Study On BmNPV Infection Of Direct Membrane Fusion Induced By Low PH

The pyosis caused by BmNPV is the most serious disease in sericulture.BmNPV belongs to group I baculovirus.Previous studies have shown that the budding virus particles of BmNPV invade the host cells in the way of macropinocytosis.Ac MNPV(Autographa California Nucleopolyhedrosis virus)and BmNPV are both representative baculoviruses of group I,and they are also two well-known viruses,Ac MNPV invades host cells by means of clathrin mediated endocytosis,and low pH induced direct membrane fusion can also promote Ac MNPV to infect host cells effectively,but whether direct membrane fusion can invade host cells by BmNPV is unclear.Chapter I: Literature review.Firstly,the classification of baculovirus,the process of virus infection and the mode of virus invasion are introduced.Secondly,the process of envelope virus invasion and the molecular mechanism of membrane fusion are summarized.Finally,the research progress and the research achievements of Ac MNPV and BmNPV at home and abroad are compared and analyzed.In Chapter II:The direct membrane fusion of BmNPV induced by low pH was studied.Firstly,the suitable pH of BmNPV GP64 was determined.The membrane fusion protein GP64 of BmNPV was expressed instantaneously in Bm N cells,and then membrane fusion was induced under different pH conditions.The optimal fusion pH of GP64 under different pH conditions was determined and analyzed by counting the number of Syncytium index.The results showed that when the pH value was 5.0,the cell membrane fused with each other to form a larger syncytium.When the pH value was 4.5,the number of syncytium was the largest.The statistical results showed that the fusion rate increased with the decrease of pH value.It is concluded that the condition of membrane fusion mediated by BmNPV GP64 is pH less than 5.0.Next,Bm N cells were treated with ammonium chloride,and then BmNPV was used;at the same time,infection was carried out,and pH was used 4.5condition induced direct membrane fusion and then cultured in normal condition,compared with infection efficiency.The results showed that under the condition of low pH,the infectivity of the virus was analyzed by cell flow cytometry.The results showed that low pH induced the decrease of infection rate of the virus.Finally,the marker virus of VP39 was fused by e GFP to trace the transport of nucleocapsid after the direct membrane fusion The results of focusing technology show that although the virus particles enter the cytoplasm,they are not transported to the nucleus,which leads to the failure of abortive infection.Chapter III: Analysis of the transcriptome sequencing of BmNPV infected cells by direct membrane fusion.In order to explore the cause of the failure of nucleocapsid transport of the virus,the normal infected cell samples of BmNPV were used as the control,and the samples of 0 / 15 / 60 minutes after invasion induced by low pH were sequenced with high-throughput to find the host factors involved in the process.According to GO/KEGG analysis of the differentialy expressed genes in the sequencing results,95 DEGs were obtained,including 22 DEGs involved in the ribosome pathway,6 DEGs involved in the endoplasmic reticulum protein processing pathway,4 in the shear pathway and 3 in the phagocytic pathway.Hsp90 was reported to be related to many insect immune pathways,calreticulin CRT and heavy chain binding protein Bi P(HSP70)were important members of the protein secretory folding pathway,and all of them were reported to be related to the invasion or release of a variety of viruses.Chapter IV: Analysis and verification of differential expressed proteins.In order to determine the role of differentially expressed proteins in the invasion process of BmNPV,the expression of Hsp90,CRT and Bi P was verified by q PCR,and the expression of Hsp90,CRT and Bi P was knocked down by RNAi technology to determine the infection efficiency of the virus.The expression of Hsp90,CRT and Bi P were confirmed by q PCR.The results were consistent with the results of transcriptome sequencing;The expression of Hsp90,CRT and Bi P in the knockdown cells was effectively reduced by RNA interference,which led to the decrease of infection efficiency of BmNPV.The infection efficiency of BmNPV was measured.When the expression of BIP and CRT was decreased,the infection efficiency of BmNPV was decreased,but the decrease of Hsp90 increased the infection rate of cells.In this study,we found that the direct membrane fusion induced by low pH can lead to the failure of BmNPV infection,which is caused by the fact that the nucleocapsid can not be transported to the nucleus.The host cytokines with different expression were selected by high-throughput sequencing analysis to participate in the invasion of BmNPV,which laid a foundation for further understanding the molecular mechanism of BmNPV invasion.