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VIROLOGICAL STUDIES ON A CASE OF EBOLA VIRUS INFECTION IN MAN AND IN MONKEYS

E.T.W. BOWEN (1), G. LLOYD (1), G. PLATT (1), L.B. McARDELL (1), P.A. WEBB (2), D.I.H. SIMPSON (3)

1. Microbiological Research Establishment, Porton Down, Salisbury SP4 OJG, Wiltshire, England.
2. Center for Disease Control, Atlanta.
3. London School of Hygiene and Tropical Medicine, London.

INTRODUCTION

On the 5th November 1976 an investigator at the Microbiological Research Establishment accidentally pricked his thumb through a protective rubber glove whilst inoculating guinea pigs with material containing Ebola virus (1,2,3). He developed an illness which was clinically similar to Ebola haemorrhagic fever cases seen during the July to November outbreaks which occurred in the Western Equatorial province of the Sudan and in the adjacent Equateur Region of Zaire (4,5).

Virological investigations were carried out on a blood sample collected about hours after he became feverish six days after the accident. Further studies were also made on specimens of blood collected daily during the acute phase of the illness and on blood, urine, throat swabs, faeces and seminal fluid collected during the convalescent phase. Although monkeys had not been implicated in transmission of the disease in Africa, it was thought important to infect keys experimentally with Ebola virus to determine whether they were susceptitible to infection, to define the pathogenesis of the infection and to determine whether this would provide a useful experimental model for evaluating methods therapy for use in human Ebola virus infection.

RESULTS

Human Studies. The first specimen of blood collected on the 11th November, hours after the patient became feverish was examined by electron microscopy and virus particles were seen which were similar to those of Ebola virus. Guinea pigs which had been inoculated with this blood specimen developed a febrile illness and electronmicroscope examinations of their blood and tissues collected on the fifth and sixth day post inoculation showed particles which were again similar to those of Ebola virus.

Studies on blood collected during the acute phase of the illness showed that highest levels of virus in the blood (10^4.5 guinea pig infectious units/ml) were recorded on the first and second days of the illness. Following treatment with interferon and convalescent serum the level dropped to 10^0.5 guinea pig infectious units/ml and remained at this level until the viraemia was undetectable on the ninth day from onset of illness (table 1). No virus was isolated from throat swabs, faeces and urine collected between days 14 and 27. Ebola virus was, however isolated from specimens of seminal fluid collected on days 39 and 61. Samples of seminal fluid collected on days 76, 92, 110 and 128, and two further specimens collected at three monthly intervals thereafter were negative.

After the infusion of 450 ml of convalescent plasma (fluorescent antibody titre of 1/128-1/256) on the second day of illness antibody levels of 1/16 were recorded in the patient's blood from days three to nine. This increased to 1/32 on day 10 and gradually increased to a fluorescent antibody titre of 1/128 by day 34. The patient was then subjected to plasmaphoresis between 16th and 25th February 1977. A total of seven units of plasma was taken which resulted in the fluorescent antibody level dropping from 1/128 to 1/32. A specimen of blood collected on the 5th May 1977 had a fluorescent antibody titre of 1/16 while a specimen collected on the 9th November almost one year from the onset of illness had a fluorescent antibody titre of 1/8.

Transmission of Ebola virus infection to the monkey. In this patient's case it was not possible to assess the relative effectiveness of either interferon or serotherapy in the treatment of the infection. We therefore considered it important to try to establish a suitable animal experimental model system to evaluate this type of therapy and preliminary studies were carried out in rhesus (Macaca mulatta) and vervet (Cercopithecus aethiops) monkeys.

Virus inoculum. The original source of virus was human acute-phase blood prototype strain E718 from the Zaire outbreak which was sent to us by Professor Pattyn. The virus inoculum was a suspension of guinea pig liver taken during the late febrile stage of the disease in the third guinea pig passage. The monkeys were sedated and inoculated intraperitoneally with 0.4ml of virus suspension and the dose of virus calculated by parallel intraperitoneal titrations in guinea pigs and expressed as guinea pig infectious units (G.P.I.U.)/ml. Monkeys received 10^3 and 10^4 (G.P.I.U.).

Clinical Observations. Monkeys became febrile on the third day after infection with temperatures ranging from 40ºC - 40.6ºC. (table 2). The pyrexia persisted until the terminal stage of the infection when the temperature became subnormal. Maculo-papular skin rashes involving the forehead and face, the medial aspects of the fore and hind limbs and the chest developed in all rhesus monkeys between the 4th and 5th day, fading slightly before death which occurred between the 5th to 8th post-inoculation day. No animal survived the infection but neither of the two vervet monkeys infected developed skin rashes. Two of the monkeys had diarrhoea and all monkeys lost about 10 per cent body weight. Virus was first detected in the blood on the second day reaching maximum virus titres of 10 5.5 to 10 6.5 /ml on the 4th and 5th day.

Necropsy was carried out on all monkeys shortly after death and various tissues removed for virological studies and histopathology. Virus infectivity titrations showed high titres of Ebola virus in most of the organ tissues examined. It was not possible to determine whether some of these organs contained Ebola virus because of the high concentration of virus present in the blood. Further details of the virological studies and histopathology will be published elsewhere.

TABLE 1


Day of Sample and Test from Onset of illness


Details & Remarks


Activity of Circulating Antibody as F.A. Titre

Recovery of Infective Virus as Guinea Pig I.P. Infective Unitsper ml or gram of Sample Tested


Positive


Negative


1


-


-


Blood, 10^4.5

 

2


Before transfusion of 450ml convalesc. Plasma


Lte 1/2


Blood, 10^4.5

 

3


11am, 6pm, 11pm


1/16


Blood, 10^0.5

 

4


-


1/16


Blood, 10^0.5

 

5


-


1/16


Blood, 10^0.5

 

6


-


1/16


Blood, 10^0.5

 

7


am pm


1/16


Blood, 10^0.5

 

8


-


1/8


Blood, 10^0.5

 

9


-


1/16

 

Blood


10, 11, 12, 13


-


1/32

 

Blood


14, 16, 20


-


1/64

 

Blood, Faeces, Urine and Throat Swab


23, 27


-


Not done

 

Blood, Faeces, Urine and Throat Swab


34


-


1/128

 

Blood


39


-


Not done


Seminal Fluid 10^0.5

 

61


-


1/128


Seminal Fluid 10^0.5


Blood, Urine


76


-


1/128

 

Blood, Urine, Seminal Fluid


92, 110, 128, 219, 310


-


Not done

 

Urine, Seminal Fluid


TABLE 2


RECTAL TEMPERATURE (ºC) AND VIRUS CONCENTRATION IN THE BLOOD (ML)

Monkey

Dose

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Day 7

Day 8

Total Weight Loss

Rhesus 6
Wt
3.686 kg

10^4

38.6
10^0.5

38.9
10^2.5

40.4
10^4.5

40.0
10^5.5

Moribund
34.5
10^4.5

     

255 g

Rhesus 7
Wt
3.629 kg

10^4

38.1
10^0.5

38.7
10^2.5

40.2
10^4.5

39.8
10^6.5

39.5
10^5.5

Moribund
34.5
10^5.5

   

340 g

Rhesus12
Wt
3.289 kg

10^3

38.8
10^0.5

38.8
10^2.5

39.6
10^4

40.6
10^5

39.8
10^5

39.6
10^6.5

39.8
10^6.0

Found Dead

396 g

Vervet 1

10^4

38.6
10^05

38.9
10^2

39.6
10^4.5

40.2
10^6

39.7
10^6

Moribund
34.5
10^5.5

   

N.D.

Vervet 3

10^4

38.2
10^0.5

38.9
10^1.5

40.1
10^4

40.6
10^6.5

39.7
10^6.5

Killed
36.1
10^5.5

   

N.D.

DISCUSSION

Details of the clinical illness in man following accidental laboratory infection with Ebola virus has been described fully by Emond et al. 5 . Virus was detected early in the disease, 10^4.5 G.P.I.U./ml being detected on the first day of illness. No detectable change in the levels of circulating virus was evident on the day following initiation of interferon therapy. However within twelve hours of administering 450ml of Ebola immune plasma the viraemia level had fallen to 10^0.5 G.P.I.U./ml. This much reduced level of circulating virus persisted throughout the acute stages of illness and virus became undetectable on the 9th day of illness.

Emond et al. (5) were unable to draw any definite conclusions as to the value of either interferon or immunotherapy. As both were administered together their respective merits cannot be assessed. There is no doubt that viraemia levels were dramatically reduced soon after the administration of immune plasma, but the patient's clinical condition deteriorated despite the low virus levels in the blood.

The discovery of virus in the semen was not unexpected since Marburg virus is known to persist in seminal fluid for several weeks after infection (6,7). Marburg virus has also been recovered from the anterior chamber of the eye two months after the onset of illness (8).

The disease produced in rhesus, vervet and squirrel (Saimiri sciureus) monkeys following infection with Marburg virus closely paralleled the illnesses caused in man (9). Fever, severe weight loss, anorexia, skin rash and haemorrhage were common factors. Ebola virus similarly produces an illness in monkeys which resembles the disease in man. The rash in rhesus monkeys is very much more marked than in vervets which often have no obvious rash at all. As with Marburg, Ebola causes a uniformly fatal illness in monkeys and this animal is considered to be eminently suitable for studying the pathogenesis and possible treatment of these unique virus infections in more detail.

SUMMARY

Ebola virus was detected in high titre in the blood of a patient at the onset of illness. Administration of interferon had no immediate effect on circulating virus levels but the additional administration of immune plasma reduced circulating virus levels dramatically within 12 hours. Virus was detected in seminal fluid for up to 61 days after the onset of illness.

Experimental infection of rhesus and vervet monkeys produced a uniformly fatal illness. The course of disease resembled that found in man with weight loss, anorexia, fever haemorrhages and skin rash being frequently seen. Viraemia was obvious within two days of infection and persisted until death which occurred between days 5 and 8.

ACKNOWLEDGEMENTS

We acknowledge with appreciation some financial support from the Wellcome Trust and from the World Health Organization.

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, i, 569.
2. Bowen, E.T.W., Platt, G.S., Lloyd, G., Baskerville, A., Harris, W.J., Vella, E.E. (1977) Viral haemorrhagic fever in southern Sudan and northern Zaire. Preliminary studies on the aetiological agent. Lancet, i, 571.
3. Pattyn, S., Jacob, J., Van der Groen, G., Piot, P., Courteille, G. (1977) Isolation of Marburg-like virus from a case of haemorrhagic fever in Zaire Lancet, i, 573.
4. World Health Organization (1976) Viral haemorrhagic fever. Weekly Epidemiological Record N' 51, 325.
5. Emond, P.T.D., Brandon Evans, Bowen, E.T.W., Lloyd, G. (1977) A case of Ebola virus infection. Brit. Med. J., 2, 541.
6. Martini, G.A., Schmidt, H.E. (1968) Spermatogene Ubertragung des "Virus Marburg" (Erreger der "Marburger Affenkrankheit") Klin. Wschr., 46, 398.
7. Martini, G.A. (1969) Marburg agent disease: in man. Trans. Roy. Soc. Trop. Med. Hyg. 63, 295.
8. Gear, J.S.S., et al. (1975) Outbreak of Marburg virus disease in Johannesburg Brit. Med. J., 4, 489.
9. Simpson. D.I.H. (1969) Marbu-g agent disease: in monkeys. Trans. Roy. Soc. Trop. Med. Hyg. 63, 303.
DISCUSSION
P . Brès : Mr. Bowen Just mentioned that in the monkey experiments no virus was found in the bile nor in the feces. I think this is of enormous importance for future control measures in human cases. Would Mr. Bowen give us more details and tell how many samples were examined ?
E.T.W. Bowen : Only three fecal samples were examined, we would like to do a few more before we would be categoric about stating that the virus was not excreted in the feces.
P. Brès : Was the blood on the skin lesions also positive ?
E.T.W. Bowen : We did not take any blood from the skin lesions.
J.G. Breman : I am very interested in seeing the high virus titer in the pancreas of the monkeys, this correlates with our clinical impression that the patients were suffering from pancreatitis. This also correlates with the German observations and one observation in South Africa where a prolonged amylasemia and amylasuria was observed. I would like to know if there are related observations in autopsy studies. Secondly, the lung positives I think are also very important. You will recall from the clinical presentation that in the Sudan there were more predominant pulmonary symptoms than in Zaire and this then may mean that this was a very important mode of spread.
J. Casals : Dr. Webb, you have shown in your serological crosstesting a systematic, consistent difference between the homologous and heterologous titers observed between the viruses in Sudan and those in Zaire. I don't know whether in your own mind that constitutes enough of a difference to talk about different strains or varieties of the same virus. In that case we would have to consider the two epidemics as two separate outbreaks of similar diseases.
P. A. Webb : I think one should remember that the fluorescence test after all is qualitative and not really quantitative, based on interpretation of degrees of fluorescence. I think that until we have a neutralization test, we can't say.
E.T.W. Bowen : We have done cross protection tests in guinea-pigs, the viruses certainly crossprotect.
C.E. Gordon Smith : You will recall in the 1967 Marburg outbreak, there was one case with seminal infection and transmission; it must have been at a rate of something like 1 in 20 and here we have one out of one studied. We also, in the South African case, had a persistent infection of the eye. Someone is going to have to explain why no such persistent infections have led to further outbreaks of the disease in the Sudan or Zaire.

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