扎伊尔型埃博拉病毒的实时荧光RT-PCR检测方法研究

目的 建立扎伊尔型埃博拉病毒的核酸检测方法,以期用于埃博拉出血热临床标本的检测.方法 针对扎伊尔型埃博拉病毒核蛋白和糖蛋白基因设计引物和探针,建立单重和双重实时荧光RT-PCR检测方法,利用体外转录病毒RNA和埃博拉病毒系列参考品RNA评价其敏感性,利用马尔堡病毒、健康人、登革热患者和发热伴血小板减少综合征患者血清评价其特异性.结果 所建立的实时荧光RT-PCR检测方法扩增效率在95％～105％,可特异性地检测扎伊尔型埃博拉病毒核蛋白和糖蛋白基因,与马尔堡病毒、登革热和发热伴血小板减少综合征病毒均无交叉反应,体外转录的病毒RNA可检出10～100拷贝/μl.双重检测方法通过细胞培养的扎伊尔型埃博拉病毒RNA验证,可检出100 pfu/ml病毒.结论 本研究建立的检测扎伊尔型埃博拉病毒的实时荧光RT-PCR方法具有良好的特异性和敏感性,可用于埃博拉出血热临床标本的检测.     

Monoclonal antibodies to Ebola virus: isolation, characteristics, and study of cross reactivity with Marburg virus

Thirteen hybridoma strains producing monoclonal antibodies (Mabs) to Ebola virus were prepared by fusion of NS-O mouse myeloma cells with splenocytes of BALB/c mice immunized with purified and inactivated Ebola virus (Mayinga strain). Mabs directed against viral proteins were selected and tested by ELISA. Protein specificity of 13 Mabs was determined by immunoblotting with SDS-PAGE proteins of Ebola virus. Of these, 11 hybridoma Mabs reacted with 116 kDa protein (NP) and 2 with Ebola virus VP35. Antigenic cross-reactivity between Ebola and Marburg viruses was examined in ELISA and immunoblotting with polyclonal and monoclonal antibodies. In ELISA, polyclonal antibodies of immune sera to Ebola or Marburg viruses reacted with heterologous filoviruses, and two anti-NP Ebola antibodies (Mabs 7E1 and 6G8) cross-reacted with both viruses. Target proteins for cross-reactivity, Ebola NP (116 kDa) and Marburg NP (96 kDa), and VP35 of both filoviruses were detected by immunoblotting with polyclonal and monoclonal antibodies (6G8) to Ebola virus.     

Complete genome sequence of an Ebola virus (Sudan species) responsible for a 2000 outbreak of human disease in Uganda

The entire genomic RNA of the Gulu (Uganda 2000) strain of Ebola virus was sequenced and compared to the genomes of other filoviruses. This data represents the first comprehensive genetic analysis for a representative isolate of the Sudan species of Ebola virus. The genome organization of the Sudan species is nearly identical to that of the Zaire species, but the presence of a gene overlap (between GP and VP30 genes) and a longer trailer sequence distinguish it from that of the Reston species. As has been observed with other filoviruses, stemloop structures were predicted to form at the 5' end of Ebola Sudan mRNA molecules, and the genomic RNA termini showed a high degree of sequence complimentarity. Comparisons of the amino acid sequences of encoded gene products shows that there is a comparable level of identity or similarity between Ebola virus species, with Sudan and Zaire actually showing a slightly closer relationship to the Reston species than to one another. These comparisons also indicated that the VP24 is the most conserved Ebola virus protein (followed closely by the VP40 and L proteins), while the GP is the least conserved gene product. The most divergent regions were seen in the C-terminus of GP1 (mucin-like region) and within the C-terminal third of the nucleoprotein sequence.     

Detection of all known filovirus species by reverse transcription-polymerase chain reaction using a primer set specific for the viral nucleoprotein gene

The filoviruses, Marburg virus (MARV) and Ebola virus (EBOV), are causative agents of severe hemorrhagic fever with high mortality rates in humans and non-human primates. Sporadic outbreaks of filovirus infection have occurred in Central Africa and parts of Asia. Identification of the natural reservoir animals that are unknown yet and epidemiological investigations are current challenges to forestall outbreaks of filovirus diseases. The filovirus species identified currently include one in the MARV group and five in the EBOV group, with large genetic variations found among the species. Therefore, it has been difficult to develop a single sensitive assay to detect all filovirus species, which would advance laboratory diagnosis greatly in endemic areas. In this study, a highly sensitive universal RT-PCR assay targeting the nucleoprotein (NP) gene of filoviruses was developed. The genomic RNAs of all known MARV and EBOV species were detected by using an NP-specific primer set. In addition, this RT-PCR procedure was verified further for its application to detect viral RNAs in tissue samples of animals infected experimentally and blood specimens of infected patients. This assay will be a useful method for diagnostics and epidemiological studies of filovirus infections.     

Comparison of the protective efficacy of DNA and baculovirus-derived protein vaccines for EBOLA virus in guinea pigs

The filoviruses Ebola virus (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever in humans for which no vaccines are available. Previously, a priming dose of a DNA vaccine expressing the glycoprotein (GP) gene of MARV followed by boosting with recombinant baculovirus-derived GP protein was found to confer protective immunity to guinea pigs (Hevey et al., 2001. Vaccine 20, 568-593). To determine whether a similar prime-boost vaccine approach would be effective for EBOV, we generated and characterized recombinant baculoviruses expressing full-length EBOV GP (GP(1,2)) or a terminally-deleted GP (GPa-) and examined their immunogenicity in guinea pigs. As expected, cells infected with the GPa- recombinant secreted more GP(1) than those infected with the GP(1,2) recombinant. In lectin binding studies, the insect cell culture-derived GPs were found to differ from mammalian cell derived virion GP, in that they had no complex/hybrid N-linked glycans or glycans containing sialic acid. Despite these differences, the baculovirus-derived GPs were able to bind monoclonal antibodies to five distinct epitopes on EBOV GP, indicating that the antigenic structures of the proteins remain intact. As a measure of the ability of the baculovirus-derived proteins to elicit cell-mediated immune responses, we evaluated the T-cell stimulatory capacity of the GPa- protein in cultured human dendritic cells. Increases in cytotoxicity as compared to controls suggest that the baculovirus proteins have the capacity to evoke cell-mediated immune responses. Guinea pigs vaccinated with the baculovirus-derived GPs alone, or in a DNA prime-baculovirus protein boost regimen developed antibody responses as measured by ELISA and plaque reduction neutralization assays; however, incomplete protection was achieved when the proteins were given alone or in combination with DNA vaccines. These data indicate that a vaccine approach that was effective for MARV is not effective for EBOV in guinea pigs.     

Cathepsins B and L activate Ebola but not Marburg virus glycoproteins for efficient entry into cell lines and macrophages independent of TMPRSS2 expression

Ebola (EBOV) and Marburg virus (MARV) cause severe hemorrhagic fever. The host cell proteases cathepsin B and L activate the Zaire ebolavirus glycoprotein (GP) for cellular entry and constitute potential targets for antiviral intervention. However, it is unclear if different EBOV species and MARV equally depend on cathepsin B/L activity for infection of cell lines and macrophages, important viral target cells. Here, we show that cathepsin B/L inhibitors markedly reduce 293T cell infection driven by the GPs of all EBOV species, independent of the type II transmembrane serine protease TMPRSS2, which cleaved but failed to activate EBOV-GPs. Similarly, a cathepsin B/L inhibitor blocked macrophage infection mediated by different EBOV-GPs. In contrast, MARV-GP-driven entry exhibited little dependence on cathepsin B/L activity. Still, MARV-GP-mediated entry was efficiently blocked by leupeptin. These results suggest that cathepsins B/L promote entry of EBOV while MARV might employ so far unidentified proteases for GP activation.     

Ebola and Marburg virus-like particles activate human myeloid dendritic cells

The filoviruses, Ebola (EBOV) and Marburg (MARV), are potential global health threats, which cause deadly hemorrhagic fevers. Although both EBOV and MARV logarithmically replicate in dendritic cells (DCs), these viruses do not elicit DC cytokine secretion and fail to activate and mature infected DCs. Here, we employed virus-like particles (VLPs) of EBOV and MARV to investigate whether these genome-free particles maintain similar immune evasive properties as authentic filoviruses. Confocal microscopy indicated that human myeloid-derived DCs readily took up VLPs. However, unlike EBOV and MARV, VLPs induced maturation of DCs including upregulation of costimulatory molecules (CD40, CD80, CD86), major histocompatibility complex (MHC) class I and II surface antigens, and the late DC maturation marker CD83. The chemokine receptors CCR5 and CCR7 were also modulated on VLP-stimulated DCs, indicating that DC could migrate following VLP exposure. Furthermore, VLPs also elicited DC secretion of the pro-inflammatory cytokines TNF-alpha, IL-8, IL-6, and MIP-1alpha. Most significantly, in stark contrast to DC treated with intact EBOV or MARV, DC stimulated with EBOV or MARV VLPs showed enhanced ability to support human T-cell proliferation in an allogenic mixed lymphocyte response (MLR). Thus, our findings suggest that unlike EBOV and MARV, VLPs are effective stimulators of DCs and have potential in enhancing innate and adaptive immune responses.     

Human recombinant antibodies to Ebola virus: preparation and characteristics

Human recombinant antibodies against a purified Ebola virus (EV) lysate were selected from a combinatorial library of scFv-antibodies using the phage display technique. Nine unique antibodies were identified after sequencing the Vh- and Vl-genes encoding the selected antibodies. Solid-phase enzyme immunoassay (EIA) indicated that these antibodies were able to bind both inactivated and native EV. Immunoblotting showed that 6 antibodies identified nucleoprotein (NP), one antibody did VP24 and another antibody did VP40. One of the selected antibodies reacted with two EP proteins: VP24 and VP40. Solid-phase EIA demonstrated cross-reactivity with Marburg virus (MAR) and defined VP24 MAR as a target protein for the antibody.     

Complex adenovirus-vectored vaccine protects guinea pigs from three strains of Marburg virus challenges

The Marburg virus (MARV), an African filovirus closely related to the Ebola virus, causes a deadly hemorrhagic fever in humans, with up to 90% mortality. Currently, treatment of disease is only supportive, and no vaccines are available to prevent spread of MARV infections. In order to address this need, we have developed and characterized a novel recombinant vaccine that utilizes a single complex adenovirus-vectored vaccine (cAdVax) to overexpress a MARV glycoprotein (GP) fusion protein derived from the Musoke and Ci67 strains of MARV. Vaccination with the cAdVaxM(fus) vaccine led to efficient production of MARV-specific antibodies in both mice and guinea pigs. Significantly, guinea pigs vaccinated with at least 5 x 10(7) pfu of cAdVaxM(fus) vaccine were 100% protected against lethal challenges by the Musoke, Ci67 and Ravn strains of MARV, making it a vaccine with trivalent protective efficacy. Therefore, the cAdVaxM(fus) vaccine serves as a promising vaccine candidate to prevent and contain multi-strain infections by MARV.     

Chimpanzee adenovirus vaccine protects against Zaire Ebola virus

This study evaluated the use of a chimpanzee-based adenovirus vaccine in mouse and Guinea pigs models of Zaire Ebola virus (ZEBOV) infection. Vaccine vector expressing the envelope glycoprotein of ZEBOV was created from the molecular clone of chimpanzee adenovirus pan7 (AdC7). AdC7 vaccine stimulated robust T and B cell responses to ZEBOV in na?ve mice inducing complete protection to an otherwise lethal challenge of ZEBOV. Complete protection to Zaire Ebola virus was also observed in Guinea pigs vaccinated with a relatively low dose of AdC7 (5 x 10(9)/kg). Pre-existing immunity to AdHu5 was generated in mice following pre-exposure to AdHu5 or administration of pooled human immune globulin. Pre-existing immunity to human adenoviruses severely compromised the efficacy of the human AdHu5 vaccine but not the chimpanzee AdC7 vaccine. These results validate further development of Chimpanzee-based vaccine and highlight the impact of pre-existing immunity to the vaccine carrier.     

The properties of Ebola virus proteins

The paper describes the structure and functions of Ebola virus properties. It also presents information on the role of structural (NP, VP40, VP35, GP, VP30, VP24, and L) and secreted (sGP, delta-peptide, GP1, GP(1,2delta), ssGP) proteins in the viral replication cycle and in the pathogenesis of Ebola hemorrhagic fever.     

Characterization of Marburg virus glycoprotein in viral entry

One major determinant of host tropism for filoviruses is viral glycoprotein (GP), which is involved in receptor binding and viral entry. Compared to Ebola GP (EGP), Marburg GP (MGP) is less well characterized in viral entry. In this study, using a human immunodeficiency virus-based pseudotyped virus as a surrogate system, we have characterized the role of MGP in viral entry. We have shown that like EGP, the mucin-like region of MGP (289-501) is not essential for virus entry. We have developed a viral entry interference assay for filoviruses, and using this assay, we have demonstrated that transfection of EGP or MGP in target cells can interfere with EGP/HIV and MGP/HIV pseudotyped virus entry in a dose-dependent manner. These results are consistent with the notion that Ebola and Marburg viruses use the same or a related host molecule(s) for viral entry. Substitutions of the non-conserved residues in MGP1 did not impair MGP-mediated viral entry. Unlike that of EGP1, individual substitutions of many conserved residues of MGP1 exerted severe defects in MGP expression, incorporation to HIV virions, and thus its ability to mediate viral entry. These results indicate that MGP is more sensitive to substitutions of the conserved residues, suggesting that MGP may fold differently from EGP.     

Filovirus budding

Family Filoviridae, which includes Ebola virus (EBOV) and Marburg virus (MARV), is a growing threat to human and non-human primate populations in central Africa. Although many facets of the filovirus life cycle remain to be deciphered, a great deal has been learned in recent years. In particular, a clearer understanding of the roles played by viral, as well as cellular, proteins in the assembly and budding processes has been achieved. This review will discuss the current state of filovirus budding research, with especial emphasis placed on the viral matrix protein VP40 and its relationship with the cellular vesicular sorting pathway. Possible budding functions of the viral glycoprotein (GP), as well as the membrane-associated viral protein 24 (VP24), will also be described, and a model for filovirus budding will be proposed.     

Protection of Nonhuman Primates against Two Species of Ebola Virus Infection with a Single Complex Adenovirus Vector

Ebola viruses are highly pathogenic viruses that cause outbreaks of hemorrhagic fever in humans and other primates. To meet the need for a vaccine against the several types of Ebola viruses that cause human diseases, we developed a multivalent vaccine candidate (EBO7) that expresses the glycoproteins of Zaire ebolavirus (ZEBOV) and Sudan ebolavirus (SEBOV) in a single complex adenovirus-based vector (CAdVax). We evaluated our vaccine in nonhuman primates against the parenteral and aerosol routes of lethal challenge. EBO7 vaccine provided protection against both Ebola viruses by either route of infection. Significantly, protection against SEBOV given as an aerosol challenge, which has not previously been shown, could be achieved with a boosting vaccination. These results demonstrate the feasibility of creating a robust, multivalent Ebola virus vaccine that would be effective in the event of a natural virus outbreak or biological threat.The filoviruses, Ebola virus (EBOV) and Marburg virus (MARV), cause outbreaks of severe hemorrhagic fever disease in humans, with case-fatality rates that range up to 90%. Among the Ebolavirus genus, there are four distinct species: Zaire ebolavirus (ZEBOV), Sudan ebolavirus (SEBOV), Reston ebolavirus (REBOV), and Cote d''Ivoire ebolavirus (CIEBOV) (10), with a possible fifth species identified in a recent outbreak in the Bundibugyo region of Western Uganda (34). Of these, ZEBOV and SEBOV are known to cause lethal disease in humans. The persistence of these viruses in nature is not well understood. Sporadic outbreaks due to EBOV have been occurring in Central Africa since the 1970s, but since the mid-1990s, the incidence of outbreaks has increased more than 4-fold (6, 7, 8, 42-45), and EBOV has spread aggressively throughout the great ape sanctuaries of West and Central Africa, decimating wild populations of gorillas and chimpanzees (2). While the filoviruses infect both humans and great apes, due to the high mortality rates of the infection, neither is thought to serve as reservoirs for these viruses but only as accidental hosts (16). Recent findings suggest that African fruit bats may serve as a reservoir host for filoviruses (2, 22); however, little is known about the nature of transmission to humans and nonhuman primates from bats or the likelihood of other reservoir species. In outbreak situations, filoviruses are believed to transmit from person to person mainly through contact with bodily fluids from infected patients. However, recent studies of Ebola outbreaks in wild apes have suggested that there could be other modes of transmission, including aerosol (2, 36). Studies in nonhuman primates have shown that EBOV and MARV can be spread through aerosolized droplets under controlled laboratory conditions (18, 21). So, despite the low incidence of infections globally, the lethality and potential airborne transmission of filoviruses in heavily populated areas makes them a significant biological threat, resulting in their placement on the Centers for Disease Control and Prevention list of Category A Bioterrorism Agents and the Department of Health and Human Services (DHHS) list of select agents and toxins. Concern is further compounded by the potential for these agents to be obtained from the wild (2, 22). In a biological threat scenario, aerosol transmission will likely be the main mode of viral dissemination, and protection against aerosolized EBOV would be of utmost importance (3). However, most previous vaccine candidates have only been evaluated for efficacy against intramuscular or intraperitoneal challenge and not against an aerosol challenge in nonhuman primates.At present, there are no licensed vaccines or specific antiviral treatments available for EBOV or MARV infections. However, significant progress has been made over the past few years in developing vaccine candidates that can protect nonhuman primates (NHPs) from lethal EBOV and MARV challenges (11, 17, 20, 23, 32, 40, 41). Most of the candidates utilize recombinant vaccine approaches that direct the protective immune response toward the surface glycoprotein (GP) of a single species of EBOV. Importantly, each species of EBOV is antigenically distinct, based on the sequences of the viral GP (10), and therefore, vaccines targeted against the GP of one species of the virus will not provide cross-protection against infection by another (19). Unique among the vaccine candidates is the recombinant complex adenovirus vaccine (CAdVax) system, which provides multivalent protection of NHPs against multiple species of filoviruses (33). The CAdVax vaccine platform is based on a complex, replication-defective adenovirus 5 (Ad5) vector (28-30, 37, 38) that allows for the incorporation of multiple gene inserts into the vector''s genome. Using this design, a bivalent vaccine vector (EBO7) was developed that expresses modified GP gene sequences of SEBOV and ZEBOV. When included in a novel pan-filovirus vaccine formulation, this vaccine was 100% protective in NHPs against two species of EBOV (ZEBOV and SEBOV) and two different strains of MARV (Musoke and Ci67) (33).In the study presented here, we further tested the protective efficacy of the CAdVax-based EBO7 vaccine in macaques by comparing aerosol to parenteral challenge. Aerosol challenge is potentially even more lethal than parenteral infection, because it induces hemorrhagic pneumonia. This is particularly true of SEBOV aerosol challenge, against which protection has not previously been demonstrated. In our studies, we have found that for either route of infection, the vaccine-induced bivalent anti-EBOV responses were protective against lethal challenge with either SEBOV or ZEBOV. This is the first report of a vaccine that is capable of protecting against aerosol SEBOV challenge. In addition, we found that EBO7 was also capable of protecting macaques with preexisting immunity to adenovirus against ZEBOV challenge. These results provide further insight into the feasibility of developing a fully protective multivalent EBOV vaccine using the CAdVax vaccine platform.     

Identification of two amino acid residues on Ebola virus glycoprotein 1 critical for cell entry

Using site-directed mutagenesis and retroviral vector pseudotyping of the wild type or mutated glycoprotein of Zaire ebolavirus (ZEBOV), we analyzed 15 conserved residues in the N-terminus of the filovirus glycoprotein 1 (GP1) in order to identify residues critical for cell entry. Results from infectivity assays and Western blot analyses identified two phenylalanine residues at positions 88 and 159 that appear to be critical for ZEBOV entry in vitro. We extended this observation by introduction of alanines at either position 88 or 159 of Ivory Coast Ebolavirus (CIEBOV) and observed the same phenotype. Further, we showed that introduction of each of the two mutations in a recombinant full-length clone of ZEBOV (Mayinga strain) that also carried the coding sequence for GFP could not be rescued, suggesting the mutants rendered the virus non-infectious. The two phenylalanines that are critical for both ZEBOV and CIEBOV entry are found in two linear domains of GP1 that are highly conserved among filoviruses, and thus could provide a target for rational development of broadly cross-protective vaccines or antiviral therapies.     

Profiling the Native Specific Human Humoral Immune Response to Sudan Ebola Virus Strain Gulu by Chemiluminescence Enzyme-Linked Immunosorbent Assay

Ebolavirus, a member of the family Filoviridae, causes high lethality in humans and nonhuman primates. Research focused on protection and therapy for Ebola virus infection has investigated the potential role of antibodies. Recent evidence suggests that antibodies can be effective in protection from lethal challenge with Ebola virus in nonhuman primates. However, despite these encouraging results, studies have not yet determined the optimal antibodies and composition of an antibody cocktail, if required, which might serve as a highly effective and efficient prophylactic. To better understand optimal antibodies and their targets, which might be important for protection from Ebola virus infection, we sought to determine the profile of viral protein-specific antibodies generated during a natural cycle of infection in humans. To this end, we characterized the profile of antibodies against individual viral proteins of Sudan Ebola virus (Gulu) in human survivors and nonsurvivors of the outbreak in Gulu, Uganda, in 2000-2001. We developed a unique chemiluminescence enzyme-linked immunosorbent assay (ELISA) for this purpose based on the full-length recombinant viral proteins NP, VP30, and VP40 and two recombinant forms of the viral glycoprotein (GP_1-294 and GP_1-649) of Sudan Ebola virus (Gulu). Screening results revealed that the greatest immunoreactivity was directed to the viral proteins NP and GP_1-649, followed by VP40. Comparison of positive immunoreactivity between the viral proteins NP, GP_1-649, and VP40 demonstrated a high correlation of immunoreactivity between these viral proteins, which is also linked with survival. Overall, our studies of the profile of immunorecognition of antibodies against four viral proteins of Sudan Ebola virus in human survivors may facilitate development of effective monoclonal antibody cocktails in the future.     

Descriptive analysis of Ebola virus proteins

The virion proteins of two strains of Ebola virus were compared by SDS-polyacrylamide gel electrophoresis (PAGE) and radioimmunoprecipitation (RIP). Seven virion proteins were described; an L (180K), GP (125K), NP (104K), VP40 (40K), VP35 (35K), VP30 (30K), and VP24 (24K). The RNP complex of the virus contained the L, the NP, and VP30, with VP35 in loose association with them. The GP was the major spike protein, with VP40 and VP24 making up the remaining protein content of the multilayered envelope.     

Productive replication of Ebola virus is regulated by the c-Abl1 tyrosine kinase

Ebola virus causes a fulminant infection in humans resulting in diffuse bleeding, vascular instability, hypotensive shock, and often death. Because of its high mortality and ease of transmission from human to human, Ebola virus remains a biological threat for which effective preventive and therapeutic interventions are needed. An understanding of the mechanisms of Ebola virus pathogenesis is critical for developing antiviral therapeutics. Here, we report that productive replication of Ebola virus is modulated by the c-Abl1 tyrosine kinase. Release of Ebola virus-like particles (VLPs) in a cell culture cotransfection system was inhibited by c-Abl1-specific small interfering RNA (siRNA) or by Abl-specific kinase inhibitors and required tyrosine phosphorylation of the Ebola matrix protein VP40. Expression of c-Abl1 stimulated an increase in phosphorylation of tyrosine 13 (Y(13)) of VP40, and mutation of Y(13) to alanine decreased the release of Ebola VLPs. Productive replication of the highly pathogenic Ebola virus Zaire strain was inhibited by c-Abl1-specific siRNAs or by the Abl-family inhibitor nilotinib by up to four orders of magnitude. These data indicate that c-Abl1 regulates budding or release of filoviruses through a mechanism involving phosphorylation of VP40. This step of the virus life cycle therefore may represent a target for antiviral therapy.