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SAMJ
------------------------------------------
J. H. LABUSCAGNE,
Summary
Neurotropic vaccine strains of African horsesickness (AHS) virus types I and 6 were iInplicated as
the possible aetiological agents in 4 cases of
encephalitis and uveochorioretinitis in laboratory
workers accidentally exposed to the freeze-dried
vaccine preparations of the virus. To date, AHS
virus has not been known to infect man. To ascertain whether or not primates were susceptible to
infection with AHS virus, vervet monkeys (Cercopithecus pygerythrus) were inoculated, either
transnasally or intracon;unctivally, with vaccine
strains of AHS virus types I and 6. The course of
infection was monitored using parameters such as
behavioural changes, febrile reaction, cerebrospinal fluid pleocytosis, serology, magnetic
resonance imaging and autopsy. Encephalitis,
manifested by varying deg-rees of fever,
behavioural changes and pleocytosis, but no
chorioretinitis was detected in all 6 transnasally
infected monkeys. This was confirmed by autopsy, where a meningo-encephalitis affecting the
medial temporal lobe but no lesions in the eyes
was demonstrated. Neither virus appeared to
infect the aniInals after intracon;unctival inoculation. These findings support the theory that the
patients were infected by the inhalation of freezedried vaccine preparations. The pathogenesis of
the eye lesions, however, remains uncertain.
S Air Med J 1992; 81: 462467.
463
SAMJ
.--------------------------------------------------
C. pygerythrus monkeys
Five newly wild-caught male monkeys, all lacking antibodies to AHS virus, were used for experimentation
after a quarantine period of 6 weeks. In addition,S wildcaught females, monkeys which had been in captivity for
up to 5 years, all lacking detectable AHS virus antibodies, were infected. The animals were housed in 0,6 x
0,6 x 0,8 m stainless steel cages suspended from a
wall. To monitor any cross- or airborne infection, an
uninfected control animal was housed in the same
room, approximately 2,0 m apart from the experimental
animals. One week before infection, the animals' temperatures were taken, lumbar punctures were performed
and blood samples collected. Serum was tested for the
presence of antibodies to AHS virus and the cerebrospinal fluid (CSF) for cell count, and protein, glucose
and chloride values. All sampling procedures were
carried out under ketamine hydrochloride (10 mg/kg)
sedation.
After infection, the animals were monitored daily for
any behavioural changes. Every 2 - 3 days post-infection, for a period of 28 days, temperatures were taken
and blood samples collected for antibody assays and
virus isolation. Thereafter, blood specimens and temperatures were taken weekly. Lumbar punctures were
performed when indicated by the occurrences of symptoms, and magnetic resonance imaging (MRI) was done
on selected animals. The animals' eyes were all examined 2 - 4' weeks post-infection. If marked clinical symptoms of encephalitis were evident, the animal was
humanely sacrificed using pentobarbitone (200 mg/kg)
and at autopsy selected samples of tissues were removed
aseptically for virus isolation and the remainder preserved in 10% phosphate-buffered formalin for
histopathological examination. The remaining animals
were all sacrificed, autopsies performed and specimens
processed (as above) 25 days after superinfection.
Infection programme
In two consecutive experiments, monkeys of different
sex and mass, were infected either intranasally or intraconjunctivally with the attenuated vaccine strains of
either AHS virus type 1 or AHS virus type 6 or a mixture of both viruses:
1. Initial infection: 3 monkeys were inoculated
intraconjunctivally with 0,2 rnl of either type 1 (CZl!84,
female, 3,6 kg), type 6 (C2l!85, female, 3,0 kg) or both
viruses (C39/89, male, 5,5 kg). Six monkeys were
instilled intranasally with 1 rnl of either type 1 (C52/85,
female, 4,2 kg; C31/89, male, 5,9 kg), type 6 (C53/85,
female, 4,3 kg; Cll/89, male, 5,9 kg) or both viruses
(C20/85, female, 4,6 kg; C44/89, male, 6,2 kg).
Results
Clinical features and CSF changes
No clinical features associated with encephalitis, i.e.
behavioural changes, febrile reaction or lymphocytic
pleocytosis or signs indicative of chorioretinitis, were
noted in the uninfected control animal or any of the
intraconjunctivally infected animals after either initial or
superinfection.
Behavioural changes, varying from mild subtle
changes, such as passiveness, lethargy, anorexia and
sleepiness (predominantly in the female monkeys) to
marked changes, including nervous hyperactivity and
startle reactions (C31/89) and extreme aggression
(C44189), were noted in all animals infected intranasally
6 - 12 days after initial infection (Table I). Only 2 animals, C31/89 and to a lesser extent C44189, showed any
behavioural changes after superinfection. Animal
C3l!89 became nervously agitated and hyperactive with
a marked startle reaction 3 days after superinfection.
Febrile reactions (temperatures above 40C) were
detected in all intranasally infected animals 8 - 14 days
after initial infection (Table I) and only in animal
C3l!89 (day 10) after superinfection. PleoCytosis (lymphoCytes > 5 mm' and neutrophilis > mm') and also
an increase in CSF protein levels were detected in the 6
intranasally infected animals 10 - 20 days after the initial
infection (Table I) and in animal C3l!89 (day 10) after
superinfection. TO evidence of chorioretinitis was
detected in any of the intranasally infected animals' eyes
either after initial or superinfection.
464
SAMJ
TABLE I.
Sex
Mass (kg)
Infection
AHS virus type
Route
Clinical (d*)
Behaviour changes
Febrile reaction
Pleocytosis
Chorioretinitis
MRI
CSF
Red blood cells (l1l1)
Neutrophils (/1l1)
Lymphocytes (l1l1)
Protein (mgll)
Glucose (mg/l)
Bacteria - propagation
AHSV antibody titre
Type 1
Type 6
Virus isolation
Blood
CSF
Seroconversion (d*)
Type 1
Type 6
'Duration in days post-infection
IN ~ intranasally; - = negative; NO
C52185
C31/89
C53/85
C11/89
C20/85
CMI89
F
4,2
M
5,9
F
4,3
M
5,9
F
4,6
M
6,2
1
IN
1
IN
6
IN
6
IN
1 &6
IN
1 &6
IN
6-14
8-10
20;32
14-23
14-18
14-20
10-16
10
10;14
20;32
9-19
10
10;14
20;32
8-14
8
10;14
20;32
12-18
14-16
14;16
20;32
NO
Day 32
0
0
5
150
2,9
Day 16
0
0
15
300
3,0
NO
Day 20
0
0
10
150
2,3
NO
Day 32
0
0
20
250
2,3
NO
Day 20
0
0
5
390
2,5
Day 20
0
0
24
340
2,3
Day 32
<10
80
Day 32
<10
40
Day 32
30
< 10
Day 48
<10
<10
48
28
48
28
48
Day 32
20
<10
NO
28
= nol done.
TABLE 11.
AHS virus
antibodies
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
Type 1
Type 6
-3
0-23
28
48
70
-4
74
0
78
4
81
7
84
10
86
12
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
.<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
640
<10
<10
<10
<10
<10
40
<10
<10
<10
<10
<10
<10
20
<10
<10
<10
<10
<10
<10
640
20
<10
<10
<10
<10
80
<10
<10
<10
<10
<10
20
20
<10
20
<10
<10
<10
<10
1280
40
<10
<10
<10
<10
80
<10
<10
<10
<10
<10
20
20
<10
80
<10
<10
<10
<10
NT
NT
<10
<10
<10
<10
NT
<10
<10
<10
<10
<10
NT
NT
<10
NT
<10
<10
<10
<10
NT
NT
<10
<10
<10
<10
NT
<10
<10
<10
<10
<10
NT
NT
<10
NT
<10
<10
<10
<10
2560
80
<10
<10
<10
<10
80
<10
<10
<10
<10
<10
20
40
<10
40
<10
<10
<10
<10
10240
80
<10
<10
<10
<10
160
<10
<10
<10
<10
<10
40
40
<10
40
<10
<10
<10
<10
NT
NT
<10
<10
<10
<10
NT
<10
<10
<10
<10
<10
NT
NT
NT
NT
88
14
92
18
99'
25t
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
10240 10240 10240
160
160
160
<10
<10
<10
<10
<10 - <10
<10
<10
<10
<10
<10
<10
320
80
80
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
80
40
80
80
40
80
<10
<10
<10
80
40
40
Innial infection.
Superinfection.
NT = nol tested.
m.
465
SAMJ
---------------------------------------------------_........::.-MRI
Abnormalities were visualised by MRI in 1 of me 2
infected animals examined. Animal C3l/89 was first
examined 36 days after the initial infection and reexamined 13 days after superinfection. At the first
examination, two small poorly circumscribed areas of
increased signal intensity were seen in the medial
aspects of me left temporal lobe in me parahippocampal
gyrus on me T2 weighted images. An even weaker signal
increase was visible in me right parahippocampal gyrus.
When compared wim equivalent sections of me control
animal (Fig. 1) this was clearly abnormal.
Histological findings
No histological evidence of infection was shown in me
eyes and optic nerves of me control animal or in any of
the infected monkeys. In animal CZ1/84, infected intraconjunctivally, hydrocephalus wim dilatation of bom
lateral ventricles was evident on gross examination.
Parasitic cYStS with the features of cysticercus were
present in me left lateral ventricle and me left parietal
conex. The control animal, C25/89, and me omer animals infected by me intraconjunctival route, did not
show any abnormalities in me brain sections.
The 6 animals infected via me intranasal route had a
marked meningo-encephalitis wim a mononuclear infiltrate in me subarachnoidal space, extending into me
Virchow-Robin space (Fig. 3). This was associated wim
a localised encephalitis affecting the olfactory tract
bilaterally.
FIG. 1.
FIG. 3.
AG.2..
FIG. 4.
466
SAMJ
Discussion
The successful transnasal infection of the vervet
monkey, albeit without the ocular lesions, with neurotropic vaccine strains of AHS virus types 1 and 6, indicates that these attenuated viral strains can, in fact,
infect primates and could therefore have been responsible for the human infections.
Infections of the central nervous system (CNS) are
usually a rare complication of an infection established
elsewhere in the body and, for reasonsthat are unclear,
only a small proportion of infected persons will develop
neural complications. 22 Viruses may penetrate the CNS
via neural, haematogenous or olfactory routesn and it
appears that cettain viruses have a predilection for particular parts of the CNS, which are often reflected in the
clinical signs of the disease. 22 During natural infections,
the direct exposure of a nerve to a virus is an unusual
event. Replication of the virus at peripheral sites may
possibly facilitate neural spread by amplifying the original inoculum. 24 In the olfactory mucosa, however, nerve
cells (receptor cells with olfactory rods) are exposed to
the exterior through the cribriform platen ,2. and experimental evidence has shown biologically inert particles to
move directly to the olfactory lobes via the olfactory
nerves. n The olfactory spread of aerosols of arboviruses
and rabies has been implicated in several laboratoryacquired CNS infections. I ',2;
The highly conserved, localised nature of the lesions
in the monkeys' brains corresponded with the predominantly temporal and orbitofrontallobe involvement seen
in patient 2" and supports the view that the patients
were infected transnasally, via the olfactory route, by
aerosol infection with pulverised freeze-dried AHS virus
vaccine from broken vials. The lack of any clinical features or histological evidence of infection in the control
and intraconjunctivally infected monkeys, fails to support the possible role of intraconjunctival infection or
casual contact or exposure in the human infections.
In the infected monkeys both AHS virus types 1 and
6, singly or in combination; were found to cause a
meningo-encephalitis with or without subsequent seroconversion. The PRN titres in the monkeys' sera varied
(Table IT) but in most cases were higher than those
demonstrated in the patients' sera." Monkey C20/85's
titre of 10 240 to AHS virus type 1 was also higher than
commonly observed in horses following natural infection with wild type virus 2 or following vaccination.
Either AHS virus type 1 or type 6 could therefore have
been responsible for the human infections. However, in
addition to the 4 patients who developed encephalitis
with subsequent seroconversion to AHS virus types 1
and 6, 10 other workers from the same vaccine-packaging facility, with no histories of clinical infection, were
shown to have antibodies to vaccine strains of AHS
virus types other than 1 and 6."'" The possibility of
another AHS virus type playing a role in the human
infections can therefore not be excluded, considering
that the monkeys with the most severe encephalitis
(C31189 and C44189) failed to seroconvert or to develop
467
SAMJ
----------------------------------------------------------9. Erasmus BJ. Preliminary observations on me value of me guineapig in derermining me innocuiry and antigeniciry of neurotropic
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10. Erasmus
The anenuation of horsesickness virus: problems and
advantages associated with the use of different host systems. In:
Bryans IT, Gerber H, eds. Proceedings of ehe Ise Internaeional
Conference on Equine Infeccious Diseases (Stresa, Italy, 11-13 Ju!
1966). Lexington, Kentucky: Grayson Foundation, 1966: 208-213.
11. Van der Meyden CH, Erasmus BJ, Swanepoel R, Prozesky OW.
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horsesickness virus infection in laboratory workers: Pan 1. Clinical
and neurological observations. S Afr Med] 1992; 81: 451-454 (this
issue).
12. Reid R, Van der Meyden CH, Erasmus BJ, Meyer H, Hamilton
AMP. Encephalitis and chorioretinitis associated wim neurotropic
African horsesickness virus infection in laboratory workers: Pan IT.
Ophmalmological findings. S Afr Med] 1992; 81: 454-458 (this
issue):
13. Swanepoel R, Erasmus BJ, Williams R, Taylor ME. Encephalitis and
chorioretinitis associated wim neurotropic African horsesickness
virus infection in laboratory workers: Part ill. Virological and serological investigations. S Afr Med] 1992; 81: 458-461 (this issue).
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McKinney RW, Work TH. Arbovirus infections in laboratory
workers. Science 1967; 158: 1283-1286.
15. Flewen TH. Safery in me virology laboratory. 1n: Waterson AP, ed.
Recene Advances in Clinical Virology. New York: Churchill
Livingstone, 1980: 169-187.
Br
;;ummary
J. C. SEEGERS,
H. J. ELS
OrrObin ' postulated that a deficiency of gammalinolenic acid (GLA) in malignant cells and that
consequently inadequate amounts of prostaglandin El (pGE,) and/or thromboxane A., (TXA.,) may
be pivotal to the metabolic abnormalities of cancer cells.
This author suggests that levels 'Of TXA., could be elevated by directing arachidonate metabolism away from
other eicosanoids of the 2-series and that PGE, could be
increased by supplementation by its immediate precursors.
The abnormality in question, common to all malignant cells according to Horrobin, I lies in the desaruration of linoleic acid. This author suggested that a key
event in malignant disease might be the loss of delta-6desarurase activity by malignant cells. He pointed out
that loss of this enzyme would lead to reduced levels of