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Detection and Molecular Characterization of Ebola Viruses Causing Disease in Human and Nonhuman Primates

  1. Anthony Sanchez,
  2. Thomas G. Ksiazek,
  3. Pierre E. Rollin,
  4. Mary E. G. Miranda,
  5. Sam G. Trappier,
  6. Ali S. Khan,
  7. Clarence J. Peters and
  8. Stuart T. Nichol
  1. Special Pathogens Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia; Research Institute for Tropical Medicine, Philippine Department of Health, Alabang, Philippines
  1. Reprints or correspondence: Dr. Anthony Sanchez, Centers for Disease Control and Prevention, 1600 Clifton Rd., Bldg. 15, Room SB611, Mailstop G14, Atlanta, GA 30333 (ans1{at}cdc.gov).
 
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Abstract

Ebola (EBO) viruses were detected in specimens obtained during the hemorrhagic fever outbreak among humans in Kikwit, Democratic Republic of the Congo (DRC), in 1995 (subtype Zaire) and during an outbreak of disease in cynomolgus macaques in Alice, Texas, and the Philippines in 1996 (subtype Reston). Reverse transcriptase—polymerase chain reaction assays were developed and proven effective for detecting viral RNA in body fluids and tissues of infected individuals. Little change was seen in the nucleotide or deduced amino acid sequences of the glycoprotein (GP) of these EBO virus subtypes compared with those of their original representatives (i.e., the 1976 Yambuku, DRC, EBO isolate [subtype Zaire] and the 1989 Philippines and Reston, Virginia, isolates [subtype Reston]). The nonstructural secreted GP (SGP), the primary product of the GP gene, was more highly conserved than the structural GP, indicating different functional roles or evolutionary constraints for these proteins. Significant amounts of SGP were detected in acutely infected humans.

The recent reemergence of Ebola (EBO) hemorrhagic fever (EHF) in Kikwit, Democratic Republic of the Congo (DRC), the introduction of a Philippine EBO virus (subtype Reston [EBO-R]) into a primate facility in Alice, Texas [1–4], and the ensuing press and media attention have prompted worldwide interest in the biology and public health threat of these viral pathogens. The EHF outbreaks were caused by 2 phylogenetically distinct species of EBO virus [5] and were reminiscent of the first discovered outbreaks in Yambuku, DRC, in 1976 and Reston, Virginia, in 1989. There are 4 known species of EBO virus: Zaire (EBO-Z), EBO-R, Sudan, and Côte d'Ivoire. These viruses, together with the closely related Marburg virus, are nonsegmented, negative-strand RNA viruses of the family Filoviridae. Because members of this family are associated with high mortality, they are all classified as biosafety level 4 agents [6]. The natural reservoirs of filoviruses remain unknown; investigations of outbreaks of disease have shed little light on this mystery [6].

Research directed at the molecular biology of these agents has provided nucleotide sequence data that have allowed development of reverse transcription-polymerase chain reaction (RT-PCR) assays [6–9]. These assays allow for the rapid detection of viral RNA in specimens, and amplified sequences are useful in assessing relatedness to known filoviruses. Genetic and biochemical studies have shown that these viruses have a single glycoprotein (GP) gene, from which is expressed a highly glycosylated protein (containing both N- and O-linked glycans) that forms the putative attachment structure (peplomer) on the surface of virions [5, 9]. Unlike the case with Marburg virus isolates, the GP genes of all known EBO viruses have an unusual organization in that the GP is encoded in 2 frames (0 and −1 frames), which become linked (expressed) by transcriptional editing in ∼25% of the transcripts [5]. As a result of this organization, a smaller, nonstructural, secreted, soluble GP (SGP) is expressed as the predominant gene product and is produced in large amounts in cell cultures. The SGP and GP share ∼300 N-terminal residues, but have unique C-termini. The roles of these molecules in the pathogenesis of EBO virus infections have not been demonstrated.

The development of sensitive diagnostic assays capable of detecting EBO virus in human and nonhuman primates is important for identifying outbreaks and supporting ensuing epidemiologic investigations. This study describes the results of RT-PCR assays in the detection of EBO-Z in specimens from the 1995 outbreak of human disease in Kikwit, DRC, and the detection of EBO-R in specimens from the 1996 outbreak in Alice, Texas, and the Philippines. In addition, the detection of EBO virus GPs in the blood of acutely infected human patients is described.

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Materials and Methods

Detection of EBO virus RNA by RT-PCR

RNA from whole blood, sera, tissue homogenates, and mucosal secretions from EBO-Z-infected humans or EBO-R-infected nonhuman primates were extracted using a commercial kit (RNAid Kit; Bio 101, La Jolla, CA). Typically, 0.1 mL of specimen was mixed with 0.4 mL of GITC solution (4 M guanidine isothiocyanate containing 25 mM sodium citrate, 0.5% n-lauryl sarcosine, and 0.1 M 2-mercaptoethanol). To this mixture was added 50 µL of 2 M sodium acetate (pH 4.0), 0.4 mL of phenol saturated with distilled water, and 0.2 mL of chloroform—isoamyl alcohol (24:1). The mixture was vortexed, placed on ice for 15 min, and then centrifuged at 8000 g for 10 min at room temperature. The supernatant was removed and mixed with 5 µL of RNAMATRIX from the kit (without RNA binding salt), according to the manufacturer's instructions. Bound RNA was eluted from the matrix into 50 µL of RNase-free water and used immediately in RT-PCR assays or stored at −70°C until needed.

For initial testing of human specimens from the 1995 Kikwit outbreak, a GeneAmp (Perkin-Elmer, Norwalk, CT) RNA-PCR kit was used. First-strand cDNA synthesis was done by mixing 10 µL of extracted RNA with 2.5 µL of primers EBO-GP1 and FILO-A (100 ng/mL; see table 1), heating at 65°C for 1 min, quick freezing in an ethanol—dry ice bath, and then thawing at room temperature. To the tube was added 1 µL of RNase inhibitor, 6 µL of 5× RT buffer, 6 µL of 5 µM dNTP, and 2 µL of murine leukemia virus RT. The reaction mixture was vortexed, briefly centrifuged, and placed at 42°C for at least 30 min. The first-strand reaction was split and transferred to two thin-walled 0.2-mL PCR tubes. To each tube was added 8.5 µL of 10× PCR buffer, 8 µL of 25 mM MgCl2, 8 µL of 2.5 mM dNTP, 3 µL of each primer pair (100 ng/µL), 54 µL of water, and 0.5 µL of Taq polymerase (total volume = 100 µL).

Figure 1
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Figure 1

Schematic representation of open-reading frames (ORFs) of EBO-Z associated with 1995 Kikwit, Democratic Republic of the Congo, outbreak and of EBO-R associated with 1996 Alice, Texas, outbreak. Location of nucleotide differences from original 1976 EBO-Z and 1989 EBO-R strains are overlaid (vertical lines) on rectangular ORFs. Lines corresponding to nucleotide differences that result in change in amino acid sequence extend from ORF, and single-letter amino acid changes are shown. Shaded amino-terminal region identifies encoded amino acid sequences that are shared by GP (glycoprotein) and SGP (nonstructural secreted GP) molecules. All other amino acid changes in nonshaded areas correspond to differences in GP C-termini. *, identifies change in EBO-R sequence that results in loss of N-linked glycosylation site. Areas of ORFs that correspond to known variable region [5] are marked.

Figure 2
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Figure 2

Western blot of lectin-purified glycoproteins (GPs) that have been reacted with specific polyclonal antiserum against EBO-Z GPs (cross-reactive with GP and SGP [nonstructural secreted GP]). Lane 1, GPs from tissue culture fluid of EBO-Z—infected rabbit RK13 cells (positive control); lane 2, GPs from normal human serum (negative control); lanes 3–10, GPs isolated from serum of 8 acutely infected patients from Kikwit, Democratic Republic of the Congo, and surrounding area. Positions where SGP (Mr = 50−70 kDa) and GP (Mr = 130 kDa) migrate are indicated at left.

Table 1
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Table 1

Primers used in reverse transcriptase—polymerase chain reaction (RT-PCR) amplification of Ebola (EBO) virus sequences and targeted genes with predicted sizes of amplified DNA products.

For amplification of EBO GP gene sequences, the primers EBO-GP1 and EBO-GP2 were used, and for filovirus polymerase sequences, the primers FILO-A and FILO-B were used (table 1). Thermocycling was done as follows: 3 cycles of denaturing at 94°C for 30 s, annealing at 37°C for 30 s, and extension at 72°C for 2 min; this was followed by 30 cycles with a 45°C annealing temperature and an extension time of 1 min and, finally, cooling to 4°C. Thermocycling (model 9600; Perkin-Elmer) was done with rapid ramping between temperatures. The primers used to detect polymerase (L) gene sequences (FILO-A and FILO-B) amplify all known filoviruses, while the EBO-GP1 and EBO-GP2 primers amplify GP gene sequences of all 4 species of EBO virus.

The nucleoprotein (NP) genes of EBO-Z and EBO-R were amplified by use of the EZ rTth RNA PCR kit (Boehringer Mannheim, Indianapolis). This assay combines cDNA synthesis and PCR amplification in a single-tube reaction. Reactions were prepared by mixing 10 µL of 5× EZ buffer (Boehringer Mannheim), 6 µL of 2.5 mM dNTP, 6 µL of 25 mM Mn(Oac)2, 1.5 µL of each primer (see table 1), and 22 µL of RNase-free water (18 µL of water for 5 µL of template) in 0.2-mL thin-walled reaction tubes. Tubes were UV-irradiated by use of a UV Stratalinker (model 1800; Stratagene, La Jolla, CA) for 20 min, and then 2 µL of rTth polymerase and 1 µL of RNA extracted from whole blood or 10% triturated tissues (5 µL of RNA from serum) were added and thoroughly mixed. Tubes were heated at 50°C for 30 min (first-strand cDNA synthesis), followed by 35 cycles of denaturing at 94°C for 15 s and annealing at 50°C for 30 s, with rapid ramping between these temperatures. The reaction was finished by heating at 60°C for 10 min and cooling to 4°C. In all cases, amplified sequences were resolved by electrophoresis in Tris-acetate-EDTA-agarose (1.5%–2.0%, containing 0.5 µg/mL ethidium bromide) and visualized by UV illumination.

Sequence analysis

Viral sequences were determined by direct sequencing of RT-PCR products, using an automated nonisotopic method (dye-terminator cycle sequencing; Perkin-Elmer). PCR products were isolated from Tris-acetate-EDTA-agarose gels by use of the QIAEX II extraction kit (QIAGEN, Hilden, Germany), eluted into 50 µL of water, and used in sequencing reactions. The nucleotide sequence for the entire open-reading frame of the EBO-R GP gene was determined using specific primers generated from previous studies [5]. Computer-assisted nucleic acid and predicted amino acid sequence analyses were done as previously described [5].

Detection of EBO virus GP in human sera

Sera from 8 acutely infected human EHF patients from the 1995 Kikwit outbreak of EBO-Z were analyzed. GPs were extracted from 111 µL of serum by mixing with 1.0 mL of 1.11× ConA binding buffer (1× = 0.01 M Tris-HCl, pH 8.0, 0.15 M NaCl, 1 mM CaCl2, 1 mM MgCl2, and 0.02% NaN3) containing 1.11% Triton X-100. ConA-sepharose (100 µL; Sigma, St. Louis) was added to this mixture, and it was rotated overnight at 4°C. Bound GPs were washed twice with 1 mL of 1× ConA buffer containing 1% Triton X-100. GPs were removed from the ConA-sepharose by resuspending in 100 µL of 2.7% SDS containing 3.3% 2-mercaptoethanol, 20% sucrose, 0.01% bromophenol blue, and 0.082 M Tris-HCl (pH 6.7). The suspension was boiled for 3 min and centrifuged in a microfuge, and the supernatant was used to load wells of a 10% SDS polyacrylamide gel. GPs were electrophoretically separated, blotted onto nylon-reinforced nitrocellulose membranes (Optitran BA-S 85; Schleicher & Schuell, Keene, NH), and reacted with a mouse polyclonal antibody to the GP of EBO-Z virus (expressed in a baculovirus system) as previously described [10].

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Results

Genetic detection of EBO virus RNA

RT-PCR assays targeting EBO virus GP gene and filovirus L gene sequences (table 1) were done on the original 14 whole blood specimens sent to the Centers for Disease Control and Prevention (Atlanta) from the Kikwit EHF outbreak in May 1995. Both assays produced predicted-size DNA products and were identical in that they detected EBO virus RNA in the same 12 of 14 specimens tested, which correlated well with antigen-detection and isolation results (see [11], in this supplement). The amount of amplified DNA appeared greater for the L-targeted RT-PCR than for the GP in every instance (data not shown). An RT-PCR assay, using the polymerase rTth and targeting NP gene sequences, was subsequently developed and found to be more sensitive in detecting EBO-Z RNA from whole blood than were assays directed at L gene sequences (>125 times more sensitive as determined by assays performed on dilutions of RNA extracted from a positive human specimen; data not shown). The primers used in this assay (EBO-Z NP1 and EBO-Z NP2) have been described previously [8]. Analysis of EBO-Z sequences from 4 of the original 12 RT-PCR—positive patient specimens (direct sequencing of GP and L gene—amplified DNA) showed no differences in their nucleotide sequences.

A similar rTth-based single-tube RT-PCR assay was developed to target EBO-R NP gene sequences. This assay was extremely rapid and sensitive in detecting viral RNA for all known strains of EBO-R. Results of assays done in parallel with antigen-capture assays on animals from the 1996 outbreak in Alice, Texas, and samples obtained during a subsequent investigation of a Philippine primate export facility (the source of the infected monkeys shipped to Alice) showed a 100% correlation with results of antigen-capture assays (see Results in [12], in this supplement, for Alice data). Table 2 shows the results of RT-PCR assays on specimens collected from 6 animals during the Philippine investigation. It is apparent from these tests that viral RNA is easily detectable in mucosal specimens (large amounts of DNA were amplified). For 1 animal, RT-PCR results for a vaginal specimen were positive, but results for a nasal swab specimen were negative; antigen detection assay results for the same animal were negative, using liver homogenate and serum that were obtained at the same time as the other specimens.

Table 2
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Table 2

Results of reverse transcriptase—polymerase chain reaction (RT-PCR) detection of EBO-R RNA in specimens collected in the Philippines.

Nucleotide sequence analysis

Genetic characterization of the GP genes of the EBO-Z strain causing human disease in Kikwit and the EBO-R strain causing disease in cynomolgus macaques in Alice was done. Figure 1 shows the differences in the open-reading frames of these 2 viruses compared with those of the original 1976 EBO-Z and 1989 EBO-R strains. The 1995 EBO-Z differed from the original 1976 virus in 1.5% of the nucleotide and 1.5% of the predicted GP amino acid sequences, and the 1996 EBO-R differed from the 1989 virus in 1.1% of the nucleotide and 1.8% of the predicted GP amino acid sequences. Differences in the SGP molecules were smaller, with 0% and 0.5% amino acid differences detected within the EBO-Z and EBO-R strains, respectively. Differences in nucleotide sequences were primarily located in the variable region of the GP coding region/frame and were responsible for about half of the amino acid differences observed between EBO-Z (44.8%) and EBO-R (56.5%) strains. In these comparisons, only one change in a predicted N-linked glycosylation site was found; it was identified as a loss of an asparagine (N>E) in the 1996 EBO-R open-reading frame. RT-PCR products generated from EBO-R—infected monkey specimens collected from late February to early June 1996 showed no changes in nucleotide sequences when the variable region of the GP gene was examined (data not shown).

Detection of SGP in infected human blood specimens

Figure 2 shows the results of Western blot analysis of GPs isolated from the serum of 8 acutely infected humans from DRC (not from the first 14 specimens tested). All were positive for the nonstructural SGP and for lesser amounts of the structural GP, and the amount of lectin-bound protein loaded into each lane corresponds to that extracted from ∼15 µL of serum.

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Discussion

The use of RT-PCR assays to detect acute EBO-Z virus disease in humans and animals complements the use of antigen-detection ELISA and isolation techniques in the diagnosis and management of EBO outbreaks. In addition, sequence information can be obtained from amplified DNA and used to genetically characterize the EBO virus detected. This may be especially important when assessing the risks involved in the management of patients. In the 1995 Kikwit outbreak, direct sequence analysis of RT-PCR products confirmed fears and predictions that the EBO-Z species was responsible, with less than a 1.6% nucleotide difference from the 1976 EBO-Z for the entire GP gene [5]. The use of a single-tube rTth polymerase—based RT-PCR reaction for amplification of NP gene sequences increased the sensitivity, decreased the reaction time (to ∼90 min), and reduced the risk of contamination by combining first-strand cDNA synthesis and DNA amplifications into a single tube. Drawbacks to the use of RT-PCR assays in diagnostic testing are that viral RNA molecules can be degraded if specimens are not transported or extracted correctly, and a high degree of expertise is needed to perform the assays without introducing contamination (DNA or RNA). Ideally, such testing should be complemented with antigen-detection assays.

As with the EBO-Z RT-PCR assays, the assay used to detect EBO-R RNA (NP gene) was very sensitive and correlated extremely well with antigen-capture assays. Detection of EBO-R in monkey mucosal specimens (table 2) suggests that the virus was present in ample amounts for RT-PCR detection (strong DNA bands by non-nested reactions), and contact with mucosal surfaces was a potential mode of transmission between monkeys. It was noted that some animals in the Philippine primate facility showed no outward signs of disease yet harbored viral RNA (and presumably infectious virus) in their mucosa. Electron microscopic examination of experimentally infected cynomolgus monkeys has shown some mucosal cell and alveolar involvement [13], and it was noted during the 1989–1990 episodes that infected monkeys showed signs of nasal discharges (Ksiazek TG, Peters CJ; unpublished observations). One female monkey that was sacrificed in the 1996 Philippine investigation was positive for viral RNA in a vaginal swab, yet RT-PCR analysis of a nasal swab, antigen-capture and serologic assays, and isolation from a serum sample were all negative for viral RNA. We cannot completely rule out the possibility that the positive RT-PCR test on the vaginal swab could have resulted from contamination during sampling or testing; however, all possible precautions were taken. It may be that this animal was in early incubation of the disease or that infection occurred through sexual activity. From studies of humans infected with EBO-Z, including the 1995 Kikwit episode, it is known that virus can be isolated from semen even several months after infection [14].

The detection of significant amounts of SGP in the sera of acutely infected patients is the first evidence that this molecule is distributed throughout the body, and the high level of expression is consistent with in vitro studies [5] (Sanchez A, unpublished data). The question that must now be answered is what effect might the SGP have on the biology of infected human and nonhuman primates, especially the immune response. We have theorized that this molecule may directly act as a decoy to the immune response by diverting responses away from infected cells or by absorbing out certain types of antibodies directed against N-terminal sequences. Alternatively, this molecule might direct immune signaling in directions that lead to ineffective virus clearance (anergy) or induce suppression of cellular immune responses [5]. However, it is possible that this molecule has no role in pathogenesis; it may be a specialized molecule whose sole purpose is to facilitate the establishment of a persistent infection in the natural host.

The SGPs of EBO viruses are more conserved than the GP molecules [5], as demonstrated by the lack of amino acid sequence changes between the 2 EBO-Z strains and the 2 EBO-R strains in figure 1. This fact suggests that SGP may be less subject to change. The variations observed between the SGPs of different EBO species may be linked to potentially different environments in their natural hosts. The variable region of EBO virus GP, the area where the majority of amino acid changes are found (figure 1), is a region that is highly glycosylated with both N- and O-linked glycans, and it is very hydrophilic. This portion of the GP appears to have less evolutionary constraint. This could be explained if the main function of this domain is to provide a backbone for the extensive glycosylation. However, the variability may be due to selective pressure by changes in receptor conformation or immune selection. It appears that some form of selection is driving this variability, since strain variation in this region does not appear to be random (i.e., the observed nucleotide changes usually lead to changes in amino acid sequence). The large amount of O-linked glycans confers a mucin-like extended structure to this area that would make it highly exposed in an aqueous environment, and, therefore, it likely plays an important role in receptor binding. The variability of this region may confer unique antigenic or receptor-binding properties that could affect the ability of the virus to replicate in a natural or unnatural host.

Throughout the Kikwit investigation, analysis of the NP, GP, and L gene sequences from the EBO-Z strain detected in human specimens failed to turn up any changes in the genetics of the virus (see [15], in this supplement). Similarly, over the 3 months in which the EBO-R strain was detected in the Philippine export facility, no sequence variation was seen. This lack of sequence variation early and late (2–4 months) in EBO-Z and EBO-R investigations is intriguing, given that the virus is replicating in a presumed unnatural host.

It has been postulated that the 1996 EBO-R episode may have been caused by a virus that was never completely eradicated from the facility, and that the strain was directly related to those viruses that caused previous outbreaks of disease in 1989 and 1992. Given that no changes were seen in the 3-month time interval in which the virus was circulating in the facility, it is interesting to note that significant differences are indeed seen between the 1989, 1992, and 1996 viruses. The genetic stability of the EBO-R strain during the 1996 episode may be explained if the virus was introduced into the facility and if this and previous EBO-R strains represent separate introductions from the wild of independently evolving lineages. This is supported by the pattern of nucleotide differences seen in the 1989, 1992, and 1996 strains of EBO-R. There is no phylogenetic evidence of the 1989 strain serving as the ancestral sequence for the subsequent 1992 and 1996 strains. This would tend to suggest that different lineages of EBO-R were either circulating in different monkey breeding pens within the compound or were introduced at different times into the facility. However, so far there is no evidence showing that EBO-R is present outside of the grounds of the single Philippine primate facility that has been the source of all the known EBO-R out-breaks.

The recent EBO-Z and EBO-R strains may not represent strains that have evolved directly from the original 1976 EBO-Z and 1989 EBO-R viruses, but the diseases caused by these species of EBO viruses do not appear to have changed appreciably, and the 1%–2% amino acid sequence variation appears not to have resulted in a dramatic biologic alteration.

In conclusion, we have developed sensitive RT-PCR assays that can detect EBO-Z and EBO-R viruses (and filoviruses in general) in tissues and body fluids of infected humans and monkeys. Analysis of the recently emerged forms of these 2 EBO virus species have shown that they have not changed appreciably over the years, and since the natural reservoirs for these viruses remain unknown, it is uncertain whether they represent related or separately evolving lineages of EBO-Z and EBO-R species. As genetic information on filoviruses accumulates, however, the evolution of these mysterious agents should become more clear. The role of the SGP and GP molecules in the pathogenesis of EHF will also be an important area of investigation, and we are currently producing reagents for the development of sensitive serologic assays capable of detecting and discriminating between these GPs. These assays will not only be important in pathogenesis studies but may also be important diagnostic tools.

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Acknowledgments

We thank M. Tamfun and G. van der Groen for providing human specimens from Kikwit, DRC, and N. Miranda and A. Calor for their assistance in obtaining monkey specimens from the Philippines.

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  1. J Infect Dis. (1999) 179 (Supplement 1): S164-S169. doi: 10.1086/514282
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