Immunofluorescence Method for Detection of Ebola Virus Immunoglobulin G, Using HeLa Cells Which Express Recombinant Nucleoprotein

The Ebola virus (EBO) is responsible for severe forms of hemorrhagic fever. The first EBO outbreak was recognized in Zaire and Sudan in 1976 (2, 9, 18, 19). Several western African countries were later struck by EBO outbreaks caused by one of the three known human-pathogenic EBO subtypes (1, 3, 4, 10, 12, 14a). Another EBO (Reston subtype [EBO-R]) outbreak occurred among captured cynomolgus monkeys (Macaca fascicularis) in the Philippines (6, 14, 15). EBO-R was imported from the Philippines to the United States by EBO-R-infected monkeys in 1989, 1990, and 1996 and was also imported to Italy in 1992 (6, 15).

We developed an indirect immunofluorescence (IF) method for the detection of immunoglobulin G (IgG) antibody to EBO, using HeLa cells expressing recombinant EBO (rEBO) nucleoprotein (NP) by gene transfer with a baculovirus system.

The baculovirus AcCMV-EBO-NP was constructed as follows. PCR was performed to add a BamHI site at each extremity of the NP gene of EBO subtype Zaire (16) using the primers EBO(Z)NP/F (5′-CAAGGATCCGAGTATGGATTCTCG-3′; theBamHI restriction site is underlined) and EBO(Z)NP/R (5′-ATGGATCCATGCTCATTCACTGATG-3′). The amplified DNA was digested with BamHI, purified, and subcloned into the BamHI site of pAcYM1CMV (17), resulting in the production of the recombinant transfer plasmid pAcYM1CMV-EBO-NP, which contains the DNA of EBO NP under the control of the cytomegalovirus immediate-early (CMV-IE) promoter. It was identified as having the correct orientation of EBO NP DNA relative to the promoter by restriction mapping. The entire insert was sequenced and was confirmed to be identical to that of the original cDNA. Tn5 cells were transfected with mixtures of purified AcNPV DNA and the transfer plasmid (11, 13), resulting in the production of the novel recombinant baculovirus AcCMV-EBO-NP. This baculovirus was expected to express EBO NP under the control of the CMV-IE promoter in mammalian cells upon abortive infection with the virus (5, 17). Ac-ΔP, a baculovirus which lacks the polyhedrin gene, was used as a negative control virus. These recombinant baculoviruses were grown in Tn5 cells as reported previously (13).

HeLa cells were infected with AcCMV-EBO-NP at a multiplicity of infection of 20 PFU/cell. The cells were incubated for 48 h in Dulbecco's minimal essential medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics. Then, the cells were harvested by trypsinization at 48 h postinfection, washed with phosphate-buffered saline (PBS), spotted on 14-well HT-Coated slide glasses (AR Brown Co., Ltd., Tokyo, Japan), air dried, and fixed with acetone at room temperature for 5 min. The slides (rEBO NP slides) were stored at −70°C until use. Negative-antigen slides were prepared similarly using Ac-ΔP-infected HeLa cells. The spot slides were thawed and dried before use.

For the IF test, twofold serial dilutions (1:25 to 1:102,400) of test sera were placed on both rEBO NP slides and negative slides, and the slides were incubated under humidified conditions at 37°C for 1 h. After a washing with PBS, the antigens were reacted with fluorescein isothiocyanate (FITC)-conjugated goat anti-human IgG antibody (1:50; Kirkegaard & Perry, Gaithersburg, Md.) and with FITC-conjugated goat anti-rabbit IgG antibody (1:50; Kirkegaard & Perry), when human and monkey sera and rabbit serum were tested, respectively. After a washing with PBS, the slides were examined for the staining pattern under a fluorescent microscope (Zeiss, Oberkochen, Germany) with appropriate barrier and excitation filters for FITC visualization. Positive and negative controls were included with each assay. The titers of tested samples were recorded as the reciprocals of the highest dilutions producing positive results.

To confirm the expression of rEBO NP in HeLa cells, an rEBO NP slide was stained with anti-EBO NP rabbit serum, which was raised against purified His-tagged rEBO NP. The AcCMV-EBO-NP-infected HeLa cells showed a granular staining pattern (Fig.1a), while the Ac-ΔP-infected HeLa cells did not show any staining (data not shown).

• Open in new tab
• Download powerpoint

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-expressing HeLa cells with serum collected from a patient with EBO infection in the convalescent phase. (c) Positive IF staining with serum collected from an EBO-R-infected monkey.

Sera collected from 14 patients with EBO subtype Zaire infections (4 in the 1976 outbreak, 1 in the 1977 outbreak, and 9 in the 1995 outbreak of EBO Zaire) in the convalescent phase and 48 sera from subjects in West African countries without a history of EBO infection were tested by the IF test. All 14 patients' sera showed positive staining with a granular pattern (Fig. 1b). The Ac-ΔP-infected HeLa cells (negative control) did not show any staining (data not shown). The IgG antibody titers determined by the IF system in these human sera ranged from 1:25 to 1:25,600 (Fig. 2). The negative slides were not stained by any of these patients' sera. Among the 48 sera from subjects without a history of EBO infections, one showed positive staining at a dilution of 1:100 (Fig. 2). We could not rule out the possibility that this serum had IgG antibodies specific to EBO. Even if the positive reaction by 1 of the 48 control sera was nonspecific, the IF system using rEBO NP-expressing HeLa cells has high efficacy in detecting EBO-specific IgG, with 100% sensitivity and 98% specificity. The number of serum samples used for the evaluation of this IF system was small; these results indicate the usefulness of this IF method for detection of EBO-specific IgG antibody.

• Open in new tab
• Download powerpoint

Fig. 2.

Titers of IgG antibody to rEBO NP in sera collected from 14 EBO-infected patients at the convalescent phase and in 48 control sera. 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 by the IF method at a titer of 1:100; the other 47 were negative by the IF method.

We wondered whether this IF system could also detect the EBO-R-specific IgG antibody, though the rEBO NP was derived from EBO subtype Zaire. To address this, sera collected from two EBO-R-infected cynomolgus monkeys (M. fascicularis) in an outbreak in 1996 (14) were tested by the IF method for the presence of EBO IgG antibody. Both sera showed typical positive staining as shown in Fig. 1c at a dilution of 1:25, while they showed no background staining with the negative slides (data not shown). The results suggest that the IgG antibody to EBO-R can be detected by the IF method using rEBO NP derived from EBO subtype Zaire.

Hofmann et al. (5) and Shoji et al. (17) reported that the novel baculovirus vector with the CMV-IE promoter could efficiently deliver and express foreign genes in mammalian cells. We confirmed that rEBO NP was efficiently expressed by gene transfer with the recombinant baculovirus AcCMV-EBO-NP in several mammalian cell lines. Among them, rEBO NP-expressing HeLa cells exhibited a unique staining pattern with anti-EBO NP rabbit serum (Fig. 1a). No cytopathic effect was observed in AcCMV-EBO-NP-inoculated HeLa cells because the virus infected the mammalian cells abortively, while EBO-infected cells showed a strong cytopathic effect which sometimes causes nonspecific reactions in the IF test. Because of the unique nature of the baculovirus-HeLa cell system, this IF method is considered efficacious in detecting EBO IgG with high sensitivity and specificity.

In previous reports on the seroepidemiology of EBO, inactivated EBO-infected cells were used as antigens for indirect IF methods (7, 8). Our IF method may offer an attractive alternative to the use of live EBO-infected cells, which should be handled in a biosafety level 4 laboratory.

• Copyright © 2001 American Society for Microbiology