Download Slow Virus Diseases of the Central Nervous System

Slow Virus Diseases of the Central Nervous System
D. CARLETON GAJDUSEK,
National Institute
of Neurological
M.D.
Diseases and Stroke, National
Bethesda, Maryland
I will focus more on
subacute and chronic degenerative diseases
of man than on animal diseases, and will
discuss a few slow infections caused by
persistent viruses in man wherein the etiology has now been proved. The degree of
involvement of the immune mechanism in
the pathogenesis of these diseases can be
assessed where we now know that a latent,
long-lasting virus is present, especially for
those diseases in which transmission to an
experimental animal is possible and the
animal models may be used in the study of
pathogenesis. We were not surprised to see,
during the past two decades of virology,
a long list of viruses which may be present
in an animal for months, even years, after
first contact with the agent. But when we
asked how many viruses had been isolated
from a human subject for a second time,
three to six months or a year after original
isolation, the list of such viruses was very
small. At the time we held the first Symposium on Slow, Latent, and Temperate
Virus Infections at the National Institutes
of Health six years ago,10 the question that
faced us was what viruses or other infectious agents came to mind when a virus
diagnostic laboratory was confronted with
specimens from a subacute, chronic, or recurrent disease. We knew that herpes simplex was found to be latent in man in the
dawn of virology. Indeed, most of us here
are carrying that virus in our buccal muIN THIS PRESENTATION
Received May 17, 1971.
Reprints of this entire Research Symposium are
available from the ASCP Secretariat, 2100 West
Harrison Street, Chicago, Illinois 60612.
Institutes
of
Health,
cosal membranes and a wide range of chemical and physical insults can provoke the
masked agent to become pathogenic. What
other agents were known five to 10 years
ago to fall into an eclipse, hidden, or
masked phase from which they could be
resurrected?
Warts may last for years in some children. Lesions of molluscum contagiosum
may persist for many months. Rabies may
develop in man after incubation periods of
well over a year, during which time the
virus is slowly spreading from cell to cell
in Schwann cells surrounding nerve fibers,
as Johnson has demonstrated by immunofluorescent staining technics.16 Moreover,
old epidemiologic evidence supported a relationship between herpes zoster and varicella. Subsequently, isolation of the viruses
responsible for the two diseases showed
them to be indistinguishable. However,
some cases of herpes zoster develop within
a few days of injury to the same dermatome
in which the lesions appear, and this has
been interpreted as strong evidence for the
latency of varicella virus in dorsal root
ganglia cells and its activation by nerve
injury.
At the time of our 1964 symposium on
slow virus infections,10 persistent infection
in infants with cytomegalovirus and rubella
virus had just been recognized. After intrauterine or neonatal infection with rubella an infant may harbor and shed rubella virus in the respiratory tract for
months and perhaps for more than a year.
Persistence of the cytomegalovirus in the
urine for similarly long periods after con-
320
September 1971
SLOW VIRUS DISEASES O F T H E CNS
genital infection was also found. Some infants will react to such persistent infections
with cumulative pathology, whereas others
show very few, if any, pathologic lesions.
Infectious hepatitis, with its recurrences
and the obvious transmission of the virus to
recipients from blood and blood products
of healthy carriers, which came to medical
attention in World War II, is another example of persistent virus infection that has
been known for decades.
Thus, in 1964 the above-mentioned list
of persistent agents in man was nearly exhaustive, unless we wanted still to refer to
the larger organisms of lymphogranuloma
venerium and trachoma as viruses. Since
1964, however, we have been able to add
a large number of other agents to the list.
I will concentrate my remarks on the four
slow and persistent diseases of the CNS in
which we have been most thoroughly involved in our laboratory and mention what
may be the possibility that, in man, an
autoimmune mechanism is involved with
the pathogenesis of these slow infections.
I do not think there are any "slow"
viruses. There may be slow virologists, but
not slow viruses. We usually refer to "slow
virus infections"; we are then talking about
subacute and chronic disease, slowly but
unremitting progressive disease, and recurrent disease in which the virus is persistent
and the tissue damage is cumulative. Viruses which may be so persistent are decidedly not "slow" viruses in vitro or in
the appropriate susceptible host. Thus, viruses such as herpes simplex virus and rubella virus have nothing slow about their
behavior in a susceptible primarily-infected
host. They produce classical examples of
acute infections with rapidly replicating
agents, which in some cell systems may be
cytolytic. Even influenza, mumps, measles,
rabies, and many other viruses causing
acute diseases may be induced to form symbiotic, persistent infections in certain cell
systems in vitro.
321
T o turn to a problem of semantics and
terminology, we named our section eight
years ago a "Laboratory of Slow, Latent,
and Temperate Virus Infections"—a bit facetiously. At that time we knew of no viral
disease in man in which a "temperate"
relationship between the virus and the
human cell had been established. On the
other hand, it has been shown that in their
oncogenic roles the SV40 virus and some
of the adenoviruses can enter into a genetic, genome-localized, "temperate" relationship with the cell. The work of Dr.
Wallace Rowe and others has beautifully
documented this. Thus, the analogies which
were drawn between mammalian viruses
and bacteriophage in the early days of
phage work are now supported fully by
dynamic studies of mammalian virus-cell
interaction.
Many terms for this sort of infection are
being bantered about. (Parenthetically, I
may add that I am sure no three virologists
would agree on a definition of these terms
as applied to a mammalian virus or a mammalian cell system.) We talk of "persistent"
or "endosymbiotic" infections; of slow, latent, and temperate infections; of masked,
hidden, eclipse-phase; integrated, lysogenic,
and repressed viruses; and also use their
antonyms: vegetative as opposed to integrated; and derepressed versus repressed;
partial, imperfect, incomplete, defective,
and incompletely assembled viruses as opposed to complete or fully formed, or
infectious virions. T h a t these mechanisms
are involved in any given human infectious
disease has yet to be fully established. The
terms usually are taken from a much more
quantitative and exact science of bacteriophage virology. Even now, in oncogenic
virus work we refer to "permissive" and
"nonpermissive" cells which are "competent" or "incompetent." T h e semantics
eventually will be worked out. No one
should imply that all other mammalian
virologists agree with him in the way he
322
GAJDUSEK
A.J.C.P.—Vol.
56
ENAGE
1
AUG 63
t
XL"
brain
ic
AUG 63
brain
ic iv
DEC 68
T
SEPT 63
•
Chimpanzee
A1f
Spider monkey
S45f
Chimpanzee
A2m
inc: 21 mot.
dur: 9 mot.
inc. 23 mot.
dur: 1}4 mot.
inc. 30 mo*.
dur: 4 mos.
Chimpanzee
A4f
inc: 20 mos.
dur: 5 mos.
IGIERAKABA
— i —
Chimpanzee
A17m
inc: 16 mot.
dur: 5 mot.
Chimpanzee
AlBm
inc: 17 mot.
dur: -6 mos.
Chimpanzee
A19m
inc: 19 mos.
dur: 7 mos.
KABUINAMPA
brain
brain
iciv
NOV 63
_1
Chimpanzee
A5f
Chimpanzee
A6f
Chimpanzee
A7m
Chimpanzee
A8f
inc: 28 mos.
dur: 4 mos.
inc: 27 mos.
dur: 4 mos.
inc: 25 mos.
dur: 5 mos.
inc: 38 mot.
dur: 6 mos.
inc: 19 mot.
dur: 6 mot.
Chimpanzee
A43m
inc: 39 mos.
dur: 2 mos.
inc. 18 mos.
dur: 8 mos.
TO"1 220 nm
ic iv
85° C/30 mint,
iciv
T
Chimpanzee
A46m
Chimpanzee
A9f
Capuchin monkey
666Wm
t
Chimpanzee
A93m
inc: 45 mos.
dur: 1 mo.
inc: 14 mos.
dur: 2 mos.
Chimpanzee
A25m
Chimpanzee
A47m
inc. 17 mos.
dur: 3 mos.
inc: 18 mot.
dur: 6 mot.
10'° icip
ivsc im
r
Chimpanzee
A90m
Chimpanzee
A92m
inc: 24 mos.
dur: 1 mo.
inc: 18 mot.
dur: 1 mo.
Squirrel monkey
SSCIm
inc: 25 mos.
dur: IK mos
Fie. 1. Transmission of kuru directly from human brain tissue of 11 different patients to
chimpanzees and to New-World monkeys, with incubation periods varying from 14 to 45
months. T h e brain tissue of patient Kupenota (lower right) produced disease in chimpanzee
A92 when inoculated at 10"B dilution.
is using any of these terms, unless he defines his usage carefully.
The disease I pick for brief description
is kuru. Dr. Vincent Zigas and I first reported kuru in 1957 and have worked on
this disease continually since that discovery.
We may soon be reporting its demise and
disappearance. None of you, and indeed
very few physicians throughout the world,
have had a chance to see patients with
kuru. Yet, in its decade and a half of medical history, more than 300 scientific papers
have dealt with it and whole symposia have
been devoted to it in London, Paris, Washington, and Port Moresby.
Kuru is an exotic, subacute, progressive,
degenerative disease of the central nervous
system in a highland neolithic population
in New Guinea, a cannibalistic Stone Age
group in which we discovered the disease,
new to Western medicine, 13 years ago.12
It certainly was that disease of man which
brought slow virus infections of man to
worldwide consideration. It is the first
chronic degenerative disease of man with a
clearly established viral etiology, transmissibility, and excellent animal model. I can
now say "models" since the disease is transmissible to several hosts.9 Furthermore, the
cause of the disease in the animal models
is certainly the same as the cause of the
disease in man.
I will return to kuru, but to advance the
discussion quickly I will point out that its
close pathologic and clinical similarities to
scrapie, a slow virus disease of sheep, led
to our transmission of kuru to chimpanzees,
spider monkeys, squirrel monkeys, and the
capuchin monkey, with incubation periods
of \y2 to 4y2 years. This degenerative,
September 1971
323
SLOW VIRUS DISEASES OF T H E CNS
spongiform encephalopathy of man, which
is a system disease of the cerebellum, presenting clinically with incoordination, truncal ataxia, tremors, dysarthria, and progressive motor incoordination, and which in
all cases is fatal, usually within $ to 9
months, has now led us to discover quite
similar disorders throughout the civilized
world, perhaps caused by very similar
agents—certainly, again, viruses. As a consequence of our studies of kuru, we have
pursued further pathologic and clinical
analogies and studied the presenile dementias which, in the Creutzfeldt-Jakob form,
present a neuropathology quite similar to
that we see in kuru in man, chimpanzees,
and New World monkeys which develop
the disease.
We were looking for a less exotic and
less geographically restricted disease in man
which has a similar, strange pathology.
This pathology cannot be easily produced
by artifacts; a spongiosis of gray matter
with intracellular vacuolation of neurons,
a generalized gliosis, and a system degeneration pattern of neuron loss. We have
now successfully transmitted CreutzfeldtJakob (C-j) disease from 11 humans to 13
chimpanzees inoculated with suspensions of
brain tissues. Each of the chimpanzees developed a clinical disease and neuropathology similar to those seen in the human
patients. We have inoculated animals witli
brain and visceral tissues obtained by surgical biopsy or autopsy from many additional cases of this disease that have occurred throughout the United States, Canada, Europe, and Australia. Most specimens
have been inoculated into New World
monkeys (Fig. 1) and chimpanzees (Fig. 2),
A.T.
M.W.
-I
biopsy
brain 5%
ic iv
autopsy
brain
I
__l
NOV 66
10% ic iv
FEB 68
20% ip iv im
MAY 69
autopsy
brain 10%
ic iv
FEB 68
autopsy
brain 10/S
ic iv
MAR 68
Chimpanzee
A54 m
Chimpanzee
A79f
Chimpanzee
A77f
Chimpanzee
A124m
Chimpanzee
A78f
Chimpanzee
A81 m
inc: 13 mos.
dur: 1mo.
inc 12 mos.
dur 2J4 mos.
inc: 12 mos.
dur: Yh mos.
inc: 16 mos.
dur: 2 mos.
inc 14 mos.
dur 2 mos.
inc: 14 mos.
dur: 1 mo.
J.J.
E.S.
autopsy
brain 10%
biopsy
brain 5%
autopsy + biopsy
brain 10%
J.D.
L.E.
D. M.
autopsy
brain 10%
.autopsy
brain 5%"
autopsy
brain 5%
IC IV
IC IV
ICIV
IC IV
IC IV
IC IV
JUNE 68
AUG 68
FEB 69
MAY 69
OCT 69
MAR 70
Chimpanzee
A106m
Chimpanzee
A114f
Chimpanzee
A119m
Chimpanzee
A123m
Chimpanzee
A142 f
Chimpanzee
A152m
inc: 13 mos.
dur: 1 mo.
inc: 12 mos.
dur: 1 mo.
inc: 12 mos.
dur: V/i mos.
inc: 11 mos.
dur: 2 mos.
inc: 14 mos.
dur: 1 mo.
inc: 11 mos
dur: 1 mo.
FIG. 2. Transmission of Creutzfeldt-Jakob disease from 10 human patients to chimpanzees,
with incubation periods of 11 to 16 months. T h e brain of patient M. W. (top row center) produced disease in chimpanzee A124 when inoculated intraperitoncally, intravenously and intramuscularly, without intracerebral inoculation.
324
GAJDUSEK
but sufficiently long incubation periods
have not yet passed to report any of these
experiments as negative.
Incubation periods observed in chimpanzees that have developed C-J disease
have ranged from 11 to 14 months. On
second passage in chimpanzees the incubation period has not changed. The disease
in all susceptible species of subhuman primates is similar to Creutzfeldt-Jakob disease
in man. All clinical features are present:
myoclonus, fasciculations, spasticity, and
apparent dementia. Most strikingly, the
neuropathologic lesions are identical.4- 20>21
Indeed, our neuropathologist collaborators
now claim that they cannot distinguish at
the cellular level C-J disease pathology of a
chimpanzee from a human brain with either
kuru or Creutzfeldt-Jakob disease. We no
longer consider kuru an exotic, isolated,
strange disease which a few physicians have
reported only from New Guinea, but rather,
a type-disease which is found to be mimicked in every major city in the world, in
the form of Creutzfeldt-Jakob presenile dementias. Although most pathologists clearly
distinguish the Creutzfeldt-Jakob type of
presenile dementia from the more common
Alzheimer type, some cases are borderline
between the two in their clinical or pathologic pictures and, at times, in both.
With two viruses of man (kuru and
Creutzfeldt-Jakob disease) causing slow formation of amyloid-containing plaques, similar to Alzheimer's and senile plaques, and
a slow generalized neuronal loss with gliosis, we have some of the features of the
aging brain produced by a slow virus infection. 8 Whether immune mechanisms are
involved in the pathogenesis of these diseases, we cannot yet specify, since no serologic typing of these viruses or, for that
matter the related diseases, scrapie and
mink encephalopathy, has yet been possible.
Yet as kuru was being elucidated, my
own laboratory and several others were simultaneously working on other diffuse
A.J.C.P.—Vol.
56
subacute degenerations of the brain that
strongly suggested to us the possibility of
a slow virus infection. One of these suddenly provided clues showing that it was,
in fact, caused by such an infection: namely,
Dawson's or subacute type A inclusion body
encephalitis, called, in Europe, Van Bogaert's subacute sclerosing leukoencephalitis, or Pette-Dbring panencephalitis. Doctors Zeman, Sever, Alpers, Adels, Gibbs,
and I decided to rename the disease because of its many different eponyms and
other synonyms when we were preparing
for the world conference we convened on
the disease three years ago at the National
Institutes of Health. 25 At that time, measles
virus had become heavily implicated as the
etiologic agent, in several laboratories including my own. We had to give the disease
a new and descriptive name to surmount
the huge synonymy, but we were unable to
make a good choice. We settled in compromise on "subacute sclerosing panencephalitis," or "SSPE," which the disease is now
generally called. It is a bad name. We had
better have called the disease "delayed and
slow measles encephalitis." In this disease
of children and young adults between the
ages of 3 years and their early 20's (most
common in children less than 10 years old),
there is progressive dementia, myoclonic
jerking, and an afebrile course of progressive motor disability, and increasing neurologic deterioration almost always fatal in 1
to 3 years. Typical periodic slow-wave discharges are found in the EEG, and the cerebrospinal fluid shows a paretic colloidal
gold curve and elevated gamma globulin.
This disease gives us a convincing case for
persistent virus-induced autoimmune damage in man. Measles virus has now been
isolated repeatedly from brain biopsies and
autopsy brain tissue of these patients. It
follows the primary episode of acute measles, often in early infancy, by a period
ranging from a few months to more than
10 years. Many patients have histories of
September 1971
SLOW VIRUS DISEASES OF T H E CNS
atypical measles, measles without rash, or
measles exposure but no diagnosed measles.
It is not the same as classical post-measles
encephalitis; rather, it is a delayed, slow
measles encephalitis with a progressive
course ranging from a few months to several years to death.
In SSPE, the final isolation of the infectious measles virus was slow in being
achieved, although we had ample indirect
evidence that it was the cause. Its study
provides a lesson for the investigation of
any disease which may have a persistent,
latent viral etiology. We had extended to
SSPE our origfnal approach to kuru and
begun to inoculate chimpanzees and other
hosts using the "Noah" approach: two or
more of every species of subhuman primate
we could obtain were inoculated and held
under observation. We had done this with
brain biopsy and early autopsy material
from several cases of SSPE obtained for us
from European and American patients by
cooperating neurosurgeons and neurologists. However, no disease developed in any
of the animals inoculated over long-term
observation. Yet, SSPE could not be removed from the 20 idiopathic, degenerative
diseases of the central nervous system of
man which we compiled in 1958 as demanding this sort of search for a transmissible
agent. Then the work of French electron
microscopists, Bouteille and co-workers,5
was brought to our attention, work in
which they reported finding tubular structures in the intranuclear inclusion bodies
in this disease, which suggested to them
myxoviruses. Others claimed that they were
merely neurotubular structures of a type
not unusual in degenerating neurons. We
immediately recognized that it is probably
expecting too much for a common myxovirus infection of man to be neurovirulent
in our exotic hosts. Thus, we started virus
serologic study of our SSPE patients and
inoculation of animals and tissue and cell
cultures in attempts to isolate measles virus
325
from the tissues we had received from patients with the disease.1
Connolly and his co-workers0 in Belfast
detected unusually high measles antibody
titers in the serum and cerebrospinal fluid
(CSF) of two patients and by direct FA test
demonstrated fluorescence of material in intranuclear inclusion bodies in the patient's
brain when stained with convalescent measles serum that had been conjugated with
isothiocyanate. We quickly confirmed these
findings in our laboratory, and at the same
time we demonstrated the specificity of the
antibody in the patient's serum and CSF
to measles virus with no unusual serologic
reactions or cross-reactions with other myxovirus antigens. 1
That SSPE is, in part at least, an autoimmune disease, is suggested by the following. There is uniformly a hyperimmune
serum response to measles virus antigens.
Specific antimeasles gamma globulin is
found in the spinal fluid. There is a plasma
cell proliferation in the brain with mononuclear cell proliferation producing a perivascular cuffing. Antibody is produced in
the brain itself by plasma cells in the inflammatory reaction. There is fluorescent
staining of the SSPE brain sections using
anti-gamma globulin and also anti-beta C'3
(third component of complement) according to the technic of Dixon and Oldstone. 7
This indicates that an antigen has coupled
to the specific antibody in these cytoplasmic
areas; ter Meulen, 28 using quickly frozen
brain obtained immediately after the death
of a child who had been carefully studied
during the course of typical SSPE, succeeded in demonstrating that the gamma
globulin extracted from washed brain fragments was measles specific antibody and not
antibody to some other myxovirus. The
gamma globulin content of the brain was
quantitatively so high that it resulted in a
very significantly higher yield of globulins
from brain tissue than could be obtained
from normal brain. Once the measles anti-
326
GAJDUSEK
body had been eluted from frozen lyophilized brain sections with 3 M thiocyanate
buffer of pH 6.0, measles antigen in the
cytoplasm was unmasked. Fluorescent staining of antigen in the cytoplasm with measles specific antiserum was then possible.
Without such pretreatment to release and
unmask the measles antigen from gamma
globulin antibody binding, only intranuclear inclusions were stained with fluorescein coupled measles antisera. Negative
controls were obtained using other virus
specific antibodies in direct and indirect
FA systems.
Do we have an experimental animal
model for the study of this disease? No. In
addition to the work we have been doing,
many other attempts have been made to
transmit the disease to ferrets, monkeys,
and other laboratory animals, using as inocula brain and other tissue suspensions
from patients with SSPE as well as the tissue culture isolated measles virus. Katz and
his co-workers17 have reported an inapparent or subclinical infection of ferrets manifested by EEG changes and a mild encephalopathy following inoculation of SSPE
brain suspensions. However, no disease
lending itself to therapeutic trials is produced.
Long before SSPE was known to be associated with measles virus, Rustigian 23>2i
succeeded, in a laboratory exercise which
has been repeated, in modifying the measles
virus through passage in tissue culture,
such that it no longer is cytolytic, but becomes completely parasymbiotic. The measles virus now, in a repressed, incompletely
assembled form, filled the cells maintained
in vitro, passing from cell to cell, and yet
was not being released to the outside medium as a hemagglutinin or an infectious
virus particle.
T h e procedures found necessary in isolating the measles virus regularly from the
biopsies of brains of SSPE patients is revealing. The fresh brain tissue is minced
A.J.C.P.—Vol.
56
into small fragments, trypsinized, and the
trypsin-dispersed cells are layered in flasks
to produce cell cultures. One may also grow
the brain tissue as an explant, using pinhead-sized fragments. In either case, some
brain cells, principally astroglia, grow well.
They may continue to survive and slowly
divide for weeks or many months. But no
measles virus is usually released, there is
no cell surface hemagglutinin, and there is
no FA-positive reaction on either the trypsinized or explant cultures, at first. However, after prolonged cultivation in vitro,
occasional giant cells appear; these are
found to contain measles antigen by FA
staining, and myxovirus microtubules by
electron microscopy. 3 -" Furthermore, when
these cells are subcultured or cocultivated
with other cell lines, such as primary rhesus
monkey kidney, stable green monkey kidney B-SC-1, and HeLa cells, they often yield
polykaryons, and in an occasional polykaryon, with immunofluorescent technics,
one can detect measles antigen at a time
when no hemagglutinating fully infectious
measles virus is detectable. There is no infectious virus released into the medium and
there is no infection caused by these cultures, at this stage, to a measles susceptible
system. However, by repeated passages of
persistently infected cells and continued
subculturing of polykaryons, that is, human
brain cells fused with measles virus permissive cells, fully infectious active measles
virus is shed, which on genetic analysis
turns out to be intermediate in its properties between vaccine strains and the wild
virus of natural acute measles.14
All the trypsinization and other manipulations through many subcultures that have
gone into the process of viral genome rescue, or unmasking of the virus, would certainly be sufficient to accomplish a genetic
alteration of the strain isolated. Therefore,
the answer to the question, "What is the
relationship of this isolated measles virus
to wild measles or attenuated live measles
September
1971
SLOW VIRUS DISEASES OF THE CNS
vaccine strains?" is not readily answerable,
since the modification that isolation technics caused cannot be predicted.
As recent work by ter Meulen indicates,
we undoubtedly have in SSPE, a situation
in which the virus is not present as fully
infectious or fully formed measles virions.
Instead, there is an asynchronous, incomplete, and uncoordinated production of
viral subunits; tubular structures without
nucleic acid or nucleic acid and viral antigen, without full assembly of EM visible
virions. This goes on in the human brain,
spreading from cell to cell, as it occurs in
model cultures infected with Rustigian's
strain of defective, incomplete measles virus. That this kind of infection may go
on for weeks, months, and years, perhaps
decades, silently, without producing significant pathology and then, for unknown reasons, may be triggered into a more rapid,
slowly pathogenic course, is the significant
fact for consideration in the study of
chronic idiopathic disease. Whether a virus
specific defect in the delayed hypersensitivity reactions may contribute to such release of latent virus is not yet established.
However, the genetic heterogeneity of the
population with respect to immunologic
competence may well account for a rare
individual whose delayed or sluggish immune responses favor the development of
such a measles infection.
Finally, we have a model of this disease
in nature, in hard-pad disease in the dog.
The closest virus serologically to measles
is the distemper virus. It is not distemper
virus in the human brain causing SSPE,
but measles. However, dogs with distemper
occasionally recover, and a small percentage develop a neurologic disease with myoclonic jerking and progressive cerebral degeneration, quite akin to SSPE. This spontaneous, natural disease is not a good
laboratory model for studying SSPE, since
we cannot produce the disease in more than
a rare animal that recovers from distemper.
327
Only after intranasal infection with the
distemper virus has the incidence of this
complication been raised, but still not sufficiently to make it a usable system for experimental trial therapy and other studies.
In these studies half of the dogs died of
their primary distemper infection and only
half of the survivors went on to develop
a chronic CNS disease. Interestingly, this
syndrome occurred only in those dogs which
developed delayed antibody responses in
their primary distemper infection. If antibody formation occurred early enough to
prevent spread of virus from lymphatic
cells to epithelial cells, these tissues were
not infected. If virus reached such cells as
epidermal cells and neurons before protective levels of antibody were attained, the
virus persisted and late encephalitis, or
hard-pad disease developed.2
Koprowski and associates18 have found
in explant cultures of SSPE brain tissue,
grown for weeks in in vitro cultures, a
papova virus identified by electron microscopy study. It is neither polyoma nor SV40,
but in size and morphology is indistinguishable from these known viruses. Neither
is it the rat papova viruses, nor any of the
morphologically similar viruses such as the
virus of warts. They hypothesize that this
virus is so often found by electron microscopy in the SSPE explant cultures that it,
in addition to the proved presence of measles virus, must be involved in the pathogenesis of the disease and, further, the two
viruses must act symbiotically, and the
papova virus must stimulate the disease
produced by the measles virus. We do not
know what to call this papova virus. I suspect that it is the same virus that ZuRhein
and Chou 2 7 and others have been finding
regularly in the lesions of progressive multifocal leukoencephalopathy, which they have
similarly been unable to distinguish morphologically from polyoma or SV40 viruses,
and which they and we have been unable
328
GAJDUSEK
to cultivate in vitro* I tend to call it "the
papova virus latent in human brain."
Progressive multifocal leukoencephalopathy (PML) is a fatal brain disease which
develops in patients who are dying of
chronic Hodgkin's disease, Boeck's sarcoid,
tuberculosis, leukemia, or other lymphomas, and in whom the subacute brain infection kills them before their primary disease. At autopsy one finds pinpoint scattered
lesions in the white matter of the brain
which are not, on pathologic examination,
caused by the primary disease. Since the
lesions are filled in their peripheries with
crystalline arrays of papova virions, we
have a good idea of what has happened:
a latent agent has been activated in the
course of the immunosuppression that accompanies these natural diseases.
Since ZuRhein's first demonstration of
intranuclear crystalline masses of papova
virus in the pinpoint lesions of this disease,
others have repeatedly confirmed her finding, but the virus still has not been identified. We have, with the aid of Dr. Wallace
Rowe of the NIH, found no evidence of
antibodies to SV40, polyoma, or to the papova rat (RV and X14) viruses, or mouse
virus (K virus) in sera from the patients,
or from animals inoculated with this disease.
How many other viruses can man silently
carry for years, like herpes simplex, measles virus, infectious hepatitis, rubella virus,
cytomegalovirus, the EB virus of infectious
mononucleosis, the varicella virus, warts,
adenoviruses, or reoviruses? How many
other agents of this sort do we have lying in
wait in our tissues for the right additional
factors to appear to stimulate them to path* More recently, this virus has been cultivated in
three laboratories. [Gardner SD, Field AM, Coleman
DV, Hulme B; New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet
1:7712, 1253-1256, 1971. Padgett BL, Walker DL,
ZuRhein GM, Eckroade RJ; Cultivation of papovalike virus from human brain with progessive multifocal leukoencephalopathy. Lancet 1:7712, 12571260, 1971]
A.J.C.P.—Vol.
56
ogenic activity? Such factors may be the
presence of a second virus from a new acute
infection or another persistent agent; they
could be a bacterial or a protozoal or helminthic infection, or the immunosuppressive effects of another chronic disease or
that induced therapeutically for organ
transplantation, or they might even be provided by certain drugs, toxins, nutritional
or dietary factors or, finally, even traumatic injury or changes in endocrine balance. Indeed, these may be the underlying
mechanisms of many chronic diseases of unknown etiology. That such latent and persistent infections may eventually produce a
hyperimmune state and lead to autoimmune tissue damage with the antibody reacting with the virus filled tissue cells is
amply demonstrated in SSPE in man and
in the persistent-tolerant infection of mice
with LCM virus which develop immunecomplex renal disease. Finally, such a
mechanism is not even excluded in diseases
known to be heredofamilial, for the genetic
abnormality of the immune system may
well predispose to autoimmune disease in
the presence of persistent virus infections,
as appears to be the case with the NZB
mice.
Now to return to kuru. The disease has
been transmitted to more than 50 chimpanzees (another 30 have been given Creutzfeldt-Jakob disease).13 Kuru is in the fifth
passage in the chimpanzee, and in the third
passage in the spider monkey. In the fourth
passage, the virus reaches titers of 107 infectious doses per ml. in the brain, which is
as high as some of the equine encephalitis
virus titers attained in human brain. It is
present also in the lymph nodes and visceral tissue, as well as the brains of affected
chimpanzees, and can be transmitted to the
chimpanzee by peripheral, non-intracerebral, routes of inoculation.
No electron microscopically visible particles identifiable as virions have been
found in any of our studies of either kuru
September 1971
329
SLOW VIRUS DISEASES OF T H E CNS
or Creutzfeldt-Jakob disease; instead, only
stacks of curled fragments of plasma membrane filling the intracellular vacuoles of
neurons have been observed.20-21
Our search for virus which we can cultivate in vitro from the surgically sterile tissues of animals affected with kuru and
Creutzfeldt-Jakob disease has yielded more
than 150 isolations of virus strains from
tissues which are bacteriologically and virologically sterile for the first weeks of
study. They are thus truly "latent" viruses.
Attempts to isolate virus by inoculating
grindings of the fresh tissue into many primary and continuous cell lines have been
negative. However, by maintaining explant
cultures of brain, thymus, lymph nodes,
kidney, etc., for weeks, months, and as long
as a year, my colleagues have been isolating
strains of latent viruses from these "sterile"
explains. 11 ' 22
These many isolated chimpanzee virus
strains have been typed into 13 different
viruses. There are few fragments of chimpanzee brain, from young or old animals,
from which we have not isolated viruses.
Sometimes, not only one virus, but viruses,
i.e., two or more strains from one explant,
have been isolated. We cannot say this yet
about human tissues as well. We have isolated only a few agents from human tissue
culture explants thus far.
This discovery of latent viruses in "sterile" tissues should not have surprised us,
had we just thought about the history of
poliovirus vaccine. We used millions of
doses of a virus vaccine which we never
would have started to use, had we thought
it contained either live or dead extraneous
unidentified virions. Yet, within a few years
of its introduction, there were dozens of
viruses known to be contaminating various lots of that vaccine. The simian viruses,
one after another, were isolated from the
healthy, surgically removed, at first virologically sterile, monkey kidney. This has
been well known for years. One of the la-
tent simian viruses, SV40, was even distributed live from the drug store shelves, uninactivated by the concentration of formalin
used to inactivate the poliovirus. However,
we continued to think "the kidney is a
'dirty' organ; the monkey a 'dirty' animal."
Our more recent work with the chimpanzees affected with kuru indicates that their
brain and thyroid are equally "dirty."
Such latency can, of course, suggest possibilities for the triggering of autoimmune
disease. Such virus infections may lead to
the alteration of cell-surface antigens and
the addition of new cell-surface antigens
and of new intracellular antigens. These
new antigens may be, at times, virus subunits which are not being assembled into
complete virions—defective viruses, if you
wish—although the whole genome is present, some of it repressed, and assembly is
not proceeding normally. The latent virus
genome may also program for the production of non-virus antigens, such as tumor
antigens, and thus produce antigenic molecules which are not virus subunits. We are
thus immediately faced with the possibility
of autoimmune mechanisms at work in cancer as well.
I leave the matter of such speculations
here, and with apologies, I will not go into
the long and involved story of kuru and its
possible origins at this time.
References
1. Adds BR, Gajclusck DC, Gibbs CJ Jr, et al.\
Attempts to transmit subacute sclerosing panencephalitis and isolate a measles related
agent, with a study of the immune response
in patients and experimental animals. Neurology 18:1 (part 2), 30-51, 1968
2. Appcl MJG: Pathogenesis of canine distemper.
Amer J Vet Res 30:7, 1167-1182, 1969
3. Baublis JV, Payne FE: Measles antigen and syncytium formation in brain cell cultures from
subacute sclerosing panencephalitis (SSPE).
Proc Soc Exp Biol Med 129:593-597, 1968
4. Beck E, Daniel PM, Gajdusek DC, et at.: Subacute degenerations of the brain transmissible
to experimental animals: A neuropathological
evaluation. Proc. Vlth Congr. Inter, de Neuropathologie. Paris, Masson et Cie, 1970, pp
858-873
330
GAJDUSEK
5. Boutcille M, Fontaine C, Vedrenne C, et al.:
Sur un cas d'enccphalite subaigue a inclusions.
Etude anatomo-clinique et ultrastructurale.
Rev Neurol 113:4, 454-458, 1965
6. Connolly GH, Allen IV, Hurwitz LJ, et al.:
Measles virus antibody and antigen in subacute sclerosing panencephalitis. Lancet 1:542,
1967
7. Dixon FR, Oldstone MBA: Lymphocytic choriomeningitis: Production of antibody by "tolerant" infected mice. Science 158:1193-1195,
1967
8. Gajdusek DC: Slow virus infections and activation of latent virus infections in aging. Advances Geront Res (in press), 1971
9. Gajdusek DC, Gibbs CJ Jr: Transmission of
two subacute spongiform encephalopathies of
man (kuru and Creutzfeldt-Jakob disease) to
New World monkeys. Nature 230:588-591,
1971
10. Gajdusek DC, Gibbs CJ Jr, Alpers M (editors):
Slow, Latent and Temperate Virus Infections.
NINDB Monograph No. 2, PHS Publication
No. 1378, U. S. Government Printing Office,
Washington, D. C , 1965, 489 pp
11. Gajdusek DC, Rogers NG, Basnight M, et al.:
Transmission experiments with kuru in chimpanzees and the isolation of latent viruses
from the explanted tissues of affected animals. Ann NY Acad Sci 162:1, 529-550, 1969;
also, Virology Abstracts, V3484, 1969
12. Gajdusek DC, Zigas V: Degenerative disease of
the central nervous system in New Guinea.
T h e endemic occurrence of "kuru" in the native population. New Eng J Med 257:30, 974978, 1957
13. Gibbs CJ Jr, Gajdusek DC: Transmission and
characterization of the agents of spongiform
virus encephalopathies: Kuru, CreutzfeldtJakob disease, scrapie and mink encephalopathy, Immunological Disorders of the Nervous
System. Baltimore, Williams and Wilkins,
A.R.N.M.D. 49 (in press): 1971
14. Horta-Barbosa L, Fuccillo DA, Hamilton R,
et al.: Some characteristics of SSPE measles
virus. Proc Soc Exp Biol Med 134:1, 17-21,
1970
15. Horta-Barbosa L, Fuccillo DA, London W T ,
et al.: Isolation of measles virus from brain
cell cultures of two patients with subacute
sclerosing panencephalitis. Proc Soc Exp Biol
Med 132:272-277, 1969
16. Johnson R T , Merger EH: Experimental rabies:
Studies of cellular vulnerability and pathogenesis using fluorescent antibody staining. J
Neuropath Exp Neurol 24:662-674, 1965
A.J.C.P.—Vol.
56
17. Katz M, Rorke LB, Masland WS, et al.: Transmission of an encephalitogenic agent from
brains of patients with subacute sclerosing
panencephalitis to ferrets. New Eng J Med
279:793-798, 1968
18. Kibrick S, Gooding GW: Pathogenesis of infection with herpes simplex virus with special
reference to nervous tissue, NINDB Monograph No. 2, Slow, Latent, and Temperate
Virus Infections. Edited by DC Gajdusek, CJ
Gibbs Jr, and M Alpers. U. S. Government
Printing Office, Washington, D. C , 1965, pp
143-154
19. Koprowski H, Barbanti-Brodano G, Katz M:
Interactions between papova-like virus and
paramyxovirus in human brain cells: A hypothesis. Nature 225:1045-1047, 1970
20. Lampert PW, Earle KM, Gibbs CJ Jr, et al.:
Electron microscopic studies on experimental
spongiform encephalopathies (kuru and Creutzfeldt-Jakob disease) in chimpanzees. Proc.
Vlth Congr. Intern, de Neuropathologie, Paris,
Masson et Cie, 1970, pp 916-930
21. Lampert PW, Gajdusek DC, Gibbs CJ, Jr: Experimental spongiform encephalopathy (Creutzfeldt-Jakob disease) in chimpanzees: Electron
microscopic studies. J Neuropath Exp Neurol
30:1, 20-32, 1971
22. Rogers NG, Basnight M, Gibbs CJ Jr, et al.:
Latent viruses in chimpanzees with experimental kuru. Nature 216:446-449, 1967
23. Rustigian R: Persistent infection of cells in
culture by measles virus. I. Development and
characteristics of HeLa sublines persistently
infected with complete virus. J Bact 92:1792,
1966a
24. Rustigian R: Persistent infection of cells in culture by measles virus. II. Effect of measles
antibody on persistently infected HeLa sublines and recovery of a HeLa clonal line persistently infected with incomplete virus. J
Bact 92:1805, 1966b
25. Sever JL, Zeman W (editors) : Conference on
"Measles Virus and Subacute Sclerosing Panencephalitis." Neurology 18:1 (Part 2), 1-200,
1968
26. ter Meulen V, Enders-Rucklc G, Miiller D, et
al.: Immunohistological, microscopical and
neurochemical studies on encephalitides. III.
Subacute progressive panencephalitis, virological and immunohistological studies. Acta
Neuropath 12:244-259, 1969
27. ZuRhein GM, Chou SM: Papova virus in progressive multifocal leukoencephalopathy, Infections of the Nervous System. Baltimore,
Williams and Wilkins, A.R.N.M.D. 64:307362, 1968
Discussion following Dr. Gajdusek's Paper
(In response to question from the audience)
DR. GAJDUSEK: At the Wistar Institute,
Doctors Hilary Koprowski and Michael
Katz have been able to produce an EEG
pattern in ferrets inoculated with suspensions of brain from patients with SSPE
similar to that seen in the patients. When
the apparently healthy animal is sacrificed
they show an inclusion-body type of panencephalitis resembling that of SSPE. When
they take the isolated measles virus together
September 1971
SLOW VIRUS DISEASES OF THE CNS
with cells in which polyoma-like virus particles are visible by electron microscopy
(which is the only way it can be demonstrated), this mixture of the two agents
produces the phenomenon more regularly
in ferrets, and now in hamsters as well.
I have purposely avoided the complex
problem of how one is ever, in chronic persistent incomplete virus infections, going to
fully investigate the matter of possible viral
pathogenesis. It is better here, to an audience rather than in press, to become wildly
speculative. If we accept the fact that defective, unassembled replication of viruses
continues over years silently, and then,
spontaneously, from factors we do not understand, can be stimulated into a different
level of misbehavior so that disease with a
cumulative, finally fatal pathology results;
if we accept this as a fact for measles virus
in SSPE and distemper virus in hard-pad
disease—what is then the next analogy we
can make with defective bacteriophage systems? We can have more extensive defectiveness; namely, the genetic information
for the whole virion is not really present,
but masked or repressed. Assembly may not
be proceeding at all. The equivalent of
only a tumor antigen is left and no infectious virions are resurrectable under any
conditions. If that sort of thing occurs, and
it surely does go on in experimental models in a test tube, it might be such a mechanism that lies behind Parkinson's disease
with a defective replication of influenza
virus, multiple sclerosis with a fully defective and incomplete bit of measles virus, or
hepatitis or some other virus. Such theories
are effortless armchair exercises; what we
need more is a means of probing in these
directions. How are we going to get at it?
The only approaches currently available,
I'm afraid, are to make a potent antisera
against each of the subviral components of
the whole gamut of myxoviruses and other
possible virus candidates for use against appropriately collected tissue specimens. Then
331
you, as clinical pathologists, collect material, not in formalin but in dimethylbutane and gluteraldehyde, and quick-frozen
thin sections at —70 C. and liquid nitrogen
temperatures. With the bulk of such tissue
we may also be able to make density gradient fractions of viral subunits in the tissues.
This should be the approach in any idiopathic disease these days. We may forget
the old hematoxylin-and-eosin stained section. For chronic degenerative central nervous system diseases the neuropathologists,
neurosurgeons, and neurologists are supplying us with well collected tissues in large
amounts and very often. We are working
intensively on rare diseases: Pick's, Alzheimer's, metachromatic leukodystrophy,
Huntington's chorea, myoclonic epilepsy,
and other rare neurologic syndromes. Tissues from many cases of each of these diseases are being accumulated as brain biopsies and as specimens from autopsies within
a few hours of death. We are having less
luck with the more common diseases, such
as multiple sclerosis and disseminated lupus
erythematosus. We need live cells for trypsinized cultivation. We do not need material taken five hours postmortem and put
into a fixative, but material collected immediately, within a few hours of death, in a
sterile manner. When it is taken from separate organs, the autopsy room must be
equipped with separate sets of sterile instruments and containers. There is no point
in opening the head with one set of instruments, and then taking specimens of liver
and spleen with the same instruments. Most
autopsy rooms are not even equipped with
separate sets of sterile instruments and
other equipment for collecting sterile specimens. So I make a real plea. If, within the
field of rheumatoid diseases, the collagen
diseases, the other degenerative idiopathic
diseases in addition to the neurologic, you
are planning to follow the immunologic
and virologic investigative approach, reequip the autopsy and the surgical biopsy
332
GAJDUSEK
rooms for the sterile collection of proper
specimens, some quickly frozen in liquid
nitrogen, others in other special tissue culture media for cultivation in tissue cultures.
(In response to question from the
audience)
DR. GAJDUSEK: The whole discipline of
virology depends on serologic identification
and the specificity of the serologic reactions. In other words, when you make the
diagnosis in any virus diagnostic laboratory
of poliomyelitis or any other specific virus
infection, you rarely do anything but decide what virus antigens or antibodies are
there, depending on the specificity of serologic reactions. If we discard this, we have
discarded neutralization, complement fixation, hemagglutination-inhibition, fluorescent antibody, and every other serologic
diagnostic method. In other words, when
I identify rabies or poliomyelitis viruses in
the laboratory, I can assure you that it is
rabies or poliomyelitis, respectively, by specific antigen-antibody reactions, or using
these serologic procedures state that it is
an unknown agent which is related antigenically to the known viruses. Now, in the
case of the measles virus in SSPE, we are
growing the infectious measles virus from
the brain. So the full genetic information
of the measles virus is there. It takes great
effort to isolate it, however. We first thought
that this was because we had to overcome
the neutralization of the virus which already had occurred in vivo. On the other
hand, dilution does not release the virus,
and it is obtained only when we produce
cell fusions. When that trick works, as it
does in many other experimental viral systems, we are inclined to think the virus
A.J.C.P.—Vol.
56
was in a genetic modification well synchronized with the host and no longer producing cytolytic effect. The antigen present
has not been identified. It may be coat antigen, or something else. The antisera
which have been made and which have
worked are crude antisera made against a
tissue culture harvest of measles virus—
not even the purified measles virus. Until
we have a more accurate antigenic tool—
antisera against component parts of measles virus—I do not think we can answer
the question of participation of the various
parts of the virus in the disease process.
(In response to questions from the
audience)
DR. GAJDUSEK: One may ask of a surgical
biopsy of any organ: "Are there virus agents
there? What state are they in? How many
agents? Do they have any role in any pathology seen?" I think those are all fair
questions. That the symbiotic relationship
may be deranged and the latent viruses
begin producing disease is already demonstrated in certain diseases. For example,
Dr. Sydney Kibrick and his colleagues in
Boston have regularly produced excretion
of herpes simplex from corneal cells, followed in some animals by herpes encephalitis, by simply injecting adrenalin into
normal animals which had stopped shedding herpes virus from their corneas for
many months after they had been given
corneal herpes simplex infections and recovered. His triggering shot to produce the
shedding of infectious herpes simplex virus,
new corneal pathology, and even acute or
chronic herpes invasion of the nervous system, is an injection of adrenalin. 18