Journal of Clinical Microbiology, January 2001, p. 1-7, Vol. 39, No. 1
Special Pathogens Laboratory, Department of
Virology 1, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan,1 and
Special Pathogens Branch, National Center for Infectious
Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia
303332
Received 3 May 2000/Returned for modification 16 August
2000/Accepted 16 October 2000
The full-length nucleoprotein (NP) of Ebola virus (EBO) was
expressed as a His-tagged recombinant protein (His-EBO-NP) by a
baculovirus system. Carboxy-terminal halves of NPs of EBO and Marburg
virus (MBG) were expressed as glutathione
S-transferase-tagged recombinant proteins in an
Escherichia coli system. The antigenic regions on the NPs
of EBO and MBG were determined by both Western blotting and
enzyme-linked immunosorbent assay (ELISA) to be located on the
C-terminal halves. The C-terminal 110 and 102 amino acids of the NPs of
EBO and MBG, respectively, possess strong antigenicity. The full-length
NP of EBO was strongly expressed in insect cells upon infection with
the recombinant baculovirus, while expression of the full-length NP of
MBG was weak. We developed an immunoglobulin G (IgG) ELISA using
His-EBO-NP and the C-terminal halves of the NPs of EBO and MBG as
antigens. We evaluated the IgG ELISA for the ability to detect IgG
antibodies to EBO and MBG, using human sera collected from EBO and MBG
patients. The IgG ELISA with the recombinant NPs showed high
sensitivity and specificity in detecting EBO and MBG antibodies. The
results indicate that ELISA systems prepared with the recombinant NPs
of EBO and MBG are valuable tools for the diagnosis of EBO and MBG
infections and for seroepidemiological field studies.
The two members of the family
Filoviridae, Ebola and Marburg viruses (EBO and MBG,
respectively), are responsible for severe forms of hemorrhagic fevers.
The first recognized outbreaks of Ebola hemorrhagic fever occurred in
Zaire and Sudan in 1976 (3, 7, 22, 23). After the
discovery of EBO in 1976, several African countries were struck by
outbreaks of Ebola hemorrhagic fevers caused by one of the three known
human-pathogenic EBO subtypes: Zaire (EBO-Z), Sudan (EBO-S), or
Côte d'Ivoire (EBO-CI) (13-15). Another outbreak
of Ebola hemorrhagic fever caused by EBO (Reston subtype [EBO-R])
occurred among captured cynomolgus macaques in the Philippines in 1989 (5). EBO-R was carried from the Philippines to the United
States by infected monkeys in 1989, 1990, and 1996, as well as to Italy
in 1992 (13). MBG was first identified in the outbreaks of
hemorrhagic disease in Germany and Yugoslavia in 1967 among technicians
and animal handlers who worked with vervet monkeys
(Cercopithecus aethiops) imported from Uganda or with
tissues from these monkeys (19, 20). Since then, there have been sporadic cases of hemorrhagic fever due to MBG infection on
three occasions (in 1975 in South Africa and Zimbabwe; in 1980 and 1987 in Kenya) (4). Recently, a relatively large outbreak of
MBG infections has occurred in the Durba region of the Democratic Republic of the Congo since 1998 (1, 24).
As EBO-R was introduced from the Philippines to the United States and
MBG was introduced from Uganda to Europe, there is always a possibility
that the deadly hemorrhagic fever viruses could be introduced to areas
previously free from outbreaks. Therefore, preparation of diagnostic
materials for EBO and MBG infections is important even in countries
without outbreaks of Ebola or Marburg hemorrhagic fevers. However, EBO
and MBG must be handled in a biosafety level 4 (BSL-4) facility. This
restriction makes it difficult to prepare diagnostic materials for EBO
and MBG infections. To overcome this difficulty, we developed
enzyme-linked immunosorbent assays (ELISA) using recombinant filovirus
nucleoproteins (NPs) to detect immunoglobulin G (IgG) antibodies to EBO
and MBG. We demonstrated that the ELISA had high sensitivity and
specificity for detection of EBO and MBG antibodies. Thus, our ELISA
systems are useful for diagnosis and epidemiological studies.
Recombinant transfer vector.
An entire cDNA clone of EBO-Z
NP was supplied by the Special Pathogens Branch, Centers for Disease
Control and Prevention (CDC), Atlanta, Ga. (18). An entire
cDNA clone of MBG NP was provided by H.-D. Klenk, Phillips University,
Marburg, Germany (2). The DNA of EBO NP was amplified by
PCR from the source using primers EBO (Z) NP/F
(5'-CAAGGATCCGAGTATGGATTCTCG-3') and EBO (Z)
NP/R (5'-ATGGATCCATGCTCATTCACTGATG-3') (the
BamHI site is underlined). The amplification conditions were
as reported previously (17). The amplified DNA of the
2.2-kbp fragment was subcloned into the BamHI site of pQE31
vector DNA (QIAGEN GmbH, Hilden, Germany) to construct pQE31-EBO-NP.
The inserted EBO NP DNA was sequenced and confirmed to be identical to
the original sequence in order to exclude PCR errors. The DNA fragment
of EBO NP with a histidine (His) tag was isolated from plasmid
pQE31-EBO-NP by digestion of the plasmid with EcoRI and
Hind III. Then it was repaired for blunting using Klenow
enzyme and was ligated into pAcYM1 (12). The resultant
recombinant transfer vector with the correct orientation to the
promoter was designated pAcYM1-His-EBO-NP.
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.1-7.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Enzyme-Linked Immunosorbent Assays for Detection of
Antibodies to Ebola and Marburg Viruses Using Recombinant
Nucleoproteins
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Sera and plasma.
The sera and plasma used in the study are
summarized in Table 1. Of 26 anti-EBO
serum samples, 14 were collected from EBO-infected patients in the
Democratic Republic of the Congo (formerly called Zaire) in 1976 (7) and 1995 (14, 15). Serum samples were also collected from animals infected with EBO-Z, EBO-CI, EBO-S, or
EBO-R and from rabbits immunized with the purified recombinant NP of
EBO-Z (His-EBO-NP).
|
Generation of recombinant baculoviruses.
High five
(Tn5) insect cells were transfected with mixtures of
purified Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA and the recombinant pAcYM1 vector
(pAcYM1-His-EBO-NP or pAcYM1-His-MBG-NP) by the procedures described by
Kitts et al. (8), with the modifications of Matsuura et
al. (12), resulting in the production of recombinant
baculoviruses. The baculoviruses, which express His-EBO-NP and
His-MBG-NP, were designated Ac-His-EBO-NP and Ac-His-MBG-NP,
respectively. A baculovirus (Ac-
P) which lacks polyhedrin expression
was used as a negative-control virus.
Expression of whole NPs of EBO and MBG. The recombinant baculoviruses were grown in Tn5 cells as reported previously (12). Tn5 insect cells, which were infected with Ac-His-EBO-NP or with Ac-His-MBG-NP, were incubated at 26°C for 72 h. Then the cells were washed twice with phosphate-buffered saline (PBS) and were lysed in cold 1% NP-40-PBS solution. The cell lysate was centrifuged at 12,000 rpm at 4°C for 10 min. The supernatant fraction was collected as a source of recombinant His-EBO-NP for purification. The His-EBO-NP was purified with a Ni2+-resin purification system (QIAGEN GmbH) according to the manufacturer's instructions. The expression of recombinant His-EBO-NP and His-MBG-NP was analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (11) gels (10% polyacrylamide) stained with Coomassie blue and was confirmed by Western blot analysis using anti-His antibody (RGS-His Antibody; QIAGEN GmbH) (17).
Expression of truncated NPs.
It was found by a preliminary
experiment that the C-terminal halves of EBO NP and MBG NP possessed
strong antigenic activities. So the C-terminal halves of EBO NP and MBG
NP were each divided into four fragments as shown in Fig.
1. The DNA corresponding to each
truncated NP fragment was amplified with the primer sets designed. Each
amplified DNA was subcloned into the cloning site of plasmid pGEX-2T
(Amersham Pharmacia Biotech, Tokyo, Japan). Each insert was sequenced
and was confirmed to be identical to the original sequence. The
glutathione S-transferase (GST)-tagged truncated NPs of EBO
and MBG, and GST alone, were expressed in an Escherichia
coli (BL21 strain) system and were purified using glutathione
Sepharose 4B (Amersham Pharmacia Biotech).
|
Western blotting. The recombinant NP fragments (His-EBO-NP, GST-EBO-NP/C-half, and GST-EBO-NP5 to -NP8; His-MBG-NP, GST-MBG-NP/C-half, and GST-MBG-NP5 to -NP8; GST) were tested for reactivity to the serum samples in Table 1 by Western blotting (17).
ELISA. ELISA was performed as described previously (10) except for the antigen preparation. For the IgG ELISA to detect antibodies to EBO and MBG in human sera, ELISA plates were coated with a predetermined optimal quantity of recombinant NP antigens (usually 25 to 50 ng/well) at 4°C overnight. The plates were inoculated with test samples (100 µl/well), which were diluted fourfold from 1:100 to 1:6,400 with 0.05% Tween 20-PBS containing 5% skim milk (T-PBS-M). The adjusted optical density (OD) measured at 410 nm was calculated by subtracting the OD of the noncoated well from that of the corresponding His-EBO-NP antigen-coated well. In experiments where GST-tagged recombinant NP fragments were used, the adjusted OD was calculated by subtracting the OD of the GST-coated well from that of the corresponding well. The means and standard deviations (SD) of the adjusted ODs were calculated from those of the 48 control sera. The cutoff value for the assay was set at the mean plus 3 SD.
Antigenic index. We defined the term "antigenic index" to compare the degrees of antigenic activity among the truncated and GST-tagged NP fragments. First, ELISA plates were coated with the GST-tagged truncated NPs (GST-EBO-NP/C-half, EBO-NP5 to -NP8, GST-MBG-NP/C-half, MBG-NP5 to -NP8, and GST) at 4°C overnight to express the equal antigenic activities of GST in ELISA using anti-GST goat serum (1:1,000; Amersham Pharmacia Biotech). Then ELISA was performed against the GST-tagged NP fragments of EBO and MBG n each test sample. The plates were inoculated with each test sample (100 µl/ml) diluted with T-PBS-M at twofold dilutions of 1:500 through 1:64,000 (see Table 2).
Examples of the calculation of the antigenic index are shown in Table 2. The antigenic index was calculated as the maximum dilution at which the OD was over the threshold divided by the dilution level at the threshold. The threshold was defined as the OD of the GST-coated well at a given dilution (Table 2). Thus, the higher the antigenic index, the stronger the antigenic activity. In every experiment, anti-GST goat serum (1:1,000; Amersham Pharmacia Biotech) was tested to confirm the equality of antigenic activity of GST among the GST-tagged NP fragments on the ELISA plates.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Expression and purification of recombinant NPs of EBO and MBG.
Expression of His-EBO-NP and His-MBG-NP in insect cells upon infection
with Ac-His-EBO-NP and Ac-His-MBG-NP, respectively, was demonstrated by
SDS-PAGE analysis (Fig. 2a and c).
Expression of His-EBO-NP was high, whereas expression of His-MBG-NP was
relatively low. Although the expression of His-MBG-NP was weak, it was
demonstrated by Western blotting (Fig. 2b). GST-EBO-NP/C-half,
GST-EBO-NP5 to -NP8, GST-MBG-NP/C-half, and GST-MBG-NP5 to -NP8 were
strongly expressed in E. coli and could be purified (data
not shown).
|
Antigenicities of truncated EBO and MBG NPs determined by Western
blotting.
All the sera reacted to His-EBO-NP in Western blotting
(Table 3). Serum sample E19 reacted to
GST and was excluded for the next evaluation. The remaining 25 EBO
serum samples reacted to GST-EBO-NP/C-half, and 24 of these (all but
E9) reacted to GST-EBO-NP8. Nineteen, 19, and 6 of 25 tested sera
reacted to GST-EBO-NP5, -NP6, and -NP7, respectively. These results
suggest that whole NP of EBO (His-EBO-NP), GST-EBO-NP/C-half, and
GST-EBO-NP8 are promising candidates for antigens in an EBO antibody
detection system.
|
|
Antigenicities of the truncated NP fragments determined by ELISA. All the EBO sera reacted to GST-EBO-NP/C-half in ELISA, with antigenic indices of 4 to >128 (Table 3). Furthermore, the antigenic indices of GST-EBO-NP8 to all EBO sera were equal to or greater than 2. All the EBO sera reacted to GST-EBO-NP8 in ELISA. Although the E9 serum did not react to GST-EBO-NP8 in Western blotting, it reacted to GST-EBO-NP8 in ELISA with an antigenic index of 16. The average of the log2 antigenic indices for each fragment is shown in Table 3. Based on these results, we conclude that GST-EBO-NP/C-half and GST-EBO-NP8 possess the highest antigenicities followed by GST-EBO-NP6, GST-EBO-NP5, and GST-EBO-NP7.
Similar results were obtained using MBG NP fragments (Table 4). All the MBG sera reacted to GST-MBG-NP/C-half and -NP8 with, antigenic indices from 8 to >128. According to the average log2 antigenic indices, GST-MBG-NP/C-half and GST-MBG-NP8 possess the highest antigenicities among the truncated MBG NP fragments.IgG ELISA for EBO antibody detection using His-EBO-NP,
GST-EBO-NP/C-half, and GST-EBO-NP8.
The mean and SD of ODs of the
48 control sera at a 1:400 dilution in ELISA using His-EBO-NP as an
antigen were 
0.004 and 0.120, respectively, resulting in a mean plus
3 SD of 0.356. According to this threshold, 13 of 14 EBO patients'
serum samples were EBO antibody positive, giving an IgG ELISA
sensitivity of 93% (Fig. 3a).
Furthermore, 50 of 51 control sera, including 3 MBG sera, were judged
negative, resulting in 98% specificity.
|
IgG ELISA for MBG antibody detection using GST-MBG-NP/C-half.
The mean and SD of ODs of the control sera at a 1:400 dilution in ELISA
using GST-MBG-NP/C-half were 0.005 and 0.045, respectively. The
threshold was set at 0.135 (the value of the mean plus 3 SD). According
to this threshold, three MBG-positive sera (M1, M2, and M3) were judged
MBG antibody positive and 62 control sera, including 14 EBO patients'
sera, were judged MBG antibody negative (Fig.
4).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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So far, ELISA and indirect immunofluorescent methods using cells infected with EBO have been developed and used for detection of EBO-specific antibody (6, 10, 21). These methods require preparation of antigens by handling live EBO and MBG. In order to make it possible to detect antibodies to EBO and MBG without handling the live viruses, we tried to develop an IgG ELISA using recombinant EBO NP and MBG NP.
We first attempted to understand the antigenicities of the recombinant EBO and MBG NPs. We revealed that EBO-NP8 and MBG-NP8, representing the C-terminal portion of NP, had the highest antigenicities by both Western blotting and ELISA. Furthermore, the C-terminal halves of the EBO and MBG NPs showed high levels of antigenicity, suggesting that these regions can be used as antigens for detection of EBO or MBG antibody. This report is the first demonstration of the antigenic regions within the NPs of filoviruses.
We evaluated the efficacy of an ELISA prepared with purified His-EBO-NP, GST-EBO-NP/C-half, or GST-EBO-NP8 as the antigen. The ODs of EBO sera, which were collected from EBO patients, by ELISA using His-EBO-NP and GST-EBO-NP/C-half at dilutions of 1:400, 1:1,600, and 1:6400 are shown in Fig. 3. We eventually set the threshold at the mean plus 3 SD of ODs of control sera tested at a 1:400 dilution. With this threshold, the ELISA system using these two recombinant EBO NPs has high sensitivity and specificity. One EBO patient's serum was judged negative in ELISA both with His-EBO-NP and with GST-EBO-NP/C-half. We included this serum as a positive-control serum because it was collected from an EBO-infected patient. Although data are not shown here, IgG antibody to EBO was detected in this serum by an indirect immunofluorescent method using recombinant EBO NP-expressing HeLa cells (submitted for publication).
Because the expression of His-MBG-NP was low in insect cells (Fig. 2), we evaluated GST-MBG-NP/C-half as an antigen for ELISA instead. Unfortunately, the sensitivity of IgG ELISA using GST-MBG-NP/C-half could not be evaluated in this study because the number of MBG antibody-positive sera was so small. Further study is needed to evaluate accurately the sensitivity of this ELISA system. However, all the MBG patients' sera tested MBG antibody positive. Furthermore, the specificity of the IgG ELISA was 100%. These results suggest that the ELISA may be useful not only for diagnosis of surviving MBG-infected patients but also for epidemiological field studies.
To determine the antigenic regions on the EBO NP, 26 sera, which contained IgG antibody to EBO-Z, EBO-S, EBO-CI, or EBO-R, were used. The IgG antibody induced by infection with EBO-S, EBO-CI, or EBO-R reacted to the recombinant EBO NP, His-EBO-NP, which was derived from EBO-Z, indicating that the recombinant EBO NP from EBO-Z can be used to detect IgG antibody not only to EBO-Z but also to other EBO subtypes.
Recently, Prehaud et al. reported the usefulness of the recombinant EBO NP and glycoprotein (Gabon 94 strain) for IgG and IgM antibody detection (16). They studied the efficacy of the recombinant EBO NP and glycoprotein expressed in an E. coli system using seven sera collected from EBO-infected patients.
Ksiazek et al. reported the limitation of EBO IgG detection for the diagnosis of EBO infection (9). Some patients with EBO infections died before the EBO IgG response. For the accurate diagnosis of acute EBO and MBG infections, some methods of detecting virus are necessary: antigen ELISA, reverse transcription-PCR, or isolation of infectious agents. IgM antibodies can also be useful for inferring recent onset of the immune response. Thus, other diagnostic systems, such as a filovirus antigen capture ELISA and an IgM capture ELISA using recombinant NPs of filoviruses, are under construction in our laboratory.
In conclusion, we have developed ELISA systems to detect IgG antibodies against filoviruses using recombinant NPs. These ELISA systems can be used for anti-filovirus antibody detection in a facility without a BSL-4 laboratory.
| |
ACKNOWLEDGMENTS |
|---|
We thank H.-D. Klenk, Phillips University, Marburg, Germany, for providing the DNA of MBG NP. We also thank T. Kurata, A. Yamada, and M. Tashiro, National Institute of Infectious Diseases, Tokyo, Japan, for their contributions to the present work, and the staff of the Special Pathogens Branch, National Center for Infectious Diseases, CDC, for their help.
This work is partly supported by a grant-in-aid from the Ministry of Health and Welfare of Japan.
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FOOTNOTES |
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* Corresponding author. Mailing address: Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Gakuen 4-7-1, Musashimurayama, Tokyo 208-0011, Japan. Phone: 81-42-561-0771, ext. 791. Fax: 81-42-564-4881. E-mail: morikawa{at}nih.go.jp.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Clinical Microbiology, January 2001, p. 1-7, Vol. 39, No. 1
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.1.1-7.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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