The Construction Of TrVLP System Modeling The Life Cycles Of Ebola Virus And Its Interaction Effects In Host Cells

Ebola virus(EBOV)is a kind of pathogenic microorganisms that can be transmitted between human and non-human primates through contact,with a fatality rate of up to 90%.EBOV is highly protected and difficult to acquire.At present,most countries in the world do not have Ebola pathogens,let alone the resources and conditions to carry out relevant research on it.Besides,the screening and evaluation of drugs related to EBOV also rely on the high-level biosafety laboratory.The experimental process has high biosafety risks,high cost and long time,which greatly limit the development and application of anti-EBOV drugs.In addition,the study on the effects of host cells induced by EBOV is still incomplete,lacking systematic exploration and summary.Recently,pseudogenes,long noncoding RNAs(lncRNAs)and circular RNAs(circRNAs)were found to act as miRNA “sponges” that affect miRNA activity.However,ceRNA studies related to EBOV have not been reported before.These ceRNAs,including circRNAs and lncRNAs,may play important roles in many human diseases such as Alzheimer's disease,pulmonary fibrosis and human breast cancer.However,the ceRNAs responsible for the effects induced by EBOV replication and transcription have not been reported.The purpose of this study was to build up a transcription and replication competent virus-like particle(trVLP)system that can model the life cycles of EBOV under Biosafety Level 2(BSL-2)conditions.Then the expression profiles of EBOV reproduction-related RNAs and the ceRNA interaction networks were constructed.Next,the exploration of the interaction mechanisms between circRNA,miRNA and mRNA associated with EBOV reproduction were also performed.Finally,a rapid screening platform for EBOV inhibitors by using the virus-like particle system was established,based on which the host-encoded miRNAs with inhibitory effects against EBOV were found.This study provides basic data and technical support for the research on the infection and pathogenesis of the EBOV,as well as the traceability research,clinical diagnosis and treatment,and disease prevention and control.This study consists of the following four parts:1.The construction and identification of trVLP System Modeling the Life Cycles of Ebola Virus.A novel Ebola virus-like particles,wich can model EBOV lifecycles under BSL-2 conditions including morphogenesis,budding,entry,genome replication,and transcription,were constructed by reverse genetics technology and eukaryotic expression system.Thus,it provides strong technical support for the basic and applied research of EBOV.The virus characterics such as morphology,gene,protein,reproduction and passage ability,and genetic stability were identified.The results showed that the virus-like particles had typical EBOV morphological structure,gene and protein specificity,certain ability of reproduction and passage,and good genetic stability,which could be used for the follow-up experimental studies.2.The construction of EBOV reproduction-related RNA expression profiles and ceRNA interaction networks.In this study,we conducted a genome-wide screen to identify the differentially expressed lncRNAs,circRNAs,miRNAs and mRNAs 0 h,24 h,72 h,96 h post virus infection using trVLP system that models the life cycles of EBOV in 293 T cells.The interaction relationships of ceRNAs were predicted through the bioinformatics analysis processes including RNA expression trend analysis,co-expression gene analysis,weighted co-expression gene analysis,and RNA targeting sequence prediction.Then the ceRNA networks based on the RNAs responsible for the effects induced by EBOV replication and transcription in human cells were constructed for the first time.3.Exploration of the interaction mechanisms between circRNA,miRNA and mRNA associated with EBOV reproduction.Based on the RNA expression profile and ceRNA interaction network successfully drawn in the second part,the potential interaction mechanism of RNAs related to the proliferation of ebola virus was further explored.The CLDN18 gene with the strongest co-expression ability in the co-expression network and the associated mir-30b-3p and circRNA-chr19 were selected as the research subjects.Through gene expression detection,RNA targeting sequence verification,gene knockdown,miRNA specific inhibition and other related experiments,the key RNAs interaction mechanism was further identidied.These results demonstrated that CLDN18 was positively correlated with the expression trend of circRNA-chr19,and there was a direct interaction between CLDN18 and miR-30b-3p.Moreover,circRNA-chr19 regulates the expression of CLDN18 by interacting with miR-30b-3p.This part provides a theoretical basis for systematically expounding the regulation process of gene expression in host cells during the process of virus reproduction and the variation rules of relevant life activities.4.Study on the host-encoded miRNAs with inhibitory effects against Ebola virus reproduction.Based on the trVLP system that can model EBOV lifecycles under BSL-2 conditions including morphogenesis,budding,entry,genome replication,and transcription,a novel platform for rapid,efficient and accurate screening and evaluation of anti-Ebola drugs was established.Furthermore,bioinformatics and virological identification methods were performed to screen host-encoded miRNAs with inhibitory effects against Ebola virus by using the transcriptome data obtained in chapter 2.Among these miRNAs,miR-150-3p had the most significant inhibitory effect.The identification results demonstrated that miR-150-3p inhibited the reproduction of trVLPs via the regulation of GP and VP40 expression by directly targeting the coding regions of GP and VP40.This part not only discovered host-encoded miRNAs that inhibit the reproduction of EBOV,but also provided a new idea,a novel method and an innovative platform for the screening and evaluation of anti-Ebola drugs.