Immunofluorescence Method for Detection of Ebola VirusImmunoglobulin G, Using HeLa Cells Which Express
Recombinant NucleoproteinMASAYUKI SAIJO, MASAHIRO NIIKURA, SHIGERU MORIKAWA,* AND ICHIRO KURANE
Special Pathogens Laboratory, Department of Virology 1, National Institute of Infectious Diseases, Gakuen 4-7-1,Musahimurayama, Tokyo 208-0011, Japan
Received 21 August 2000/Returned for modification 29 September 2000/Accepted 15 November 2000
A novel recombinant baculovirus which expresses Ebola virus (EBO) nucleoprotein (NP) under the controlof the cytomegalovirus immediate-early promoter was constructed. HeLa cells abortively infected with thebaculovirus expressed EBO NP, and this was used as an immunofluorescent (IF) antigen to detect EBOimmunoglobulin G (IgG) antibody. This IF method has high efficacy in detecting EBO IgG antibody in clinicalspecimens, indicating its usefulness in the diagnosis of EBO infections and seroepidemiological studies.
The Ebola virus (EBO) is responsible for severe forms ofhemorrhagic fever. The first EBO outbreak was recognized inZaire and Sudan in 1976 (2, 9, 18, 19). Several western Africancountries were later struck by EBO outbreaks caused by one ofthe three known human-pathogenic EBO subtypes (1, 3, 4, 10,12, 14a). Another EBO (Reston subtype [EBO-R]) outbreakoccurred among captured cynomolgus monkeys (Macaca fas-cicularis) in the Philippines (6, 14, 15). EBO-R was importedfrom the Philippines to the United States by EBO-R-infectedmonkeys in 1989, 1990, and 1996 and was also imported to Italyin 1992 (6, 15).
We developed an indirect immunofluorescence (IF) methodfor the detection of immunoglobulin G (IgG) antibody toEBO, using HeLa cells expressing recombinant EBO (rEBO)nucleoprotein (NP) by gene transfer with a baculovirus system.
The baculovirus AcCMV-EBO-NP was constructed as fol-lows. PCR was performed to add a BamHI site at each extrem-ity of the NP gene of EBO subtype Zaire (16) using the primersEBO(Z)NP/F (59-CAAGGATCCGAGTATGGATTCTCG-39;the BamHI restriction site is underlined) and EBO(Z)NP/R(59-ATGGATCCATGCTCATTCACTGATG-39). The ampli-fied DNA was digested with BamHI, purified, and subclonedinto the BamHI site of pAcYM1CMV (17), resulting in theproduction of the recombinant transfer plasmid pAcYM1CMV-EBO-NP, which contains the DNA of EBO NP under thecontrol of the cytomegalovirus immediate-early (CMV-IE)promoter. It was identified as having the correct orientation ofEBO NP DNA relative to the promoter by restriction mapping.The entire insert was sequenced and was confirmed to beidentical to that of the original cDNA. Tn5 cells were trans-fected with mixtures of purified AcNPV DNA and the transferplasmid (11, 13), resulting in the production of the novel re-combinant baculovirus AcCMV-EBO-NP. This baculoviruswas expected to express EBO NP under the control of the
CMV-IE promoter in mammalian cells upon abortive infectionwith the virus (5, 17). Ac-DP, a baculovirus which lacks thepolyhedrin gene, was used as a negative control virus. Theserecombinant baculoviruses were grown in Tn5 cells as reportedpreviously (13).
HeLa cells were infected with AcCMV-EBO-NP at a mul-tiplicity of infection of 20 PFU/cell. The cells were incubatedfor 48 h in Dulbecco’s minimal essential medium supple-mented with 10% heat-inactivated fetal bovine serum and an-tibiotics. Then, the cells were harvested by trypsinization at48 h postinfection, washed with phosphate-buffered saline(PBS), spotted on 14-well HT-Coated slide glasses (AR BrownCo., Ltd., Tokyo, Japan), air dried, and fixed with acetone atroom temperature for 5 min. The slides (rEBO NP slides) werestored at 270°C until use. Negative-antigen slides were pre-pared similarly using Ac-DP-infected HeLa cells. The spotslides were thawed and dried before use.
For the IF test, twofold serial dilutions (1:25 to 1:102,400) oftest sera were placed on both rEBO NP slides and negativeslides, and the slides were incubated under humidified condi-tions at 37°C for 1 h. After a washing with PBS, the antigenswere reacted with fluorescein isothiocyanate (FITC)-conju-gated goat anti-human IgG antibody (1:50; Kirkegaard &Perry, Gaithersburg, Md.) and with FITC-conjugated goat an-ti-rabbit IgG antibody (1:50; Kirkegaard & Perry), when hu-man and monkey sera and rabbit serum were tested, respec-tively. After a washing with PBS, the slides were examined forthe staining pattern under a fluorescent microscope (Zeiss,Oberkochen, Germany) with appropriate barrier and excita-tion filters for FITC visualization. Positive and negative con-trols were included with each assay. The titers of tested sam-ples were recorded as the reciprocals of the highest dilutionsproducing positive results.
To confirm the expression of rEBO NP in HeLa cells, anrEBO NP slide was stained with anti-EBO NP rabbit serum,which was raised against purified His-tagged rEBO NP. TheAcCMV-EBO-NP-infected HeLa cells showed a granularstaining pattern (Fig. 1a), while the Ac-DP-infected HeLa cellsdid not show any staining (data not shown).
* Corresponding author. Mailing address: Special Pathogens Labo-ratory, Department of Virology 1, National Institute of InfectiousDiseases, Gakuen 4-7-1, Musahimurayama, Tokyo 208-0011, Japan.Phone: 81-42-561-0771 (791). Fax: 81-42-564-4881. E-mail: [email protected].
Sera collected from 14 patients with EBO subtype Zaireinfections (4 in the 1976 outbreak, 1 in the 1977 outbreak, and9 in the 1995 outbreak of EBO Zaire) in the convalescentphase and 48 sera from subjects in West African countrieswithout a history of EBO infection were tested by the IF test.All 14 patients’ sera showed positive staining with a granularpattern (Fig. 1b). The Ac-DP-infected HeLa cells (negativecontrol) did not show any staining (data not shown). The IgG
antibody titers determined by the IF system in these humansera ranged from 1:25 to 1:25,600 (Fig. 2). The negative slideswere not stained by any of these patients’ sera. Among the 48sera from subjects without a history of EBO infections, oneshowed positive staining at a dilution of 1:100 (Fig. 2). Wecould not rule out the possibility that this serum had IgGantibodies specific to EBO. Even if the positive reaction by 1 ofthe 48 control sera was nonspecific, the IF system using rEBONP-expressing HeLa cells has high efficacy in detecting EBO-specific IgG, with 100% sensitivity and 98% specificity. Thenumber of serum samples used for the evaluation of this IFsystem was small; these results indicate the usefulness of thisIF method for detection of EBO-specific IgG antibody.
We wondered whether this IF system could also detect theEBO-R-specific IgG antibody, though the rEBO NP was de-rived from EBO subtype Zaire. To address this, sera collectedfrom two EBO-R-infected cynomolgus monkeys (M. fascicu-laris) in an outbreak in 1996 (14) were tested by the IF methodfor the presence of EBO IgG antibody. Both sera showedtypical positive staining as shown in Fig. 1c at a dilution of 1:25,while they showed no background staining with the negativeslides (data not shown). The results suggest that the IgG an-tibody to EBO-R can be detected by the IF method usingrEBO NP derived from EBO subtype Zaire.
Hofmann et al. (5) and Shoji et al. (17) reported that thenovel baculovirus vector with the CMV-IE promoter couldefficiently deliver and express foreign genes in mammaliancells. We confirmed that rEBO NP was efficiently expressed bygene transfer with the recombinant baculovirus AcCMV-EBO-NP in several mammalian cell lines. Among them, rEBONP-expressing HeLa cells exhibited a unique staining patternwith anti-EBO NP rabbit serum (Fig. 1a). No cytopathic effectwas observed in AcCMV-EBO-NP-inoculated HeLa cells be-cause the virus infected the mammalian cells abortively, whileEBO-infected cells showed a strong cytopathic effect whichsometimes causes nonspecific reactions in the IF test. Becauseof the unique nature of the baculovirus-HeLa cell system, this
FIG. 1. IF staining patterns of rEBO NP-expressing HeLa cells. (a)IF staining pattern of rEBO NP-expressing HeLa cells with the anti-EBO NP rabbit serum. (b) Positive IF staining of rEBO NP-expressingHeLa cells with serum collected from a patient with EBO infection inthe convalescent phase. (c) Positive IF staining with serum collectedfrom an EBO-R-infected monkey.
FIG. 2. Titers of IgG antibody to rEBO NP in sera collected from14 EBO-infected patients at the convalescent phase and in 48 controlsera. All the 14 EBO sera showed a positive reaction by the IF method.Of the 48 control sera, only 1 showed an EBO IgG-positive reaction bythe IF method at a titer of 1:100; the other 47 were negative by the IFmethod.
IF method is considered efficacious in detecting EBO IgG withhigh sensitivity and specificity.
In previous reports on the seroepidemiology of EBO, inac-tivated EBO-infected cells were used as antigens for indirectIF methods (7, 8). Our IF method may offer an attractivealternative to the use of live EBO-infected cells, which shouldbe handled in a biosafety level 4 laboratory.
We thank R. F. Meyer, C. J. Peters, and T. G. Ksiazek, SpecialPathogens Branch, Centers for Disease Control and Prevention, At-lanta, Ga., for providing us with the cDNA clone of EBO and humansera used in this study. We also thank M. E. Miranda, ResearchInstitute of Tropical Medicine, The Philippines, for providing us withsera from EBO-R-infected monkeys.
This study was supported by a grant from the Ministry of Health andWelfare, Japan.
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