THE MORPHOLOGY OF VIRAL INCLUSIONS AND THEIR
PRACTICAL IMPORTANCE IN T H E DIAGNOSIS OF
HUMAN DISEASE*
HENRY PINKERTON, M.D.
From the Department of Pathology, St. Louis University School of Medicine,
St. Louis, Missouri
Although the terms "inclusion" and "inclusion body" are applied by cytologists to a wide variety of intracellular structures, their use by the pathologist
usually implies the presence of a virus. The less ambiguous term "viral inclusion"
may be used for intracellular bodies which are known to be caused by the
presence or activity of a virus.
Viral inclusions occur either in the cytoplasm or in the nucleus, a few viruses
causing both cytoplasmic and nuclear inclusions in the same cell. They may be
eosinophilic or basophilic, depending on their nature and also to some extent
on the staining procedure used. In size they range from 1 to 20 microns in greatest dimension but most often approximate the diameter of a red blood cell.
They are usually spherical or ovoid, but occasionally quite irregular in shape.
When situated in the cytoplasm close to the nucleus, they are often indented
by the nuclear membrane or tend to curve around it. Within the nucleus they
are centrally located, and their shape frequently corresponds to that of the
nucleus. Characteristically they are surrounded by a clear zone or "halo".
They may be single or multiple in either cytoplasm or nucleus but are more
often multiple in the cytoplasm. Evidence of associated cellular damage is
present to a variable extent.
Most inclusions are well brought out by staining with hematoxylin and eosin
but use of Giemsa's stain is essential for demonstrating those of the psittacosislymphogranuloma venereum group. Special stains often aid in identification and
study and may provide better contrast with the surrounding cytoplasm or
nucleoplasm.
Internally some viral inclusions are composed of discrete coccoid or bacillary
bodies and others are finely granular, while those associated with the smaller
viruses are, in general, homogeneous but may contain vacuoles.
To one familiar with their appearance, many viral inclusions are definitely
diagnostic and, in at least five viral diseases, diagnosis is completely dependent
on their recognition. To illustrate this point, let us consider the picture of
salivary gland virus infection (the so-called "inclusion disease"). 3,6 Generalized
infection with this virus is a fairly common cause of death in infants and a rare
cause in adults. The diagnostic features are (1) the gigantic size of the affected
cell as compared with its normal neighbors, and (2) the combination of a single
huge nuclear inclusion, usually eosinophilic, with numerous small cytoplasmic
* Presented at, the Twenty-Eighth Annual Meeting of the American Society of Clinical
Pathologists, in Chicago, on October 14, 1949.
Received for publication, October 13, 1949.
201
202
PINKERTON
bodies which tend to be basophilic. This picture could not possibly be mistaken
for that of any other known human viral infection. At present, diagnosis of this
disease can be made only by finding the inclusions. Antemortem diagnosis has
never been made but, since there is marked desquamation of renal epithelium,
these cells could probably be found in the urine.
The importance of this viral infection is not generally recognized. I t is not
included in recent text books of pediatrics or in text books covering the viral
infections of man, probably because it has not been transmitted to animals or
otherwise isolated and the diagnosis must, therefore, be based entirely on the
presence of the inclusions. The same may be said of leprosy, however, which we
diagnose without hesitation on the basis of the presence of the characteristic
bacilli in lepra cells. Under certain conditions, purely morphologic criteria may
have diagnostic significance.
There is some overlapping in the .cytologic pictures of viral infections. For
example, psittacosis and lymphogranuloma venereum cannot be distinguished
morphologically. Such overlapping is, however, usually associated with close
TABLE 1
D I S E A S E S IN W H I C H D I A G N O S I S I S COMPLETELY D E P E N D E N T ON I N C L U S I O N S
L E S I O N S N O T TRANSMITTED TO ANIMALS
1.
2.
3.
4.
5.
Molluscum contagiosum, cytoplasmic
Inclusion disease (salivary gland virus), nuclear and cytoplasmic
Adam's infantile pneumonia, cytoplasmic
Goodpasture's infantile pneumonia, nuclear
H e c h t ' s giant cell pneumonia, cytoplasmic and nuclear
antigenic relationship; in fact, the study of the immunologic relationship between psittacosis and lymphogranuloma venereum was first suggested by the
morphologic similarity in the associated inclusions.
Regarding the nature of viral inclusions, those caused by many large viruses
are known to be simply intracellular clusters or colonies of small microorganisms.
For no really good reason, it is customary to call such microorganisms elementary
bodies if the agent involved is classed as a virus. Similar intracellular clusters
are formed by bartonellae and rickettsiae. The characteristic intracytoplasmic
clusters of Bartonella bacillijormis, seen post mortem in the reticuloendothelial
cells of fatal human infections of Oroya fever,12 differ in no essential way from
certain of the cytoplasmic viral inclusions. When stained by hematoxylin-eosin,
they appear homogeneous. They were observed by pathologists long before
the individual organisms were seen. Rickettsia bumeti, the cause of Q-fever, forms
compact intracellular clusters in the tissues of infected guinea pigs.2 Intranuclear
clusters of rickettsiae are diagnostic of spotted fever in the tissues of ticks,11
and are occasionally seen in mammalian tissues. 9,10 Such intranuclear structures
greatly resemble viral intranuclear inclusions, particularly when they are not
stained sharply enough to bring out their internal structure.10
Bearing in mind, then, the fact that certain minute intracellular bacteria and
VIRAL INCLUSIONS IN HUMAN DISEASE
203
rickettsiae may form compact intracytoplasmic or intranuclear clusters, we may
pass on to those infective agents which are classed, rather arbitrarily, as viruses.
I t is logical to consider next a group of viral diseases in which cytoplasmic inclusions, composed of discrete microorganisms, are seen—namely, the so-called
psittacosis-lymphogranuloma group. This includes trachoma and inclusion conjunctivitis, as well as psittacosis and lymphogranuloma venereum, and a number
of viral infections of lower animals not yet shown to affect man. Inclusions in
this group are basophilic, and best seen in preparations stained by Giemsa's
method, Castaneda's method, or any other staining method suitable for showing
rickettsiae. The organisms or elementary bodies forming these inclusions are
embedded in a matrix which stains differentially with certain technics.16 Whether
this matrix is contributed by the organism or by the host cell is not clear. Homogeneous plaques, 1 to 10 microns in diameter, have been observed in the early
stages of intracellular multiplication, and elementary bodies apparently separate
from these plaques in the evolution of the mature inclusion. The mature inTABLE 2
D I S E A S E S IN W H I C H D I A G N O S I S D E P E N D S LARGELY ON INCLUSIONS
1.
2.
3.
4.
5.
6.
7.
8.
Trachoma, cytoplasmic
Inclusion conjunctivitis (swimming pool), cytoplasmic
Lymphogranuloma venereum, cytoplasmic
Psittacosis (after animal injection), cytoplasmic
Herpes encephalitis, nuclear
Chronic encephalitis with herpes-like inclusions, nuclear
B virus infection, nuclear
Louping ill, nuclear
elusion stains deep purple by the Giemsa's method, and is composed of closely
packed but still discrete elementary bodies which are rickettsia-like in appearance.
No one would deny that inclusions of this type are pathognomonic for this
group of viral infections. The specific virus involved must be determined by evidence, such as the clinical picture and serologic tests.
Another group of cytoplasmic inclusions composed of definite organisms is
that represented in man by the structures seen in smallpox, vaccinia, and
molluscum contagiosum and in animals, by the poxes. These are eosinophilic,
and have a matrix composed of lipoid material usually surrounded by a capsulelike membrane.5 When freed from their capsule and matrix, the elementary bodies
of these viral infections, which are only slightly smaller than rickettsiae, have
been shown by electron microscopy to be similar to rickettsiae and bacteria
in their morphology.4 Again we may say that inclusions of this type are specific
for this group of viruses.
As we pass on to the homogeneous inclusions associated with the smaller
viruses, analogic reasoning would lead to the conclusion that they also may be
composed of exceedingly minute elementary bodies and their secretions, with
204
PINKERTON
or without the addition of a matrix formed from cytologic constituents. I t is
safer, however, to reserve judgment, particularly in view of what I shall say
later about structures resembling the homogeneous viral inclusions which appear
in cells after injury by heavy metals and other poisons.
The nuclear inclusions containing granules are those which Cowdry1 has called
type A. They usually cause severe damage to the cells and are rarely, if ever,
found in the absence of a virus. Cowdry's type B nuclear inclusions are homogeneous, often cause little visible damage to cells, and are not so easily distinguished from non-viral inclusions. This classification has proved helpful in
interpreting nuclear inclusions, but an entirely satisfactory classification of all
viral inclusions has not yet been proposed. For convenience, I am presenting
them in groups which are morphologically similar or identical, and which are
caused by biologically related viruses.
The recognition of specific viral inclusions, like many other diagnostic procedures, requires experience. The diagnosis of rabies in the dog brain should be
TABLE 3
D I S E A S E S I N W H I C H INCLUSIONS ARE P R E S E N T AND O F T E N H E L P F U L IN D I A G N O S I S
1.
2.
3.
4.
5.
6.
7.
S.
Yellow fever, nuclear
Herpes simples dermatitis, nuclear
Herpes zoster, nuclear
Smallpox, cytoplasmic and nuclear
Vaccinia, cytoplasmic
Chicken-pox, cytoplasmic and nuclear
Poliomyelitis, nuclear (?)
Foot-and-mouth disease, nuclear
eft to experts, because of the danger of confusing the inclusions of distemper
with those of rabies. To the expert, however, the Negri body, with its vacuoles
containing basophilic granules, is pathognomonic. It is easy to overlook viral
inclusions if one does not look specifically for them. When only a few are present,
they can often be located best by examination under low or medium power,
but the oil immersion lens is, of course, necessary for detail study.
Structures most often confused with cytoplasmic viral inclusions are ingested
erythrocytes and nuclear lobes of neutrophils, secretion granules, droplets of
hyaline, fatty, mucoid, glycogenic, or calcific nature, Russell fuchsin bodies, and
even the ovoid chromosomes of certain mitotic cells and swollen Nissl bodies
in ganglion cells. These are distinguished from viral inclusions by morphology,
color, location, presence of a halo, occurrence in adjacent cells and other features.
Location is of particular importance. Structures which are normal in liver cord
cells, for example, would be entirely out of place in bile duct epithelium, the
cytoplasm of which normally should be clear and homogeneous.
Structures most often confused with nuclear viral inclusions are fluid vacuoles,
glycogen droplets, and large eosinophilic nucleoli. Although in a few instances
there is some evidence that the nucleolus participates in the formation of viral
inclusions, it is usually present and intact in inclusion-bearing nuclei.
VIRAL INCLUSIONS IN HUMAN DISEASE
205
There can be no doubt that a variety of injurious agents may produce intranuclear structures closely simulating nuclear inclusions of viral origin.7 These
include notably a number of metallic poisons, and the toxins liberated from
burned tissue. The inclusions caused in renal epithelium by lead poisoning perhaps most closely simulate viral inclusions. Wachstein14 recently has shown that
these structures, in contrast to several viral inclusions, are acid-fast, an observation which may prove helpful.
The observation of these intranuclear bodies in non-viral intoxications is
believed by some workers to cast doubt on the diagnostic importance of nuclear
inclusions, in the absence of evidence of experimental transmission of a viral
disease. I can only say that all circumstances must be considered in any given
situation. In ascribing diagnostic significance to the inclusions of the salivary
TABLE 4
V I R A L I N F E C T I O N S W I T H N O INCLUSIONS
1.
2.
3.
4.
5.
6.
7.
8.
0.
10.
11.
12.
13.
14.
15.
16.
Von Eeononio's encephalitis
St. Louis encephalitis
Equine encephalitis
Japanese B encephalitis
Lymphatic choriomeningitis
Russian far-cast encephalitis
Post-infection encephalitis
Measles (?)
German measles
Mumps
Infectious and scrum hepatitis
Epidemic keratoconjunctivitis
Dengue
Influenza
Common cold
P r i m a r y atypical pneumonia
gland virus, for example, we are greatly helped by the fact that entirely similar
inclusions in guinea pigs, mice, and other animals are inseparably associated
with serially transmissible viral infections. In cases of human encephalitis, I
think we are justified in believing that the presence of definite nuclear inclusions
gives strong presumptive evidence that the encephalitis is of viral origin. Nuclear
inclusions in the brain have not been produced by non-viral agents.
Although I have not had a wide experience with inclusions caused by heavy
metals and other known chemicals, I believe that if careful morphologic studies
are made, and all of the attending circumstances studied in each case, the clanger
of confusing non-viral inclusions with viral inclusions is rather slight. In morphologic studies, it is important to study the various stages in the formation of the
inclusions. Thus, while individual inclusions in lead intoxication may resemble
viral inclusions, other forms are present which are quite different from any
known stage in the formation of true viral inclusions.
Nuclear inclusions have been described in gliomas. I have not been convinced
206
PINKERTON
that these structures show any marked resemblance to viral inclusions/Many
of the nuclear inclusions described in human tumors are probably abnormal
nucleoli. The "bird's eye" inclusions described years ago by Leyden in carcinoma
are cytoplasmic. The nature of these bodies and of many other structures described in malignant cells has not been established. It is only in certain tumors
of lower animals, transmissible by cell-free filtrates, that definite viral inclusions
are found. A notable example is Luck6's carcinoma of the frog kidney which
contains nuclear inclusions.
Just a word about the occurrence of inflammation and necrosis in association
with inclusion as related to the problem of their viral etiology. These reactions
are present in variable degree in viral infections and may be completely absent.
In studying the tissues of minks dying in epidemics of distemper, the only
lesion noted in about 10 per cent of the animals was the presence of the inclusions
in otherwise normal cells, and often the inclusions were so rare that one had to
search for five minutes or more to find them. 8 1 have never found any structures
resembling these inclusions in minks dying of other causes, and I am sure that
others have had this same experience. The presence of the inclusions without
other pathologic lesions may, therefore, in some cases be sufficient for the
diagnosis of a fatal viral infection.
Table 1 lists five viral diseases in which the recognition of specific inclusions
is at present the only means of establishing diagnosis. In Table 2, I have listed
eight more diseases in which diagnosis usually depends largely on cytologic
observations. Herpes encephalitis is encephalitis with typical herpetic nuclear
inclusions, from which herpes virus may be transmitted to animals.13 Chronic
encephalitis with herpes-like inclusions is a condition diagnosed solely on the
presence of inclusions. In some of the diseases included in Table 2 animal
transmission is impractical; mothers, the diagnosis is usually made microscopically
on the autopsy material and material for inoculation often is not available.
This brings up the point that in doubtful cases at autopsy, it is a simple
matter to "deep-freeze" the tissues. If inclusions are later found microscopically,
the frozen material becomes most valuable. If the diagnosis becomes clear by
finding an obviously non-viral cause of the death, the material may be discarded.
Table 3 lists eight viral diseases of man in which the recognition of inclusions
is often helpful in diagnosis, and in Table 4 I have named sixteen viral diseases
in which definite inclusions have not been found. I have included post-infection
encephalitis and primary atypical pneumonia, neither of which is of established
viral etiology. I believe the absence of inclusions in viral diseases may be explained in some cases by assuming that the elementary bodies are not clumped
but are diffusely distributed in cells. In other instances relatively small numbers
of elementary bodies may be present. We should also keep in mind the possibility
that some of the smaller viruses may be complex protein molecules. Electron
microscopy will in the future add much to our knowledge of elementary bodies
and their relation to viral inclusions.
In conclusion, I would like to emphasize the value to the pathologist, of a work-
VIRAL INCLUSIONS IN HUMAN DISEASE
207
ing knowledge of the morphology of viral inclusions. I have brought out the fact
that the diagnosis of many viral diseases is based largely or entirely on the recognition of specific cytologic changes. Moreover, the observation of inclusion bodies
in tissues by the pathologist has not infrequently been the first step in the
recognition of a new disease entity. The next step is, of course, the isolation of
the virus, and until this is accomplished there will always be skepticism regarding
the viral nature of any given condition. I have attempted to make the point,
however, that such skepticism is not always justified.
REFERENCES
1. COWDRY, E . V.: T h e problem of intranuclear inclusions in virus diseases. Arch. P a t h . ,
18: 527-542, 1934.
2. D A V I S , G. E . , AND C O X , H . R . : A filter-passing infectious agent isolated from t i c k s ;
isolation from Dermacentor undersoni, reaction in animals, and filtration experiments.
P u b . Health R e p . , 53: 2259-2267, I93S.
3. F A R B E H , S., AND WOLBACH, S. B . : Intranuclear and cytoplasmic inclusions ("protozoanlike bodies") in salivary glands and other organs of infants. Am. J . P a t h . , 8: 123—
136, 1932.
4. G R E E N , R . H . , A N D E R S O N , T . F . , AND S.MADEL, J . E . : Morphological s t r u c t u r e of t h e
virus of vaccinia. J . Ex per. Med., 75: 651-656, 1942.
5. H I M M E L W E I T , F . : Observations on living vaccinia and ectromelia viruses by high power
microscopy. Brit. J . Exper. P a t h . , 19: 10S-123, 1938.
6. MCCORDOCK, H . A., AND SMITH, M . G.: Intranuclear inclusions; incidence and possible
significance in whooping cough and in a variety of other conditions. Am. J . D i s . Child.,
47: 771-779, 1934.
7. OLITSKY, P . K., AND H A R F O R D , C. G . : Intranuclear inclusion bodies in t h e tissue reactions produced by injections of certain foreign substances. Am. J . P a t h . , 13: 729748, 1937.
8. P I N K E R T O N , H . : Immunological and histological studies on mink distemper. J . A m .
Vet. M . A., 96: 347-355, 1940.
9. P I N K E R T O N , H . : T h e pathogenic rickettsiae with particular reference t o their n a t u r e ,
biological properties, and classification. Bact. R e v . , 6 : 37-78, p . 66, 1942.
10. P J N K E R T O N , H., AND H A S S , G. M . : Spotted fever; intranuclear rickettsiae in spotted
fever studied in tissue culture. J . Exper. Med., 56: 151-156, 1932.
11. P I N K E R T O N , I I . , AND H A S S , G. M . : Spotted fever; t h e identification of Dermacentroxenus rickcttsi and its differentiation from non-pathogenic rickettsiae in ticks.
J . Exper. Mod., 66: 729-739, 1937.
12. P I N K E R T O N , H . , AND W E I N M A N , D . : Carrion's disease; comparative morphology of t h e
etiological agent in oroya fever and Verruga P e r u a n a . P r o c . Soc. Exper. Biol, a n d
Med., 37:591-593, 1937.
13. SMITH, M . G., L E N E T T E , E . H . , AND R E A M E S , H . R . : Isolation of t h e virus of herpes
simplex and the demonstration of intranuclear inclusions in a case of acute encephalitis. Am. J . P a t h . , 17: 55-6S, 1941.
14. WACHSTEIN, M . : Studies of inclusion bodies; acid-fastness of nuclear inclusion bodies
t h a t arc induced by ingestion of lead and bismuth. Am. J . Clin. P a t h . , 19: 60S-614,
1949.
15. YANAMURA, H . Y . , AND M E Y E R , K . P . : Studies on t h e virus of psittacosis cultivated
in vitro. J. Infect. Dis., 68: 1-15, 1941.