J. gen. Virol. (1983), 64, 1427-1432. Printed in Great Britain
1427
Key words: visna virus~antigenic variants~slow infection/pathogenesis
The Emergence of Antigenic Variants is a Rare Event in Long-term Visna
Virus Infection in vivo
By H A L L D O R
T H O R M A R , * M A R C R. B A R S H A T Z K Y ,
AND P I O T R B. K O Z L O W S K I
KEITH
ARNESEN
New York State Institute for Basic Research in Mental Disabilities, 1050 Forest Hill Road,
Staten Island, N e w York 10314, U.S.A.
(Accepted 24 February 1983)
SUMMARY
Six sheep persistently infected with visna virus were studied for 4 1/2 to 5 3/4 years
until they became ill. Virus was isolated at intervals from peripheral blood leukocytes
and cerebrospinal fluid (CSF), and at the time of sacrifice from various parts of the
brain and the lungs. Both brain and lungs showed lesions typical of advanced
visna/maedi. All the sheep formed antibodies in sera and CSF. Virus isolates from each
sheep were tested in neutralization tests against sera and CSF collected from the same
animal. In one sheep all isolates were found to be identical to the inoculated virus by
this test. In each of the other sheep an antigenic variant emerged from 1 to 3 years after
inoculation and remained in circulation even after the formation of autologous
antibodies. In one case a variant was isolated from the lungs, whereas in all cases the
virus isolated from the brain was identical to the inoculated virus. The results show that
antigenic variants are rare in visna and do not seem to have a role in the pathogenesis of
the disease.
INTRODUCTION
Several studies have recently been done on the pathogenesis of visna in experimentally
infected sheep, but most have concentrated on the early events of the infection, occurring during
the first months after inoculation (P6tursson et al., 1976; Georgsson et al., 1977; N a t h a n s o n et
al., 1976). However, visna virus is known to persist in infected sheep for 8 years or even longer
(Thormar & P~lsson, 1967; Gudnad6ttir, 1974) and to cause lesions in the central nervous
system (CNS) or the lungs which may or may not lead to clinical signs during the lifetime of the
animals. It has been suggested that a continuous antigenic drift of visna virus may explain its
ability to replicate and cause damage in the infected host in the face of high titres of neutralizing
antibodies (Gudnad6ttir, 1974; N a r a y a n et al., 1978).
In this paper we report observations of Icelandic and Dorset sheep inoculated with visna
virus, and showing persistent viral infection in their CNS and lungs when sacrificed up to 6
years later. We confirm earlier findings that antigenic variants develop in sheep during the
course of the infection. However, our study found such variants to be rare and did not support
the notion that they are important in the pathogenesis of the disease.
METHODS
Animal experiments. Five 1-year-oldfemale Icelandic sheep and a castrated male lamb of Dorset breed were
inoculated intracerebrally with 106.oto 106.8TCIDso of visna virus strain K796 or with 105.6 TCIDs0 of strain
K485 (sheep no. 14). Samples of clotted and heparinized blood were collected from the jugular vein, at first biweekly and then monthly or bi-monthly. Peripheral blood leukocytes (PBL) were separated from the heparinized
blood by incubation with a solution of dextran T250 (Pharmacia), followed by washing in Eagle's basal medium
(BME) (Thormar et al., 1980). Suspensionsof at least 107 PBL were inoculated onto monolayers of sheep choroid
plexus (SCP) cells in 30 ml Falcon flasks for isolation of virus. Cerebrospinal fluids (CSF) were collected by lumbar
puncture 3 or 4 times a year. T~e fluids were centrifuged and the cells, suspended in 0.5ml CSF, were inoculated
onto SCP cultures for virus isolation. The clarified fluids were frozen at - 86 °C for later determination of anti0022-1317/83/0000-5572 $02.00 © 1983 SGM
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1428
H. T H O R M A R A N D O T H E R S
body. The brain was removed at the time of sacrifice and explant tissue cultures were m a d e from various separate
areas, i.e. the right cerebral hemisphere, midbrain, cerebellum and medulla. Lung tissue was similarly explanted.
Explant cultures were incubated with growth m e d i u m (BME with 15 % lamb serum) until widespread cellular outgrowth had formed (Brown & Thormar, 1975); they were then changed to maintenance m e d i u m (BME with 2%
lamb serum). All explant and inoculated cultures were kept for at least 2 m o n t h s and examined for cytopathic
effect. Blind passages of fluid were made every 2 to 3 weeks.
The left half of each brain, the spinal cord and a piece of lung tissue were fixed in 10% buffered formalin.
Paraffin-embedded brains were cut by coronal sections at five levels and stained with haematoxylin-eosin (HE),
Kluver-Barrera, Loyez, Holtzer and combined Bodian-PAS techniques. Sections of lung tissue were stained with
HE.
Virus neutralization. Neutralization tests were done as described earlier (Thormar, 1963) using 100 TCID50 of
virus mixed with doubling dilutions of serum or CSF in maintenance medium. The mixtures were incubated at
22 °C for 20 h and then inoculated onto monolayers of SCP cells in 96-well Linbro microtitre tissue culture plates.
One-tenth ml of each mixture was inoculated into each of two wells. Cytopathic effect was read at 7 a n d [4 days.
Titres were expressed as the highest dilution showing complete neutralization of virus. All sera were heated at
56 °C for 30 min before use.
Hyperimmunization of rabbits. Visna virus was purified by the method of Lin & T h o r m a r (1971). A 50 to 100 p.g
amount of virus in phosphate-buffered saline pH 7.2 was mixed with an equal volume of complete Freund's
adjuvant and injected intradermally into the hind footpads of New Zealand white male rabbits. A booster was
given 6 to 10 weeks later by intramuscular injection into the hind legs. The rabbits received three or four boosters
and were bled t0 days after the last injection.
RESULTS
Clinical signs and histological lesions in infected sheep
Of the six sheep included in this study, one (no. 13) was found dead without previously
showing noticeable signs of illness. The other five sheep became ill from 4 1/2 to 5 3/4 years
following inoculation. They developed weakness of the legs which progressed into a severe
paralysis in a few days to weeks. All showed loss of weight for some time before they became
paralysed, but otherwise no signs of a slowly progressing visna infection were noticed.
In neuropathological examination changes in the myelin of the brain was the most striking
feature. In the cerebral hemisphere large areas of centrum semiovale and the white matter of
gyri showed diffuse pallor of myelin, but with no sharp borders between the intact myelin and
those undergoing demyelination. The small, sharp-bordered demyelinating foci were found in
capsula interna and in striopallidal bundles, giving the picture of 'moth-eaten' foci.
Demyelination was more intensive in centrum semiovale and in white matter of frontal cortex
gyri, while corpus callosum and white matter of parietal and temporal cortex were less damaged.
There was a marked fibrillary gliosis in the areas of demyelination. Similar gliosis was seen in
subependymal areas adjacent to the fourth and lateral ventricles and around the central canal in
the spinal cord. Neuronal changes consisted of dispersed loss of neurons in cerebral cortex with
numerous pictures of neuronophagia and satellitosis as well as microfoci of necrosis in
hippocampus and in the depth of cerebral sulci. No inflammatory infiltrations were observed in
any of the brain, cerebellum or spinal cord.
In the lungs a chronic, interstitial inflammation was found. The alveolar walls were
thickened, and numerous lymphocytes and mononuclear cells were seen within the septae or
forming peribronchial cuffs. In some areas an almost total consolidation of tissue was seen,
while in other areas, in the lungs of the same sheep, the alveolar walls were broken, forming
spikes and large bullae that gave a picture of compensatory emphysema.
Virus isolations
Table l summarizes the results of attempts to isolate virus from the six sheep during the
persistent infection and at the time of sacrifice. Virus was isolated from the brains of all four
sheep from which explant cultures were successfully grown. The virus was found to be
widespread in the brain, since it was in most cases isolated from several areas, such as the
cerebral hemisphere, cerebellum, medulla and choroid plexus. Virus isolation was made from
the lungs of two out of four sheep. In all cases, virus cytopathic effect appeared in the primary
cultures from 12 to 18 days after explantation. Virus isolations were made from 10% or less of
spinal fluids tested and from 5% (sheep no. 6) to 20% (sheep no. 13) of the PBL samples.
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Antigenic variants in visna
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Fig. 1
Fig. 2
Fig. I. Sheep no. 3. Titre against: (a) the inoculating virus strain K796; (b) virus isolated from PBL ( ~ ),
CSF ( ~ ) and brain (B) at the indicated times; (c) virus isolated from PBL ( ~ ) and lung (L). O, Sera; O,
CSF; f, time of death.
Fig. 2. Sheep no. 1. Titre against: (a) the inoculating virus strain K796; (b) virus isolated from PBL ( ~ )
and CSF ( ~ ) during the infection and at the time of death (t)- O, Sera; O, CSF.
T a b l e 1. Virus isolations from infected sheep
Time of sacrifice r
Sheep no.
(years)
PBL
1
5 1/4
6/46*
3
5 3/4
7/46
6
5 3/4
2/43
13
5 1/2
10/46
14
4 1/2
6/36
D12
4 1/2
2/37
Virus isolations
*
CSF
Brain Lungs
2/17"
Not
ND
2/18
+
+
0/14
+
-2/20
ND
ND
0/17
+
+
0/4
+
--
* No. positive/no, tested.
"~ND, Not done.
Neutralizing antibodies in sera and CSF
Virus isolates from PBL, CSF, b r a i n a n d lungs were passaged 2 or 3 times in S C P cultures a n d
tested in n e u t r a l i z a t i o n tests against sheep serum a n d C S F samples. T h e virus strains used for
i n o c u l a t i o n of the sheep were also included in the tests. T h e results are s h o w n in Fig. 1 to 4. First,
all the sheep formed neutralizing serum a n t i b o d i e s against the i n o c u l a t e d virus w i t h i n 1 to 4
m o n t h s post-inoculation. The antibodies a p p e a r e d a little later in the C S F b u t reached
significant titres in most of the a n i m a l s a n d r e m a i n e d elevated for the d u r a t i o n of the persistent
infection. I n one sheep (no. 3) the C S F titres were equal to or greater t h a n the serum titres (Fig.
1 a, b). W h e n P B L virus isolates from each sheep were tested a g a i n s t a series o f sera from the
same sheep collected at intervals throughout the d u r a t i o n of the infection various p a t t e r n s were
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Time after inoculation (years)
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Time after inoculation (years)
Fig. 3
Fig. 4
Fig. 3. Sheep no. 6. Titre against: (a) the inoculating virus (plaque-purified strain K796) and a brain
isolate (B); (b) virus isolated from PBL 10 months and 4 1/3 years respectively, after inoculation ( q ).
O, Sera; 0 , CSF; t, time of death.
Fig. 4. Sheep no. 13. Titre against: (a) the inoculating virus strain K796; (b) virus isolated from PBL ( ~ )
and CSF (~) during the first 2 t/2 years of infection; (c) virus isolated from PBL ( ~ ) during late
infection. O, Sera; 0 , CSF; t, time of death.
observed. In one sheep (no. 1) all five PBL virus strains isolated either within 2 years postinoculation or as late as 5 1/2 years post-inoculation were neutralized by the sera in a pattern
identical to that of the inoculated virus (Fig. 2). In another sheep (no. 14) there was no
significant formation of neutralizing antibodies until about 3 1/2 years post-inoculation. There
was then a similar increase in titre against virus isolates from PBL, brain and lungs as against the
inoculated virus strain K485 (data not shown). I n sheep no. 6 (Fig. 3) the two P B L isolates were
identical, although separated in time by more than 3 1/2 years, but were slightly different from
the inoculated virus. Virus isolated from various parts of the brain at the time of sacrifice
showed a pattern identical to that of the inoculum. Similar results were o b t a i n e d for Dorset
sheep no. D12 (data not shown). Two PBL virus strains isolated from sheep no. 13 within 2 1/2
years after inoculation were identical to the inoculum (Fig. 4a, b). However, four strains isolated
at 3 years post-inoculation or later were different from the inoculum and the early PBL isolates,
although they were all identical to one another (Fig. 4c). Finally, four PBL strains isolated from
sheep no. 3 at 1 to 2 years and 4 to 5 years post-inoculation were identical to the inoculum and to
virus isolated from the brain of the sheep (Fig. 1 a, b). On the other hand, two PBL isolates
obtained at 2 1/2 and 4 years post-inoculation and virus isolated from the sheep's lungs were
identical but different from the inoculum and the other PBL and brain isolates. In this sheep,
therefore, two serotypes of visna virus co-existed for 3 1/2 years. One was located in the lungs
and the other one in the brain at the time of death. A series of spinal fluids were tested in
neutralization tests against virus strains isolated from C S F of the same sheep. In all cases the
CSF isolates were identical to the virus strain used for inoculation (Fig. I to 4). Therefore, virus
was apparently circulating in the C S F as well as in the blood for years after the formation of
autologous neutralizing antibodies.
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Antigenic variants in visna
1431
Table 2. Cross-reaction between inoculating virus strain and brain and lung isolates
Titres of antisera
1,
¢
Virus strain Anti-K796 Anti-no. 3B Anti-no. 3I~
K796"
512
512
256
No. 3Bt
1024
1024
128
No. 3L:~
64
16
128
*Strain used for inoculation.
t Brain isolate.
Lung isolate.
Cross-reactions between virus isolates
Cross-neutralization tests with virus isolates from sheep no. 3 using hyperimmune sera raised
in rabbits showed that the brain isolate and the inoculated virus strain were identical. On the
other hand, hyperimmune sera against these strains had 8- to 64-fold lower titres against the lung
isolate than against the autologous virus (Table 2). This confirms the difference found in tests
with sheep sera (Fig. 3).
DISCUSSION
Our study agrees with previously published results (Narayan et al., 1978) which showed that
antigenic variants of visna virus emerge in sheep during the slow infection, probably by
mutation. However, we did not observe a continuous progression of antigenic variants in any of
our sheep. On the contrary, the appearance of antigenic variants was rare in the persistently
infected sheep and occurred only once in any one of them during the almost 6 years of infection.
The discrepancy in our observations and those of N arayan et al. (1978) may be due to differences
in virus strains used or to the fact that these investigators used 10- to 100-fold larger inocula in
some of their sheep, resulting in a greatly increased number of virus isolations from the PBL.
Our results are similar to those of P&ursson and co-workers (P6tursson et al., 1981 ; Lutley et al.,
1983) who found antigenic variants to be rare in sheep that had been persistently infected with
visna for several years. They also agree with earlier studies (Thormar & Helgad6ttir, 1965)
which found that a few antigenic variants occurred among virus strains isolated from the lungs
of natural cases of maedi, but most of the strains were antigenically closely related to one
another, and even to a visna virus strain isolated from the brain of a natural case of visna a
decade earlier and passaged many times both in sheep and in tissue cultures. Similarly, Icelandic
visna and maedi virus strains were neutralized by sera from Dutch sheep naturally affected with
Zwoegerziekte (Thormar, 1966), a lung disease caused by visna/maedi virus (De Boer, 1975).
Since the Icelandic and Dutch strains of the virus had been separated geographically for at least
three to four decades and probably much longer, this demonstrates the antigenic stability of the
virus in nature. This stability is remarkable in view of the fact that the virus persists in sheep for
several years and most of this time in the presence of high concentrations of circulating
antibodies. However, our present study does not rule out minor antigenic differences among the
visna virus isolates.
At the time of paralysis and death, virus identical to the inoculated strain was found to be
widespread in the brains of sheep showing lesions typical of advanced visna. Similarly, virus
isolated from lungs with typical maedi lesions was identical to a variant which was present in the
sheep's blood 4 years earlier. Therefore, this study does not support the idea that a continuous
antigenic drift of visna virus has an important role in the pathogenesis of the slow disease.
We thank Patricia Brophyfor technical assistance and Edith M. Riedel and LucilleS. Donadiofor preparation
of the manuscript. Dr Richard S. Trowbridge generouslyprovided the plaque-purified strain of K796. This work
was supported in part by grant RG-817 from the National Multiple Sclerosis Society.
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H. T H O R M A R A N D O T H E R S
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