The Ebola virus (EBOV), a member of the Filoviridae family, is a highly pathogenic agent responsible for severe hemorrhagic fever in humans. Understanding the molecular mechanisms governing its replication is critical for developing effective antiviral strategies. VP35-TurboID immunosuppression coupled with quantitative mass spectrometry identified Septin9, the host GTP-binding protein which played a role in cytoskeletal regulation, as a novel interactor of VP35. Western blotting and Far-Western blotting confirmed the direct interaction and demonstrated that the C-terminal region of VP35 was the critical binding domain. Functionally, EBOV replication as well as the formation of viral inclusion bodies (VIBs) was demonstrated to be significantly suppressed by Septin9 knockdown and depletion, as shown by the EBOV minigenome (EBOV MG) and the transcription- and replication-competent virus-like particles (trVLPs) system. This study reveals that VP35 engages in a specific interaction with the GTP-binding protein Septin9, thereby impeding EBOV replication through the disruption of inclusion bodies. The overarching objective of this study is to significantly enhance our understanding about the pathogenic mechanism of EBOV and offer a robust theoretical foundation and solid empirical support for the formulation of innovative therapeutic strategies against EBOV.
Virus Replication/physiology* ; Septins/physiology* ; Humans ; Ebolavirus/physiology* ; Inclusion Bodies, Viral/metabolism* ; Viral Regulatory and Accessory Proteins/metabolism* ; Hemorrhagic Fever, Ebola/virology*

Adenoviridae ; genetics ; Ebolavirus ; genetics ; pathogenicity ; Gene Transfer Techniques ; Genetic Vectors ; therapeutic use ; Glycoproteins ; genetics ; HEK293 Cells ; Hemorrhagic Fever, Ebola ; genetics ; pathology ; virology ; Humans ; Inflammation ; genetics ; pathology ; virology ; Mucins ; genetics ; Transfection ; Viral Envelope Proteins ; genetics

The quality control process throughout the Ebola virus nucleic acid detection in Sierra Leone-China Friendship Biological Safety Laboratory (SLE-CHN Biosafety Lab) was described in detail, in order to comprehensively display the scientific, rigorous, accurate and efficient practice in detection of Ebola virus of first batch detection team in SLE-CHN Biosafety Lab. Firstly, the key points of laboratory quality control system was described, including the managements and organizing, quality control documents and information management, instrument, reagents and supplies, assessment, facilities design and space allocation, laboratory maintenance and biosecurity. Secondly, the application of quality control methods in the whole process of the Ebola virus detection, including before the test, during the test and after the test, was analyzed. The excellent and professional laboratory staffs, the implementation of humanized management are the cornerstone of the success; High-level biological safety protection is the premise for effective quality control and completion of Ebola virus detection tasks. And professional logistics is prerequisite for launching the laboratory diagnosis of Ebola virus. The establishment and running of SLE-CHN Biosafety Lab has landmark significance for the friendship between Sierra Leone and China, and the lab becomes the most important base for Ebola virus laboratory testing in Sierra Leone.
China ; Ebolavirus ; classification ; genetics ; isolation & purification ; Hemorrhagic Fever, Ebola ; diagnosis ; virology ; Humans ; Laboratories ; manpower ; standards ; Laboratory Infection ; Quality Control ; RNA, Viral ; genetics ; Sierra Leone

The Ebola virus is highly infectious and can result in death in ≤ 90% of infected subjects. Detection of the Ebola virus and diagnosis of infection are extremely important for epidemic control. Presently, Chinese laboratories detect the nucleic acids of the Ebola virus by real-time reverse transcription-polymerase chain reaction (RT-PCR). However, such detection takes a relatively long time and necessitates skilled personnel and expensive equipment. Enzyme-linked immunosorbent assay (ELISA) of serum is simple, easy to operate, and can be used to ascertain if a patient is infected with the Ebola virus as well as the degree of infection. Hence, ELISA can be used in epidemiological investigations and is a strong complement to detection of nucleic acids. Cases of Ebola hemorrhagic fever have not been documented in China, so quality-control material for positive serology is needed. Construction and expression of human-mouse chimeric antibodies against the nucleoprotein of the Ebola virus was carried out. Genes encoding variable heavy (VH) and variable light (VL) chains were extracted and amplified from murine hybridoma cells. Genes encoding the VH and VL chains of monoclonal antibodies were amplified by RT-PCR. According to sequence analyses, a primer was designed to amplify functional sequences relative to VH and VL chain. The eukaryotic expression vector HL51-14 carrying some human antibody heavy chain- and light chain-constant regions was used. IgG antibodies were obtained by transient transfection of 293T cells. Subsequently, immunological detection and immunological identification were identified by ELISA, immunofluorescence assay, and western blotting. These results showed that we constructed and purified two human- mouse chimeric antibodies.
Animals ; Antibodies, Monoclonal ; genetics ; immunology ; Cloning, Molecular ; Ebolavirus ; genetics ; immunology ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Immunoglobulin Heavy Chains ; genetics ; immunology ; Mice ; Nucleoproteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.
Animals ; Antibodies, Viral ; immunology ; Ebolavirus ; genetics ; immunology ; Female ; Gene Expression ; Hemorrhagic Fever, Ebola ; immunology ; virology ; Humans ; Immunization ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics ; immunology

Given the Ebola outbreak in West Africa and the risks of spread to other regions, a rapid, sensitive and simple method for the detection of the Ebola virus (EBOV) is of great significance for the prevention and control of Ebola. We developed a simple colorimetric isothermal multiple self-matching initiated amplification (IMSA) for rapid detection of the Zaire subtype of the Ebola virus (EBOV-Z). This method employed six primers that recognized seven sites of the EBOV-Z nucleoprotein gene for amplification of nucleic acids under isothermal conditions at 63 degrees C for 1 h. Amplification products were detected through visual inspection of color change by pre-addition of hydroxyl naphthol blue dye. Relative sensitivity was validated by detection of serial tenfold dilutions of virus-like particles containing the partial EBOV-Z nucleoprotein gene and mock clinical sample. Specificity of IMSA was validated by detection of the plasma of 30 healthy volunteers, the dengue virus, and Japanese encephalitis virus. IMSA had comparable sensitivity to Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and cross-reaction with human plasma or other viruses was not observed. Reverse transcription-isothermal multiple self-matching initiated amplification (RT-IMSA) was also evaluated and compared in parallel with the commercial RT-qPCR kit for detection of EBOV-suspected samples of human blood in Sierra Leone. Sensitivity and specificity of the RT-IMSA was 91.4% and 100%, respectively. These data suggest that RT-IMSA is a valuable tool for the detection of the EBOV with the distinct advantages of simplicity and low cost compared with RT-qPCR.
Colorimetry ; methods ; DNA Primers ; genetics ; Ebolavirus ; genetics ; isolation & purification ; Hemorrhagic Fever, Ebola ; diagnosis ; virology ; Humans ; Nucleic Acid Amplification Techniques ; methods

Ebolavirus ; pathogenicity ; Endemic Diseases ; Hemorrhagic Fever, Ebola ; epidemiology ; virology ; Humans

The Ebola virus belongs to the Filovirus family, which causes Ebola hemorrhagic fever (mortality, 25%-90%). An outbreak of infection by the Ebola virus is sweeping across West Africa, leading to high mortality and worldwide panic. The Ebola virus has caused a serious threat to public health, so intensive scientific studies have been carried out. Several vaccines (e.g., rVSV-ZEBOV, ChAd3-ZEBOV) have been put into clinical trials and antiviral drugs (e.g., TKM-Ebola, ZMAPP) have been administered in the emergency setting to patients infected by the Ebola virus. Here, recent advances in vaccines and drugs against the Ebola virus are reviewed.
Animals ; Antiviral Agents ; administration & dosage ; Ebola Vaccines ; administration & dosage ; genetics ; immunology ; Ebolavirus ; drug effects ; genetics ; immunology ; physiology ; Hemorrhagic Fever, Ebola ; drug therapy ; prevention & control ; virology ; Humans

We aimed to develop a real-time polymerase chain reaction (PCR) detection method for the Reston subtype of the Ebola virus. The NP gene of the Reston subtype of the Ebola virus was selected as the detection object. Sequences of different subtypes of Ebola viruses were aligned using Clustal W software. The most unique and conserved regions of the Reston subtype of the Ebola virus were recruited as candidate sequences for specific primers. Primer Express and Primer Premier 5. 0 software were used to filter the optimal pair of primers for detection. Real-time PCR was carried out using optimized parameters and positive DNA prepared by serial (tenfold) dilution of a recombinant plasmid and by plotting a standard curve. In addition, the reproducibility, accuracy, and specificity of the assay were tested. Results showed that the sensitivity of detection of the Reston subtype of the Ebola virus by real-time PCR could reached 10(2) copies/microL. The linear relationship (R2) reached 0.997, the slope of the standard curve was -0.3101, and amplification efficiency was 110.145%. A sharp and narrow melting peak appeared at 79.94 degrees C for all standards in different dilutions. In conclusion, a fast and sensitive real-time PCR detection system for the Reston subtype of the Ebola virus was developed. This system could be used as a supplementary diagnostic and monitoring approach for basic and clinical studies on the Reston subtype of the Ebola virus. The detection system does not require expensive technology or specialist operators.
DNA Primers ; genetics ; Ebolavirus ; classification ; genetics ; isolation & purification ; Hemorrhagic Fever, Ebola ; virology ; Humans ; Real-Time Polymerase Chain Reaction ; methods ; Sensitivity and Specificity

No abstract available.
Antiviral Agents/therapeutic use ; Hemorrhagic Fever, Ebola/drug therapy/*prevention & control/virology ; Humans ; Protective Devices ; Vaccines/immunology