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SOME OBSERVATIONS ON THE PROPERTIES OF EBOLA VIRUS

P.A. WEBB, K.M. JOHNSON, H. WULFF, J.V. LANGE
Center for Disease Control, Public Health Service, U.S. Department of Health, Education, and Welfare, Atlanta, Georgia 30333, U.S.A.

Ebola virus, morphologically a twin to Marburg virus in electron micrograph was found to be immunologically distinct by fluorescent antigen and antibody techniques (FA).

The number and variety of observations we have been able to make have been severely limited by restricted facilities in our currently functional Class IV Laboratory. Also, because work was done in occasional moments stolen from our primary task of providing sero-epidemiological support to the field team in Zaire, most of these observations are incomplete.

All attempts in the CDC laboratory to isolate viruses from human material submitted from Zaire have been made in Vero cell cultures. Cytopathic effect (CPE), while not dramatic or diagnostically definitive, nevertheless occurs (1). Harvests of cell cultures which show CPE all contain fluorescent antigen. However, when very small amounts of virus were inoculated, fluorescent antigen was not observed until 9 days later, at which time CPE was not present. This finding indicates that CPE alone cannot be used as an end point to diagnose Ebola virus infection in field samples inoculated into cell cultures.

We had difficulty in producing CF antigen in cell cultures to both Marburg and Ebola viruses. High virus concentrations are required, and since the homologous systems for Marburg showed that FA is far more sensitive than CF (2), we used the FA test exclusively.

Mr. E. Bowen of the Microbiological Research Establishment, Porton, provided us with a Sudanese strain of Ebola virus which was isolated in a guinea pig (GP), and convalescent human sera from Sudan survivors. Results of cross-FA tests on human antisera from the Sudan and Zaire and antigens prepared from Sudan and Zaire Ebola strains are shown in Table 1. In most instances homologous titers were twofold to fourfold higher. The same pattern of antibody response occurred with single-injection GP immune sera as depicted in Table 2.

We tried to determine whether Ebola virus is sensitive to interferon (IF) in vitro in a study carried out in consultation with Dr. T. Merrigan, Stanford University Medical Center, who kindly provided the human IF. A single screening test with various doses of human IF and Vero cell cultures was performed.

TABLE 1
IMMUNOFLUORESCENT ANTIBODIES IN HUMAN SERA, ZAIRE AND SUDAN STRAINS OF EBOLA VIRUS



immunofluorescent antigen


Human Sera


Zaire str.


Sudan str.


Zaire 1


1024 (x)


256


Zaire 2


16


16


Sudan 164


64


256


Sudan 2


16


64


Normal


< 4


< 4


(x) Reciprocal of endpoint serum dilution.

TABLE 2
IMMUNOFLUORESCENT ANTIBODIES IN GUINEA PIG SERA, ZAIRE AND SUDAN STRAINS OF EBOLA VIRUS


 

Zaire Str.


Sudan Str.


Zaire (Guinea Pig)


1


512 (x)


256



2


1024


256


Sudan (Guinea Pig)


1 (xx)


64


64



2


64


256



3


64


128



4


32


128


Marburg (Guinea Pig)

 

4


4


Normal (Guinea Pig)

 

< 4


< 4


(x) Reciprocal of endpoint serum dilution.

(xx) This animal was infected in Porton and its serum was sent to CDC.

Maintenance medium (E-MEM with 2% fetal calf serum) with and without IF was placed on the cultures 24 hours before they were inoculated with virus. Media were removed, and one precalculated virus dose was inoculated into replicate tubes. CPE was used as an end point for VSV-infected cultures and fluorescent antigen for Ebola-virus cultures. Results shown in Table 3 reveal that VSV was 100 times more sensitive than Ebola virus to IF. Obviously these results are not definitive. A more suitable cell culture system for IF assay such as the HR 202 human diploid line should be tried.

TABLE 3
IN VITRO SENSITIVITY OF EBOLA VIRUS TO HUMAN INTERFERON


Virus


Dose


Interferon Dosage and Virus Growth



TCID 50


10


100


1.000


10.000 IU


VSV


50


+


-


-


-


Ebola


10


+


+


+


-


+ = Virus grew. - = Culture protected.

In vivo attempts to challenge Ebola-immune GP's with a high dose of Ebola or Marburg virus failed because we were unable to reliably kill 100% of animals with either agent regardless of dose. However, Ebola-immune GP's challenged with Marburg virus did develop FA titers to both viruses, which is useful for testing unknown field specimens for both agents using a single serum.

We have been unable to inactivate Ebola virus completely in infected Vero cells with ultraviolet light. Usually about 50% of the tubes inoculated with an "inactivated" cell suspension are positive.

Finally, in this parade of negative data, all of our attempts to neutralize Ebola virus with in vitro test systems have so far been unsuccessful. Table 4 summarizes three different approaches. We suspect that such will continue to be the case until we can purify virus suspensions and separate specific proteins to use as antigen or infective material, a procedure which must await the availability of our new maximum containment "suit" laboratory and distant horizons.

SUMMARY

Ebola virus is morphologically identical to and immunologically distinct from Marburg virus. Vero cell cultures have been used to isolate virus, and fluorescent antigen and antibody techniques have been used to confirm isolates and test strain differences. A twofold to fourfold higher titer was seen in the homologous antibody response pattern in sera from convalescent humans and immune GP's between the Zaire and Sudan strain of Ebola virus. VSV was 100 times more sensitive to interferon in vitro than Ebola virus. Preliminary attempts to neutralize Ebola virus have been unsuccessful.

TABLE 4
EBOLA VIRUS - IN VITRO NEUTRALIZATION ATTEMPTS


A


CPE Vero Cell Cultures - Serum Dilution

 

G.P. Immune Serum


Homolog. FA Titer


Serum Dilution

1:4, 1:16 vs 1,000 TCID50

 

Eb-Zaire


1024


No neutralization

 

Eb-Sudan


64

 

Marburg


1024

 

Normal


< 4


B


Fluorescent-Focus Inhibition Test

 

G.P. Immune Serum


Human FA Titer


Serum Dilution

4 - 16 - 64 - 256

 

Eb-Zaire 1


1024


25-30 foci

 

Eb-Zaire 2


256


15 foci

 

Eb-Sudan


64


15-20 foci

 

Normal


< 4


15-20 foci


C


Precipitation of Virus-Serum Complexes with Staphylococcal Protein A.

 

Virus + Diluent + Staph. A

 
 

Virus + Eb. Human + Staph A


CPE Observed in All Cell Cultures at the Same Time.

 

Virus + Normal Human + Staph A

 
 

Virus + Eb. Human + Staph B

 
 

Virus + Diluent Only

 

REFERENCES
1. Johnson, K.M., Webb, P.A., Lange, J.V., Murphy, F.A. (1977) Isolation and partial characterization of a new virus causing acute haemorrhagic fever in Zaire, Lancet, 1, 569-571.
2. Wulff, H., Conrad, J.L. (1977) Marburg virus disease, in Comparative Diagnosis of Viral Diseases, edited by E &C Kurstak, Academic Press, New York, N.Y., p. 29.

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