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THE EFFECT OF INTERFERON ON EXPERIMENTAL EBOLA VIRUS INFECTION IN RHESUS MONKEYS

E.T.W. BOWEN (1), A. BASKERVILLE (1), K. CANTELL (2), G.F. MANN (3), D.I.H. SIMPSON (3), A.J. ZUCKERMAN (3)
1. Microbiological Research Establishment, Porton Down, Salisbury SP4 OJG, Wiltshire, England.
2. Central Public Health Laboratory, Helsinki.
3. London School of Hygiene and Tropical Medicine, London.

INTRODUCTION

The potential use of interferon preparations for the treatment of viral infections is receiving increasing attention, for example for the treatment of recurrent herpes keratitis, infection with herpes viruses in immunosuppressed patients and more recently for persistent infection with hepatitis B virus. Infection with Marburg or with Ebola or with Lassa viruses presents an acute lifethreatening situation. The case of a patient in London with Ebola virus infection who recovered after treatment with human leucocyte interferon and convalescent serum (1) stressed the need to investigate the effect of interferon on the course of this infection in an experimental nonhuman primate model.

The properties and mode of action of interferon. Interferons are small molecular weight glycoproteins which inhibit the replication of a wide range of animal viruses and are released from animal cells after induction. Interferons differ from other less specific inhibitors on the basis of the following criteria: their activity against many unrelated viruses, they are usually species specific but without tissue specificity, interferons are not toxic to cells and act via an intracellular mechanism requiring functional host cell metabolism and finally interferons are usually stable at pH 2. Interferons are not antigenic in the homologous species but specific antibodies can be obtained by immunization of heterologous hosts.

The chemical structure of interferons is not known but all contain polysaccharide residues, although the latter do not play a measurable role in antiviral activity. The human leucocyte interferon is a stable molecule known to contain at least one disulphide bond.

Interferon does not prevent adsorption or cell penetration by the virus but blocks viral replication at some point during assembly. The primary site of action of interferon appears to be on the cell membrane where it interacts with an interferon specific receptor system. The receptor is near the thyrotrophin, gonadotrophin, cholera and diphtheria toxin receptors, and in man it may be controlled by chromosome 21. The receptor system consists of a binding site made of gangliosides and an activator site consisting probably of glycoproteins. An interferon specific modification of the cell membrane components is required as a primary step for antiviral activity. The cells produce an inhibitory protein and other products including a protein kinase, a nuclease and other proteins. The inhibitory protein apparently interferes with the translation of viral mRNA at the ribosome inhibiting the synthesis of virus specific protein.

In addition to the antiviral effect, interferon preparations can decrease cell replication and may inhibit tumour growth, inhibit the replication of intracellular organisms such as rickettsia and protozoa, alter the sensitivity of cells to hormones and toxins, inhibit humoral antibody formation and cell mediated immunity but may enhance phagocytosis and cytotoxicity of T-lymphocytes. Some of these effects may be due to biologically active impurities in the interferon preparations.

Simply stated, therefore, the interferon system is part of the normal host defence against virus infection. Interferon is synthesized in response to infection and actively secreted from the cells. When interferon reacts with the membrane of uninfected cells an antiviral state is activated within the cells. All these properties make the clinical application of interferon particularly attractive for prophylaxis or for treatment early in the course of infection, although it may be effective therapeutically when the pathogenesis of the disease is dependent on slow and continued replication of the virus.

EXPERIMENTAL DESIGN

Animals. These were young adults rhesus monkeys (Macacca mulatta) of either sex weighing between 3 and 4 kgms. The monkeys were anaesthetised by intramuscular injection of ketamine hydrochloride for sampling of blood and for inoculation. Rectal temperature was recorded daily.

Virus inoculum. The virus was the prototype strain E 718 isolated from the patient who died in Zaire from Ebola haemorrhagic fever during the epidemic in 1976. The virus was passaged by intraperitoneal inoculation into DunkinHartley guinea pigs weighing 250 gms. The inoculum was prepared as a 10% suspension in phosphate-buffered saline containing 0.75% bovine albumin from a guinea pig liver at the third passage of the virus. The suspension was diluted and the dose of virus was calculated by titration in guinea pigs and expressed as guinea pig infective units. The monkeys received 10^4 guinea pig infective units in 0.4 ml by intraperitoneal inoculation.

Human leucocyte interferon. Interferon was produced by induction with Sendai virus of leucocytes in suspension culture obtained from buffy coats of human blood donations at the Central Public Health Laboratory, Helsinki. The specific activity of the final preparation was 2x10^6 units/mg protein. The monkeys received 3x10^6 units of interferon intramuscularly once daily.

Treatment schedule. Two of the rhesus monkeys received interferon for 2 days before infection and the treatment was continued daily thereafter. Two monkeys were inoculated with the virus suspension and interferon was administered 1 hr later and daily thereafter. The third group of monkeys was given interferon when fever developed on the third day after inoculation with the virus. A group of 3 other rhesus monkeys was inoculated with the virus only and these served as controls.

Sampling of blood and tissues. Samples of blood were obtained daily by femoral venepuncture. Biopsies were not carried out during life in view of the risk of uncontrolled haemorrhage. Autopsy was carried out on all monkeys shortly after death. Portions of the following organs were removed for examination: lungs, heart, liver, spleen, adrenals, kidneys, intestinal tract, mesenteric lymph nodes and testes. Bile and faeces were also collected for virus titration.

A 10% suspension of each tissue, bile and faeces was prepared for virus titration in guinea pigs.

Tissues were also fixed in 10% buffered neutral formalin and embedded in paraffine wax. Sections cut at 5 microm and stained with haematoxylin and eosin. Selected sections were stained by Verhoeff-van Gieson method, by Machiavello's technique for inclusion bodies, by the periodic acid - Schiff and Gordon and Sweet's methods and with Mallory's phosphotungstic acid.

RESULTS

Clinical observations. There were no marked differences in the clinical course of infection between the monkeys treated with interferon and the controls. Yet an impression was obtained that in the interferon treated group the introduction of measures to combat disseminated intravascular coagulation, such as anticoagulant therapy and replacement of platelets and coagulation factors and fluid replacement may have prolonged survival. The monkeys became febrile on or about the third day after infection and the pyrexia persisted until death. Maculo-papular skin rashes with petechiae developed in all the animals on the 4th or 5th day and remained until death, although in 3 of the monkeys treated with interferon the rash was fading by the 6th or 7th day. The time of death of the monkeys in the different groups is shown in Table 1, and it is noted that survival may have been somewhat prolonged in 3 of the animals treated with interferon.

TABLE 1 DAY ON WHICH DEATH OCCURRED


Day


Controls


Interferon -2 days


+1 hr


+3 days


5


+

     

6


+


+

   

7


+

   

++


8

 

+


+

 

9

   

+

 

TABLE 2 TITRE OF EBOLA VIRUS IN BLOOD (Log 10/ml)
Time in days after inoculation


Rhesus Monkeys


1


2


3


4


5


6


7


8


Controls


<0.5


2.5


5;4.5


5.5


4.5


-

   

<0.5


2.5


54.5


6.5


5.5


5.5

   

<0.5


2.5


4.0


5.0


5.0


6.5

   

-2 Days


<0.5


<0.5


3.5


5.5


5.5


6.5


lte 6.5

 

<0.5


<0.5


1.5


5.5


6.5


7.5


-

 

Interferon +1 Hr


<0.5


<0.5


1.5


3.5


2.5


3.5


6.5


6.5


<0.5


2.5


3.5


6.5


2.5


3.5


5.5


-


+3 Days


<0.5


2.5


4.0


2.5


6.5


6.5

   

<0.5


1.5


5.0


6.5


6.0


6.5

   

Viraemia . The titre of Ebola virus in the blood samples collected daily from each monkey was calculated by parallel titration in guinea pigs. The titres are shown in Table 2. Viraemia was not detected on the second day after infection in 3 out of 4 monkeys treated with interferon, whereas viraemia was evident in the 3 control monkeys and in the 2 monkeys treated with interferon 3 days after infection.

Findings at autopsy. Petechial and maculo-papular skin rashes of variable severity were present involving the forehead, face and cheecks, the medial aspects of the limbs and the chest. Visceral petechiae were present in all the animals. Peritonitis was evident -in-mo-st monkeys and intestinal haemorrhages and mucosal ulceration were found in many of the animals. The mesenteric lymph nodes were enlarged and haemorrhagic. The liver was pale and mottled. Petechiae and small haemorrhages were found on the pleural and cut surface of the lungs. Congestion and inflammatory changes were found in the tunica vaginalis and tunica albuginea of the testes.

There were no differences between the gross pathological findings between the treated and untreated animals.

The histological lesions in rhesus monkeys infected with Ebola virus have been described in detail elsewhere (2). Briefly, the findings in the present series were numerous small foci of parenchymal cell necrosis distributed randomly throughout the lobules of the liver. Areas of coagulative necrosis of variable size were common in some of the animals. Single or multiple intracytoplasmic inclusion bodies ranging in size from 5-25 microm were found in hepatocytes. These inclusion bodies stained a bright red colour by Machiavello's method. In the more severely affected livers, thrombosis occurred in some of the central and portal veins. There was little cellular infiltration in the portal tracts. Subcapsular haemorrhages were present. Necrosis of lymphoid follicles and depletion of lymphocytes, which were widespread in some monkeys, were found in the spleen and lymph nodes. Multiple small haemorrhages were present in the medulla and cortex of the adrenal glands. The kidneys were severely affected with widespread haemorrhages, fibrin thrombi in the glomeruli and tubules and cellular and fibrin casts were present in the tubules and collecting ducts. Cellular infiltration was absent. The histological changes in the intestines ranged from congestion and focal haemorrhages in the submucosa and serosa to severe haemorrhagic necrotising enteris with necrosis of Peyer's patches. The lesions in the lungs varied in their severity and included thrombosis of vessels, small foci of necrosis of the alveolar septae, oedema, intra-alveolar haemorrhages and areas of coagulative necrosis. The histological changes in the testes consisted of haemorrhages between seminiferous tubules and acute inflammatory changes with necrosis in the tunica vaginalis and tunical albuginea.

There were no consistent differences in the nature or severity of the lesions between the monkeys treated with interferon and the untreated animals.


DISCUSSION

Vaccines against Marburg, Ebola and Lassa viruses have not yet been developed and there is no specific therapy for these serious life-threatening infections. The interferons and antiviral chemotherapy should, therefore, be explored as an alternative to immunization, even for limited field application in any future localised outbreaks. Bowen, et al. have shown (3) the illness induced in rhesus and vervet monkeys by infection with the Zaire strain of Ebola virus was very similar to the disease which occurs in man. Our preliminary findings with a small number of rhesus monkeys treated prophylactically with human leucocyte interferon and at the time of infection are not discouraging. Clinically, survival appeared to be enhanced and an impression was gained that life-support measures to combat disseminated intravascular coagulation and fluid replacement in the animals treated with interferon would have favoured recovery. Viraemia was delayed and there is obviously a need to establish the optimal dose and duration of treatment with interferon either prophylactically or early in the course of the infection. Combination therapy with infusion of convalescent serum (when available) is also being explored in the experimental rhesus monkey model.

At present, the availability of interferon is very limited. However, several chemical inducers of interferon have been investigated. Polyinosinic polycytidylic acid (Poly 1. Poly C), a double-stranded RNA, is a potent inducer of interferon in mice and has been found to be effective against various virus infections both prophylactically and therapeutically. It is, however, a poor inducer of interferon in man and in nonhuman primates and it does not induce any detectable serum interferon. This may be related to the presence of an enzyme in the serum of primates which hydrolyses and inactivates Poly I. Poly C.

Poly I. Poly C has been complexed with poly l-lysine and carboxymethyl cellulose (Poly. ICLC) by Levy, et al. (4). This stabilised derivative is partially resistant to hydrolysis by primate serum and induces the formation of several thousand units of interferon per ml. of serum in primates. This compound has been tested in non-human primates against several serious virus diseases. Thus, Poly. ICLC was effective prophylactically in simian haemorrhagic fever. When this compound was given to monkeys 6 hours after a large inoculum of yellow fever virus, about 75% of the treated animals survived and developed good serum antibody titres, in contrast to deaths of all the untreated monkeys (5). The use of Poly. ICLC in experimental Ebola virus infection in nonhuman primates is being explored, since it can induce high levels of serum interferon with few major toxic side-effects.

Finally, there is an urgent need to investigate the use of chemical antiviral agents both for the prophylaxis and treatment of these serious virus diseases. For example, ribavirin, a synthetic nucleoside analogue of guanosine, has been shown to be active in vitro against a wide range of both DNA and RNA viruses. Bivavirin appears to be particularly effective in vivo against influenza A and B and parainfluenza, all RNA viruses, and studies of such antiviral drugs in experimental Ebola virus infection are being planned.

SUMMARY

Rhesus monkeys were treated with human leucocyte interferon prophylactically and after experimental infection with the Zaire strain of Ebola virus. Viraemia was delayed and clinically survival appeared to be enhanced. There were no consistent differences in the pathological changes and the outcome of the infection beween the animals treated with interferon and those not receiving interferon. Nevertheless, there are clear indications for further investigation on the use of interferon alone and in combination with serotherapy, and for the use of interferon inducers.

ACKNOWLEDGEMENT

This work was supported in part by a grant from the World Health Organization.

REFERENCES
1. Emond, R.T.D., Evans, B., Bowen, E.T.W., Lloyd, G. (1977) A case of Ebola virus infection. Brit. med. J., 2, 541544.
2. Baskerville, A., Bowen, E.T.W., Platt, G.S., McArdle, L.B. Simpson, D.I.H. (1978) The pathology of experimental Ebola virus infection in monkeys. In press.
3. Bowen, E.T.W., Lloyd, G., Platt, G.S., McArdell, L.B., Webb, P.A., Simpson, D.I.H. (1978) Virological studies on a case of Ebola virus infection in man and in monkeys. In press.
4. Levy, J.B., Baer, G., Baron, S., Buckler, C.E., Gibbs, C.J., Iadarola, M.J., London, W.T., Rice, J. (1975) A modified polyriboinosinic-polyribocytidylic acid complex that induces interferon in primates. J. infect. Dis., 132, 434439.
5. WHO Technical Report Series. Interferon and antiviral agents with special reference to influenza, 1978. In press.
DISCUSSION
G.A. Eddy : I would like to ask Dr. Zuckerman if he measured the titers of interferon that resulted following inoculation in these monkeys. I would also like to point out our experience using interferon inducer Poly 1:c which was a total failure. In our control monkeys inoculated with Machupo virus we found that the untreated monkeys which received no interferon inducer developed high titers of interferon on about day three or four, which is about four days prior to the onset of viremia or illness. This rather discouraged us from pursuing interferon with respect to Arena viruses. There is obviously plenty of interferon before the monkeys were even sick or had viremia.
A.J. Zuckerman : The levels of interferon in these monkeys are being assayed at the moment. As far as your second comment is concerned, you are right but there may be a very important difference between an endogenous and an exogenous interferon. We are trying to differentiate between the two, because we suspect that in the rhesus monkeys after infection interferon is produced anyway but the critical question is whether we measure endogenous or exogenous.
J. De Smyter : I don't think it will make much sense to administer interferon to an animal or to a person with circulating interferon. Thus it will be very important to know this spontaneous titer of interferon in this disease in monkeys. This still does not mean that exogenous interferon would be useless. Perhaps the disease could be avoided by giving interferon or an inducer after exposure i.e. after an accident like that which occurred in Porton Down. As far as there is a possible comparison with rabies, in this infection it is possible in monkeys to stop the infection if interferon or an inducer are given immediately after exposure, the production of interferon may even be the mechanism of action of post-exposure vaccine in rabies. So there could be a useful post-exposure immediate administration of interferon.
A.J. Zuckerman : I agree with you entirely. What we are trying to do in fact in this experiment is to mimic conditions in the field, one group has preexposure prophylaxis, the second immediate post-exposure prophylaxis and the third is comparable to a patient with a febrile illness. The impression was gained that if interferon is going to be used at all, it has to be used either prophylactically or immediately after exposure rather than after the full symptoms have developed.

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