Studies On The Important Host Factors Related To The Life Cycle Of Ebola Virus And Anti-Ebola Virus Neutralizing Antibodies

Ebola virus disease(EVD)causes deadly hemorrhagic fever in humans and non-human primates resulting from infection with the Ebola virus(EBOV)genus of the family Filoviridae.Although many studies and treatments on EVD have made significant progress,there are still many unclear molecular mechanisms related to the life cycle of EBOV in host cells,and limited therapeutic methods for patients with suspected or confirmed EVD.We produced EBOV transcription-and replication-competent virus-like particles(trVLPs)to mimic the EBOV life cycle under biosafety level 2 conditions,synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle(including viral entry,uncoating,fusion,replication,assembly,and budding),screened host factors associated with the trVLP life cycle by RNAi.Then,we assessed interactions of host proteins with trVLPs glycoprotein(GP),VP40,and RNA by co-immunoprecipitation(Co-IP)and chromatin immunoprecipitation(ChIP).Meanwhile,we synthesized anti-Zaire EBOV GP and anti-Zaire EBOV VP40 monoclonal antibodies(mAbs)by hybridoma technology,to study the antiviral effect of single and combined application of anti-EBOV GP and anti-VP40 mAbs against EBOV-trvlps in vitro.In the study,we found host chaperones,membrane trafficking and signal transduction proteins are closely related to the life cycle of EBOV-trvlps,and have interactions with GP,VP40 protein or nucleic acid of trVLPs;and proved the combination of anti-GP and anti-vp40 mAb,or combination of anti-vp40 mAbs have synergistic antiviral effect.Part 1 Establishment of Ebola virus transcription-and replication-competent Virus-like particlesAimThe identification of EBOV wild strain with biosafety level 4 conditions greatly limited researchers' study of the virus.Therefore,this study intends to construct EBOV-trvlps under biosafety level 2 condition to simulate the life cycle and basie functions of EBOV wild strain.Material and methodUnder biosafety level 2 conditions,we produced EBOV transcription-and replication-competent Virus-like particles(trVLPs)on the basis of the principle of reverse genetics,by using EBOV related plasmids transfection in 293T cells.trVLPs were evaluated by detecting reporter activity,electron microscopy observation;and the invasion ability of DiI labeled trVLPs into 293T cells was used to observe fluorescence microscopy.ResultsThe reporter activities of trVLPs from p0 to p5 generations showed a gradually increasing trend,which proved the system had stable replication ability.TrVLPs present as filamentous virus-like particles under electron microscope,and highly simulated EBOV wild strain in terms of size and morphology.Meanwhile,DiI labeled trVLPs showed the ability to invade living cells and transport within cells.ConclusionTrVLPs can replicate stably in vitro and simulate the life cycle and basic functions of EBOV wild strain under biosafety level 2 conditions.TrVLP is in favour of EBOV research as it has no biological hazard and has the characteristics of stability and monitoring.Part 2 Screening of important host factors related to EBOV-trvlps life cycle and study of the interactionsAimEbola virus(EBOV)life cycle involves numerous host factors and probably interacts with them,but there are still many unclear aspects concerning host factors,pathogenetic mechanism and interaction relations.Material and methodOn the basis of trVLPs system,we synthesized 65 siRNAs targeting host genes mostly connected with aspects of the negative-sense RNA virus life cycle(including viral entry,uncoating,fusion,replication,assembly,and budding),then screening host factors associated with the trVLPs life cyele by by RNA interference technology(RNAi).Later,we assessed interactions of the candidate host proteins with trVLPs glycoprotein(GP),VP40,and nucleic acid by co-immunoprecipitation(Co-IP),chromatin immunoprecipitation(ChIP),western blot and other methods.ResultsThe results demonstrate that RNAi silencing with 11 siRNAs(ANXA5,ARFGAP1,FLT4,GRP78,HSPAIA,HSP90AB1,HSPA8,MAPK11,MEK2,NTRK1 and YWHAZ)decreased the replication efficiency of trVLPs.Co-IP revealed nine candidate host proteins(FLT4,GRP78,HSPAIA,HSP90AB1,HSPA8,MAPK11,MEK2,NTRK1 and YWHAZ)potentially interacting with trVLP GP,and four(ANXA5,GRP78,HSPA1A,and HSP90AB1)potentially interacting with trVLP VP40.Ch-IP identified nine candidate host proteins(ANXA5,ARFGAP1,FLT4,GRP78,HSPA1A,HSP90AB1,MAPK11,MEK2 and NTRK1)interacting with trVLPs nucleic acid.ConclusionWe found host chaperones,membrane trafficking and signal transduction proteins which likely to affect EBOV-trVLPs life cycle and interact with trVLP GP,VP40 proteins or trVLPs nucleic acid.Part 3 Studies and Characterization of Neutralizing Antibodies against Ebolavirus Glycoprotein and Protein 40AimMonoclonal antibody(mAb)is one of the effective methods to treat EVD.Up to now,the anti-EBOV antibodies are mostly limited to anti-GP antibodies,and the combined application of antibodies is also limited to the combination of anti-GP antibodies.There is no research characterizing a neutralizing Abs cocktail against Zaire EBOV GP and VP40.Therefore,we intend to study the anti-virus effect of anti-GP and anti-VP40 antibodies on the basis of EBOV-trVLPs.Material and methodWe synthesized anti-EBOV GP and VP40 mAbs by hybridoma technology.On the basis of trVLPs system,we verify the neutralizing efficacy of each anti-GP or anti-VP40 mAb,and report promising cocktail of anti-GP and anti-VP40 mAbs,or cocktail of two anti-VP40 mAbs by RT-PCR,western blot,immunofluorescence assay and other methods.ResultsThe results demonstrate that anti-GP or anti-VP40 mAbs effectively inhibit trVLPs replication at the dose of 5-10?g/ml(P<0.05).Compared with antibody alone,the cocktails of anti-GP and anti-VP40 mAbs,or between anti-VP40 mAbs,had more significant antiviral effect,which demonstrated synergistic anti-trVLPs efforts.ConclusionThe study verifies neutralizing efficacy of anti-GP or anti-VP40 mAb,report promising cocktail of anti-GP and anti-VP40 mAb,or cocktail of two anti-VP40 mAbs.This paper reports the important anti-viral effect of cocktails of anti-GP and anti-VP40 mAbs in vitro.