Skip Navigation

In Vitro and In Vivo Characterization of Recombinant Ebola Viruses Expressing Enhanced Green Fluorescent Protein

  1. Hideki Ebihara1,2,3,4,a,
  2. Steven Theriault4,5,a,
  3. Gabriele Neumann7,
  4. Judie B. Alimonti4,
  5. Joan B. Geisbert8,
  6. Lisa E. Hensley8,
  7. Allison Groseth4,5,
  8. Steven M. Jones6,
  9. Thomas W. Geisbert9,b,
  10. Yoshihiro Kawaoka1,2,3,7 and
  11. Heinz Feldmann4,5
  1. 1 Department of Special Pathogens, International Research Center for Infectious Diseases, University of Tokyo, Tokyo
  2. 2 Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo
  3. 3 Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Saitama, Japan
  4. 4 Special Pathogens Program, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba, Canada
  5. 5 Departments of Medical Microbiology, University of Manitoba, Winnipeg, Manitoba, Canada
  6. 6 Departments of Immunology, University of Manitoba, Winnipeg, Manitoba, Canada
  7. 7 Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison
  8. 8 US Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland
  9. 9 Integrated Research Facility at Fort Detrick, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Fort Detrick, Maryland
  1. Reprints or correspondence: Dr. Heinz Feldmann, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E 3R2 (Heinz_Feldmann{at}phacaspc.gc.ca); or Dr. Hideki Ebihara, Public Health Agency of Canada, 1015 Arlington St., Winnipeg, Manitoba, Canada R3E 3R2 (Hideki_Ebihara{at}phac-aspc.gc.ca).

  • ↵a H.E. and S.T. contributed equally to this work.

  • ↵b All work was completed while T.W.G. was employed at the US Army Medical Research Institute of Infectious Diseases.

 
Next Section

Abstract

To facilitate an understanding of the molecular aspects of the pathogenesis of Zaire ebolavirus (ZEBOV) infection, we generated 2 different recombinant viruses expressing enhanced green fluorescent protein (eGFP) from additional transcription units inserted at different positions in the virus genome. These viruses showed in vitro phenotypes similar to that of wild-type ZEBOV (wt-ZEBOV) and were stable over multiple passages. Infection with one of the viruses expressing eGFP produced only mild disease in rhesus macaques, demonstrating a marked attenuation in this animal model. However, in mice lacking signal transducer and activator of transcription 1, both viruses expressing eGFP caused lethal cases of disease that were moderately attenuated, compared with that caused by wt-ZEBOV. In mice, viral replication could be easily tracked by the detection of eGFP-positive cells in tissues, by use of flow cytometry. These findings demonstrate that the incorporation of a foreign gene will attenuate ZEBOV in vivo but that these viruses still have potential for in vitro and in vivo research applications.

Over the past decade, reverse-genetics systems have been developed for many of the negative-stranded RNA viruses [1]. This technology has enabled the generation of artificial replication systems and recombinant mutant viruses that can be used to study different aspects of virus biology. In particular, the de novo synthesis of RNA viruses from cloned cDNA has provided researchers with powerful tools to investigate the viral life cycle, determine protein function, and study viral pathogenesis through genetic manipulation of virus genomes, using molecular cloning techniques. In particular, recombinant viruses expressing reporter proteins have been instrumental in tracking viral infection and spread in vivo and in the development of novel antiviral drug—screening systems [2–4]. Furthermore, reverse-genetics systems have been successfully used to develop replication-deficient and live-attenuated vaccine candidates that express foreign antigens [1, 5–7].

More recently, reverse-genetics systems have been developed for members of the genera Ebolavirus and Marburgvirus in the family Filoviridae [4, 8–12]. As has been done with other negative-sense single-stranded RNA viruses, the infectious-clone system for the species Zaire ebolavirus (ZEBOV) has provided valuable information regarding viral protein function and the molecular basis of ZEBOV virulence, through in vivo examination of the phenotypes of genetically engineered virus mutants [4, 9, 12–16]. These studies included the generation of recombinant ZEBOV expressing enhanced green fluorescent protein (eGFP) and its in vitro characterization [4]. In contrast to several other members of the order Mononegavirales, eGFP-expressing ZEBOV (eGFP-ZEBOV) has not yet been examined for its potential in the study of pathogenesis, viral tropism, and in vivo screening of antiviral drugs. To evaluate and further characterize its in vitro and in vivo applications, we generated 2 variants of eGFP-ZEBOV from full-length ZEBOV cDNA clones possessing an individual transcription unit located close to either the 3′ end of the genome, between the nucleoprotein (NP) and the VP35 genes, or the 5′ end of the genome, between the VP30 and VP24 genes, and we examined their growth in cell culture and their virulence in 2 animal models, type I interferon (IFN)-deficient mice and rhesus macaques.

Previous SectionNext Section

Materials and Methods

Cells and viruses. Vero E6 (monkey kidney), 293T (human embryonic kidney), and U937 (human monocytic) cells were grown and maintained at 37°C in Dulbecco's MEM (DMEM) or RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum, 2 mmol/L l-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Primary human macrophages derived from blood samples from healthy donors were isolated by use of Ficoll gradient (Amersham) and were incubated at 37°C in RPMI 1640 supplemented with 5% human type AB serum (Sigma). The Mayinga strain of recombinant wild-type ZEBOV (wt-ZEBOV) was grown and passaged twice in Vero E6 cells [9]. Virus infectivity titers (in focus-forming units) were obtained by counting the number of infected cell foci, by use of an indirect IFA using rabbit polyclonal anti-ZEBOV/VP40 or murine monoclonal anti—ZEBOV/glycoprotein (GP) antibodies for surface staining under nonpermeabilized conditions and a goat anti-rabbit IgG-fluorescein isothiocyanate conjugate or a goat anti-mouse IgG-Cy3 conjugate, as described elsewhere [17, 18]. All work with infectious ZEBOV was performed in the biosafety level (BSL) 4 laboratories at the National Microbiology Laboratory of the Public Health Agency of Canada in Winnipeg, Manitoba (tissue culture and mouse experiments), and the US Army Medical Research Institute for Infectious Diseases at Fort Detrick, MD (nonhuman primates).

Generation of infectious cDNA clones of ZEBOV that contain an eGFP gene. Two infectious cDNA clones of ZEBOV that express the eGFP reporter protein from an additional transcription unit as an eighth gene were constructed by use of conventional molecular cloning techniques. To create the first eGFP-ZEBOV clone, designated “NP/35-eGFP,” the eGFP open reading frame (ORF) was engineered with flanking ZEBOV NP transcription start and termination signals and was inserted between the NP and VP35 genes by use of newly created restriction-enzyme sites within the NP/VP35 intergenic region (IGR; figure 1A). The second eGFP-ZEBOV clone, designated “VP30/24-eGFP,” was constructed by insertion of the eGFP ORF between the VP30 and VP24 genes as an additional transcription unit composed of the 5′ noncoding region (NCR) of the VP24 gene, the eGFP ORF, the 3′ NCR of the VP24 gene, and the VP30/VP24 IGR (figure 1A). The eGFP expression cassette of VP30/24-eGFP possesses a copy of the VP30/VP24 IGR, to mimic the mechanism of VP24 gene transcription (figure 1A). Both full-length clones were assembled as plasmids containing the full-length ZEBOV genome flanked by the T7 promoter sequence and the hepatitis D virus (HDV) ribozyme- T7 terminator sequence [9]. By use of the resulting full-length clones, the recombinant NP/35-eGFP and VP30/24-eGFP ZEBOV variants were rescued in accordance with established protocols [9, 15]. Cytopathic effect (CPE) and eGFP expression were monitored to confirm the recovery of recombinant viruses. To prepare working stocks, all viruses were amplified twice in Vero E6 cells.

Figure 1
View larger version:
Figure 1

Generation of 2 Zaire ebolavirus (ZEBOV) variants expressing enhanced green fluorescent protein (eGFP), with different eGFP coding strategies. A, Schematic diagram depicting strategies for insertion of the eGFP transcription unit into the full-length genome of a wild-type ZEBOV clone. B, Detection of eGFP and viral glycoprotein (GP) expression in Vero E6 cells infected with the NP/35-eGFP variant on day 5 after infection. Left, eGFP fluorescence; middle, immunostaining using an anti-ZEBOV/GP monoclonal antibody; right, merged images. IGR, intergenic region; Le, leader; NP, nucleoprotein; sGP, soluble glycoprotein; Tr, trailer; tss, transcription start signal; tts, transcription termination signal.

Infection of Vero E6, 293T, and U937 cells and primary human macrophages with recombinant eGFP-ZEBOV variants. Confluent Vero E6 and 293T cells and primary human macrophages were prepared in 6-well plates and were infected with eGFP-ZEBOV at an MOI of 0.05 (2.4×105 ffu). After 1 h at 37°C, the inoculum was removed, and the cells were washed with DMEM or RPMI 1640. Infected cells were incubated for 4 days and then harvested into 4% paraformaldehyde (PFA) for fixation before analysis for eGFP expression using an LSRII flow cytometer with FACSDiva software (BD Biosciences). To examine the growth kinetics of the viruses, Vero E6 and U937 cells were infected at an MOI of 0.05 for 1 h at 37°C, followed by a washing step with DMEM. Supernatants were collected on days 1–3 and 5 after infection, and virus titration was done on Vero E6 cells by use of an assay to measure focus-forming units, as described above.

Experiments with mice. Female knockout mice lacking signal transducer and activator of transcription 1 (STAT1−/−; 5–6 weeks old) were obtained from a commercial supplier (Charles River Laboratories). All mice were housed in groups in microisolater cages and were allowed to acclimate for a minimum of 5 days prior to use in experiments. To assay the virulence of the eGFP-ZEBOV variants, groups of 3–6 mice were injected intraperitoneally, at 2 different sites (100 µL per site), with virus dilutions ranging from 104 to 2×10−1 ffu/mL. After infection, mice were scored for clinical signs, and weight loss was recorded for a minimum of 11 days after infection. For all surviving animals, observation was continued until day 21 after infection. To assess disease progression based on eGFP expression, groups of 9 mice were infected intraperitoneally with 1000 ffu of either NP/35-eGFP or VP30/24-eGFP. Spleen and liver samples were collected from 1 uninfected and 4 infected mice on days 3 and 5 after infection. The collected organ samples were homogenized and fixed in 4% PFA. The tissue samples were analyzed on a LSRII flow cytometer with FACSDiva software. All mouse experiments were performed according to “Animal Use Documents H01-001 and H06-015,” approved by the Animal Care Committee, Canadian Science Centre for Human and Animal Health, Winnipeg, and according to the guidelines of the Canadian Council on Animal Care, Ottawa, and were performed in the BSL4 facility at the National Microbiology Laboratory, Winnipeg.

Experiments with nonhuman primates. Two filovirus-seronegative rhesus macaques (Macaca mulatta) weighing 4–5 kg were inoculated in the caudal thigh with 1000 pfu of VP30/24-eGFP. Analysis of hematology and serum biochemistry was done as described elsewhere [19]. Total white and red blood cell counts, platelet counts, hematocrit values, total hemoglobin, mean cell volume, mean corpuscular volume, and mean corpuscular hemoglobin concentration were determined from blood samples, collected in EDTA tubes, by use of a hematologic analyzer (Coulter Electronics). Titers of infectious VP30/24-eGFP from all blood samples were determined by plaque assay on Vero E6 cells. Both animals were rechallenged with 1000 pfu of the Kikwit strain of wt-ZEBOV at 28 days after primary infection, and follow-up was as described above. Research was conducted in compliance with the Animal Welfare Act and other federal statues and regulations relating to animals and experiments involving animals and adhered to the principles stated in the Guide for the Care and Use of Laboratory Animals [20]. The facility where this research was conducted is fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (Rockville, MD).

Statistical analysis. All virus titers in the growth-kinetics experiments and the percentages of eGFP-positive cells are shown as mean±SE. P values for mean time to death in the mouse experiments were calculated by use of Student's t test (2-tailed distribution and 2-sample unequal variance). P <.05 was considered to be statistically significant.

Previous SectionNext Section

Results

Generation of 2 different recombinant eGFP-ZEBOV variants. To examine the effects of inserting an additional transcription unit expressing a foreign gene on the biological properties of ZEBOV, we engineered 2 viruses with an eGFP gene at different positions in the genome, based on the background of the Mayinga strain of wt-ZEBOV (figure 1A) [9]. Rescue of viruses from cDNA was confirmed by the appearance of CPE and eGFP expression in Vero E6 cells (first passage; figure 1B, left and right panels) within 5–7 days after transfer of supernatants from a mixed culture of 293T/Vero E6 cells transfected with the plasmids needed to create infectious ZEBOV [9]. To confirm that eGFP was expressed in eGFP-ZEBOV-infected cells, we simultaneously detected ZEBOV GP surface expression and eGFP expression by IFA (figure 1B, middle and right panels).

In vitro characterization of eGFP-ZEBOV. We first examined the stability of the eGFP transcription units by passaging both eGFP-ZEBOV variants 10 times in Vero E6 cells. eGFP expression remained stable in all passages, confirming the stability of the additional transcription unit in vitro (data not shown). To demonstrate the utility of eGFP-ZEBOV, we examined cell tropism in vitro by using 1 of the 2 recombinant viruses (NP/35-eGFP). Vero E6 cells and primary human macrophages, which are permissive for ZEBOV infection, and 293T cells, which are nonpermissive for ZEBOV infection and served as a negative control, were infected with NP/35-eGFP at an MOI of 0.05, and eGFP-positive cells were quantified by flow cytometry at day 4 after infection. Infected Vero E6 and macrophage cultures showed eGFP expression levels of 94% and 13%, respectively, indicating different levels of susceptibility in these target cell types (figure 2B and 2D). In contrast, 293T cells were resistant to NP/35-eGFP infection (figure 2E and 2F). These data correspond with the tropism of wt-ZEBOV (data not shown).

Figure 2
View larger version:
Figure 2

In vitro cell tropism of Zaire ebolavirus (ZEBOV) expressing enhanced green fluorescent protein (eGFP). Vero E6 cells, primary human macrophages, and 293T cells were infected with the NP/35-eGFP variant at an MOI of 0.05. On day 4 after infection, the number of eGFP-positive cells was quantified by flow cytometry. The horizontal line in each panel denotes the eGFP-positive gate used for the analysis; the no. above the horizontal line is the percentage of eGFP-positive cells.

We next compared the growth characteristics of NP/35-eGFP, VP30/24-eGFP, and wt-ZEBOV in Vero E6 and U937 cells. The growth kinetics for all 3 viruses in Vero E6 cells were indistinguishable (figure 3A), which is consistent with results reported previously [4]. In human monocytic U937 cells, which are capable of inducing a strong antiviral state, we observed a minor but statistically significant attenuation with the eGFP-expressing viruses, particularly NP/35-eGFP (1-log difference on day 5 after infection), compared with wt-ZEBOV (figure 3B). Thus, unexpectedly, the localization of the eGFP-expression cassette in the ZEBOV genome did not have a dramatic impact on viral replication in vitro.

Figure 3
View larger version:
Figure 3

In vitro growth characteristics of Zaire ebolavirus (ZEBOV) variants expressing enhanced green fluorescent protein (eGFP). Vero E6 cells (A) and U937 (human monocytic) cells (B) were infected, at an MOI of 0.05, with wild-type (wt) ZEBOV, NP/35-eGFP, or VP30/24-eGFP. Supernatantswere collected on days 1–3 and 5 after infection, and titration was done on Vero E6 cells by use of a focus-forming unit assay. Infectivity titers are presented as log10 ffu/mL.

In vivo characterization of eGFP-ZEBOV. We first examined the virulence of the 2 eGFP-ZEBOV variants in a small-animal model. Since wt-ZEBOV does not produce lethal infection in immunocompetent mice [21], we used STAT1−/− mice [22, 23]. It has been shown previously that, because of their inability to activate a type I IFN-mediated antiviral response [23, 24], STAT1−/− mice are highly susceptible to infection with wt-ZEBOV, which often leads to a lethal outcome [21]. As expected, in mice challenged with either a high or a low dose (1000 or 0.2 ffu per mouse, respectively), wt-ZEBOV infection resulted in 100% lethality (figure 4), with a disease course very similar to that in immunocompetent mice infected with mouse-adapted ZEBOV [13, 25]. In comparison with wt-ZEBOV, both eGFP-expressing viruses showed altered virulence in mice after challenge with a high or low dose (figure 4A and 4B, respectively). With a low-dose challenge (0.2 ffu per mouse), both NP/35-eGFP and VP30/24-eGFP caused lethal infection, but NP/35-eGFP-infected mice in particular demonstrated a significantly prolonged time to death, compared with wt-ZEBOV-infected mice (P = .01; figure 4B). Attenuation of the eGFP-expressing viruses, especially NP/35-eGFP, became more apparent after a high-dose challenge (1000 ffu per mouse), with NP/35-eGFP and VP30/24-eGFP infection resulting in 33% and 67% lethality, respectively (figure 4A), and a significantly prolonged time to death (for NP/35-eGFP vs. wt-ZEBOV, P = .01; for VP30/24-eGFP vs. wt-ZEBOV, P = .05). Similar to the in vitro results (figure 3), NP/35-eGFP was found to be more attenuated in STAT1−/− mice than was VP30/24-eGFP, although the difference in time to death between the 2 eGFP-expressing viruses was not statistically significant.

Figure 4
View larger version:
Figure 4

Virulence of Zaire ebolavirus (ZEBOV) variants expressing enhanced green fluorescent protein (eGFP) in mice lacking signal transducer and activator of transcription 1 (STAT1−/−). STAT1−/− mice were challenged with either a high dose (1000 ffu per mouse; A) or a low dose (0.2 ffu per mouse; B) of VP30/24-eGFP, NP/35-eGFP, or wild-type (wt) ZEBOV, by intraperitoneal injection. Following infection, mice were observed daily for signs of disease, and weight was recorded for 11 days after infection. All surviving animals were observed until day 21 after infection. The graphs show the percentage survival in a Kaplan-Meier curve.

To evaluate the utility of eGFP-ZEBOV in studies of pathogenesis, we attempted to track the replication and spread of the eGFP-expressing viruses in STAT1−/− mice by detecting eGFP-positive cells in infected tissues. As a primary attempt, we detected eGFP-positive cells in organ smears from mice infected with NP/35-eGFP and VP30/24-eGFP (figure 5A). eGFP expression was detected in liver (figure 5A, top), spleen (figure 5A, bottom), and kidney (data not shown) cells, as visualized by florescence microscopy, whereas mock-infected organ smears only showed background fluorescence. Next, we performed flow cytometry to quantify eGFP expression in organs collected from virus- and mock-infected animals at days 3 and 5 after infection (figure 5B and 5C). The percentage of infected (eGFP-positive) cells in liver and spleen increased as infection progressed (figure 5B). As indicated in figure 5C, the percentage of eGFP-positive cells seemed higher in liver than in spleen at day 3 after infection, whereas viral replication in spleen increased dramatically by day 5. Thus, in vivo viral replication and spread can be easily monitored by use of eGFPZEBOV.

Figure 5
View larger version:
Figure 5

Detection and tracking of Zaire ebolavirus (ZEBOV) expressing enhanced green fluorescent protein (eGFP) in mice lacking signal transducer and activator of transcription 1 (STAT1−/−). Mice were infected with 1000 ffu per mouse of NP/35-eGFP or VP30/24-eGFP or were mock infected. A, eGFP fluorescence in organ smears. B, Histograms representing either NP/35-eGFP or VP30/24-eGFP in the tissues. The pink line depicts the eGFP-positive gate used for the analysis. C, Percentage of cells expressing eGFP in each tissue. The level of viral spread in the liver and spleen was determined by following eGFP expression on days 3 and 5 after infection, by use of flow cytometry. pi, postinfection.

Finally, we examined the virulence of eGFP-ZEBOV in a rhesus macaque model, which closely resembles Ebola hemorrhagic fever in humans [19, 26, 27]. We chose VP30/24-eGFP as the challenge virus, since this virus was slightly less attenuated than NP/35-eGFP in STAT1−/− mice (figure 4). Two healthy animals (subjects 1 and 2) were challenged by intramuscular injection with 1000 ffu of VP30/24-eGFP. Surprisingly, in contrast to rhesus macaques with wt-ZEBOV infection, which causes severe disease with a lethality of 190% [19, 26, 27], both VP30/24-eGFP-infected macaques displayed only mild clinical symptoms (slightly more severe in subject 1 than in subject 2), including anorexia, transient lymphopenia, thrombocytopenia, and increased levels of d-dimers in blood (data not shown); they fully recovered by day 10 after infection (figure 6). Although both animals were viremic at day 6 after infection (titers of 104.52 pfu/mL for subject 1 and 103.06 pfu/mL for subject 2), both had completely cleared viremia by day 10 (figure 6), in contrast to rhesus macaques infected with 1000 pfu of wt-ZEBOV (historical control animals, n = 14) [19, 26, 27]. The VP30/24-eGFP-infected animals were rechallenged 28 days later by intramuscular injection with 1000 pfu of wt-ZEBOV, strain Kikwit. Both animals were completely protected against this uniformly lethal challenge dose without evidence of clinical illness, viremia, or pathologic changes in hematology and clinical chemistry (data not shown).

Figure 6
View larger version:
Figure 6

Pathogenesis of Zaire ebolavirus (ZEBOV) expressing enhanced green fluorescent protein (eGFP) in rhesus macaques. Two rhesus macaques (subjects 1 and 2) were infected by intramuscular (i.m.) injection with 1000 pfu of VP30/24-eGFP and were followed over a period of 28 days. Blood samples were collected on days 3, 6, 10, and 14 after infection. Selected hematology data for lymphopenia and thrombocytopenia are given as percentage of lymphocytes and platelet counts, respectively. Values for wild-type (wt) ZEBOV-infected animals (wt-inf.) were derived from historical controls infected with the same dose of wt-ZEBOV (n = 14) Virus infectivity titers in blood are shown as log10 pfu/mL. Virus titers for wt-ZEBOV-infected animals were derived from historical controls infected with the same dose of wt-ZEBOV (n = 14 for days 3 and 6 after infection; n = 10 for day 8 after infection). Animals infected with wt-ZEBOV normally succumb to infection before day 10.

Previous SectionNext Section

Discussion

In this study, we generated 2 eGFP-ZEBOV variants expressing eGFP from a separate transcription unit located close to either the 3′ end (between NP and VP35) or the 5′ end (between VP30 and VP24) of the genome (figure 1A). Similar to other nonsegmented negative-stranded (NNS) RNA viruses, in vitro characterization demonstrated that the 2 eGFP-ZEBOV variants were stable and showed the same in vitro cell tropism as wt-ZEBOV (figure 2) [4, 28–30]. Both viruses were only slightly attenuated in vitro but showed a more obvious attenuation in vivo, compared with wt-ZEBOV. NP/35-eGFP, which carries the additional transcription unit closer to the 3′ end of the genome, showed the least virulent phenotype, which may be explained by altered transcription of the 6 downstream viral genes (VP35, VP40, GP, VP30, VP24, and L), rather than the altered transcription of only 2 genes (VP24 and L) in VP30/24-eGFP (figures 3B and 4). A similar genome position-dependent effect on gene transcription, from the introduction of a foreign gene, has been reported for other NNS RNA viruses and is consistent with the concept of a transcriptional gradient along the single-stranded NNS RNA genome [3, 31–33].

In contrast to the moderate attenuation of the eGFP-ZEBOV variants in the mouse model, severe attenuation was observed in the rhesus macaque model. Both VP30/24-eGFP-infected animals survived challenge and showed onlymild clinical symptoms (figure 6), which is in contrast to wt-ZEBOV infection in rhesus macaques, which usually results in severe hemorrhagic fever with lethality of >90% when animals are challenged with 1000 pfu of virus [19, 26, 27]. The attenuation in virulence correlated with a transient and lower-level viremia in the 2 animals, which was 1–1.5 log10 lower than that in wt-ZEBOV-infected rhesus macaques. Levels of viremia and d-dimers (data not shown) correlated with severity of disease in the 2 eGFPZEBOV-infected animals and were similar to those in a previous report of severe attenuation of mouse-adapted ZEBOV in rhesus macaques [34]. On the basis of the results with VP30/24-eGFP, an ethical decision was made to not challenge the rhesus macaques with NP/VP35-eGFP, since this virus was even more attenuated than VP30/24-eGFP in vitro (figure 3) and in the model using STAT1−/− mice (figure 4). Currently, why attenuation is more severe in nonhuman primates than in STAT1−/− mice is not known, but the defect in type I IFN signaling in these mice might be a logical explanation for this phenomenon. Additional studies of viral replication and tropism of eGFP-ZEBOV in the 2 animal models will help identify viral and host factors associated with attenuation.

With the exception of studies of large DNA viruses and retroviruses [35, 36], to date no experiments with nonhuman primates and recombinant viruses expressing foreign genes have been reported. Unfortunately, the severe attenuation of eGFPZEBOV in rhesus macaques makes this first attempt less suitable for pathogenesis studies. Thus, additional attempts will be made to generate ZEBOV mutants expressing marker proteins that remain lethal for nonhuman primates, the reference standard of animal models of Ebola hemorrhagic fever. Nevertheless, even attenuated mutant eGFP-ZEBOV variants, such as those reported in this study, will be instrumental for in vivo studies of viral tropism and replication. They also might help in the investigation of immunologic parameters important for surviving Ebola hemorrhagic fever.

An advantage of viruses expressing reporter genes is the ability to easily track viral infection and spread in vitro and in vivo [2, 3, 28, 37, 38]. In this study, we were able to demonstrate the usefulness of the 2 eGFP-ZEBOV variants for in vivo tracking and pathogenesis studies in a mouse model (figure 5). Quantification of eGFP-expressing cells in tissues, by flow cytometry, on day 3 after infection showed fewer eGFP-positive cells in spleen than in liver (figure 5B and 5C). This observation did not correlate with virus titers from the same organs harvested from immunocompetent mice infected with mouse-adapted ZEBOV, which showed higher virus titers in spleen [13, 25]. However, histopathologic analysis of the liver from the same BALB/c mice demonstrated the presence of viral antigen and RNA in several liver cell types, such as hepatocytes, Kupffer cells, and endothelial cells, at day 3 after infection, whereas only mononuclear cells in the spleen were positive for viral antigen and RNA at day 3 after infection [39]. These findings suggest that primary target cells in the spleen (macrophages, monocytes, and dendritic cells) have greater potential to support production of infectious virus particles than do those in the liver, a hypothesis supported by the increased number of eGFP-positive spleen cells, compared with the number of liver cells, in STAT1−/− mice on day 5 after infection (figure 5). Further characterization of eGFP-positive target cells, using cell-sorting techniques, will reveal the tropism of ZEBOV in the mouse model. Nevertheless, despite similar clinical symptoms and disease course in both STAT1−/− and BALB/c mice, differences may exist because of the lack of a normal type I IFN response in STAT1−/− mice [13, 21, 23, 25]. In addition, we observed resistance in STAT1−/− mice infected with highdose eGFP-ZEBOV (figure 4A), similar to that reported for immunocompetent mice infected with mouse-adapted ZEBOV [13]. This resistance in type I IFN signaling—deficient animals was unexpected and demonstrates that this phenomenon most likely is not associated with a type I IFN signaling—mediated antiviral state. A better understanding of the mechanism behind this phenomenon may help explain protective host responses against lethal ZEBOV infection.

Another application for eGFP-ZEBOV will be in vivo antiviral drug screening. To date, several experimental oligonucleotide-based approaches [40], such as those using small interfering RNA and phosphorodiamidate morpholino oligomers, have been reported to control ZEBOV infection in vitro and in vivo [41–44]. However, efficacy testing, particularly for approaches using siRNA, has been difficult in mice because of unexpected siRNA-induced type I IFN responses, which led to nonspecific protection of mice after virus challenge (authors' unpublished data) [42]. These observations may explain why antiviral drugs can be protective in mice but fail to protect or show little efficacy in nonhuman primates. This problem could be avoided by infection of type I IFN signaling-deficient STAT1−/− mice with a eGFP-ZEBOV [21] that can be easily tracked by flow cytometry, as demonstrated in this study (figure 5).

In conclusion, this study presents the engineering, rescue, and in vitro and in vivo characterization of 2 eGFP-expressing wt-ZEBOV—based viruses.We could demonstrate the usefulness of these viruses for in vivo studies in a small-animal model based on immunodeficient STAT1−/− mice, although severe attenuation of virulence was observed in nonhuman primates. Future plans involve virus tracking of virulent eGFP-ZEBOV in larger animals, such as rhesus macaques, and the development of eGFP-ZEBOV for use with immunocompetent mice and guinea pigs, for which adapted ZEBOV strains exist [13, 45, 46]. The use of virus mutants expressing reporter genes will greatly facilitate future pathogenesis studies.

Previous SectionNext Section

Acknowledgments

The authors thank Friederike Feldmann and Andrea Paille (National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Manitoba) and Elizabeth A. Fritz (US Army Medical Research Institute for Infectious Diseases, Fort Detrick, MD) for assistance with the mouse and rhesus macaque experiments at biosafety level 4; Lisa Fernando (National Microbiology Laboratory, Public Health Agency of Canada) for technical assistance with the flow cytometry analysis; and Thomas Hoenen (National Microbiology Laboratory, Public Health Agency of Canada) for discussion and editing of the manuscript.

Supplement sponsorship. This article was published as part of a supplement entitled “Filoviruses: Recent Advances and Future Challenges,” sponsored by the Public Health Agency of Canada, the National Institutes of Health, the Canadian Institutes of Health Research, Cangene, CUH2A, Smith Carter, Hemisphere Engineering, Crucell, and the International Centre for Infectious Diseases.

Previous Section
 

References

  1. 1.↵
    1. Ebihara H,
    2. Groseth A,
    3. Neumann G,
    4. Kawaoka Y,
    5. Feldmann H
    . The role of reverse genetics systems in studying viral hemorrhagic fevers [review]. Thromb Haemost 2005;94:240-53.
    MedlineWeb of Science
  2. 2.↵
    1. Rudd PA,
    2. Cattaneo R,
    3. von Messling V
    . Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion. J Virol 2006;80:9361-70.
    Abstract/FREE Full Text
  3. 3.↵
    1. von Messling V,
    2. Milosevic D,
    3. Cattaneo R
    . Tropism illuminated: lymphocyte- based pathways blazed by lethal morbillivirus through the host immune system. Proc Natl Acad Sci USA 2004;101:14216-21.
    Abstract/FREE Full Text
  4. 4.↵
    1. Towner JS,
    2. Paragas J,
    3. Dover JE,
    4. et al
    . Generation of eGFP expressing recombinant Zaire ebolavirus for analysis of early pathogenesis events and high-throughput antiviral drug screening. Virology 2005;332:20-7.
    CrossRefMedlineWeb of Science
  5. 5.↵
    1. Jones SM,
    2. Feldmann H,
    3. Stroher U,
    4. et al
    . Live attenuated recombinant vaccine platform protects non-human primates against lethal challenge with either Ebola virus or Marburg virus. Nature Med 2005;11:786-90.
    CrossRefMedlineWeb of Science
  6. 6.
    1. Geisbert TW,
    2. Jones S,
    3. Fritz EA,
    4. et al
    . Development of a new vaccine for the prevention of Lassa fever. PLoS Med 2005;2:e183.
    CrossRefMedline
  7. 7.↵
    1. Hevey M,
    2. Negley D,
    3. Pushko P,
    4. Smith J,
    5. Schmaljohn A
    . Marburg virus vaccines based upon alphavirus replicons protect guinea pigs and nonhuman primates. Virology 1998;251:28-37.
    CrossRefMedlineWeb of Science
  8. 8.↵
    1. Enterlein S,
    2. Volchkov V,
    3. Weik M,
    4. et al
    . Rescue of recombinant Marburg virus from cDNA is dependent on nucleocapsid protein VP30. J Virol 2006;80:1038-43.
    Abstract/FREE Full Text
  9. 9.↵
    1. Neumann G,
    2. Feldmann H,
    3. Watanabe S,
    4. Lukashevich I,
    5. Kawaoka Y
    . Reverse genetics demonstrates that proteolytic processing of the Ebola virus glycoprotein is not essential for replication in cell culture. J Virol 2002;76:406-10.
    Abstract/FREE Full Text
  10. 10.
    1. Muhlberger E,
    2. Lotfering B,
    3. Klenk HD,
    4. Becker S
    . Three of the four nucleocapsid proteins of Marburg virus, NP, VP35, and L, are sufficient to mediate replication and transcription of Marburg virus-specific monocistronic minigenomes. J Virol 1998;72:8756-64.
    Abstract/FREE Full Text
  11. 11.
    1. Muhlberger E,
    2. Weik M,
    3. Volchkov VE,
    4. Klenk HD,
    5. Becker S
    . Comparison of the transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems. J Virol 1999;73:2333-42.
    Abstract/FREE Full Text
  12. 12.↵
    1. Volchkov VE,
    2. Volchkova VA,
    3. Muhlberger E,
    4. et al
    . Recovery of infectious Ebola virus from complementary DNA: RNA editing of the GP gene and viral cytotoxicity. Science 2001;291:1965-9.
    Abstract/FREE Full Text
  13. 13.↵
    1. Ebihara H,
    2. Takada A,
    3. Kobasa D,
    4. et al
    . Molecular determinants of Ebola virus virulence in mice. PLoS Pathog 2006;2:e73-74.
    CrossRefMedline
  14. 14.
    1. Hartman AL,
    2. Dover JE,
    3. Towner JS,
    4. Nichol ST
    . Reverse genetic generation of recombinant Zaire Ebola viruses containing disrupted IRF-3 inhibitory domains results in attenuated virus growth in vitro and higher levels of IRF-3 activation without inhibiting viral transcription or replication. J Virol 2006;80:6430-40.
    Abstract/FREE Full Text
  15. 15.↵
    1. Neumann G,
    2. Ebihara H,
    3. Takada A,
    4. et al
    . Ebola virus VP40 late domains are not essential for viral replication in cell culture. J Virol 2005;79:10300-7.
    Abstract/FREE Full Text
  16. 16.↵
    1. Neumann G,
    2. Geisbert TW,
    3. Ebihara H,
    4. et al
    . Proteolytic processing of Ebola virus glycoprotein is not critical for Ebola virus replication in nonhuman primates. J Virol 2007;81:2995-8.
    Abstract/FREE Full Text
  17. 17.↵
    1. Takada A,
    2. Ebihara H,
    3. Jones S,
    4. Feldmann H,
    5. Kawaoka Y
    . Protective efficacy of neutralizing antibodies against Ebola virus infection. Vaccine 2006;25:993-9.
    CrossRefMedlineWeb of Science
  18. 18.↵
    1. Takada A,
    2. Feldmann H,
    3. Ksiazek TG,
    4. Kawaoka Y
    . Antibody-dependent enhancement of Ebola virus infection. J Virol 2003;77:7539-44.
    Abstract/FREE Full Text
  19. 19.↵
    1. Geisbert TW,
    2. Hensley LE,
    3. Larsen T,
    4. et al
    . Pathogenesis of Ebola hemorrhagic fever in cynomolgus macaques: evidence that dendritic cells are early and sustained targets of infection. Am J Pathol 2003;163:2347-70.
    MedlineWeb of Science
  20. 20.↵
    Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources Commission of Life Sciences, National Research Council. Washington, DC: National Academy Press; 1996. Guide for the care and use of laboratory animals.
  21. 21.↵
    1. Bray M
    . The role of the type I interferon response in the resistance of mice to filovirus infection. J Gen Virol 2001;82:1365-73.
    Abstract/FREE Full Text
  22. 22.↵
    1. Schindler C,
    2. Darnell JE Jr.
    . Transcriptional responses to polypeptide ligands: the JAK-STAT pathway [review]. Annu Rev Biochem 1995;64:621-51.
    MedlineWeb of Science
  23. 23.↵
    1. Durbin JE,
    2. Hackenmiller R,
    3. Simon MC,
    4. Levy DE
    . Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 1996;84:443-50.
    CrossRefMedlineWeb of Science
  24. 24.↵
    1. Garcia-Sastre A,
    2. Durbin RK,
    3. Zheng H,
    4. et al
    . The role of interferon in influenza virus tissue tropism. J Virol 1998;72:8550-8.
    Abstract/FREE Full Text
  25. 25.↵
    1. Bray M,
    2. Davis K,
    3. Geisbert T,
    4. Schmaljohn C,
    5. Huggins J
    . A mouse model for evaluation of prophylaxis and therapy of Ebola hemorrhagic fever. J Infect Dis 1998;178:651-61.
    Abstract/FREE Full Text
  26. 26.↵
    1. Geisbert TW,
    2. Hensley LE
    . Ebola virus: new insights into disease aetiopathology and possible therapeutic interventions. Expert Rev Mol Med 2004;6:1-24.
    Medline
  27. 27.↵
    1. Geisbert TW,
    2. Young HA,
    3. Jahrling PB,
    4. Davis KJ,
    5. Kagan E,
    6. Hensley LE
    . Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event. J Infect Dis 2003;188:1618-29.
    Abstract/FREE Full Text
  28. 28.↵
    1. Hashimoto K,
    2. Ono N,
    3. Tatsuo H,
    4. et al
    . SLAM (CD150)-independent measles virus entry as revealed by recombinant virus expressing green fluorescent protein. J Virol 2002;76:6743-9.
    Abstract/FREE Full Text
  29. 29.
    1. Quinones-Kochs MI,
    2. Schnell MJ,
    3. Buonocore L,
    4. Rose JK
    . Mechanisms of loss of foreign gene expression in recombinant vesicular stomatitis viruses. Virology 2001;287:427-35.
    CrossRefMedline
  30. 30.↵
    1. He B,
    2. Paterson RG,
    3. Ward CD,
    4. Lamb RA
    . Recovery of infectious SV5 from cloned DNA and expression of a foreign gene. Virology 1997;237:249-60.
    CrossRefMedline
  31. 31.↵
    1. Tokusumi T,
    2. Iida A,
    3. Hirata T,
    4. Kato A,
    5. Nagai Y,
    6. Hasegawa M
    . Recombinant Sendai viruses expressing different levels of a foreign reporter gene. Virus Res 2002;86:33-8.
    CrossRefMedlineWeb of Science
  32. 32.
    1. Wertz GW,
    2. Moudy R,
    3. Ball LA
    . Adding genes to the RNA genome of vesicular stomatitis virus: positional effects on stability of expression. J Virol 2002;76:7642-50.
    Abstract/FREE Full Text
  33. 33.↵
    1. Cattaneo R,
    2. Rebmann G,
    3. Schmid A,
    4. Baczko K,
    5. ter Meulen V,
    6. Billeter MA
    . Altered transcription of a defective measles virus genome derived from a diseased human brain. EMBO J 1987;6:681-8.
    MedlineWeb of Science
  34. 34.↵
    1. Bray M,
    2. Hatfill S,
    3. Hensley L,
    4. Huggins JW
    . Haematological, biochemical and coagulation changes in mice, guinea-pigs and monkeys infected with a mouse-adapted variant of Ebola Zaire virus. J Comp Pathol 2001;125:243-53.
    CrossRefMedlineWeb of Science
  35. 35.↵
    1. Chang WL,
    2. Tarantal AF,
    3. Zhou SS,
    4. Borowsky AD,
    5. Barry PA
    . A recombinant rhesus cytomegalovirus expressing enhanced green fluorescent protein retains the wild-type phenotype and pathogenicity in fetal macaques. J Virol 2002;76:9493-504.
    Abstract/FREE Full Text
  36. 36.↵
    1. Alexander L,
    2. Veazey RS,
    3. Czajak S,
    4. et al
    . Recombinant simian immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys. AIDS Res Hum Retroviruses 1999;15:11-21.
    CrossRefMedlineWeb of Science
  37. 37.↵
    1. von Messling V,
    2. Svitek N,
    3. Cattaneo R
    . Receptor (SLAM [CD150]) recognition and the V protein sustain swift lymphocyte-based invasion of mucosal tissue and lymphatic organs by a morbillivirus. J Virol 2006;80:6084-92.
    Abstract/FREE Full Text
  38. 38.↵
    1. Duprex WP,
    2. McQuaid S,
    3. Roscic-Mrkic B,
    4. Cattaneo R,
    5. McCallister C,
    6. Rima BK
    . In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein. J Virol 2000;74:7972-9.
    Abstract/FREE Full Text
  39. 39.↵
    1. Gibb TR,
    2. Bray M,
    3. Geisbert TW,
    4. et al
    . Pathogenesis of experimental Ebola Zaire virus infection in BALB/c mice. J Comp Pathol 2001;125:233-42.
    CrossRefMedlineWeb of Science
  40. 40.↵
    1. Warfield KL,
    2. Panchal RG,
    3. Aman MJ,
    4. Bavari S
    . Antisense treatment for biothreat agents [review]. Curr Opin Mol Ther 2006;8:93-103.
    MedlineWeb of Science
  41. 41.↵
    1. Enterlein S,
    2. Warfield KL,
    3. Swenson DL,
    4. et al
    . VP35 knockdown inhibits Ebola virus amplification and protects against lethal infection in mice. Antimicrob Agents Chemother 2006;50:984-93.
    Abstract/FREE Full Text
  42. 42.↵
    1. Geisbert TW,
    2. Hensley LE,
    3. Kagan E,
    4. et al
    . Postexposure protection of guinea pigs against a lethal Ebola virus challenge is conferred by RNA interference. J Infect Dis 2006;193:1650-7.
    Abstract/FREE Full Text
  43. 43.
    1. Warfield KL,
    2. Swenson DL,
    3. Olinger GG,
    4. et al
    . Gene-specific countermeasures against Ebola virus based on antisense phosphorodiamidate morpholino oligomers. PLoS Pathog 2006;2:e1.
    CrossRefMedline
  44. 44.↵
    1. Hensley LE,
    2. Jones SM,
    3. Feldmann H,
    4. Jahrling PB,
    5. Geisbert TW
    . Ebola and Marburg viruses: pathogenesis and development of countermeasures [review]. Curr Mol Med 2005;5:761-72.
    CrossRefMedlineWeb of Science
  45. 45.↵
    1. Connolly BM,
    2. Steele KE,
    3. Davis KJ,
    4. et al
    . Pathogenesis of experimental Ebola virus infection in guinea pigs. J Infect Dis 1999;179((Suppl 1)):S203-17.
    Abstract/FREE Full Text
  46. 46.↵
    1. Volchkov VE,
    2. Chepurnov AA,
    3. Volchkova VA,
    4. Ternovoj VA,
    5. Klenk HD
    . Molecular characterization of guinea pig-adapted variants of Ebola virus. Virology 2000;277:147-55.
    CrossRefMedline
| Table of Contents

This Article

  1. J Infect Dis. (2007) 196 (Supplement 2): S313-S322. doi: 10.1086/520590
  1. AbstractFree
  2. » Full Text (HTML)Free
  3. Full Text (PDF)Free

Classifications

Share

  1. Email this article

Navigate This Article

Current Issue

  1. March 1, 2012 205 (5)
  1. Journal of Infectious Diseases
  1. Alert me to new issues

Most