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IDENTIFICATION OF INCLUSIONS IN VIRUS
DISEASES*
E. V. COWDRY
Washington University and the Barnard Free Skin and Cancer Hospital, St. Louis
Scarcely a year has passed in the last decade without the recognition of one or more hitherto unsuspected virus diseases of man.
The profession has become acutely virus conscious. We even
hear of "Virologists" and we read, with profit, "Alice in Virusland." 1 Clinical pathologists are immediately concerned because
their advice is quickly sought in dealing with cases presenting
unusual features and which cannot be easily diagnosed, for they
have at their disposal laboratory facilities not possessed by most
practitioners and they employ experimental methods.
The viruses have made themselves known in unexpected ways
and it is quite possible that still others lurk in our bodies or may
invade us from the great animal reservoir of disease with which
we are in contact.
Who would have supposed innocent looking love-birds and
worldly-wise parrots to harbor the deadly virus of psittacosis?
Who would have expected that house mice carry the equally dangerous virus of lymphocytic choriomeningitis? In some parts
of the world sheep are a source of infection. Thus, investigators
working on a virus disease (louping ill), observed in Scotch sheep,
have become mildly affected by it. While I was in Kenya Colony
the lambs in a nearby farm commenced to die off in large numbers.
Blood from them was brought to the laboratory by Daubney 2
and a virus isolated. All who first worked with it became severely
but not dangerously ill. In Australia a virus of sheep origin has
become established in humans often with fatal results. The
* Received for publication July 1, 1939.
Read before the Eighteenth Annual Convention of The American Society
of Clinical Pathologists, May 12-14, 1939, St. Louis, Missouri.
133
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B. V. COWDEY
condition is called " X disease" and resembles infantile paralysis
slightly but sheep are not susceptible to infantile paralysis virus.
There are points of resemblance to louping ill; but, for humans,
it is much more fatal.
When B was bitten by a normal looking monkey, and after
the local lesion appeared to be clearing up, a virus gradually
ascended his nervous system with fatal results. The virus was
isolated3 without difficulty and proved to be new to science but
in the animal host, which showed no symptoms, it may have
been operating for thousands of years.
Human virus diseases of animal origin are fairly numerous;
for example:
Cowpox-vaccinia from cattle
Encephalomyelitis from horses (by mosquito)
Foot and mouth disease from cattle and pigs
Swine-herd disease from pigs (Switzerland)
Rabies from dogs
Influenza from swine (?)
Psittacosis from parrots, etc.
Rift valley fever from sheep
Louping ill from sheep
Australian X disease from sheep
Lymphocytic choromeningitis from mice and others
Lines are laid to some viruses by the discovery of nuclear, or
cytoplasmic inclusions, or both, in biopsy or autopsy specimens.
I t was the observation of such bodies in the liver cells of monkeys
experimentally infected with yellow fever4 that intensified the
search for a virus. And, when similar nuclear inclusions were
observed in human lesions both from the West Coast of Africa
and from the Americas,6 belief that there are not two brands of
yellow fever, an African and an American, but one, and that
probably caused by a virus, increased.
1. HUMAN VIRUS DISEASES
It is likely that there are several thousand viruses which prey
on animals and plants, not to mention the bacteriophages. But
space is limited and we are primarily interested in human diseases.
INCLUSIONS IN VlKUS DISEASES
135
To make a list of human virus diseases acceptable to everyone
is not feasible because there is little agreement as to what viruses
are. Particularly ill defined is the line between Rickettsiae and
viruses and Rickettsioses are hence not included in the following
list of examples:
No characteristic inclusions: mumps, common colds, measles, dengue, sandfly fever, epidemic influenza, encephalitis (St. Louis), Encephalitis lethargica,
poliomyelitis, Australian X disease, infectious anemia (?), encephalomyelitis,
herpes Zoster (?).
Nuclear inclusions: yellow fever, chickenpox, herpes febrilis, lymphogranuloma inguinale, laryngeal papilloma, B virus disease, salivary gland disease,
whooping cough (?).
Cytoplasmic inclusions: rabies, vaccinia, molluscum contagiosum, trachoma,
Inclusion blennorrhea (swimming bath conjunctivitis).
Both nuclear and cytoplasmic inclusions: smallpox, alastrim.
2. NUCLEAK INCLUSIONS
1. Technic. For routine purposes it is not feasible to study nuclear inclusions
in living cells. It is necessary to fix the tissues, embed them in paraffin, cut
sections and stain them. Nuclear inclusions are easilyfixedand are not noticeably soluble in alcohol, dilute acetic acid or other chemicals in general use.
Consequently almost any good fixative gives satisfactory results. Zenker's
fluid (with 5 per cent of acetic), or Zenker's fluid in which the acetic acid is
replaced by 10 per cent of commercial formalin, is recommended. But the
ground substance of the nucleus, which contains the inclusions, will appear
more homogeneous and lifelike after fixation in osmic acid mixtures.6
Experience shows that hematoxylin and eosin technic is the best staining
method for the demonstration of nuclear inclusions. In general, they are
acidophilic and color well with eosin in contrast with the basophilic, hematoxylin
staining chromatin. What hematoxylin and eosin preparations lose in brilliancy
they make up for by permanence. If the balsam is of good quality and not too
acid, and if they are not over exposed to the sun, the preparations will last twentyfive years or more. The thymonucleic acid of the nucleus, which makes up
most of the basophilic chromatin, can be clearly shown by the Feulgen reaction7 by which it is colored an enduring purplish blue and the inclusions can
be counterstained with acid violet (light green) but the acid violet fades in the
course of a few months. Another beautiful combination is safranin for the
basic chromatin, and light green for the inclusions, but the contrasting colors,
especially the shades of green, become decolorized comparatively quickly.
Some investigators favor eosin, or phloxin and methylene blue, or Giemsa's
blood stain but these are not permanent, and, in addition, the degree of red
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E. V. COWDRY
and of blue coloration is more dependent upon the fixative than in the case of
hematoxylin and eosin. Nuclear inclusions can often be brilliantly colored
with fuchsin and the basophilic chromatin counterstained with methyl green,
or with something else; but this is not advised because the fuchsin stains so
many other materials besides the inclusions. More depends upon the topographic and morphological features of the inclusions than upon slight differences
in staining reaction. The latter are more likely to be due to slight, unconscious
variations in the use of the technics mentioned than when hematoxylin and
eosin are employed. Iron hematoxylin can be substituted for the usual Harris'
or Delafield's hematoxylin, in which case the staining will be even more permanent, but the contrast will be less satisfactory.
2. Caused by viruses. When nuclear inclusions are encountered
in a hematoxylin and eosin preparation of a tissue submitted for
examination what conclusions are justified? There is much
difference of opinion as to how this question should be answered.
First, we must form an estimate of the probability that the particular inclusions are caused by virus action. We use the word
"probability" because nuclear inclusions, however typical of
virus action, do not themselves constitute proof. The only
proof of virus action is to demonstrate experimentally the presence
of a virus. When all of the following features are noted in a
section of tissue it is likely that a virus has been at work:
a) A considerable number of inclusion-laden nuclei which can be arranged in
series representing stages in development. This indicates an active process in
which the nuclei exhibiting the most advanced alterations were affected first
and the others in succession.
b) A change in which the accumulation of acidophilic material, forming the
inclusion, is accompanied by margination of basophilic chromatin on the nuclear
membrane, a disappearance of nucleoli and ultimate death and disintegration of
the cells. This suggests that the inclusion formation is not merely an intranuclear heaping up of material effected without injury.
c) A cellular reaction characterized by hyperplasia, hypertrophy or necrosis.
It will be helpful to compare the unknown nuclear inclusions
with preparations of others unquestionably caused by a half a
dozen or more different viruses. When actual preparations are
not available, the next best thing is to examine good colored illustrations of known nuclear inclusions.6' 8
It is seldom necessary to hazard an opinion, from the detailed
appearance of the nuclear inclusions, which virus has caused them.
INCLUSIONS IN VIBUS DISEASES
137
Clues to most of the known viruses will be supplied by the clinical
history except when the viruses belong to the group called "inapparent" comprising those ordinarily not accompanied by
distinctive clinical symptoms. 9 For these we should be on the
lookout. It has been shown that the submaxillary virus of mice,
ordinarily innocuous, can become invasive and cause death, 10
and an unusually potent submaxillary virus of guinea pigs has
been discovered.11 Also, there is some evidence that the submaxillary virus of children, with perhaps bacterial cooperation,
may be involved in whooping cough.12 Moreover, there is reason
to think that superposed infection with a second virus may, on
occasion, make the primary disease more severe. Dr. Downey
L. Harris reports in a child, not yet recovered from the mumps,
that the virus of rabies spread to the central nervous system from
a small bite on the finger with unprecedented speed and so irresistibly that Pasteur treatment was unavailing, although others,
not having mumps, and more extensively bitten by the same dog,
on similar treatment, all recovered.* Several cases have been
described of the complication of virus infections by encephalomyelitis,13 and the viruses themselves often exist in different
strains subject to slight and even to marked variations.14
There are, however, some differences in nuclear inclusions
caused by particular viruses. It is not difficult to distinguish
between those in liver cells due to yellow fever and to herpes5
and slight differences can be noted between those caused by
herpes and virus III; 15 but nuclear inclusions in different virus
diseases are usually much alike. We would not expect, and
we do not find, anything approaching the diversity among them
that is characteristic of cytoplasmic inclusions. This is probably
due to restriction in character of available material for their
development within nuclei coupled with limitation in modes of
nuclear response to environmental stimuli owing to the buffer
afforded by the surrounding cytoplasm. But there may be a
third factor, namely, the penetrability of cytoplasm and nucleus
to virus particles.
Because nuclear inclusions do not exhibit all of the three criteria
* Personal communication.
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E. V. COWDRY
for their identification does not mean that they are not manifestations of virus action. For example, in the case of nuclear inclusions occasionally found in the salivary glands of several species
including man the involved cells, though never numerous, are
greatly hypertrophied and are of uniform appearance with little
or no accompanying tissue reaction. Yet, in the salivary glands
of guinea pigs and of several other species showing these inclusions, viruses have been demonstrated to cause them. Consequently, it is likely that wherever such cells are found in the
salivary glands viruses will also be found.
In the kidneys of a number of species of mammals a few enlarged cells are encountered, possessed of nuclear inclusions,
bearing a striking resemblance to the cells just mentioned in the
salivary glands. But in this locality the search for viruses has
been less intensive. Therefore, we say with less assurance that
they indicate the probability of virus action.
When we come to nuclear inclusions of another sort—which
I have called type B 16 because, as they are formed, the basophilic
chromatin does not marginate on the nuclear membrane and the
nuclear structure does not disintegrate—we have to proceed
warily. Such inclusions are merely droplet-like masses of acidophilic material surrounded by clear halos. They have been
reported in Borna disease, in poliomyelitis and in several other
conditions. When observed in routine preparations they are
seldom conspicuous structures. It is only when strongly stained
with fuchsin, for instance, that they catch the eye. I sometimes
wonder whether careful search of controls not subjected to virus
with equally intense stains would not reveal a few similar bodies.
I t is possible that they are simply an over-development, under
the influence of a virus, of nuclear components already present.
If so, their diagnostic value would be slightly different.
S. Not caused by viruses. To make us feel more secure in the
interpretation of nuclear inclusions actually or probably formed
by viruses, it is desirable also to approach the problem from the
opposite direction, that is to say, to complete the picture by a
brief discussion of nuclear inclusions that are, to the best of our
knowledge, not attributable directly or indirectly to viruses.
INCLUSIONS IN VIRUS DISEASES
139
These are divisible into two groups, those that occur normally,
and those produced as the result of some experimental procedure
without virus.
As inclusions brought about by the inapparent viruses tend to
occur in the salivary glands and possibly in the kidneys, so nuclear
inclusions not caused by viruses are more prone to make their
appearance in certain special parts of the body, the liver, the
kidney and the epididymis, than in others.
Some nuclear inclusions in the hepatic cells of dogs not showing
signs of disease, are acidophilic and are surrounded by halos of
clear nucleoplasm but they do not constitute a source of confusion
because they are crystals and can always be surely identified by
their angular shape. Excellent illustrations are available.17
In human livers examples of what appear to be hypertrophies
nuclei and nucleoli have been reported. 5 The latter are not
associated with any nuclear disintegration, yet they are faintly
acidophilic. They should not be confused with nuclear inclusions
occasioned by viruses.
Sections of the kidneys from 1012 routine autopsies have been
examined.18 Inclusions of type B were found in only 17 or
1.67 per cent. • No type A inclusions were seen. But in animals
nuclear inclusions are often met with. There is evidence that
those present in the kidneys of apparently normal monkeys are
greatly increased in number by administration of irradiated ergosterol.19
In the epididymis many investigators have described and illustrated intranuclear acidophilic masses which do resemble
type B inclusions.20-23 They occur, however, in 100 per cent
of individuals (presumably adults) of the species mentioned and
are obviously not due to viruses. Almost all other organs of the
body occasionally show similar intranuclear bodies and therefore
exhibit the same phenomenon but to a less degree. I am not
inclined to attach any special significance to them, as far as the
search for viruses is concerned, unless they happen to be unusually
numerous and are accompanied by traces of local tissue reaction.
The observations of Scharrer, 24 ' 26 on the nuclear peculiarities
of certain groups of cells in the midbrain, are important and
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E. V. COWDRY
relate, among other things, to invaginations of the nuclear membrane, cross sections of which look like nuclear inclusions, and
on which we shall soon report together. Nuclear inclusions described in gliomas26 do not appear to be of virus etiology.
In ordinary postmortem degeneration the diameter of nuclei
is frequently reduced so that the acidophilic components are
crowded within a smaller space with the result that, relative to
the size'of the nuclei, they seem to be more prominent. But such
oxychromatic masses are fairly uniform in contrast with most
nuclear inclusions caused by virus action.
4- Nature of nuclear inclusions. Before concluding this
account of nuclear inclusions it is desirable to review in a few
words what is known and suspected concerning the nature of the
inclusions which are formed under the influence of viruses.
In 1923 Goodpasture and Teague27 expressed the opinion that
in herpes "the intranuclear bodies are essentially masses of virus."
Two years later, Goodpasture 28 reported: " I t seems evident,
however, that the material which constitutes the 'inclusion' may
partly, at least, be composed of coagulated nucleoplasm which
may impart the acidophilic staining property of the inclusions.
It is to be noted, however, that when minute granules are discrete enough to be recognized as such they stain faintly basophilically, whereas the precipitate from the nucleoplasm of normal
cells is more acidophilic. They are to be regarded at present
as elementary bodies taking part in the structure of the herpetic
inclusions." Very recently Markham 29 has supported Goodpasture insofar that he has concluded that the salivary gland
inclusions of guinea pigs are aggregates of "elementary bodies"
of the virus, but he said nothing about basophilia.
Not the findings, but the interpretations of these investigators
are questionable. There is as yet no convincing experimental
evidence that any virus enters the nuclei of affected cells. That
in some conditions the Rickettsiae of Rocky Mountain spotted
fever do80 is beside the point. 31 That some minute granules
within the herpetic inclusions stain faintly basophilically is scant
reason for believing that they are elementary bodies of the virus.
INCLUSIONS IN VIBUS DISEASES
141
It is difficult to be sure, in the first place, that a slight degree of
basophilia is not occasioned by mordanting, overstaining with
basic dyes or other deviations in technic. And, in the second
place, it is not easy to distinguish between basophilia due to
invading virus and the basophilia of chromatin in injured nuclei.
Most nuclear inclusions in virus diseases are acidophilic, give
negative reactions for both thymonucleic acid and masked iron
and yield little or no ash after microincineration. 7 ' 31 - 32 But
there are exceptions. For instance, the inclusions in the salivary
glands of moles are basophilic and do leave a mineral residue.33
The cytoplasmic inclusions in fowl pox, which we admit are made
up of elementary bodies of virus, are rich in mineral matter 34 but
it has not been shown whether the minerals are actually in the
elementary bodies or in some cytoplasmic or other material which
binds them together and exists between them. Markham was
influenced in his conclusion by the fact that inclusions in the
guinea pig's submaxillaries are aggregates of very fine particles
and give a positive Millon's reaction for protein. Neither of
these considerations justifies his contention that they are made
up of elementary bodies of virus; but the possibility cannot be
denied. It has simply not been proved for any nuclear inclusions
in virus diseases. Of course all nuclear inclusions are not alike.
In some, elementary bodies may be demonstrated by further work
and in others not.
It is important in this connection to compare, with the most
delicate methods available, nuclear inclusions actually caused
by viruses with others produced experimentally in the absence of
virus.
Those who hold that nuclear inclusions are masses of virus, or
elementary bodies of virus held together in some way by cytoplasmic material, will be the last to admit that they can be
produced experimentally by means other than viruses. Yet there
have been numerous reports recently of the production of nuclear
inclusions by chemical means. 36-39 Such observations usually
fail to supply crucial evidence. It is conceivable that an inapparent virus, resident in the tissue, might have been activated,
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E. V. COWDRY
or tissue resistance to it lowered, by a substance like irradiated
ergosterol, with great increase in nuclear inclusions not directly
produced by the substance in question.
Olitsky and Harford 38 have properly pointed out that in no
instance, save in their own experiments of inclusion formation
occasioned by a variety of foreign substances, was systematic
search made for a virus. The "characteristic nuclear inclusions"
which they induced by the injection of normal brain substance 39
merit very serious consideration. It is to be hoped that these
investigators will supply further details. If the said inclusions
are finely particulate, and the particles are even faintly basophilic,
then the evidence on which Goodpasture and Teague, Goodpasture, and Markham concluded that elementary bodies of virus
are present in the inclusions must be given less weight.
Finally Belt40 has described inclusions in the liver cells of three
men and one woman who died of severe burns, which, he states,
"are morphologically indistinguishable from yellow fever inclusions." He points out that the toxemia in certain cases of
burns produces an effect on the liver similar to that observed in
certain virus diseases and suggests, "that the noxious agent in
both instances, whatever it may be, whether virus or toxins, is of
similar character."
This appears to me to be a very significant suggestion. But
it does not necessarily mean that the yellow fever virus and the
toxic substance produced in burns are of similar character.
Obviously, the virus is capable of inciting a transmissible disease
which the toxic substance in burns cannot. It indicates, however,
that in this instance, and possibly in others, the nuclear inclusions
are not masses of virus but rather manifestations of injury initiated by virus on the one hand, and a physical agent (heat) on
the other. The sequences of events between different primary
causes lead to the same, or to similar results in the shape of
nuclear inclusions. At what stages forces are generated, sufficiently alike to bring about, when they go into action, similar
nuclear injuries, we have no idea. After all, as we have already
insisted, nuclear responses and nuclear materials are limited
so that it would be unsafe to assume that the forces are identical.
INCLUSIONS IN VIRUS DISEASES
143
Injuries produced by somewhat different forces may have features
in common.
3 . CYTOPLASMIC INCLUSIONS
In humans no cytoplasmic inclusions, indicative of virus
action, have thus far been found in the absence of clinical symptoms. That is to say, we have no information on "inapparent"
cytoplasmic inclusion producing viruses but such may exist.
Since the materials available for building cytoplasmic inclusions are more diversified, the responses of the cytoplasm to
environmental changes are more varied, and liability to invasion
by organisms is greater, there is a wider range in properties of
the inclusions. While some of them can be seen after routine
staining with hematoxylin and eosin, special technics are often
required. Cytoplasmic inclusions, as indicators of virus action,
may be grouped under three headings. For the first organismal
nature is undemonstrated and unlikely; for the second, it is likely
and for the third, it can be regarded as proved.
In the first group we include the Negri bodies of rabies and
certain cytoplasmic inclusions in infectious myxomatosis of
rabbits. The evidence provided by Goodpasture41 that Negri
bodies are formed from mitochondria and neurofibrils is not convincing. They remain enigmas. When of considerable size,
they are made up of an acidophilic ground substance in which
basophilic particles are embedded. But it would be premature
to conclude that the particles are themselves tiny organisms of
the category of elementary bodies. The cytoplasmic inclusions
in infectious myxomatosis are finaly particulate and acidophilic.
There is no reason to think that they are elementary bodies which
are usually basophilic.
The second group includes, among others, the cytoplasmic
inclusions in molluscum contagiosum, cowpox and trachoma.
All of these are aggregates of tiny granules that look like elementary bodies. Whether they actually are viruses themselves, or
particular stages in their life cycle (if any), remains to be demonstrated. I am aware also that the Guarnieri bodies in vaccinia
are said to be clumps of elementary bodies of virus, and admit
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E. V. COWDRY
that in some cases they look particulate; but an investigation,
which I made several years ago of their development with the aid
of vital stains, seemed to indicate their formation by overdevelopment of material present in small amounts in uninfected cells.
The nature of the particles in lymphocystis disease of fish42
awaits determination.
The third group consists of intracytoplasmic colonies of
Rickettsiae.43 In these clumps of Rickettsiae, especially in those
of heartwater, 44 the individual Rickettsiae, making up a clump,
are ordinarily of very uniform size but occasionally some organisms are seen which are larger and remind us of the so-called
"initial bodies." In trachoma we frequently encounter, even
in neighboring cells, Prowazek bodies consisting chiefly or solely
of tiny elementary bodies on the one hand and of the larger initial
bodies on the other, though both may occur in a single Prowazek
body. Julianelle's illustrations of Prowazek bodies should be
examined.45
Now when we pass to organisms which are unquestionably
protozoa, like Theileria parva of East Coast fever,46 we observe
that in a certain limited stage of their life cycle they form spherical
cytoplasmic inclusion bodies made up of many individual parasites the appearance of which is reminiscent of the elementary
bodies in the above mentioned virus diseases of group 2 and of the
Rickettsiae, which, before they became well known, were classified as viruses.
It is unwise to reach any conclusion; but, it is evident that the
viruses are a very heterogeneous group. To repeat, many produce no inclusions (poliomyelitis and encephalitis). A few
produce nuclear inclusions in some hosts and not in others
(Louping ill and mad itch). Others produce nuclear inclusions
in different degrees (in humans the yellow fever virus sometimes
does but not always). Others apparently invariably produce
nuclear inclusions (herpes and the salivary gland viruses) in
some of which a few investigators claim to find the virus itself.
Others produce both cytoplasmic and nuclear inclusions (smallpox
and paravaccinia). Others produce cytoplasmic inclusions in
which there are apparently no traces of elementary bodies
INCLUSIONS IN VIRUS DISEASES
145
(Rabies and infectious myxomatosis). Others produce cytoplasmic inclusions which are probably aggregates of elementary bodies
(Molluscum contagiosum and perhaps vaccinia). Others produce
cytoplasmic inclusions which are probably masses of elementary
bodies (trachoma and inclusion blennorrhoea). Others, not
strictly viruses, produce cytoplasmic bodies that are certainly
masses of organisms (the Rickettsioses). And finally there are
inclusions made up of organisms that are surely protozoa (East
Coast Fever).
All this is consistent either with the idea that, under the
heading of viruses, we have many different agents grading from
pathogenic to relatively benign, of different sizes and of different
invasive powers, or that the viruses are essentially parasites which
have become so adapted to an intracellular habitat that they have
progressively lost in different degrees the structural components
necessary for functions concerned with everything except reproduction in terms of Green's hypothesis.47 Perhaps all or most
components, except the giant molecules to which attention has
been frequently directed, become useless and are discarded with
increasing dependence on the cellular hosts.
4. SUMMARY
Clinical pathologists occupy a strategic position in the investigation of virus diseases as they perform almost all of the autopsies.
In the routine histological study of autopsy tissues by the ordinary hematoxylin and eosin technic they will certainly encounter
nuclear inclusions brought about by inapparent viruses in the
absence of reported clinical symptoms. The tissues of special
interest are the salivary glands and kidneys. A search for nuclear
inclusions in infants dead of whooping cough is indicated. Diagnosis of almost all known virus diseases will, however, be made
before autopsy so that the likelihood of meeting with either
nuclear inclusions, cytoplasmic inclusions or no inclusions will be
appreciated in advance. Clinical pathologists will perform a
useful service if they report cases in which the usual course of any
particular virus diseases has been modified by a second virus,
bacterial infections or in other ways. Because they know that
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E. V. CO\fa>ftf
viruses are subject to variation and even to mutation, they will be
on the lookout for the unusual. Biopsies by the thousands are
made by them, particularly of the skin. Dermatology is a
medical specialty now coming of age and these biopsies are likely
to yield some surprising lesions, for the skin is after all the
ancestral home of most viruses.
REFERENCES
Owing to their number, references are omitted but will appear in the author's
reprints.