found that FAdV-4 required about 60 min to complete the invasion of LMH cells.?2?FAdV-4 invading LMH cells does not depend on the intracellular acidic environment but requires the involvement of dynamin.LMH cells were treated with intracellular acidic environmental inhibitors such as NH₄Cl,chloroquineand Dynamin inhibitor Dynasore for 2 h,then inoculated with FAdV-4 for laser confocal microscopy and The detection of Hexon protein expression and virus copy numbers.The results showed that compared with the control group,the specific green fluorescence representing virions in the NH₄Cl and Chloroquine treatment groups showed no obvious membrane aggregation,and the Hexon protein expression and virus copy number did not decrease significantly.This indicates that FAdV-4 invades LMH cells independent of the intracellular acidic environment.The specific green fluorescence representing virions in the Dynasore treatment group showed significant membrane aggregation,and the Hexon protein expression and virus copy number also decreased significantly.It indicated that FAdV-4invaded LMH cells required Dynamin.?2?FAdV-4 invasion of LMH cells is dependent on caveolin-mediated endocytisis.After infecting LMH cells with FAdV-4,transmission electron microscopy showed the presence of endocytosis.LMH cells were treated with clathrin inhibitor chlorpromazine for 2 h,and then inoculated with FAdV-4 for laser confocal observation and detection of Hexon protein expression and virus copy numbers.The results showed that the location of virions was not significantly different from that of the control group,and the expression of Hexon protein and virus copy numbers did not change significantly.This indicates that FAdV-4 invades LMH cells independent of clathrin-mediated endocytosis.LMH cells were treated with caveolin inhibitors Nystatin and M?CD for 2 h,then inoculated with FAdV-4for laser confocal observation and detection of Hexon protein expression and virus copy numbers.The results showed that the specific green fluorescence representing virions showed significant membrane aggregation,and the Hexon protein expression and virus copy number decreased significantly,indicating that FAdV-4 invasion of LMH cells is dependent on caveolin-mediated endocytosis.?3?FAdV-4 invasion of LMH cells also depends on the macrocytoplasmic pathway and requires the participation of PI3K molecules.LMH cells were treated with EIPA,an inhibitor of the macropinocytosis,and Wortmannin,a related inhibitor of PI3K,for 2 h,and then inoculated with FAdV-4 for laser confocal observation and detection of Hexon protein expression and virus copy numbers.The results showed that compared with the control group,the green fluorescence representing virions was significantly concentrated in the membrane,and the expression of Hexon protein and the copy number of virus were significantly decreased,indicating that FAdV-4 invaded LMH cells also depended on the macropinocytosis and required PI3K molecules.Taken together,it is shown that FAdV-4 invasion of LMH is dependent on caveolin-mediated endocytic pathway and macrocytoplasmic pathway and requires the participation of Dynamin and PI3K molecules,but no PH dependence.This provides a new idea for the target selection of new antiviral drugs..